WO2000063375A1

WO2000063375A1 - Dna encoding a kinesin-like protein (hklp) comprising biallelic markers

Info

Publication number: WO2000063375A1
Application number: PCT/IB2000/000562
Authority: WO
Inventors: Lydie Bougueleret; Isabelle Dufaure-Gare; Pascale Grel
Original assignee: Genset
Priority date: 1999-04-20
Filing date: 2000-04-20
Publication date: 2000-10-26
Also published as: AU3984800A

Abstract

The present invention is directed to polynucleotides encoding a human kinesin-like polypeptide as well as a regulatory region located at the 3'-end of said coding region. The invention also concerns polypeptides encoded by the kinesin-like gene. The invention also deals with antibodies directed specifically against such polypeptides that are useful as diagnostic reagents. The invention further encompasses biallelic markers of the HKLP gene useful in genetic analysis.

Description

DNA ENCODING A KINESIN-LIKE PROTEIN (HKLP ) COMPRISING BIALLELIC MARKERS

FIELD OF THE INVENTION

The present invention is directed to polynucleotides encoding a human kinesin-like polypeptide as well as a regulatory region located at the 3 '-end of said coding region. The invention also concerns polypeptides encoded by the kinesin-like gene. The invention also deals with antibodies directed specifically against such polypeptides that are useful as diagnostic reagents. The invention further encompasses biallelic markers ofthe HKLP gene useful in genetic analysis.

BACKGROUND OF THE INVENTION

The kinesins are mechanochemical proteins utilizing chemical energy from ATP hydrolysis to generate mechanical force. The kinesins can bind to and move on microtubules in the presence of ATP. The ability to move on microtubules has led to the classification of kinesins as microtubule motor proteins. The kinesins play important roles in intracellular transport and cell division.

Several kinesin proteins are involved in vesicle/organelle transport in neurons, and mutants of kinesin in Drosphila show impaired neuronal function. In humans, defects in kinesin-encoding genes could cause neurological disorders or syndromes of clinical importance.

The kinesin proteins carry out or facilitate movements ofthe chromosomes and spindle in meiosis and mitosis. Defective meiotic kinesins in humans may be the causes of infertility, spontaneous abortion, neonatal chromosome disorders, and aneuploidy. In mitotically dividing cells, mutations in kinesin proteins could cause somatic abnormalities or cellular transformation, including neoplasia.

Finally, the kinesins could be involved in developmental processes as the localization of some morphogens has been shown to be microtubule-dependent.

The KIF kinesin superfamily proteins have been identified as candidate motor proteins involved in organelle transport. Among the KJFs, the murine KJF1 A protein has been proposed as a transporter of synaptic vesicle precursors. KIF1A disruption assays in mice allowed to show that KIF1 A is involved in the transport of a synaptic vesicle precursor and that KIF1 A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons (Yonekawa et al., 1998). The murine KIF IB protein is co-localized with mitochondria in vivo and could be 0 involved in the transport of mitochondria (Nangaku et al., 1994).

SUMMARY OF THE INVENTION

The present invention pertains to nucleic acid molecules comprising the genomic sequence of a novel human gene which encodes a kinesin-like protein and which has been named HKLP by the inventors. The HKLP presents homology with murine KIF1A and KIF IB. The HKLP genomic sequence comprises regulatory sequence located downstream (3 '-end) ofthe transcribed portion of said gene, these regulatory sequences being also part ofthe invention.

The invention also deals with the complete cDNA sequence encoding the HKLP protein, as well as with the corresponding translation product. Oligonucleotide probes or primers hybridizing specifically with a HKLP genomic or cDNA sequence are also part ofthe present invention, as well as DNA amplification and detection methods using said primers and probes.

A further object ofthe invention consists of recombinant vectors comprising any ofthe nucleic acid sequences described above, and in particular of recombinant vectors comprising a HKLP regulatory sequence or a sequence encoding a HKLP protein, as well as of cell hosts and transgenic non human animals comprising said nucleic acid sequences or recombinant vectors.

The invention is also directed to biallelic markers that are located within the HKLP genomic sequence or that are in linkage disequilibrium with the HKLP gene, these biallelic markers representing useful tools in order to identify a statistically significant association between specific alleles of HKLP gene and diseases, for example cancer and neurological disorders. These association methods are within the scope ofthe invention.

Finally, the invention is directed to methods for the screening of substances or molecules that inhibit the expression of HKLP, as well as with methods for the screening of substances or molecules that interact with a HKLP polypeptide or that modulate the activity of a HKLP polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a block diagram of an exemplary computer system.

Figure 2 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.

Figure 3 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous.

Figure 4 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence.

BRIEF DESCRIPTION OF THE SEQUENCES PROVTOED IN THE SEQUENCE

LISTING

SEQ ID Nos 1 and 2 contain the genomic sequence ofthe HKLP gene comprising the exons and introns, and the 3' regulatory region (downstream untranscribed region). SEQ ID No 3 contains a cDNA sequence ofthe HKLP gene. SEQ ID No 4 contains the amino acids sequence encoding by the cDNA of SEQ ID No 3. SEQ ID Nos 5, 6, 7 and 8 respectively contain the nucleotide sequence ofthe amplicons 10- 265, 10-266, 12-592 and 12-783.

SEQ ID No 9 contains a primer containing the additional PU 5' sequence described further in Example 2. SEQ ID No 10 contains a primer containing the additional RP 5' sequence described further in Example 2.

In accordance with the regulations relating to Sequence Listings, the following codes have been used in the Sequence Listing to indicate the locations of biallelic markers within the sequences and to identify each ofthe alleles present at the polymorphic base. The code "r" in the sequences indicates that one allele ofthe polymorphic base is a guanine, while the other allele is an adenine. The code "y" in the sequences indicates that one allele ofthe polymorphic base is a thymine, while the other allele is a cytosine. The code "m" in the sequences indicates that one allele ofthe polymorphic base is an adenine, while the other allele is an cytosine. The code "k" in the sequences indicates that one allele ofthe polymorphic base is a guanine, while the other allele is a thymine. The code "s" in the sequences indicates that one allele ofthe polymorphic base is a guanine, while the other allele is a cytosine. The code "w" in the sequences indicates that one allele ofthe polymorphic base is an adenine, while the other allele is an thymine.

The nucleotide code ofthe original allele for each biallelic marker is the following table:

DETAILED DESCRD?TION OF THE INVENTION

The present invention concerns polynucleotides and polypeptides related to the HKLP gene. Oligonucleotide probes and primers hybridizing specifically with a genomic or a cDNA sequence of HKLP are also part ofthe invention. A further object ofthe invention consists of recombinant vectors comprising any ofthe nucleic acid sequences described in the present invention, and in particular recombinant vectors comprising a regulatory region of HKLP or a sequence encoding the HKLP protein, as well as cell hosts comprising said nucleic acid sequences or recombinant vectors. The invention also encompasses methods of screening of molecules which inhibit the expression of the HKLP gene or which modulate the activity ofthe HKLP protein. The invention also deals with antibodies directed specifically against such polypeptides that are useful as diagnostic reagents. The invention also concerns HKXP-related biallelic markers which can be used in any method of genetic analysis including linkage studies in families, linkage disequilibrium studies in populations and association studies of case-control populations. An important aspect ofthe present invention is that biallelic markers allow association studies to be performed to identify genes involved in complex traits.

Definitions

Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope ofthe terms used to describe the invention herein.

The terms "HKLP gene", when used herein, encompasses genomic, mRNA and cDNA sequences encoding the HKLP protein, including the untranslated regulatory regions ofthe genomic DNA.

The term "heterologous protein", when used herein, is intended to designate any protein or polypeptide other than the ΗKLP protein. More particularly, the heterologous protein is a compound which can be used as a marker in further experiments with a HKLP regulatory region. The term "isolated" requires that the material be removed from its original environment (e. g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all ofthe coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.

The term "purified" does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. As an example, purification from 0.1 % concentration to 10 % concentration is two orders of magnitude. The term "purified" is used herein to describe a polynucleotide or polynucleotide vector ofthe invention which has been separated from other compounds including, but not limited to other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis ofthe polynucleotide), or the separation of covalently closed polynucleotides from linear polynucleotides. A polynucleotide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polynucleotide sequence and conformation (linear versus covalently close). A substantially pure polynucleotide typically comprises about 50%, preferably 60 to 90% weight/weight of a nucleic acid sample, more usually about 95%, and preferably is over about 99% pure. Polynucleotide purity or homogeneity is indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.

The term "polypeptide" refers to a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

The term "recombinant polypeptide" is used herein to refer to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.

The term "purified" is used herein to describe a polypeptide ofthe invention which has been separated from other compounds including, but not limited to nucleic acids, lipids, carbohydrates and other proteins. A polypeptide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polypeptide sequence. A substantially pure polypeptide typically comprises about 50%, preferably 60 to 90% weight/weight of a protein sample, more usually about 95%, and preferably is over about 99% pure. Polypeptide purity or homogeneity is indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art. As used herein, the term "non-human animal" refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used herein, the term "animal" is used to refer to any vertebrate, preferable a mammal. Both the terms "animal" and "mammal" expressly embrace human subjects unless preceded with the term "non-human".

As used herein, the term "antibody" refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where an antibody binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen. Antibodies include recombinant proteins comprising the binding domains, as wells as fragments, including Fab, Fab', F(ab)₂, and F(ab')₂ fragments. As used herein, an "antigenic determinant" is the portion of an antigen molecule, in this case a HKLP polypeptide, that determines the specificity ofthe antigen-antibody reaction. An "epitope" refers to an antigenic determinant of a polypeptide. An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 6 such amino acids, and more usually at least 8-10 such amino acids. Methods for determining the amino acids which make up an epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method described by Geysen et al. 1984; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506.

Throughout the present specification, the expression "nucleotide sequence" may be employed to designate indifferently a polynucleotide or a nucleic acid. More precisely, the expression "nucleotide sequence" encompasses the nucleic material itself and is thus not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically characterizes a specific DNA or RNA molecule.

As used interchangeably herein, the terms "nucleic acids", "oligonucleotides", and "polynucleotides" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term "nucleotide" as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single- stranded or duplex form. The term "nucleotide" is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term "nucleotide" is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO 95/04064. The polynucleotide sequences ofthe invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art. A sequence which is "operably linked" to a regulatory sequence such as a promoter means that said regulatory element is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression ofthe nucleic acid of interest. As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription ofthe coding sequence.

The terms "trait" and "phenotype" are used interchangeably herein and refer to any visible, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example. Typically the terms "trait" or "phenotype" are used herein to refer to symptoms of, or susceptibility to a disease, a beneficial response to or side effects related to a treatment. Preferably, said trait can be, without to be limited to, cancers, developmental diseases, and neurological diseases.

The term "allele" is used herein to refer to variants of a nucleotide sequence. A biallelic polymorphism has two forms. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.

The term "heterozygosity rate" is used herein to refer to the incidence of individuals in a population which are heterozygous at a particular allele. In a biallelic system, the heterozygosity rate is on average equal to 2P_a(l-P_a), where P_a is the frequency ofthe least common allele. In order to be useful in genetic studies, a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.

The term "genotype" as used herein refers the identity ofthe alleles present in an individual or a sample. In the context ofthe present invention, a genotype preferably refers to the description ofthe biallelic marker alleles present in an individual or a sample. The term "genotyping" a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.

The term "haplotype" refers to a combination of alleles present in an individual or a sample. In the context ofthe present invention, a haplotype preferably refers to a combination of biallelic marker alleles found in a given individual and which may be associated with a phenotype. The term "polymorphism" as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. "Polymorphic" refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A "polymorphic site" is the locus at which the variation occurs. A single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide or insertion of a single nucleotide also gives rise to single nucleotide polymorphisms. In the context ofthe present invention, "single nucleotide polymorphism" preferably refers to a single nucleotide substitution. Typically, between different individuals, the polymorphic site may be occupied by two different nucleotides.

The term "biallelic polymorphism" and "biallelic marker" are used interchangeably herein to refer to a single nucleotide polymorphism having two alleles at a fairly high frequency in the population. A "biallelic marker allele" refers to the nucleotide variants present at a biallelic marker site. Typically, the frequency ofthe less common allele ofthe biallelic markers ofthe present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (i.e. heterozygosity rate of at least 0.42). A biallelic marker wherein the frequency ofthe less common allele is 30% or more is termed a "high quality biallelic marker".

The location of nucleotides in a polynucleotide with respect to the center ofthe polynucleotide are described herein in the following manner. When a polynucleotide has an odd number of nucleotides, the nucleotide at an equal distance from the 3' and 5' ends ofthe polynucleotide is considered to be "at the center" ofthe polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be "within 1 nucleotide ofthe center." With an odd number of nucleotides in a polynucleotide any ofthe five nucleotides positions in the middle ofthe polynucleotide would be considered to be within 2 nucleotides ofthe center, and so on. When a polynucleotide has an even number of nucleotides, there would be a bond and not a nucleotide at the center ofthe polynucleotide. Thus, either ofthe two central nucleotides would be considered to be "within 1 nucleotide ofthe center" and any ofthe four nucleotides in the middle ofthe polynucleotide would be considered to be "within 2 nucleotides ofthe center", and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is "at the center" of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides ofthe polymorphism and the 3' end ofthe polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides ofthe polymorphism and the 5' end ofthe polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be "within 1 nucleotide ofthe center." If the difference is 0 to 5, the polymorphism is considered to be "within 2 nucleotides ofthe center." If the difference is 0 to 7, the polymorphism is considered to be "within 3 nucleotides ofthe center," and so on.

The terms "complementary" or "complement thereof are used herein to refer to the sequences of polynucleotides which is capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety ofthe complementary region. For the purpose ofthe present invention, a first polynucleotide is deemed to be complementary to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base. Complementary bases are, generally, A and T (or A and U), or C and G. "Complement" is used herein as a synonym from "complementary polynucleotide", "complementary nucleic acid" and "complementary nucleotide sequence". These terms are applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.

Variants and Fragments

1- Polynucleotides

The invention also relates to variants and fragments ofthe polynucleotides described herein, particularly of a HKLP gene containing one or more biallelic markers according to the invention.

Variants of polynucleotides, as the term is used herein, are polynucleotides that differ from a reference polynucleotide. A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants ofthe polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences ofthe reference and the variant are closely similar overall and, in many regions, identical.

Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences which are at least 95% identical to a polynucleotide selected from the group consisting ofthe nucleotide sequences of SEQ ID Nos 1-3 or to any polynucleotide fragment of at least 8 consecutive nucleotides of a polynucleotide selected from the group consisting ofthe nucleotide sequences of SEQ ID Nos 1-3, and preferably at least 99% identical, more particularly at least 99.5% identical, and most preferably at least 99.8% identical to a polynucleotide selected from the group consisting ofthe nucleotide sequences of SEQ ID Nos 1-3 or to any polynucleotide fragment of at least 8 consecutive nucleotides of a polynucleotide selected from the group consisting ofthe nucleotide sequences of SEQ ID No 1-3. Nucleotide changes present in a variant polynucleotide may be silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.

In the context ofthe present invention, particularly preferred embodiments are those in which the polynucleotides encode polypeptides which retain substantially the same biological function or activity as the mature HKLP protein, or those in which the polynucleotides encode polypeptides which maintain or increase a particular biological activity, while reducing a second biological activity A polynucleotide fragment is a polynucleotide having a sequence that is entirely the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a HKLP gene, and variants thereof. The fragment can be a portion of an intron or an exon of a HKLP gene. It can also be a portion ofthe regulatory regions of HKLP. Preferably, such fragments comprise at least one ofthe biallelic markers Al to A32 or the complements thereto or a biallelic marker in linkage disequilibrium therewith.

Such fragments may be "free-standing", i.e. not part of or fused to other polynucleotides, or they may be comprised within a single larger polynucleotide of which they form a part or region. Indeed, several of these fragments may be present within a single larger polynucleotide. Optionally, such fragments may consist of, or consist essentially of a contiguous span of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length. 2- Polypeptides

The invention also relates to variants, fragments, analogs and derivatives ofthe polypeptides described herein, including mutated HKLP proteins. The variant may be 1) one in which one or more ofthe amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more ofthe amino acid residues includes a substituent group, or 3) one in which the mutated HKLP is fused with another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or 4) one in which the additional amino acids are fused to the mutated HKLP, such as a leader or secretory sequence or a sequence which is employed for purification ofthe mutated HKLP or a preprotein sequence. Such variants are deemed to be within the scope of those skilled in the art.

A polypeptide fragment is a polypeptide having a sequence that entirely is the same as part but not all of a given polypeptide sequence, preferably a polypeptide encoded by a HKLP gene and variants thereof.

In the case of an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several amino acids can be replaced by "equivalent" amino acids. The expression "equivalent" amino acid is used herein to designate any amino acid that may be substituted for one ofthe amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged. Generally, the following groups of amino acids represent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, He, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His. A specific embodiment of a modified HKLP peptide molecule of interest according to the present invention, includes, but is not limited to, a peptide molecule which is resistant to proteolysis, is a peptide in which the -CONH- peptide bond is modified and replaced by a (CH2NH) reduced bond, a (NHCO) retro inverso bond, a (CH2-O) methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO-CH2) cetomethylene bond, a (CHOH-CH2) hydroxyethylene bond), a (N-N) bound, a E-alcene bond or also a -CH=CH- bond. The invention also encompasses a human HKLP polypeptide or a fragment or a variant thereof in which at least one peptide bond has been modified as described above.

Such fragments may be "free-standing", i.e. not part of or fused to other polypeptides, or they may be comprised within a single larger polypeptide of which they form a part or region. However, several fragments may be comprised within a single larger polypeptide.

As representative examples of polypeptide fragments ofthe invention, there may be mentioned those which have a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids.

Identity Between Nucleic Acids Or Polypeptides

The terms "percentage of sequence identity" and "percentage homology" are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion ofthe polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment ofthe two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any ofthe variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLΛSTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al., 1990; Thompson et al., 1994; Higgins et al., 1996; Altschul et al., 1990; Altschul et al., 1993). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well known in the art (see, e.g., Karlin and Altschul, 1990; Altschul et al., 1990, 1993, 1997). In particular, five specific BLAST programs are used to perform the following task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992; Henikoff and Henikoff, 1993). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user- specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990).

Stringent Hybridization Conditions

For the purpose of defining such a hybridizing nucleic acid according to the invention, the stringent hybridization conditions are the followings : the hybridization step is realized at 65°C in the presence of 6 x SSC buffer, 5 x Denhardt's solution, 0,5% SDS and lOOμg/ml of salmon sperm DNA.

The hybridization step is followed by four washing steps :

- two washings during 5 min, preferably at 65°C in a 2 x SSC and 0.1%SDS buffer;

- one washing during 30 min, preferably at 65°C in a 2 x SSC and 0.1% SDS buffer,

- one washing during 10 min, preferably at 65°C in a 0.1 x SSC and 0.1%SDS buffer, these hybridization conditions being suitable for a nucleic acid molecule of about 20 nucleotides in length. There is no need to say that the hybridization conditions described above are to be adapted according to the length ofthe desired nucleic acid, following techniques well known to the one skilled in the art. The suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgins (1985).

Genomic Sequences Of The HKLP Gene

The present invention concerns the genomic sequence of HKLP comprising the 2 genomic contigs of SEQ ID Nos 1 and 2. The present invention encompasses HKLP gene, or HKLP genomic sequences consisting of, consisting essentially of, or comprising a sequence selected from the group consisting of SEQ ID Nos 1 and 2, a sequence complementary thereto, as well as fragments and variants thereof. These polynucleotides may be purified, isolated, or recombinant. The invention also encompasses a purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with a nucleotide sequence selected from the group consisting of SEQ ID Nos 1 and 2 or a complementary sequence thereto or a fragment thereof. The nucleotide differences as regards to the nucleotide sequences of SEQ ID Nos 1 and 2 may be generally randomly distributed throughout the entire nucleic acid. Nevertheless, preferred nucleic acids are those wherein the nucleotide differences as regards to the nucleotide sequences of SEQ ID Nos 1 and 2 are predominantly located outside the coding sequences contained in the exons. These nucleic acids, as well as their fragments and variants, may be used as oligonucleotide primers or probes in order to detect the presence of a copy of the HKLP gene in a test sample, or alternatively in order to amplify a target nucleotide sequence within the HKLP sequences.

Another object ofthe invention consists of a purified, isolated, or recombinant nucleic acids that hybridizes with a nucleotide sequence selected from the group consisting of SEQ ID Nos 1 and 2 or a complementary sequence thereto or a variant thereof, under the stringent hybridization conditions as defined above.

Particularly preferred nucleic acids ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, 10, 20, 30, 40 or 50 ofthe following nucleotide positions of SEQ ID No 1 : 1-39624, 39705-40589, 40666-43629, 43710-44203, 4431 1- 45125, 45210-45440, 45622-45717, 45791-68580, 68675-70246, 70396-72421, 72601-73295, 73434-74648, 74898-83055, 83175-85192, 85279-85609, 85740-85906, 86070-88304, 88396- 90585, 90705-91767, 91824-94380, 94490-96296, 96364-97184, 97270-101 167, 101274-109465, 109581-110228, 110363-11 1819, 1 11882-1 13636, 1 13783-1 13945, 1 14186-1 17002, 1 17075- 1 19676, and 119677-121162.

Additional preferred nucleic acids ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659-3829 and 8885-10884.

Additional preferred nucleic acids ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1, or 2, or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 nucleotide positions of any one of the following ranges of nucleotide positions of

(a) SEQ ID No 1 : 1-1000, 1001-2000, 2001-3000, 3001-4000, 4001-5000, 5001-6000, 6001-7000, 7001-8000, 8001-9000, 9001-10000, 10001-1 1000, 1 1001-12000, 12001-13000, 13001- 14 14000, 14001-15000, 15001-16000, 16001-17000, 17001-18000, 18001-19000, 19001-20000, 20001-21000, 21001-22000, 22001-23000, 23001-24000, 24001-25000, 25001-26000, 26001- 27000, 27001-28000, 28001-29000, 29001-30000, 30001-31000, 31001-32000, 32001-33000, 33001-34000, 34001-35000, 35001-36000, 36001-37000, 37001-38000, 38001-39000, 39001-

5 39624, 39705-40589, 40666-43629, 43710-44203, 44311-45125, 45210-45440, 45622-45717, 45791-68580, 68675-70246, 70396-72421, 72601-73295, 73434-74648, 74898-83055, 83175- 85192, 85279-85609, 85740-85906, 86070-88304, 88396-90585, 90705-91767, 91824-94380, 94490-96296, 96364-97184, 97270-101167, 101274-109465, 109581-110228, 110363-1 1 1819, 111882-113636, 1 13783-113945, 114186-117002, 1 17075-1 19676, and 119677-121162; and 0 (b) SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659-3829 and 8885-10884.

Additional preferred nucleic acids ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises a G at position 7159 of SEQ ID No 1. Further preferred 5 nucleic acids ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises a C either at position 2551 or 4500 of SEQ ID No 2. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this 0 section.

The HKLP genomic nucleic acid comprises at least 48 exons. The exon positions in SEQ ID Nos 1 and 2 are detailed below in the Table A. The first exon which has been identified in the cDNA ofthe present invention is not comprised in the genomic sequence described in the present invention. The sequence ofthe first exon begins at the position 1 of SEQ ID No 3 and ends at the 5 position 292. The genomic sequence of SEQ ID Nos 1 and 2 comprises respectively 44 and 4 exons. Thus, the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting ofthe exons ofthe HKLP gene, or a sequence complementary thereto. The invention also deals with purified, isolated, or recombinant nucleic acids comprising a combination of at least two exons ofthe HKLP gene, wherein the 0 polynucleotides are arranged within the nucleic acid, from the 5 '-end to the 3 '-end of said nucleic acid, in the same order as in SEQ ID Nos 1 and 2.

The position ofthe introns is detailed in Table A. Thus, the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting ofthe introns ofthe HKLP gene, or a sequence complementary thereto. 5 Thus, the present invention deals with a purified or isolated nucleic acid encoding a HKLP protein having the amino acid sequence of SEQ ID No 4 or a peptide fragment or variant thereof. In a specific embodiment, such a purified or isolated nucleic acid comprises a polynucleotide selected from the group consisting of SEQ ID Nos 1 and 2, or a complementary sequence thereto or a fragment or a variant thereof.

Table A

The HKLP genomic sequence is covered by two fragments. Indeed, one segment is unknown in the intron 44. The inventors think that this segment, which seems to comprise 20 to 30 nucleotides, forms a superstructure which prevents the sequencing. This superstructure comprises two polyG at each end ofthe segment. While this section is entitled "Genomic Sequences of HKLP," it should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of HKLP on either side or between two or more such genomic sequences.

HKLP cDNA Sequences The expression ofthe HKLP gene has been shown to lead to the production of at least one mRNA species, the nucleic acid sequence of which is set forth in SEQ ID No 3.

Another object ofthe invention is a purified, isolated, or recombinant nucleic acid comprising the nucleotide sequence of SEQ ID No 3, complementary sequences thereto, as well as allelic variants, and fragments thereof. Moreover, preferred polynucleotides ofthe invention include purified, isolated, or recombinant HKLP cDNAs consisting of, consisting essentially of, or comprising the sequence of SEQ ID No 3. Particularly preferred embodiments ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 3: 391-1619 and 6988-10682. Additional preferred embodiments ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises a nucleotide selected in the group consisting of a C at position 5487, and a C at position 6265 of SEQ ID No 3.

The invention also pertains to a purified or isolated nucleic acid having at least 95% of nucleotide identity with the nucleotide sequence of SEQ ID No 3 or a fragment thereof or a complementary sequence thereto, advantageously 99 %, preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with the nucleotide sequence of SEQ ID No 3 or a fragment thereof or a complementary sequence thereto.

Another object ofthe invention consists of a purified, isolated, or recombinant nucleic acids that hybridizes with the nucleotide sequence of SEQ ID No 3 or a complementary sequence thereto or a variant thereof, under the stringent hybridization conditions as defined above.

The cDNA of SEQ ID No 3 includes a 5'-UTR region starting from the nucleotide at position 1 and ending at the nucleotide in position 186 of SEQ ID No 3. The cDNA of SEQ ID No 3 includes a 3'-UTR region starting from the nucleotide at position 5638 and ending at the nucleotide at position 10682 of SEQ ID No 3. The polyadenylation site starts from the nucleotide at position 10631 and ends at the nucleotide in position 10636 of SEQ ID No 3.

Consequently, the invention concerns a purified, isolated, and recombinant nucleic acids comprising a nucleotide sequence ofthe 3'UTR ofthe HKLP cDNA, a sequence complementary thereto, or an allelic variant thereof.

While this section is entitled " HKLP cDNA Sequences," it should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of HKLP on either side or between two or more such genomic sequences.

Coding Regions

The HKLP open reading frame is contained in the corresponding mRNA of SEQ ID No 3. More precisely, the effective HKLP coding sequence (CDS) includes the region between nucleotide position 187 (first nucleotide ofthe ATG codon) and nucleotide position 5637 (end nucleotide ofthe TGA codon) of SEQ ID No 3. The present invention also embodies isolated, purified, and recombinant polynucleotides which encode a polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 or 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 ofthe SEQ ID No 4. The above disclosed polynucleotide that contains the coding sequence ofthe HKLP gene may be expressed in a desired host cell or a desired host organism, when this polynucleotide is placed under the control of suitable expression signals. The expression signals may be either the expression signals contained in the regulatory regions in the HKLP gene ofthe invention or in contrast the signals may be exogenous regulatory nucleic sequences. Such a polynucleotide, when placed under the suitable expression signals, may also be inserted in a vector for its expression and/or amplification.

Regulatory Sequences Of HKLP

As mentioned, the genomic sequence ofthe HKLP gene contains regulatory sequences in the non-coding 3'-flanking region that border the HKLP coding region. The 3'-regulatory sequence of the HKLP gene is localized between nucleotide position 8885 and nucleotide position 10884 of SEQ ID No 2. Polynucleotides derived from the 3' regulatory region are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID No 2 or a fragment thereof in a test sample.

In order to identify the relevant biologically active polynucleotide fragments or variants of the 3 'regulatory region from SEQ ID No 2, the one skill in the art will refer to the book of Sambrook et al.(Sambrook, 1989) which describes the use of a recombinant vector carrying a marker gene (i.e. beta galactosidase, chloramphenicol acetyl transferase, etc.) the expression of which will be detected when placed under the control of a biologically active polynucleotide fragments or variants of SEQ ID No 2. The level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site. The presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a biologically active polynucleotide in the insert.

Polynucleotides carrying the regulatory elements located at the 3' end ofthe HKLP coding region may be advantageously used to control the transcriptional and translational activity of an heterologous polynucleotide of interest. Thus, the present invention also concerns a purified or isolated nucleic acid comprising a polynucleotide ofthe 3' regulatory regions, or a sequence complementary thereto or a biologically active fragment or variant thereof.

Preferred fragments ofthe 3' regulatory region are at least 50, 100, 150, 200, 300 or 400 bases in length. By "biologically active" polynucleotide derivatives of SEQ ID No 2 are polynucleotides comprising or alternatively consisting in a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host. It could act either as an enhancer or as a repressor.

For the purpose ofthe invention, a nucleic acid or polynucleotide is "functional" as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcriptional and translational regulatory information, and such sequences are "operably linked" to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide.

The regulatory polynucleotides ofthe invention may be prepared from the nucleotide sequence of SEQ ID No 2 by cleavage using suitable restriction enzymes, as described for example in the book of Sambrook et al.(1989). The regulatory polynucleotides may also be prepared by digestion of SEQ ID No 2 by an exonuclease enzyme, such as Bal31 (Wabiko et al., 1986). These regulatory polynucleotides can also be prepared by nucleic acid chemical synthesis, as described elsewhere in the specification. The regulatory polynucleotides according to the invention may be part of a recombinant expression vector that may be used to express a coding sequence in a desired host cell or host organism. The recombinant expression vectors according to the invention are described elsewhere in the specification.

A preferred 3'-regulatory polynucleotide ofthe invention includes the 3 '-untranslated region (3'-UTR) ofthe HKLP cDNA, or a biologically active fragment or variant thereof. Polynucleotide Constructs

The terms "polynucleotide construct" and "recombinant polynucleotide" are used interchangeably herein to refer to linear or circular, purified or isolated polynucleotides that have been artificially designed and which comprise at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their initial natural environment.

DNA Construct That Enables Directing Temporal And Spatial HKLP Gene Expression In Recombinant Cell Hosts And In Transgenic Animals.

In order to study the physiological and phenotypic consequences of a lack of synthesis ofthe HKLP protein, both at the cell level and at the multi cellular organism level, the invention also encompasses DNA constructs and recombinant vectors enabling a conditional expression of a specific allele ofthe HKLP genomic sequence or cDNA and also of a copy of this genomic sequence or cDNA harboring substitutions, deletions, or additions of one or more bases as regards to the HKLP nucleotide sequence of SEQ ID Nos 1-3, or a fragment thereof, these base substitutions, deletions or additions being located either in an exon, an intron or a regulatory sequence, but preferably in an exon ofthe HKLP genomic sequence or within the HKLP cDNA of SEQ ID No 3. In a preferred embodiment, the HKLP sequence comprises a biallelic marker ofthe present invention. In a preferred embodiment, the HKLP sequence comprises a biallelic marker ofthe present invention, preferably one ofthe biallelic markers Al to A32.

The present invention embodies recombinant vectors comprising any one ofthe polynucleotides described in the present invention.

A first preferred DNA construct is based on the tetracycline resistance operon tet from E. coli transposon TnlO for controlling the HKLP gene expression, such as described by Gossen et al.(1992, 1995) and Furth et al.(1994). Such a DNA construct contains seven tet operator sequences from TnlO (tetop) that are fused to a minimal promoter, said minimal promoter being operably linked to a polynucleotide of interest that codes either for a sense or an antisense oligonucleotide or for a polypeptide, including a HKLP polypeptide or a peptide fragment thereof. This DNA construct is functional as a conditional expression system for the nucleotide sequence of interest when the same cell also comprises a nucleotide sequence coding for either the wild type (tTA) or the mutant (rTA) repressor fused to the activating domain of viral protein VP16 of heφes simplex virus, placed under the control of a promoter, such as the HCMVIE1 enhancer/promoter or the MMTV-LTR. Indeed, a preferred DNA construct ofthe invention comprise both the polynucleotide containing the tet operator sequences and the polynucleotide containing a sequence coding for the tTA or the rTA repressor.

In a specific embodiment, the conditional expression DNA construct contains the sequence encoding the mutant tetracycline repressor rTA, the expression ofthe polynucleotide of interest is silent in the absence of tetracycline and induced in its presence. DNA Constructs Allowing Homologous Recombination: Replacement Vectors

A second preferred DNA construct will comprise, from 5 '-end to 3 '-end: (a) a first nucleotide sequence that is comprised in the HKLP genomic sequence; (b) a nucleotide sequence comprising a positive selection marker, such as the marker for neomycine resistance (neo); and (c) a 5 second nucleotide sequence that is comprised in the HKLP genomic sequence, and is located on the genome downstream the first HKLP nucleotide sequence (a).

In a preferred embodiment, this DNA construct also comprises a negative selection marker located upstream the nucleotide sequence (a) or downstream the nucleotide sequence (c). Preferably, the negative selection marker consists ofthe thymidine kinase (tk) gene (Thomas et al.,

10 1986), the hygromycine beta gene (Te Riele et al., 1990), the hprt gene ( Van der Lugt et al., 1991; Reid et al., 1990) or the Diphteria toxin A fragment (Dt-A) gene (Nada et al., 1993; Yagi et al.1990). Preferably, the positive selection marker is located within a HKLP exon sequence so as to interrupt the sequence encoding a HKLP protein. These replacement vectors are described, for example, by Thomas et al.(1986; 1987), Mansour et al.(1988) and Koller et al.(1992).

15 The first and second nucleotide sequences (a) and (c) may be indifferently located within a

HKLP regulatory sequence, an intronic sequence, an exon sequence or a sequence containing both regulatory and/or intronic and/or exon sequences. The size ofthe nucleotide sequences (a) and (c) ranges from 1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.

20 DNA Constructs Allowing Homologous Recombination: Cre-LoxP System.

These new DNA constructs make use ofthe site specific recombination system ofthe PI phage. The PI phage possesses a recombinase called Cre which interacts specifically with a 34 base pairs loxP site. The loxP site is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et al., 1986). The recombination by the Cre enzyme between two

25 /O P sites having an identical orientation leads to the deletion ofthe DNA fragment.

The Cre-/oj P system used in combination with a homologous recombination technique has been first described by Gu et al.(1993, 1994). Briefly, a nucleotide sequence of interest to be inserted in a targeted location ofthe genome harbors at least two loxV sites in the same orientation and located at the respective ends of a nucleotide sequence to be excised from the recombinant

30 genome. The excision event requires the presence ofthe recombinase (Cre) enzyme within the nucleus ofthe recombinant cell host. The recombinase enzyme may be brought at the desired time either by (a) incubating the recombinant cell hosts in a culture medium containing this enzyme, by injecting the Cre enzyme directly into the desired cell, such as described by Araki et al.(1995), or by lipofection ofthe enzyme into the cells, such as described by Baubonis et al.(1993); (b) transfecting

35 the cell host with a vector comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter being optionally inducible, said vector being introduced in the recombinant cell host, such as described by Gu et al.(1993) and Sauer et al.(1988); (c) introducing in the genome ofthe cell host a polynucleotide comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter is optionally inducible, and said polynucleotide being inserted in the genome ofthe cell host either by a random insertion event or an homologous recombination event, such as described by Gu et al.(1994).

In a specific embodiment, the vector containing the sequence to be inserted in the HKLP gene by homologous recombination is constructed in such a way that selectable markers are flanked by loxP sites ofthe same orientation, it is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while leaving the HKLP sequences of interest that have been inserted by an homologous recombination event. Again, two selectable markers are needed: a positive selection marker to select for the recombination event and a negative selection marker to select for the homologous recombination event. Vectors and methods using the Cre-loxP system are described by Zou et al .(1994).

Thus, a third preferred DNA construct ofthe invention comprises, from 5'-end to 3'-end: (a) a first nucleotide sequence that is comprised in the HKLP genomic sequence; (b) a nucleotide sequence comprising a polynucleotide encoding a positive selection marker, said nucleotide sequence comprising additionally two sequences defining a site recognized by a recombinase, such as a loxP site, the two sites being placed in the same orientation; and (c) a second nucleotide sequence that is comprised in the HKLP genomic sequence, and is located on the genome downstream ofthe first HKLP nucleotide sequence (a).

The sequences defining a site recognized by a recombinase, such as a loxP site, are preferably located within the nucleotide sequence (b) at suitable locations bordering the nucleotide sequence for which the conditional excision is sought. In one specific embodiment, two lox? sites are located at each side ofthe positive selection marker sequence, in order to allow its excision at a desired time after the occurrence ofthe homologous recombination event.

In a preferred embodiment of a method using the third DNA construct described above, the excision ofthe polynucleotide fragment bordered by the two sites recognized by a recombinase, preferably two loxP sites, is performed at a desired time, due to the presence within the genome of the recombinant host cell of a sequence encoding the Cre enzyme operably linked to a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al.(1994).

The presence ofthe Cre enzyme within the genome ofthe recombinant cell host may result ofthe breeding of two transgenic animals, the first transgenic animal bearing the HKXR-derived sequence of interest containing the loxP sites as described above and the second transgenic animal bearing the Cre coding sequence operably linked to a suitable promoter sequence, such as described by Gu et al.(1994). Spatio-temporal control ofthe Cre enzyme expression may also be achieved with an adenovirus based vector that contains the Cre gene thus allowing infection of cells, or in vivo infection of organs, for delivery ofthe Cre enzyme, such as described by Anton and Graham (1995) and Kanegae et al.(1995). The DNA constructs described above may be used to introduce a desired nucleotide sequence ofthe invention, preferably a HKLP genomic sequence or a HKLP cDNA sequence, and most preferably an altered copy of a HKLP genomic or cDNA sequence, within a predetermined location ofthe targeted genome, leading either to the generation of an altered copy of a targeted gene (knock-out homologous recombination) or to the replacement of a copy ofthe targeted gene by another copy sufficiently homologous to allow an homologous recombination event to occur (knock- in homologous recombination). In a specific embodiment, the DNA constructs described above may be used to introduce a HKLP genomic sequence or a HKLP cDNA sequence. Optionally, said sequence comprises at least one biallelic marker ofthe present invention, preferably at least one biallelic marker selected from the group consisting of Al to A32.

Nuclear Antisense DNA Constructs

Other compositions containing a vector ofthe invention comprising an oligonucleotide fragment ofthe nucleic sequence SEQ ID No 3, preferably a fragment including the start codon of the HKLP gene, as an antisense tool that inhibits the expression ofthe corresponding HKLP gene. Preferred methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al.(1995) or those described in PCT Application No WO 95/24223.

Preferably, the antisense tools are chosen among the polynucleotides (15-200 bp long) that are complementary to the 5 'end ofthe HKLP mRNA. In one embodiment, a combination of different antisense polynucleotides complementary to different parts ofthe desired targeted gene are used.

Preferred antisense polynucleotides according to the present invention are complementary to a sequence ofthe mRNAs of HKLP that contains either the translation initiation codon ATG or a splicing site. Further preferred antisense polynucleotides according to the invention are complementary ofthe splicing site ofthe HKLP mRNA. Preferably, the antisense polynucleotides ofthe invention have a 3' polyadenylation signal that has been replaced with a self-cleaving ribozyme sequence, such that RNA polymerase II transcripts are produced without poly(A) at their 3' ends, these antisense polynucleotides being incapable of export from the nucleus, such as described by Liu et al.(1994). In a preferred embodiment, these HKLP antisense polynucleotides also comprise, within the ribozyme cassette, a histone stem-loop structure to stabilize cleaved transcripts against 3'-5' exonucleolytic degradation, such as the structure described by Eckner et al.(1991). Oligonucleotide Probes And Primers

Polynucleotides derived from the HKLP gene are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID Nos 1-3, or a fragment, complement, or variant thereof in a test sample.

5 Particularly preferred probes and primers ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 a nucleotide of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 1 : 1-39624, 39705-40589, 40666-43629, 43710-44203, 4431 1-45125,

10 45210-45440, 45622-45717, 45791-68580, 68675-70246, 70396-72421, 72601-73295, 73434- 74648, 74898-83055, 83175-85192, 85279-85609, 85740-85906, 86070-88304, 88396-90585, 90705-91767, 91824-94380, 94490-96296, 96364-97184, 97270-101 167, 101274-109465, 109581- 1 10228, 1 10363-1 11819, 1 11882-1 13636, 113783-113945, 1 14186-117002, 1 17075-1 19676, and 1 19677-121162. Additional preferred probes and primers ofthe invention include isolated, purified,

15 or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659-3829 and 8885-10884.

Additional preferred probes and primers ofthe invention include isolated, purified, or

20 recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No lor 2, or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 nucleotide positions of any one ofthe following ranges of nucleotide positions of:

(a) SEQ ID No 1 : 1-1000, 1001-2000, 2001-3000, 3001-4000, 4001-5000, 5001-6000,

25 6001-7000, 7001-8000, 8001-9000, 9001-10000, 10001-1 1000, 1 1001-12000, 12001-13000, 13001- 14000, 14001-15000, 15001-16000, 16001-17000, 17001-18000, 18001-19000, 19001-20000, 20001-21000, 21001-22000, 22001-23000, 23001-24000, 24001-25000, 25001-26000, 26001- 27000, 27001-28000, 28001-29000, 29001-30000, 30001-31000, 31001-32000, 32001-33000, 33001-34000, 34001-35000, 35001-36000, 36001-37000, 37001-38000, 38001-39000, 39001-

30 39624, 39705-40589, 40666-43629, 43710-44203, 44311-45125, 45210-45440, 45622-45717, 45791-68580, 68675-70246, 70396-72421, 72601-73295, 73434-74648, 74898-83055, 83175- 85192, 85279-85609, 85740-85906, 86070-88304, 88396-90585, 90705-91767, 91824-94380, 94490-96296, 96364-97184, 97270-101167, 101274-109465, 109581-1 10228, 110363-111819, 111882-113636, 113783-1 13945, 114186-117002, 1 17075-119676, and 1 19677-121162; and

35 (b) SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659-3829 and 8885-10884.

Additional preferred probes and primers ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises a G at position 7159 of SEQ ID No 1. Further preferred probes and primers ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises a C either at position 2551 or 4500 of SEQ ID No 2.

Another preferred probes and primers ofthe invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 3: 391-1619 and 6988-10682. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises a nucleotide selected in the group consisting of a C at position 5487, and a C at position 6265 of SEQ ID No 3.

Thus, the invention also relates to nucleic acid probes characterized in that they hybridize specifically, under the stringent hybridization conditions defined above, with a nucleic acid selected from the group consisting ofthe nucleotide sequences: a) 1-39624, 39705-40589, 40666-43629, 43710-44203, 4431 1-45125, 45210-45440, 45622-

45717, 45791-68580, 68675-70246, 70396-72421, 72601-73295, 73434-74648, 74898-83055, 83175-85192, 85279-85609, 85740-85906, 86070-88304, 88396-90585, 90705-91767, 91824- 94380, 94490-96296, 96364-97184, 97270-101 167, 101274-109465, 109581-1 10228, 1 10363- 1 1 1819, 1 1 1882-1 13636, 113783-113945, 1 14186-1 17002, 1 17075-1 19676, and 119677-121 162 of SEQ ID No 1 or a variant thereof or a sequence complementary thereto; b) 1-1600, 1751-2138, 2332-2539, 2659-3829 and 8885-10884 of SEQ ID No 2 or a variant thereof or a sequence complementary thereto; and c) 391-1619 and 6988-10682 of SEQ ID No 3 or a variant thereof or a sequence complementary thereto. Additionally, another preferred embodiment of a probe according to the invention consists of a nucleic acid comprising a biallelic marker selected from the group consisting of Al to A32 or the complements thereto, for which the receptive locations in the sequence listing are provided in Table 2.

The invention also relates to a purified and/or isolated nucleotide sequence comprising a polymorphic base of a HKEP-related biallelic marker, preferably of a biallelic marker selected from the group consisting of A 1 to A32, and the complements thereof. The sequence has between 8 and 1000 nucleotides in length, and preferably comprises at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 contiguous nucleotides, to the extent that such lengths are consistent with the specific sequence, of a nucleotide sequence selected from the group consisting of SEQ ID Nos 1-3 and 5-8 or a variant thereof or a complementary sequence thereto. In one embodiment the invention encompasses isolated, purified, and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos 1 - 3 and 5-8 and the complement thereof, wherein said span includes a HΛZ -related biallelic marker in said sequence; optionally, wherein said HAZ -related biallelic marker is selected from the group consisting of Al to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said HA-LP-related biallelic marker is selected from the group consisting of Al to A22 and A25 to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said HKLP-τelated biallelic marker is selected from the group consisting of A23 and A24, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; These nucleotide sequences comprise the polymorphic base of either allele 1 or allele 2 ofthe considered biallelic marker. Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides ofthe center of said polynucleotide or at the center of said polynucleotide; optionally, wherein said contiguous span is 18 to 35 nucleotides in length and said biallelic marker is within 4 nucleotides ofthe center of said polynucleotide; optionally, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide; optionally, wherein the 3' end of said contiguous span is present at the 3' end of said polynucleotide; and optionally, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide and said biallelic marker is present at the 3' end of said polynucleotide. Optionally, said polynucleotide may further comprise a label. Optionally, said polynucleotide can be attached to solid support. In a further embodiment, the polynucleotides defined above can be used alone or in any combination. In a preferred embodiment, said probes consists of, or consists essentially of a sequence selected from the following sequences: PI to P30 and the complementary sequences thereto.

In another embodiment the invention encompasses isolated, purified and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of SEQ ID Nos 1-3 and 5-8 or the complements thereof, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide, and wherein the 3' end of said polynucleotide is located or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of a HAZR-related biallelic marker in said sequence, preferably within within 20 nucleotides upstream of a HKZR-related biallelic marker in said sequence; optionally, wherein said HΛ-LP-related biallelic marker is selected from the group consisting of Al to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said HKEP-related biallelic marker is selected from the group consisting of Al to A22 and A25 to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said HAZ -related biallelic marker is selected from the group consisting of A23 and A24, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein the 3' end of said polynucleotide is located 1 nucleotide upstream of said HAX -related biallelic marker in said sequence; and optionally, wherein said polynucleotide consists essentially of a sequence selected from the following sequences: Dl to D30 and El to E30.

In a further embodiment, the invention encompasses isolated, purified, or recombinant polynucleotides comprising, consisting of, or consisting essentially of a sequence selected from the following sequences: Bl to B25 and Cl to C25.

In an additional embodiment, the invention encompasses the use of any polynucleotide for, or polynucleotides for use in determining the identity ofthe nucleotide at a HAZR-related biallelic marker or the complements thereof, as well as polynucleotides for use or use of polynucleotides in amplifying segments of nucleotides comprising a HKLP-τe\ated biallelic marker or the complements thereof; Optionally, said determining may be performed in hybridization assay, sequencing assays, and enzyme-based mismatch detection assays; Optionally, said amplifying may be performed by a PCR or LCR. optionally, wherein said HAXP-related biallelic marker is selected from the group consisting of Al to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said HKZP-related biallelic marker is selected from the group consisting of Al to A22 and A25 to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said HKZR-related biallelic marker is selected from the group consisting of A23 and A24, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said polynucleotide may be attached to a solid support, array, or addressable array; Optionally, said polynucleotide may be labeled.

The invention concerns the use ofthe polynucleotides according to the invention for determining the identity ofthe nucleotide at a HKZ -related biallelic marker, preferably in hybridization assay, sequencing assay, microsequencing assay, or an enzyme-based mismatch detection assay and in amplifying segments of nucleotides comprising a HAZP-related biallelic marker. In addition, the polynucleotides ofthe invention for use or the use of polynucleotides in determining the identity of one or more nucleotides at a HA P-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination.

The primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al.(1979), the phosphodiester method of Brown et al.(1979), the diethylphosphoramidite method of Beaucage et al.(1981) and the solid support method described in EP 0 707 592. The disclosures of all these documents are incoφorated herein by reference.

The formation of stable hybrids depends on the melting temperature (Tm) ofthe DNA. The Tm depends on the length ofthe primer or probe, the ionic strength ofthe solution and the G+C content. The higher the G+C content ofthe primer or probe, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The GC content in the probes ofthe invention usually ranges between 10 and 75 %, preferably between 35 and 60 %, and more preferably between 40 and 55 %.

Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed in International Patent Application WO 92/20702, moφholino analogs which are described in U.S. Patents Numbered 5,185,444; 5,034,506 and 5,142,047. The probe may have to be rendered "non-extendable" in that additional dNTPs cannot be added to the probe. In and of themselves analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3' end ofthe probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3' hydroxyl group simply can be cleaved, replaced or modified, U.S. Patent Application Serial No. 07/049,061 filed April 19, 1993 describes modifications, which can be used to render a probe non-extendable. A probe or a primer according to the invention has between 8 and 1000 nucleotides in length, or is specified to be at least 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 nucleotides in length. More particularly, the length of these probes and primers can range from 8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferably from 15 to 30 nucleotides. Shorter probes and primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes and primers are expensive to produce and can sometimes self-hybridize to form haiφin structures. The appropriate length for primers and probes under a particular set of assay conditions may be empirically determined by one of skill in the art. A preferred probe or primer consists of a nucleic acid comprising a polynucleotide selected from the group ofthe nucleotide sequences of PI to P30 and the complementary sequence thereto, Bl to B25, Cl to C25, Dl to D30, El to E30, for which the respective locations in the sequence listing are provided in Tables 1, 2, 3 and 4.

Any ofthe polynucleotides ofthe present invention can be labeled, if desired, by incoφorating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive substances ( P, S, H, I), fluorescent dyes (5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin) or biotin. Preferably, polynucleotides are labeled at their 3' and 5' ends. Examples of non-radioactive labeling of nucleic acid fragments are described in the French patent No. FR-7810975 or by Urdea et al (1988) or Sanchez-Pescador et al (1988). In addition, the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. in 1991 or in the European patent No. EP 0 225 807 (Chiron).

A label can also be used to capture the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support. A capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, primers or probes provided herein, may, themselves, serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase. In cases where a polynucleotide probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or "tail" that is not complementary to the target. In the case where a polynucleotide primer itself serves as the capture label, at least a portion ofthe primer will be free to hybridize with a nucleic acid on a solid phase. DNA Labeling techniques are well known to the skilled technician. The probes ofthe present invention are useful for a number of puφoses. They can be notably used in Southern hybridization to genomic DNA. The probes can also be used to detect PCR amplification products. They may also be used to detect mismatches in the HKLP gene or mRNA using other techniques.

Any ofthe polynucleotides, primers and probes ofthe present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others. The solid support is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding ofthe capture reagent to a solid support material before the performance ofthe assay or during the performance ofthe assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes® and other configurations known to those of ordinary skill in the art. The polynucleotides ofthe invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support. In addition, polynucleotides other than those ofthe invention may be attached to the same solid support as one or more polynucleotides ofthe invention.

Consequently, the invention also deals with a method for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos 1-3, a fragment or a variant thereof and a complementary sequence thereto in a sample, said method comprising the following steps of: a) bringing into contact a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ID Nos 1-3, a fragment or a variant thereof and a complementary sequence thereto and the sample to be assayed; and b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample. The invention further concerns a kit for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos 1 -3, a fragment or a variant thereof and a complementary sequence thereto in a sample, said kit comprising: a) a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting ofthe nucleotide sequences of SEQ ID Nos 1-3, a fragment or a variant thereof and a complementary sequence thereto; and b) optionally, the reagents necessary for performing the hybridization reaction. In a first preferred embodiment of this detection method and kit, said nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule. In a second preferred embodiment of said method and kit, said nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate. In a third preferred embodiment, the nucleic acid probe or the plurality of nucleic acid probes comprise either a sequence which is selected from the group consisting ofthe nucleotide sequences of PI to P30 and the complementary sequence thereto, Bl to B25, Cl to C25, Dl to D30, El to E30 or a biallelic marker selected from the group consisting of Al to A32 and the complements thereto. Oligonucleotide Arrays

A substrate comprising a plurality of oligonucleotide primers or probes ofthe invention may be used either for detecting or amplifying targeted sequences in the HKLP gene and may also be used for detecting mutations in the coding or in the non-coding sequences ofthe HKLP gene. Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support. Alternatively the polynucleotides ofthe invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region ofthe solid support which does not overlap with the attachment site of any other polynucleotide. Preferably, such an ordered array of polynucleotides is designed to be "addressable" where the distinct locations are recorded and can be accessed as part of an assay procedure. Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge ofthe precise location of each polynucleotides location makes these "addressable" arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the polynucleotides ofthe invention. One particular embodiment of these polynucleotide arrays is known as the Genechips™, and has been generally described in US Patent 5,143,854; PCT publications WO 90/15070 and 92/10092. These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods which incoφorate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., 1991). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as "Very Large Scale Immobilized Polymer Synthesis" (VLSIPS™) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPS™ technologies are provided in US Patents 5,143,854; and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/1 1995, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.

In another embodiment ofthe oligonucleotide arrays ofthe invention, an oligonucleotide probe matrix may advantageously be used to detect mutations occurring in the HKLP gene and in its regulatory region. For this particular puφose, probes are specifically designed to have a nucleotide sequence allowing their hybridization to the genes that carry known mutations (either by deletion, insertion or substitution of one or several nucleotides). By known mutations, it is meant, mutations on the HKLP gene that have been identified according, for example to the technique used by Huang et al.(1996) or Samson et al.(1996). Another technique that is used to detect mutations in the HKLP gene is the use of a high- density DNA array. Each oligonucleotide probe constituting a unit element ofthe high density DNA array is designed to match a specific subsequence ofthe HKLP genomic DNA or cDNA. Thus, an array consisting of oligonucleotides complementary to subsequences ofthe target gene sequence is used to determine the identity ofthe target sequence with the wild gene sequence, measure its amount, and detect differences between the target sequence and the reference wild gene sequence of the HKLP gene. In one such design, termed 4L tiled array, is implemented a set of four probes (A, C, G, T), preferably 15-nucleotide oligomers. In each set of four probes, the perfect complement will hybridize more strongly than mismatched probes. Consequently, a nucleic acid target of length L is scanned for mutations with a tiled array containing 4L probes, the whole probe set containing all the possible mutations in the known wild reference sequence. The hybridization signals ofthe 15- mer probe set tiled array are perturbed by a single base change in the target sequence. As a consequence, there is a characteristic loss of signal or a "footprint" for the probes flanking a mutation position. This technique was described by Chee et al. in 1996, which is herein incoφorated by reference.

Consequently, the invention concerns an array of nucleic acid molecules comprising at least one polynucleotide described above as probes and primers. Preferably, the invention concerns an array of nucleic acid comprising at least two polynucleotides described above as probes and primers. A further object ofthe invention consists of an array of nucleic acid sequences comprising either at least one ofthe sequences selected from the group consisting of PI to P30, Bl to B25, Cl to C25, Dl to D30, El to E30, the sequences complementary thereto, a fragment thereof of at least 8 consecutive nucleotides thereof, and at least one sequence comprising a biallelic marker selected from the group consisting of Al to A32 and the complements thereto.

The invention also pertains to an array of nucleic acid sequences comprising either at least two ofthe sequences selected from the group consisting of PI to P30, Bl to B25, Cl to C25, Dl to D30, El to E30, the sequences complementary thereto, a fragment thereof of at least 8 consecutive nucleotides thereof, and at least two sequences comprising a biallelic marker selected from the group consisting of Al to A32 and the complements thereof.

Amplification of the HKLP gene.

1. DNA extraction

As for the source ofthe genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples which can be tested by the methods ofthe present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions ofthe respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supematants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the context ofthe present invention is from peripheral venous blood of each donor.

The techniques of DNA extraction are well-known to the skilled technician. Such 5 techniques are described notably by Mackey et al. (1998).

2. DNA amplification

DNA amplification techniques are well-known to those skilled in the art. Amplification techniques that can be used in the context ofthe present invention include, but are not limited to, the ligase chain reaction (LCR) described in EP-A- 320 308, WO 9320227 and EP-A-439 182, the

10 disclosures of which are incoφorated herein by reference, the polymerase chain reaction (PCR, RT- PCR) and techniques such as the nucleic acid sequence based amplification (NASBA) described in Guatelli JC, et al. (1990) and in Compton J. (1991), Q-beta amplification as described in European Patent Application no 4544610, strand displacement amplification as described in Walker et al. (1996) and EP A 684 315 and, target mediated amplification as described in PCT Publication WO

15 9322461 , the disclosure of which is incoφorated herein by reference.

LCR and Gap LCR are exponential amplification techniques, both depend on DNA ligase to join adjacent primers annealed to a DNA molecule. In Ligase Chain Reaction (LCR), probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target. The first probe hybridizes to a first segment ofthe

20 target strand and the second probe hybridizes to a second segment ofthe target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate- 3 'hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion ofthe first probe and a fourth (secondary) probe can hybridize to a portion ofthe second probe in a similar abutting fashion.

25 Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization

30 and ligation, amplification ofthe target sequence is achieved. A method for multiplex LCR has also been described (WO 9320227). Gap LCR (GLCR) is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.

For amplification of mRNAs, it is within the scope ofthe present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single

35 enzyme for both steps as described in U.S. Patent No. 5,322,770 or, to use Asymmetric Gap LCR (RT-AGLCR) as described by Marshall et al. (1994). AGLCR is a modification of GLCR that allows the amplification of RNA.

The PCR technology is the preferred amplification technique used in the present invention. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see White (1997) and the publication entitled "PCR Methods and Applications" (1991, Cold Spring Harbor Laboratory Press). In each of these PCR procedures, PCR primers on either side ofthe nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including US Patents 4,683,195, 4,683,202 and 4,965,188. Each of these publications is incoφorated by reference.

One ofthe aspects ofthe present invention is a method for the amplification ofthe human HKLP gene, particularly ofthe genomic sequences of SEQ ID No 1 and 2 or ofthe cDNA sequence of SEQ ID No 3, or a fragment or a variant thereof in a test sample, preferably using the PCR technology. The method comprises the steps of contacting a test sample suspected of containing the target HKLP encoding sequence or portion thereof with amplification reaction reagents comprising a pair of amplification primers, and eventually in some instances a detection probe that can hybridize with an internal region of amplicon sequences to confirm that the desired amplification reaction has taken place.

Thus, the present invention also relates to a method for the amplification of a human HKLP gene sequence, particularly of a portion ofthe genomic sequences of SEQ ID Nos 1 and 2 or ofthe cDNA sequence of SEQ ID No 3, or a variant thereof in a test sample, said method comprising the steps of: a) contacting a test sample suspected of containing the targeted HKLP gene sequence comprised in a nucleotide sequence selected from a group consisting of SEQ ID Nos 1-3, or fragments or variants thereof with amplification reaction reagents comprising a pair of amplification primers as described above and located on either side ofthe polynucleotide region to be amplified; and b) optionally, detecting the amplification products.

The invention also concerns a kit for the amplification of a human HKLP gene sequence, particularly of a portion ofthe genomic sequences of SEQ ID No 1 and 2 or ofthe cDNA sequence of SEQ ID No 3, or a variant thereof in a test sample, wherein said kit comprises: a) a pair of oligonucleotide primers located on either side ofthe HKLP region to be amplified; and b) Optionally, the reagents necessary for performing the amplification reaction.

In a first preferred embodiment ofthe above amplification method or kit, the amplification product is detected by hybridization with a labeled probe having a sequence which is complementary to the amplified region.

The primers are more particularly characterized in that they have sufficient complementarity with any sequence of a strand ofthe genomic sequence close to the region to be amplified, for example with a non-coding sequence adjacent to exons to amplify. In a second preferred embodiment, the nucleic acid primers comprise a sequence which is selected from the group consisting ofthe nucleotide sequences of Bl to B25, Cl to C25, Dl to D30, and El to E30.

HKLP Proteins and Polypeptide Fragments:

The term "HKLP polypeptides" is used herein to embrace all ofthe proteins and polypeptides ofthe present invention. Also forming part ofthe invention are polypeptides encoded by the polynucleotides ofthe invention, as well as fusion polypeptides comprising such polypeptides. The invention embodies HKLP proteins from humans, including isolated or purified HKLP proteins consisting, consisting essentially, or comprising the sequence of SEQ ID No 4. The HKLP protein has 1816 amino acids in length. The 700 first amino acids ofthe HKLP protein present 97 % of homology with the murine KIF IB protein (Nangaku et al., 1994). The HKLP protein presents 60-70% of homology with the murine KIF1 A protein, and more particularly the 390 first amino acids ofthe HKLP protein have 85 % of homology therewith.

The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 ofthe SEQ ID No 4. In other preferred embodiments the contiguous stretch of amino acids comprises the site of a mutation or functional mutation, including a deletion, addition, swap or truncation ofthe amino acids in the HKLP protein sequence. The invention also encompasses a purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence having at least 70, 75, 80, 85, 90, 95, 98 or 99% nucleotide identity with a nucleotide sequence of SEQ ID No 4 or a complementary sequence thereto or a fragment thereof.

HKLP proteins are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes. The HKLP polypeptides ofthe invention can be made using routine expression methods known in the art. The polynucleotide encoding the desired polypeptide is ligated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems is used in forming recombinant polypeptides, and a summary of some ofthe more common systems. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like. See, for example, Methods in Enzymology for a variety of methods for purifying proteins.

In addition, shorter protein fragments is produced by chemical synthesis. Alternatively the proteins ofthe invention is extracted from cells or tissues of humans or non-human animals. Methods for purifying proteins are known in the art, arid include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation ofthe polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.

Any HKLP cDNA, including SEQ ID No 3, is used to express HKLP proteins and polypeptides. The nucleic acid encoding the HKLP protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology. The HKLP insert in the expression vector may comprise the full coding sequence for the HKLP protein or a portion thereof. For example, the HKLP derived insert may encode a polypeptide comprising at least 10 consecutive amino acids ofthe HKLP protein of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 of the amino acid positions 1-478 ofthe SEQ ID No 4.

The expression vector is any ofthe mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, MA), Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego, California). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing ofthe sequence is optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Patent No. 5,082,767.

In one embodiment, the entire coding sequence ofthe HKLP cDNA through the poly A signal ofthe cDNA are operably linked to a promoter in the expression vector. Alternatively, if the nucleic acid encoding a portion ofthe HKLP protein lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon ofthe nucleic acid using conventional techniques. Similarly, if the insert from the HKLP cDNA lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using Bgll and Sail restriction endonuclease enzymes and incoφorating it into the mammalian expression vector pXTl (Stratagene). pXTl contains the LTRs and a portion ofthe gag gene from Moloney Murine Leukemia Virus. The position ofthe LTRs in the construct allow efficient stable transfection. The vector includes the Heφes Simplex Thymidine Kinase promoter and the selectable neomycin gene. The nucleic acid encoding the HKLP protein or a portion thereof is obtained by PCR from a bacterial vector containing the HKLP cDNA of SEQ ID No 3 using oligonucleotide primers complementary to the HKLP cDNA or portion thereof and containing restriction endonuclease sequences for Pst I incoφorated into the 5 'primer and Bgffl at the 5' end ofthe corresponding cDNA 3' primer, taking care to ensure that the sequence encoding the HKLP protein or a portion thereof is positioned properly with respect to the poly A signal. The purified fragment obtained from the resulting PCR reaction is digested with Pstl, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXTl, now containing a poly A signal and digested with Bglϋ.

The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, New York) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600ug/ml G418 (Sigma, St. Louis, Missouri).

Alternatively, the nucleic acids encoding the HKLP protein or a portion thereof is cloned into pED6dpc2 (Genetics Institute, Cambridge, MA). The resulting pED6dpc2 constructs is transfected into a suitable host cell, such as COS 1 cells. Methotrexate resistant cells are selected and expanded.

The above procedures may also be used to express a mutant HKLP protein responsible for a detectable phenotype or a portion thereof.

The expressed proteins are purified using conventional purification techniques such as ammonium sulfate precipitation or chromatographic separation based on size or charge. The protein encoded by the nucleic acid insert may also be purified using standard immunochromatography techniques. In such procedures, a solution containing the expressed HKLP protein or portion thereof, such as a cell extract, is applied to a column having antibodies against the HKLP protein or portion thereof is attached to the chromatography matrix. The expressed protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound expressed protein is then released from the column and recovered using standard techniques.

To confirm expression ofthe HKLP protein or a portion thereof, the proteins expressed from host cells containing an expression vector containing an insert encoding the HKLP protein or a portion thereof can be compared to the proteins expressed in host cells containing the expression vector without an insert. The presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the HKLP protein or a portion thereof is being expressed. Generally, the band will have the mobility expected for the HKLP protein or portion thereof. However, the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.

Antibodies capable of specifically recognizing the expressed HKLP protein or a portion thereof are described below. If antibody production is not possible, the nucleic acids encoding the HKLP protein or a portion thereof is incoφorated into expression vectors designed for use in purification schemes employing chimeric polypeptides. In such strategies the nucleic acid encoding the HKLP protein or a portion thereof is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera is β-globin or a nickel binding polypeptide encoding sequence. A chromatography matrix having antibody to β-globin or nickel attached thereto is then used to purify the chimeric protein. Protease cleavage sites is engineered between the β-globin gene or the nickel binding polypeptide and the HKLP protein or portion thereof. Thus, the two polypeptides ofthe chimera is separated from one another by protease digestion. One useful expression vector for generating β-globin chimerics is pSG5 (Stratagene), which encodes rabbit β-globin. Intron II ofthe rabbit β-globin gene facilitates splicing ofthe expressed transcript, and the polyadenylation signal incoφorated into the construct increases the level of expression. These techniques are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al., (1986) and many ofthe methods are available from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro Express™ Translation Kit (Stratagene).

Thus, the present invention also concerns a method for producing one ofthe polypeptides described herein, and especially a polypeptide of SEQ ID No 4 or a fragment or a variant thereof, wherein said method comprises the steps of : a) culturing, in an appropriate culture medium, a cell host previously transformed or transfected with the recombinant vector comprising a nucleic acid encoding a HKLP polypeptide, or a fragment or a variant thereof; b) harvesting the culture medium thus conditioned or lyse the cell host, for example by sonication or by an osmotic shock; c) separating or purifying, from the said culture medium, or from the pellet ofthe resultant host cell lysate the thus produced polypeptide of interest. d) Optionally characterizing the produced polypeptide of interest.

In a specific embodiment ofthe above method, step a) is preceded by a step wherein the nucleic acid coding for a HKLP polypeptide, or a fragment or a variant thereof, is inserted in an appropriate vector, optionally after an appropriate cleavage of this amplified nucleic acid with one or several restriction endonucleases. The nucleic acid coding for a HKLP polypeptide or a fragment or a variant thereof may be the resulting product of an amplification reaction using a pair of primers according to the invention (by SDA, TAS, 3SR NASBA, TMA etc.). Antibodies That Bind HKLP Polypeptides of the Invention

Any HKLP polypeptide or whole protein may be used to generate antibodies capable of specifically binding to expressed HKLP protein or fragments thereof as described. The antibody compositions ofthe invention are capable of specifically binding or specifically bind to the HKLP protein. For an antibody composition to specifically bind to the HKLP protein it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for full length HKLP protein than for any full length protein in an ELISA, RIA, or other antibody-based binding assay.

In a preferred embodiment ofthe invention antibody compositions are capable of selectively binding, or selectively bind to an epitope-containing fragment of a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said epitope comprises at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 ofthe SEQ ID No 4, wherein said antibody composition is optionally either polyclonal or monoclonal.

The present invention also contemplates the use of polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 50, or 100 amino acids of a HKLP polypeptide in the manufacture of antibodies, wherein said contiguous span comprises at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 ofthe SEQ ID No 4. In a preferred embodiment such polypeptides are useful in the manufacture of antibodies to detect the presence and absence ofthe HKLP protein. Non-human animals or mammals, whether wild-type or transgenic, which express a different species of HKLP than the one to which antibody binding is desired, and animals which do not express HKLP (i.e. a HKLP knock out animal as described in herein) are particularly useful for preparing antibodies. HKLP knock out animals will recognize all or most of the exposed regions of HKLP as foreign antigens, and therefore produce antibodies with a wider array of HKLP epitopes. Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to the HKLP protein. In addition, the humoral immune system of animals which produce a species of HKLP that resembles the antigenic sequence will preferentially recognize the differences between the animal's native HKLP species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence. Such a technique will be particularly useful in obtaining antibodies that specifically bind to the HKLP protein.

Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.

The antibodies ofthe invention may be labeled, either by a radioactive, a fluorescent or an enzymatic label. Consequently, the invention is also directed to a method for detecting specifically the presence of a human HKLP polypeptide according to the invention in a biological sample, said method comprising the following steps : a) bringing into contact the biological sample with a polyclonal or monoclonal antibody directed against the HKLP polypeptide ofthe amino acid sequence of SEQ ID No 4, or to a peptide fragment or variant thereof; b) detecting the antigen-antibody complex formed.

The invention also concerns a diagnostic kit for detecting in vitro the presence of a human HKLP polypeptide according to the present invention in a biological sample, wherein said kit comprises : a) a polyclonal or monoclonal antibody directed against the HKLP polypeptide ofthe amino acid sequence of SEQ ID No 4, or to a peptide fragment or variant thereof, optionally labeled; b) a reagent allowing the detection ofthe antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.

HKLP -related Biallelic Markers

Advantages Of The Biallelic Markers Of The Present Invention

The HKZP-related biallelic markers ofthe present invention offer a number of important advantages over other genetic markers such as RFLP (Restriction fragment length polymoφhism) and VNTR (Variable Number of Tandem Repeats) markers.

The first generation of markers, were RFLPs, which are variations that modify the length of a restriction fragment. But methods used to identify and to type RFLPs are relatively wasteful of materials, effort, and time. The second generation of genetic markers were VNTRs, which can be categorized as either minisatellites or microsatellites. Minisatellites are tandemly repeated DNA sequences present in units of 5-50 repeats which are distributed along regions ofthe human chromosomes ranging from 0.1 to 20 kilobases in length. Since they present many possible alleles, their informative content is very high. Minisatellites are scored by performing Southern blots to identify the number of tandem repeats present in a nucleic acid sample from the individual being tested. However, there are only 10 potential VNTRs that can be typed by Southern blotting.

Moreover, both RFLP and VNTR markers are costly and time-consuming to develop and assay in large numbers.

Single nucleotide polymoφhism or biallelic markers can be used in the same manner as

RFLPs and VNTRs but offer several advantages. SNP are densely spaced in the human genome and represent the most frequent type of variation. An estimated number of more than 10⁷ sites are scattered along the 3xl0⁹ base pairs ofthe human genome. Therefore, SNP occur at a greater frequency and with greater uniformity than RFLP or VNTR markers which means that there is a greater probability that such a marker will be found in close proximity to a genetic locus of interest. SNP are less variable than VNTR markers but are mutationally more stable. Also, the different forms of a characterized single nucleotide polymoφhism, such as the biallelic markers ofthe present invention, are often easier to distinguish and can therefore be typed easily on a routine basis. Biallelic markers have single nucleotide based alleles and they have only two common alleles, which allows highly parallel detection and automated scoring. The biallelic markers ofthe present invention offer the possibility of rapid, high throughput genotyping of a large number of individuals.

Biallelic markers are densely spaced in the genome, sufficiently informative and can be assayed in large numbers. The combined effects of these advantages make biallelic markers extremely valuable in genetic studies. Biallelic markers can be used in linkage studies in families, in allele sharing methods, in linkage disequilibrium studies in populations, in association studies of case-control populations or of trait positive and trait negative populations. An important aspect of the present invention is that biallelic markers allow association studies to be performed to identify genes involved in complex traits. Association studies examine the frequency of marker alleles in unrelated case- and control-populations and are generally employed in the detection of polygenic or sporadic traits. Association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families (linkage studies). Biallelic markers in different genes can be screened in parallel for direct association with disease or response to a treatment. This multiple gene approach is a powerful tool for a variety of human genetic studies as it provides the necessary statistical power to examine the synergistic effect of multiple genetic factors on a particular phenotype, drug response, sporadic trait, or disease state with a complex genetic etiology.

Candidate Gene Of The Present Invention

Different approaches can be employed to perform association studies: genome-wide association studies, candidate region association studies and candidate gene association studies. Genome-wide association studies rely on the screening of genetic markers evenly spaced and covering the entire genome. The candidate gene approach is based on the study of genetic markers specifically located in genes potentially involved in a biological pathway related to the trait of interest. In the present invention, HKLP is the candidate gene. The candidate gene analysis clearly provides a short-cut approach to the identification of genes and gene polymoφhisms related to a particular trait when some information concerning the biology ofthe trait is available. However, it should be noted that all ofthe biallelic markers disclosed in the instant application can be employed as part of genome-wide association studies or as part of candidate region association studies and such uses are specifically contemplated in the present invention and claims.

HfiXP-Related Biallelic Markers And Polynucleotides Related Thereto

The invention also concerns HKLP-related biallelic markers. As used herein the term "HAZR-related biallelic marker" relates to a set of biallelic markers in linkage disequilibrium with the HKLP gene. The term HΛZ -related biallelic marker includes the biallelic markers designated Al to A32.

A portion ofthe biallelic markers ofthe present invention are disclosed in Table 2. Their location on the HKLP gene is indicated in Table 2 and also as a single base polymoφhism in the features of in the related SEQ ID Nos 1-3 and 5-8. The pairs of primers allowing the amplification of a nucleic acid containing the polymoφhic base of one HKLP biallelic marker are listed in Table 1 of Example 2.

27 HKLP-related biallelic markers, Al to A27, are located in the genomic sequence of HKLP. Four of them are located in exonic sequence, namely Al, A23, A24 and A25. The other HAZ -related biallelic markers are located in intronic region of HKLP. Additionally, 5 biallelic markers are located in intergenic region and are in linkage desiquilibirum with the HKLP gene. The primers for amplification or sequencing reaction of a polynucleotide comprising a biallelic marker ofthe invention may be designed from the disclosed sequences for any method known in the art. A preferred set of primers are fashioned such that the 3' end ofthe contiguous span of identity with a sequence selected from the group consisting of SEQ ID Nos 1 -3 and 5-8 or a sequence complementary thereto or a variant thereof is present at the 3' end ofthe primer. Such a configuration allows the 3' end ofthe primer to hybridize to a selected nucleic acid sequence and dramatically increases the efficiency ofthe primer for amplification or sequencing reactions. Allele specific primers may be designed such that a polymoφhic base of a biallelic marker is at the 3' end ofthe contiguous span and the contiguous span is present at the 3' end ofthe primer. Such allele specific primers tend to selectively prime an amplification or sequencing reaction so long as they are used with a nucleic acid sample that contains one ofthe two alleles present at a biallelic marker. The 3' end ofthe primer ofthe invention may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of a HXLR-related biallelic marker in said sequence or at any other location which is appropriate for their intended use in sequencing, amplification or the location of novel sequences or markers. Thus, another set of preferred amplification primers comprise an isolated polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides in a sequence selected from the group consisting of SEQ ID Nos 1-3 and 5-8 or a sequence complementary thereto or a variant thereof, wherein the 3' end of said contiguous span is located at the 3 'end of said polynucleotide, and wherein the 3 'end of said polynucleotide is located upstream of a HKLP-related biallelic marker in said sequence. Preferably, those amplification primers comprise a sequence selected from the group consisting ofthe sequences Bl to B25 and Cl to C25. Primers with their 3' ends located 1 nucleotide upstream of a HA-LP-related biallelic marker have a special utility in microsequencing assays. Preferred microsequencing primers are described in Table 4. Optionally, microsequencing primers are selected from the group consisting ofthe nucleotide sequences Dl to D30 and El to E30.

The probes ofthe present invention may be designed from the disclosed sequences for any method known in the art, particularly methods which allow for testing if a marker disclosed herein is present. A preferred set of probes may be designed for use in the hybridization assays ofthe invention in any manner known in the art such that they selectively bind to one allele of a biallelic marker, but not the other allele under any particular set of assay conditions. Preferred hybridization probes comprise the polymoφhic base of either allele 1 or allele 2 ofthe specific biallelic marker. Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides ofthe center ofthe hybridization probe or at the center of said probe.

It should be noted that the polynucleotides ofthe present invention are not limited to having the exact flanking sequences surrounding the polymoφhic bases which are enumerated in Sequence Listing. Rather, it will be appreciated that the flanking sequences surrounding the biallelic markers may be lengthened or shortened to any extent compatible with their intended use and the present invention specifically contemplates such sequences. The flanking regions outside ofthe contiguous span need not be homologous to native flanking sequences which actually occur in human subjects. The addition of any nucleotide sequence which is compatible with the nucleotides intended use is specifically contemplated.

Primers and probes may be labeled or immobilized on a solid support as described in "Oligonucleotide probes and primers".

The polynucleotides ofthe invention which are attached to a solid support encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said polynucleotides may be specified as attached individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides ofthe invention to a single solid support. Optionally, polynucleotides other than those ofthe invention may attached to the same solid support as polynucleotides ofthe invention. Optionally, when multiple polynucleotides are attached to a solid support they may be attached at random locations, or in an ordered array. Optionally, said ordered array may be addressable.

The present invention also encompasses diagnostic kits comprising one or more polynucleotides ofthe invention with a portion or all ofthe necessary reagents and instructions for genotyping a test subject by determining the identity of a nucleotide at a HKLP-related biallelic marker. The polynucleotides of a kit may optionally be attached to a solid support, or be part of an array or addressable array of polynucleotides. The kit may provide for the determination ofthe identity ofthe nucleotide at a marker position by any method known in the art including, but not limited to, a sequencing assay method, a microsequencing assay method, a hybridization assay method, or an enzyme-based mismatch detection method.

Methods For De Novo Identification Of Biallelic Markers

Any of a variety of methods can be used to screen a genomic fragment for single nucleotide polymoφhisms such as differential hybridization with oligonucleotide probes, detection of changes in the mobility measured by gel electrophoresis or direct sequencing ofthe amplified nucleic acid. A preferred method for identifying biallelic markers involves comparative sequencing of genomic DNA fragments from an appropriate number of unrelated individuals.

In a first embodiment, DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced. The nucleotide sequences thus obtained are then analyzed to identify significant polymoφhisms. One ofthe major advantages of this method resides in the fact that the pooling ofthe DNA samples substantially reduces the number of DNA amplification reactions and sequencing reactions, which must be carried out. Moreover, this method is sufficiently sensitive so that a biallelic marker obtained thereby usually demonstrates a sufficient frequency of its less common allele to be useful in conducting association studies.

In a second embodiment, the DNA samples are not pooled and are therefore amplified and sequenced individually. This method is usually preferred when biallelic markers need to be identified in order to perform association studies within candidate genes. Preferably, highly relevant gene regions such as promoter regions or exon regions may be screened for biallelic markers. A biallelic marker obtained using this method may show a lower degree of informativeness for conducting association studies, e.g. if the frequency of its less frequent allele may be less than about 10%. Such a biallelic marker will, however, be sufficiently informative to conduct association studies and it will further be appreciated that including less informative biallelic markers in the genetic analysis studies ofthe present invention, may allow in some cases the direct identification of causal mutations, which may, depending on their penetrance, be rare mutations.

The following is a description ofthe various parameters of a preferred method used by the inventors for the identification ofthe biallelic markers ofthe present invention.

Genomic DNA Samples The genomic DNA samples from which the biallelic markers ofthe present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background. The number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, preferably from about 50 to about 200 individuals. It is usually preferred to collect DNA samples from at least about 100 individuals in order to have sufficient polymoφhic diversity in a given population to identify as many markers as possible and to generate statistically significant results. As for the source ofthe genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples, which can be tested by the methods ofthe present invention described herein, and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions ofthe respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supematants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the present invention is from peripheral venous blood of each donor. Techniques to prepare genomic DNA from biological samples are well known to the skilled technician. Details of a preferred embodiment are provided in Example 1. The person skilled in the art can choose to amplify pooled or unpooled DNA samples.

DNA Amplification

The identification of biallelic markers in a sample of genomic DNA may be facilitated through the use of DNA amplification methods. DNA samples can be pooled or unpooled for the amplification step. DNA amplification techniques are well known to those skilled in the art. Various methods to amplify DNA fragments carrying biallelic markers are further described hereinbefore in "Amplification ofthe HKLP gene". The PCR technology is the preferred amplification technique used to identify new biallelic markers. A typical example of a PCR reaction suitable for the puφoses ofthe present invention is provided in Example 2.

In a first embodiment ofthe present invention, biallelic markers are identified using genomic sequence information generated by the inventors. Sequenced genomic DNA fragments are used to design primers for the amplification of 500 bp fragments. These 500 bp fragments are amplified from genomic DNA and are scanned for biallelic markers. Primers may be designed using the OSP software (Hillier L. and Green P., 1991). All primers may contain, upstream ofthe specific target bases, a common oligonucleotide tail that serves as a sequencing primer. Those skilled in the art are familiar with primer extensions, which can be used for these puφoses.

Preferred primers, useful for the amplification of genomic sequences encoding the candidate genes, focus on promoters, exons and splice sites ofthe genes. A biallelic marker presents a higher probability to be an eventual causal mutation if it is located in these functional regions ofthe gene. Preferred amplification primers ofthe invention include the nucleotide sequences Bl to B25 and Cl to C25, detailed further in Example 2, Table 1.

Sequencing Of Amplified Genomic DNA And Identification Of Single Nucleotide Polymorphisms The amplification products generated as described above, are then sequenced using any method known and available to the skilled technician. Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Sambrook et al.(1989). Alternative approaches include hybridization to high-density DNA probe arrays as described in Chee et al.(1996). Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. The products ofthe sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis. The polymoφhism search is based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallelic site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands are sequenced and a comparison between the peaks is carried out. In order to be registered as a polymorphic sequence, the polymoφhism has to be detected on both strands.

The above procedure permits those amplification products, which contain biallelic markers to be identified. The detection limit for the frequency of biallelic polymoφhisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known allelic frequencies. However, more than 90% ofthe biallelic polymoφhisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallelic markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele. Preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.

In another embodiment, biallelic markers are detected by sequencing individual DNA samples, the frequency ofthe minor allele of such a biallelic marker may be less than 0.1.

Validation Of The Biallelic Markers Of The Present Invention

The polymoφhisms are evaluated for their usefulness as genetic markers by validating that both alleles are present in a population. Validation ofthe biallelic markers is accomplished by genotyping a group of individuals by a method ofthe invention and demonstrating that both alleles are present. Microsequencing is a preferred method of genotyping alleles. The validation by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group can be as small as one individual if that individual is heterozygous for the allele in question. Preferably the group contains at least three individuals, more preferably the group contains five or six individuals, so that a single validation test will be more likely to result in the validation of more ofthe biallelic markers that are being tested. It should be noted, however, that when the validation test is performed on a small group it may result in a false negative result if as a result of sampling error none ofthe individuals tested carries one ofthe two alleles. Thus, the validation process is less useful in demonstrating that a particular initial result is an artifact, than it is at demonstrating that there is a bonafide biallelic marker at a particular position in a sequence. All ofthe genotyping, haplotyping, association, and interaction study methods ofthe invention may optionally be performed solely with validated biallelic markers.

Evaluation Of The Frequency Of The Biallelic Markers Of The Present Invention The validated biallelic markers are further evaluated for their usefulness as genetic markers by determining the frequency ofthe least common allele at the biallelic marker site. The higher the frequency ofthe less common allele the greater the usefulness ofthe biallelic marker is association and interaction studies. The determination ofthe least common allele is accomplished by genotyping a group of individuals by a method ofthe invention and demonstrating that both alleles are present. This determination of frequency by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group must be large enough to be representative ofthe population as a whole. Preferably the group contains at least 20 individuals, more preferably the group contains at least 50 individuals, most preferably the group contains at least 100 individuals. Of course the larger the group the greater the accuracy ofthe frequency determination because of reduced sampling error. A biallelic marker wherein the frequency ofthe less common allele is 30% or more is termed a "high quality biallelic marker." All ofthe genotyping, haplotyping, association, and interaction study methods ofthe invention may optionally be performed solely with high quality biallelic markers.

The invention also relates to methods of estimating the frequency of an allele in a population comprising: a) genotyping individuals from said population for said biallelic marker according to the method ofthe present invention; b) determining the proportional representation of said biallelic marker in said population. In addition, the methods of estimating the frequency of an allele in a population ofthe invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination; optionally, wherein said HKZP-related biallelic marker is selected from the group consisting of A 1 to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said HAZR-related biallelic marker is selected from the group consisting of Al to A22 and A25 to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said HKLR-related biallelic marker is selected from the group consisting of A23 and A24; optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by determining the identity ofthe nucleotides for both copies of said biallelic marker present in the genome of each individual in said population and calculating the proportional representation of said nucleotide at said HAZ -related biallelic marker for the population; optionally, determining the proportional representation may be accomplished by performing a genotyping method ofthe invention on a pooled biological sample derived from a representative number of individuals, or each individual, in said population, and calculating the proportional amount of said nucleotide compared with the total.

Methods For Genotyping An Individual For Biallelic Markers

Methods are provided to genotype a biological sample for one or more biallelic markers of the present invention, all of which may be performed in vitro. Such methods of genotyping comprise determining the identity of a nucleotide at a HKLP biallelic marker site by any method known in the art. These methods find use in genotyping case-control populations in association studies as well as individuals in the context of detection of alleles of biallelic markers which are known to be associated with a given trait, in which case both copies ofthe biallelic marker present in individual's genome are determined so that an individual may be classified as homozygous or heterozygous for a particular allele.

These genotyping methods can be performed on nucleic acid samples derived from a single individual or pooled DNA samples.

Genotyping can be performed using similar methods as those described above for the identification ofthe biallelic markers, or using other genotyping methods such as those further described below. In preferred embodiments, the comparison of sequences of amplified genomic fragments from different individuals is used to identify new biallelic markers whereas microsequencing is used for genotyping known biallelic markers in diagnostic and association study applications.

In one embodiment the invention encompasses methods of genotyping comprising determining the identity of a nucleotide at a HKZR-related biallelic marker or the complement thereof in a biological sample; Optionally, said HKLP-re\ated biallelic marker is selected from the group consisting of Al to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, said HΛEP-related biallelic marker is selected from the group consisting of Al to A 17, and A20 to A22, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, said HKZ -related biallelic marker is selected from the group consisting of A23 and A24, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said biological sample is derived from a single subject; optionally, wherein the identity ofthe nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome; optionally, wherein said biological sample is derived from multiple subjects; optionally, further comprising amplifying a portion of said sequence comprising the biallelic marker prior to said determining step; optionally, wherein said amplifying is performed by PCR; optionally, wherein said determining is performed by a hybridization assay, a sequencing assay, a microsequencing assay, or an enzyme-based mismatch detection assay.

Source of DNA for genotyping Any source of nucleic acids, in purified or non-purified form, can be utilized as the starting nucleic acid, provided it contains or is suspected of containing the specific nucleic acid sequence desired. DNA or RNA may be extracted from cells, tissues, body fluids and the like as described above. While nucleic acids for use in the genotyping methods ofthe invention can be derived from any mammalian source, the test subjects and individuals from which nucleic acid samples are taken are generally understood to be human.

Amplification Of DNA Fragments Comprising Biallelic Markers

Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallelic marker ofthe present invention. It will be appreciated that amplification of DNA fragments comprising biallelic markers may be used in various methods and for various puφoses and is not restricted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification ofthe DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker ofthe present invention. Amplification of DNA may be achieved by any method known in the art. Amplification techniques are described above in the section entitled, "Amplification ofthe HKLP gene".

Some of these amplification methods are particularly suited for the detection of single nucleotide polymorphisms and allow the simultaneous amplification of a target sequence and the identification ofthe polymoφhic nucleotide as it is further described below. The identification of biallelic markers as described above allows the design of appropriate oligonucleotides, which can be used as primers to amplify DNA fragments comprising the biallelic markers ofthe present invention. Amplification can be performed using the primers initially used to discover new biallelic markers which are described herein or any set of primers allowing the amplification of a DNA fragment comprising a biallelic marker ofthe present invention. In some embodiments the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers ofthe present invention. Preferred amplification primers are listed in Example 2. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers ofthe present invention. The spacing ofthe primers determines the length ofthe segment to be amplified. In the context ofthe present invention, amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification primers may be labeled or immobilized on a solid support as described in "Oligonucleotide probes and primers".

Methods of Genotyping DNA samples for Biallelic Markers

Any method known in the art can be used to identify the nucleotide present at a biallelic marker site. Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques. Many genotyping methods require the previous amplification ofthe DNA region carrying the biallelic marker of interest. While the amplification of target or signal is often preferred at present, ultrasensitive detection methods which do not require amplification are also encompassed by the present genotyping methods. Methods well-known to those skilled in the art that can be used to detect biallelic polymoφhisms include methods such as, conventional dot blot analyzes, single strand conformational polymoφhism analysis (SSCP) described by Orita et al.(1989), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield et al.(1991), White et al.(1992), Grompe et al.(1989 and 1993). Another method for determining the identity ofthe nucleotide present at a particular polymoφhic site employs a specialized exonuclease-resistant nucleotide derivative as described in US patent 4,656,127.

Preferred methods involve directly determining the identity ofthe nucleotide present at a biallelic marker site by sequencing assay, enzyme-based mismatch detection assay, or hybridization assay. The following is a description of some preferred methods. A highly preferred method is the microsequencing technique. The term "sequencing" is used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing. 1 ) Sequencing Assays

The nucleotide present at a polymoφhic site can be determined by sequencing methods. In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing as described above. DNA sequencing methods are described in "Sequencing Of Amplified Genomic DNA And Identification Of Single Nucleotide Polymoφhisms".

Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. Sequence analysis allows the identification ofthe base present at the biallelic marker site. 2) Microsequencing Assays

In microsequencing methods, the nucleotide at a polymoφhic site in a target DNA is detected by a single nucleotide primer extension reaction. This method involves appropriate microsequencing primers which, hybridize just upstream ofthe polymoφhic base of interest in the target nucleic acid. A polymerase is used to specifically extend the 3' end ofthe primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymoφhic site. Next the identity ofthe incoφorated nucleotide is determined in any suitable way.

Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity ofthe incoφorated nucleotide as described in EP 412 883. Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously. An example of a typical microsequencing procedure that can be used in the context ofthe present invention is provided in Example 4.

Different approaches can be used for the labeling and detection of ddNTPs. A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (1997) and Chen et al.(1997). In this method, amplified genomic DNA fragments containing polymoφhic sites are incubated with a 5'-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase. The dye- labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end ofthe genotyping reaction, the fluorescence intensities ofthe two dyes in the reaction mixture are analyzed directly without separation or purification. All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time. Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry. The base at the polymoφhic site is identified by the mass added onto the microsequencing primer (see Haff and Smimov, 1997).

Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof. Alternative methods include several solid-phase microsequencing techniques. The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogeneous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support. To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension. The 5' ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation. If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incoφorated terminator regent. This eliminates the need of physical or size separation. More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used. This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction. The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template. For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin- coated microtitration wells or avidin-coated polystyrene particles. In the same manner, oligonucleotides or templates may be attached to a solid support in a high-density format. In such solid phase microsequencing reactions, incoφorated ddNTPs can be radiolabeled (Syvanen, 1994) or linked to fluorescein (Livak and Hainer, 1994). The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as -nitrophenyl phosphate). Other possible reporter- detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712). As yet another alternative solid-phase microsequencing procedure, Nyren et al.(l 993) described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).

Pastinen et al.(1997) describe a method for multiplex detection of single nucleotide polymoφhism in which the solid phase minisequencing principle is applied to an oligonucleotide array format. High-density arrays of DNA probes attached to a solid support (DNA chips) are further described below.

In one aspect the present invention provides polynucleotides and methods to genotype one or more biallelic markers ofthe present invention by performing a microsequencing assay. Preferred microsequencing primers include the nucleotide sequences Dl to D30 and El to E30. It will be appreciated that the microsequencing primers listed in Example 4 are merely exemplary and that, any primer having a 3' end immediately adjacent to the polymoφhic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers ofthe present invention. One aspect ofthe present invention is a solid support which includes one or more microsequencing primers listed in Example 4, or fragments comprising at least 8, 12, 15, 20, 25, 30, 40, or 50 consecutive nucleotides thereof and having a 3' terminus immediately upstream ofthe corresponding biallelic marker, for determining the identity of a nucleotide at a biallelic marker site.

3) Mismatch detection assays based on polvmerases and ligases

In one aspect the present invention provides polynucleotides and methods to determine the allele of one or more biallelic markers ofthe present invention in a biological sample, by mismatch detection assays based on polymerases and/or ligases. These assays are based on the specificity of polymerases and ligases. Polymerization reactions places particularly stringent requirements on correct base pairing ofthe 3' end ofthe amplification primer and the joining of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the ligation site, especially at the 3' end. Methods, primers and various parameters to amplify DNA fragments comprising biallelic markers ofthe present invention are further described above in "Amplification Of DNA Fragments Comprising Biallelic Markers".

Allele Specific Amplification Primers

Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one ofthe alleles is amplified without amplification ofthe other allele. This is accomplished by placing the polymoφhic base at the 3' end of one ofthe amplification primers. Because the extension forms from the 3'end ofthe primer, a mismatch at or near this position has an inhibitory effect on amplification. Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele. Determining the precise location ofthe mismatch and the corresponding assay conditions are well with the ordinary skill in the art.

Ligation/Amplification Based Methods .

The "Oligonucleotide Ligation Assay" (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. OLA is capable of detecting single nucleotide polymoφhisms and may be advantageously combined with PCR as described by Nickerson et al.(1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.

Other amplification methods which are particularly suited for the detection of single nucleotide polymoφhism include LCR (ligase chain reaction), Gap LCR (GLCR) which are described above in "Amplification ofthe HKLP gene". LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences ofthe same strand ofthe target. Such hybridization forms a substrate for a template-dependant ligase. In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site. In one embodiment, either oligonucleotide will be designed to include the biallelic marker site. In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallelic marker on the oligonucleotide. In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a "gap" is created as described in WO 90/01069. This gap is then "filled" with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification ofthe desired sequence is obtained. Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271). This method involves the incoφoration of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide. The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution. 4) Hybridization Assay Methods

A preferred method of determining the identity ofthe nucleotide present at a biallelic marker site involves nucleic acid hybridization. The hybridization probes, which can be conveniently used in such reactions, preferably include the probes defined herein. Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid- phase hybridization (see Sambrook et al., 1989).

Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one ofthe alleles. Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., 1989). Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Although such hybridizations can be performed in solution, it is preferred to employ a solid- phase hybridization assay. The target DNA comprising a biallelic marker ofthe present invention may be amplified prior to the hybridization reaction. The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA. The detection of hybrid duplexes can be carried out by a number of methods. Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection ofthe hybrid duplexes. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes. Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., 1998). The TaqMan assay takes advantage of the 5' nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product. TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage ofthe TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allelic variants can be assembled at the beginning ofthe reaction and the results are monitored in real time (see Livak et al., 1995). In an alternative homogeneous hybridization based procedure, molecular beacons are used for allele discriminations. Molecular beacons are haiφin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., 1998). The polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of biallelic marker alleles in biological samples. These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker ofthe present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation. A particularly preferred probe is 25 nucleotides in length. Preferably the biallelic marker is within 4 nucleotides ofthe center ofthe polynucleotide probe. In particularly preferred probes, the biallelic marker is at the center of said polynucleotide. Preferred probes comprise a nucleotide sequence selected from the group consisting of amplicons listed in Table 1 and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymoφhic base. In preferred embodiments the polymoφhic base is within 5, 4, 3, 2, 1, nucleotides ofthe center ofthe said polynucleotide, more preferably at the center of said polynucleotide.

Preferably the probes ofthe present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in "Oligonucleotide Probes and Primers". The probes can be non-extendable as described in "Oligonucleotide Probes and Primers". By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample. High-Throughput parallel hybridizations in array format are specifically encompassed within "hybridization assays" and are described below. 5) Hybridization To Addressable Arrays Of Oligonucleotides

Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants. Efficient access to polymoφhism information is obtained through a basic structure comprising high- density arrays of oligonucleotide probes attached to a solid support (e.g., the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.

The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., 1996; Shoemaker et al., 1996; Kozal et al., 1996). Chips of various formats for use in detecting biallelic polymoφhisms can be produced on a customized basis by Affymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.

In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymoφhic marker. EP 785280 describes a tiling strategy for the detection of single nucleotide polymorphisms. Briefly, arrays may generally be "tiled" for a large number of specific polymoφhisms. By "tiling" is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members ofthe basis set of monomers, i.e. nucleotides. Tiling strategies are further described in PCT application No. WO 95/1 1995. In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences. In particular, the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers. For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymoφhism. To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker. In addition to the probes differing at the polymoφhic base, monosubstituted probes are also generally tiled within the detection block. These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymoφhism, substituted with the remaining nucleotides (selected from A, T, G, C and U). Typically the probes in a tiled detection block will include substitutions ofthe sequence positions up to and including those that are 5 bases away from the biallelic marker. The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization. Upon completion of hybridization with the target sequence and washing ofthe array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data from the scanned array is then analyzed to identify which allele or alleles ofthe biallelic marker are present in the sample. Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and US patent No. 5,424,186.

Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length. In further embodiments, the chip may comprise an array including at least one ofthe sequences selected from the group consisting of amplicons listed in table 1 and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymoφhic base. In preferred embodiments the polymoφhic base is within 5, 4, 3, 2, 1, nucleotides ofthe center ofthe said polynucleotide, more preferably at the center of said polynucleotide. In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides ofthe invention. Solid supports and polynucleotides ofthe present invention attached to solid supports are further described in "oligonucleotide probes and primers". 6^") Integrated Systems Another technique, which may be used to analyze polymoφhisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. An example of such technique is disclosed in US patent 5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips. Integrated systems can be envisaged mainly when microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements ofthe samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts. For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser- induced fluorescence detection.

Methods Of Genetic Analysis Using The Biallelic Markers Of The Present Invention

Different methods are available for the genetic analysis of complex traits (see Lander and Schork, 1994). The search for disease-susceptibility genes is conducted using two main methods: the linkage approach in which evidence is sought for cosegregation between a locus and a putative trait locus using family studies, and the association approach in which evidence is sought for a statistically significant association between an allele and a trait or a trait causing allele (Khoury et al., 1993). In general, the biallelic markers ofthe present invention find use in any method known in the art to demonstrate a statistically significant correlation between a genotype and a phenotype. The biallelic markers may be used in parametric and non-parametric linkage analysis methods. Preferably, the biallelic markers ofthe present invention are used to identify genes associated with detectable traits using association studies, an approach which does not require the use of affected families and which permits the identification of genes associated with complex and sporadic traits. The genetic analysis using the biallelic markers ofthe present invention may be conducted on any scale. The whole set of biallelic markers ofthe present invention or any subset of biallelic markers ofthe present invention corresponding to the candidate gene may be used. Further, any set of genetic markers including a biallelic marker ofthe present invention may be used. A set of biallelic polymoφhisms that could be used as genetic markers in combination with the biallelic markers ofthe present invention has been described in WO 98/20165. As mentioned above, it should be noted that the biallelic markers of the present invention may be included in any complete or partial genetic map ofthe human genome. These different uses are specifically contemplated in the present invention and claims.

Linkage Analysis

Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. Thus, the aim of linkage analysis is to detect marker loci that show cosegregation with a trait of interest in pedigrees. Parametric Methods

When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci. Estimates ofthe recombination fraction enable loci to be ordered and placed onto a genetic map. With loci that are genetic markers, a genetic map can be established, and then the strength of linkage between markers and traits can be calculated and used to indicate the relative positions of markers and genes affecting those traits (Weir, 1996). The classical method for linkage analysis is the logarithm of odds (lod) score method (see Morton, 1955; Ott, 1991). Calculation of lod scores requires specification ofthe mode of inheritance for the disease (parametric method). Generally, the length ofthe candidate region identified using linkage analysis is between 2 and 20Mb. Once a candidate region is identified as described above, analysis of recombinant individuals using additional markers allows further delineation ofthe candidate region. Linkage analysis studies have generally relied on the use of a maximum of 5,000 microsatellite markers, thus limiting the maximum theoretical attainable resolution of linkage analysis to about 600 kb on average.

Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance (i.e., the ratio between the number of trait positive carriers of allele a and the total number of a carriers in the population). However, parametric linkage analysis suffers from a variety of drawbacks. First, it is limited by its reliance on the choice of a genetic model suitable for each studied trait. Furthermore, as already mentioned, the resolution attainable using linkage analysis is limited, and complementary studies are required to refine the analysis ofthe typical 2Mb to 20Mb regions initially identified through linkage analysis. In addition, parametric linkage analysis approaches have proven difficult when applied to complex genetic traits, such as those due to the combined action of multiple genes and/or environmental factors. It is very difficult to model these factors adequately in a lod score analysis. In such cases, too large an effort and cost are needed to recruit the adequate number of affected families required for applying linkage analysis to these situations, as recently discussed by Risch, N. and Merikangas, K. (1996).

Non-Parametric Methods

The advantage ofthe so-called non-parametric methods for linkage analysis is that they do not require specification ofthe mode of inheritance for the disease, they tend to be more useful for the analysis of complex traits. In non-parametric methods, one tries to prove that the inheritance pattern of a chromosomal region is not consistent with random Mendelian segregation by showing that affected relatives inherit identical copies ofthe region more often than expected by chance. Affected relatives should show excess "allele sharing" even in the presence of incomplete penetrance and polygenic inheritance. In non-parametric linkage analysis the degree of agreement at a marker locus in two individuals can be measured either by the number of alleles identical by state (IBS) or by the number of alleles identical by descent (IBD). Affected sib pair analysis is a well- known special case and is the simplest form of these methods.

The biallelic markers ofthe present invention may be used in both parametric and non- parametric linkage analysis. Preferably biallelic markers may be used in non-parametric methods which allow the mapping of genes involved in complex traits. The biallelic markers ofthe present invention may be used in both IBD- and IBS- methods to map genes affecting a complex trait. In such studies, taking advantage ofthe high density of biallelic markers, several adjacent biallelic marker loci may be pooled to achieve the efficiency attained by multi-allelic markers (Zhao et al., 1998).

Population Association Studies

The present invention comprises methods for identifying if the HKLP gene is associated with a detectable trait using the biallelic markers ofthe present invention. In one embodiment the present invention comprises methods to detect an association between a biallelic marker allele or a biallelic marker haplotype and a trait. Further, the invention comprises methods to identify a trait causing allele in linkage disequilibrium with any biallelic marker allele ofthe present invention.

As described above, alternative approaches can be employed to perform association studies: genome-wide association studies, candidate region association studies and candidate gene association studies. In a preferred embodiment, the biallelic markers ofthe present invention are used to perform candidate gene association studies. The candidate gene analysis clearly provides a short-cut approach to the identification of genes and gene polymoφhisms related to a particular trait when some information concerning the biology ofthe trait is available. Further, the biallelic markers ofthe present invention may be incoφorated in any map of genetic markers ofthe human genome in order to perform genome-wide association studies. Methods to generate a high-density map of biallelic markers has been described in US Provisional Patent application serial number 60/082,614. The biallelic markers ofthe present invention may further be incoφorated in any map of a specific candidate region ofthe genome (a specific chromosome or a specific chromosomal segment for example).

As mentioned above, association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. Association studies are extremely valuable as they permit the analysis of sporadic or multifactor traits.

Moreover, association studies represent a powerful method for fine-scale mapping enabling much finer mapping of trait causing alleles than linkage studies. Studies based on pedigrees often only narrow the location ofthe trait causing allele. Association studies using the biallelic markers ofthe present invention can therefore be used to refine the location of a trait causing allele in a candidate region identified by Linkage Analysis methods. Moreover, once a chromosome segment of interest has been identified, the presence of a candidate gene such as a candidate gene ofthe present invention, in the region of interest can provide a shortcut to the identification ofthe trait causing allele. Biallelic markers ofthe present invention can be used to demonstrate that a candidate gene is associated with a trait. Such uses are specifically contemplated in the present invention.

Determining The Frequency Of A Biallelic Marker Allele Or Of A Biallelic Marker

Haplotype In A Population

Association studies explore the relationships among frequencies for sets of alleles between loci.

Determining The Frequency Of An Allele In A Population Allelic frequencies ofthe biallelic markers in a populations can be determined using one of the methods described above under the heading "Methods for genotyping an individual for biallelic markers", or any genotyping procedure suitable for this intended puφose. Genotyping pooled samples or individual samples can determine the frequency of a biallelic marker allele in a population. One way to reduce the number of genotypings required is to use pooled samples. A major obstacle in using pooled samples is in terms of accuracy and reproducibility for determining accurate DNA concentrations in setting up the pools. Genotyping individual samples provides higher sensitivity, reproducibility and accuracy and; is the preferred method used in the present invention. Preferably, each individual is genotyped separately and simple gene counting is applied to determine the frequency of an allele of a biallelic marker or of a genotype in a given population. Determining The Frequency Of A Haplotype In A Population

The gametic phase of haplotypes is unknown when diploid individuals are heterozygous at more than one locus. Using genealogical information in families gametic phase can sometimes be inferred (Perlin et al., 1994). When no genealogical information is available different strategies may be used. One possibility is that the multiple-site heterozygous diploids can be eliminated from the analysis, keeping only the homozygotes and the single-site heterozygote individuals, but this approach might lead to a possible bias in the sample composition and the underestimation of low- frequency haplotypes. Another possibility is that single chromosomes can be studied independently, for example, by asymmetric PCR amplification (see Newton et al, 1989; Wu et al., 1989) or by isolation of single chromosome by limit dilution followed by PCR amplification (see Ruano et al., 1990). Further, a sample may be haplotyped for sufficiently close biallelic markers by double PCR amplification of specific alleles (Sarkar, G. and Sommer S. S., 1991). These approaches are not entirely satisfying either because of their technical complexity, the additional cost they entail, their lack of generalization at a large scale, or the possible biases they introduce. To overcome these difficulties, an algorithm to infer the phase of PCR-amplified DNA genotypes introduced by Clark, A.G.(1990) may be used. Briefly, the principle is to start filling a preliminary list of haplotypes present in the sample by examining unambiguous individuals, that is, the complete homozygotes and the single-site heterozygotes. Then other individuals in the same sample are screened for the possible occurrence of previously recognized haplotypes. For each positive identification, the complementary haplotype is added to the list of recognized haplotypes, until the phase information for all individuals is either resolved or identified as unresolved. This method assigns a single haplotype to each multiheterozygous individual, whereas several haplotypes are possible when there are more than one heterozygous site. Alternatively, one can use methods estimating haplotype frequencies in a population without assigning haplotypes to each individual. Preferably, a method based on an expectation-maximization (EM) algorithm (Dempster et al., 1977) leading to maximum- likelihood estimates of haplotype frequencies under the assumption of Hardy- Weinberg proportions (random mating) is used (see Excoffier L. and Slatkin M., 1995). The EM algorithm is a generalized iterative maximum-likelihood approach to estimation that is useful when data are ambiguous and/or incomplete. The EM algorithm is used to resolve heterozygotes into haplotypes. Haplotype estimations are further described below under the heading "Statistical Methods." Any other method known in the art to determine or to estimate the frequency of a haplotype in a population may be used.

The invention also encompasses methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of: a) genotyping at least one HKLP- related biallelic marker according to a method ofthe invention for each individual in said population; b) genotyping a second biallelic marker by determining the identity ofthe nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome of each individual in said population; and c) applying a haplotype determination method to the identities ofthe nucleotides determined in steps a) and b) to obtain an estimate of said frequency. In addition, the methods of estimating the frequency of a haplotype ofthe invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said HKEP-related biallelic marker is selected from the group consisting of Al to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, said HKLP-related biallelic marker is selected from the group consisting of Al to A 17, and A20 to A22, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, said HA-LR-related biallelic marker is selected from the group consisting of A23 and A24, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said haplotype determination method is performed by asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark algorithm, or an expectation-maximization algorithm.

Linkage Disequilibrium Analysis Linkage disequilibrium is the non-random association of alleles at two or more loci and represents a powerful tool for mapping genes involved in disease traits (see Ajioka R.S. et al., 1997). Biallelic markers, because they are densely spaced in the human genome and can be genotyped in greater numbers than other types of genetic markers (such as RFLP or VNTR markers), are particularly useful in genetic analysis based on linkage disequilibrium. When a disease mutation is first introduced into a population (by a new mutation or the immigration of a mutation carrier), it necessarily resides on a single chromosome and thus on a single "background" or "ancestral" haplotype of linked markers. Consequently, there is complete disequilibrium between these markers and the disease mutation: one finds the disease mutation only in the presence of a specific set of marker alleles. Through subsequent generations recombination events occur between the disease mutation and these marker polymoφhisms, and the disequilibrium gradually dissipates. The pace of this dissipation is a function ofthe recombination frequency, so the markers closest to the disease gene will manifest higher levels of disequilibrium than those that are further away. When not broken up by recombination, "ancestral" haplotypes and linkage disequilibrium between marker alleles at different loci can be tracked not only through pedigrees but also through populations. Linkage disequilibrium is usually seen as an association between one specific allele at one locus and another specific allele at a second locus.

The pattern or curve of disequilibrium between disease and marker loci is expected to exhibit a maximum that occurs at the disease locus. Consequently, the amount of linkage disequilibrium between a disease allele and closely linked genetic markers may yield valuable information regarding the location ofthe disease gene. For fine-scale mapping of a disease locus, it is useful to have some knowledge ofthe patterns of linkage disequilibrium that exist between markers in the studied region. As mentioned above the mapping resolution achieved through the analysis of linkage disequilibrium is much higher than that of linkage studies. The high density of biallelic markers combined with linkage disequilibrium analysis provides powerful tools for fine- scale mapping. Different methods to calculate linkage disequilibrium are described below under the heading "Statistical Methods".

Population-Based Case-Control Studies Of Trait-Marker Associations

As mentioned above, the occurrence of pairs of specific alleles at different loci on the same chromosome is not random and the deviation from random is called linkage disequilibrium. Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium. If a specific allele in a given gene is directly involved in causing a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population. As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls. Therefore, association between the trait and any allele (specifically a biallelic marker allele) in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region. Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele. As any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait. Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymoφhisms (specifically biallelic markers) to be analyzed as an alternative to screening all possible functional polymoφhisms in order to find trait-causing alleles. Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits. Case-Control Populations (Inclusion Criteria)

Population-based association studies do not concern familial inheritance but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations. They are case-control studies based on comparison of unrelated case (affected or trait positive) individuals and unrelated control (unaffected, trait negative or random) individuals. Preferably the control group is composed of unaffected or trait negative individuals. Further, the control group is ethnically matched to the case population. Moreover, the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age- matched for an age-dependent trait). Ideally, individuals in the two samples are paired in such a way that they are expected to differ only in their disease status. The terms "trait positive population", "case population" and "affected population" are used interchangeably herein. An important step in the dissection of complex traits using association studies is the choice of case-control populations (see Lander and Schork, 1994). A major step in the choice of case- control populations is the clinical definition of a given trait or phenotype. Any genetic trait may be analyzed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenotypic groups. Four criteria are often useful: clinical phenotype, age at onset, family history and severity. The selection procedure for continuous or quantitative traits (such as blood pressure for example) involves selecting individuals at opposite ends ofthe phenotype distribution ofthe trait under study, so as to include in these trait positive and trait negative populations individuals with non-overlapping phenotypes. Preferably, case-control populations consist of phenotypically homogeneous populations. Trait positive and trait negative populations consist of phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20%) ofthe total population under study, and preferably selected among individuals exhibiting non-overlapping phenotypes. The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers. The selection of those drastically different but relatively uniform phenotypes enables efficient comparisons in association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes ofthe populations under study are significant enough. In preferred embodiments, a first group of between 50 and 300 trait positive individuals, preferably about 100 individuals, are recruited according to their phenotypes. A similar number of control individuals are included in such studies. Association Analysis

The invention also comprises methods of detecting an association between a genotype and a phenotype, comprising the steps of: a) determining the frequency of at least one HΛXR-related biallelic marker in a trait positive population according to a genotyping method ofthe invention; b) determining the frequency of said HAZP-related biallelic marker in a control population according to a genotyping method ofthe invention; and c) determining whether a statistically significant association exists between said genotype and said phenotype. In addition, the methods of detecting an association between a genotype and a phenotype ofthe invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said HKEP-related biallelic marker is selected from the group consisting of Al to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, said HAZR-related biallelic marker is selected from the group consisting of Al to A17, and A20 to A22, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, said HKEP-related biallelic marker is selected from the group consisting of A23 and A24, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said control population may be a trait negative population, or a random population; Optionally, each of said genotyping steps a) and b) may be performed on a pooled biological sample derived from each of said populations; Optionally, each of said genotyping of steps a) and b) is performed separately on biological samples derived from each individual in said population or a subsample thereof.

The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers ofthe present invention in both groups. If a statistically significant association with a trait is identified for at least one or more ofthe analyzed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (i.e. the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele. The specific characteristics ofthe associated allele with respect to the candidate gene function usually give further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium). If the evidence indicates that the associated allele within the candidate gene is most probably not the trait causing allele but is in linkage disequilibrium with the real trait causing allele, then the trait causing allele can be found by sequencing the vicinity ofthe associated marker, and performing further association studies with the polymoφhisms that are revealed in an iterative manner. Association studies are usually run in two successive steps. In a first phase, the frequencies of a reduced number of biallelic markers from the candidate gene are determined in the trait positive and control populations. In a second phase ofthe analysis, the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region. However, if the candidate gene under study is relatively small in length, as is the case for HKLP, a single phase may be sufficient to establish significant associations. Haplotype Analysis

As described above, when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype. This haplotype can be tracked through populations and its statistical association with a given trait can be analyzed.

Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies. Thus, a haplotype association study allows one to define the frequency and the type ofthe ancestral carrier haplotype. A haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.

In a first stage of a haplotype frequency analysis, the frequency ofthe possible haplotypes based on various combinations ofthe identified biallelic markers ofthe invention is determined. The haplotype frequency is then compared for distinct populations of trait positive and control individuals. The number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150. The same considerations apply to the number of unaffected individuals (or random control) used in the study. The results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odd ratio are calculated. If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated. An additional embodiment ofthe present invention encompasses methods of detecting an association between a haplotype and a phenotype, comprising the steps of: a) estimating the frequency of at least one haplotype in a trait positive population, according to a method ofthe invention for estimating the frequency of a haplotype; b) estimating the frequency of said haplotype in a control population, according to a method ofthe invention for estimating the frequency of a haplotype; and c) determining whether a statistically significant association exists between said haplotype and said phenotype. In addition, the methods of detecting an association between a haplotype and a phenotype ofthe invention encompass methods with any further limitation described in this disclosure, or those following: Optionally, said HKLP-τe\ated biallelic marker is selected from the group consisting of Al to A32, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, said HKLP-re\ated biallelic marker is selected from the group consisting of Al to A 17, and A20 to A22, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, said HA R-related biallelic marker is selected from the group consisting of A23 and A24, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said control population is a trait negative population, or a random population. Optionally, said method comprises the additional steps of determining the phenotype in said trait positive and said control populations prior to step c). Interaction Analysis

The biallelic markers ofthe present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions. The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein. The analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis. Interaction analysis consists in stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.

Statistical methods used in association studies are further described below. Testing For Linkage In The Presence Of Association

The biallelic markers ofthe present invention may further be used in TDT (transmission/disequilibrium test). TDT tests for both linkage and association and is not affected by population stratification. TDT requires data for affected individuals and their parents or data from unaffected sibs instead of from parents (see Spielmann S. et al., 1993; Schaid D.J. et al., 1996, Spielmann S. and Ewens W.J., 1998). Such combined tests generally reduce the false - positive errors produced by separate analyses.

Statistical methods

In general, any method known in the art to test whether a trait and a genotype show a statistically significant correlation may be used.

1) Methods In Linkage Analysis

Statistical methods and computer programs useful for linkage analysis are well-known to those skilled in the art (see Terwilliger J.D. and Ott J., 1994; Ott J., 1991).

2) Methods To Estimate Haplotype Frequencies In A Population As described above, when genotypes are scored, it is often not possible to distinguish heterozygotes so that haplotype frequencies cannot be easily inferred. When the gametic phase is not known, haplotype frequencies can be estimated from the multilocus genotypic data. Any method known to person skilled in the art can be used to estimate haplotype frequencies (see Lange K., 1997; Weir, B.S., 1996) Preferably, maximum-likelihood haplotype frequencies are computed using an Expectation- Maximization (EM) algorithm (see Dempster et al., 1977; Excoffier L. and Slatkin M., 1995). This procedure is an iterative process aiming at obtaining maximum-likelihood estimates of haplotype frequencies from multi-locus genotype data when the gametic phase is unknown. Haplotype estimations are usually performed by applying the EM algorithm using for example the EM-HAPLO program (Hawley M. E. et al., 1994) or the Arlequin program (Schneider et al., 1997). The EM algorithm is a generalized iterative maximum likelihood approach to estimation and is briefly described below.

Please note that in the present section, "Methods To Estimate Haplotype Frequencies In A Population, " of this text, phenotypes will refer to multi-locus genotypes with unknown phase. Genotypes will refer to known-phase multi-locus genotypes. A sample of N unrelated individuals is typed for K markers. The data observed are the unknown-phase K-locus phenotypes that can categorized in F different phenotypes. Suppose that we have H underlying possible haplotypes (in case of K biallelic markers, H=2^K).

For phenotype j, suppose that C_j genotypes are possible. We thus have the following equation ^CJ ^CJ

Pj = ∑ pr (genotype ι) = ∑ pr(h_k ,h_{) Equation 1 i=\ i=ϊ where Pj is the probability ofthe phenotype j, A* and h_t are the two haplotypes constituent the genotype /. Under the Hardy- Weinberg equilibrium, pr(h_hhi) becomes: pr(h_k ,h[) = pr(h_k ) if h_k = A/ , pr(h_k , A/ ) = 2pr(h_k ).pr(hι ) if Λ^- ≠ A . Equation 2 The successive steps ofthe E-M algorithm can be described as follows:

Starting with initial values ofthe of haplotypes frequencies, noted

, /?₂ ⁰⁾ , pff , these initial values serve to estimate the genotype frequencies (Expectation step) and then estimate another set of haplotype frequencies (Maximization step), noted p_λ ^m ,p , p⁽ , these two steps are iterated until changes in the sets of haplotypes frequency are very small. A stop criterion can be that the maximum difference between haplotype frequencies between two iterations is less than 10^"7. These values can be adjusted according to the desired precision of estimations.

At a given iteration s, the Expectation step consists in calculating the genotypes frequencies by the following equation: pr(genotypeiy^s' = pr(phenotype j).pr (genotype ^phenotype ) nj pr(h_k ,h_l )^<'^s) Equation 3

^~ TV ^" n(*)

J where genotype occurs in phenotype j, and where h_k and hi constitute genotype . Each probability is derived according to eq. 1, and eq. 2 described above.

Then the Maximization step simply estimates another set of haplotype frequencies given the genotypes frequencies. This approach is also known as the gene-counting method (Smith, 1957).

1 F c ^■ Pt^S+1) = - ∑ ∑S _it . pr (genotype , )^(s Equation 4

² 7=lι=l

Where δ_jt is an indicator variable which count the number of time haplotype t in genotype i.

It takes the values of 0, 1 or 2.

To ensure that the estimation finally obtained is the maximum-likelihood estimation several values of departures are required. The estimations obtained are compared and if they are different the estimations leading to the best likelihood are kept. 3) Methods To Calculate Linkage Disequilibrium Between Markers

A number of methods can be used to calculate linkage disequilibrium between any two genetic positions, in practice linkage disequilibrium is measured by applying a statistical association test to haplotype data taken from a population. Linkage disequilibrium between any pair of biallelic markers comprising at least one ofthe biallelic markers ofthe present invention (M„ M_j) having alleles (a,/b,) at marker M, and alleles (a/b,) at marker M_j can be calculated for every allele combination (a,^ a,,^ b„a, and b„b,), according to the Piazza formula:

Δa_iaj= Vθ4 - (Θ4 + Θ3) (Θ4 +Θ2), where: Θ4= - - = frequency of genotypes not having allele a, at M, and not having allele a, at M_j

Θ3= - + = frequency of genotypes not having allele a, at M, and having allele a, at M_j

Θ2= + - = frequency of genotypes having allele a, at M, and not having allele a, at M_j

Linkage disequilibrium (LD) between pairs of biallelic markers (M„ M,) can also be calculated for every allele combination (a^aj_, ai,bj_j b„a_j and b„b,), according to the maximum- likelihood estimate (MLE) for delta (the composite genotypic disequilibrium coefficient), as described by Weir (Weir B. S., 1996). The MLE for the composite linkage disequilibrium is: D_aιaj= (2n_! + n₂ + n₃ + n^/N - 2(pr(a,). pr(a,))

Where ni = Σ phenotype (a,/a„ a a,), n₂ = Σ phenotype (a,/a„ a/b,), n₃= Σ phenotype (a,/b„ a a,), n4= Σ phenotype (a/b„ a/b,) and N is the number of individuals in the sample.

This formula allows linkage disequilibrium between alleles to be estimated when only genotype, and not haplotype, data are available.

Another means of calculating the linkage disequilibrium between markers is as follows. For a couple of biallelic markers, M, (a/b,) and M, (a/b_j), fitting the Hardy- Weinberg equilibrium, one can estimate the four possible haplotype frequencies in a given population according to the approach described above.

The estimation of gametic disequilibrium between ai and aj is simply: ^Dai j = pr(haplotype(a Uj )) - pr(a_t ).pr(<tj ). Where prfaj is the probability of allele a, and pr(aj is the probability of allele α, and where prihaplotype (a„ aj) is estimated as in Equation 3 above.

For a couple of biallelic marker only one measure of disequilibrium is necessary to describe the association between M, and M_}.

Then a normalized value ofthe above is calculated as follows: D'_ai_a, = D_aιaj / max (-pr(a,). pr(a_j) , -pr(b,). pr(b,)) with D_aιaj<0

D'_aιaj = D_aιaj / max (pr(b,). pr(a,) , pr(a,). pr(b,)) with D_aιaj>0 The skilled person will readily appreciate that other linkage disequilibrium calculation methods can be used.

Linkage disequilibrium among a set of biallelic markers having an adequate heterozygosity rate can be determined by genotyping between 50 and 1000 unrelated individuals, preferably between 75 and 200, more preferably around 100.

4) Testing For Association

Methods for determining the statistical significance of a correlation between a phenotype and a genotype, in this case an allele at a biallelic marker or a haplotype made up of such alleles, may be determined by any statistical test known in the art and with any accepted threshold of statistical significance being required. The application of particular methods and thresholds of significance are well with in the skill ofthe ordinary practitioner ofthe art.

Testing for association is performed by determining the frequency of a biallelic marker allele in case and control populations and comparing these frequencies with a statistical test to determine if their is a statistically significant difference in frequency which would indicate a correlation between the trait and the biallelic marker allele under study. Similarly, a haplotype analysis is performed by estimating the frequencies of all possible haplotypes for a given set of biallelic markers in case and control populations, and comparing these frequencies with a statistical test to determine if their is a statistically significant correlation between the haplotype and the phenotype (trait) under study. Any statistical tool useful to test for a statistically significant association between a genotype and a phenotype may be used. Preferably the statistical test employed is a chi-square test with one degree of freedom. A P-value is calculated (the P-value is the probability that a statistic as large or larger than the observed one would occur by chance). Statistical Significance

In preferred embodiments, significance for diagnosis puφoses, either as a positive basis for further diagnostic tests or as a preliminary starting point for early preventive therapy, the p value related to a biallelic marker association is preferably about 1 x 10^"2 or less, more preferably about 1 x 10^"4 or less, for a single biallelic marker analysis and about 1 x 10^"3 or less, still more preferably 1 x 10^"6 or less and most preferably of about 1 x 10^"8 or less, for a haplotype analysis involving two or more markers. These values are believed to be applicable to any association studies involving single or multiple marker combinations.

The skilled person can use the range of values set forth above as a starting point in order to carry out association studies with biallelic markers ofthe present invention. In doing so, significant associations between the biallelic markers ofthe present invention and a trait can be revealed and used for diagnosis and drug screening puφoses. Phenotvpic Permutation

In order to confirm the statistical significance ofthe first stage haplotype analysis described above, it might be suitable to perform further analyses in which genotyping data from case-control individuals are pooled and randomized with respect to the trait phenotype. Each individual genotyping data is randomly allocated to two groups, which contain the same number of individuals as the case-control populations used to compile the data obtained in the first stage. A second stage haplotype analysis is preferably run on these artificial groups, preferably for the markers included in the haplotype ofthe first stage analysis showing the highest relative risk coefficient. This experiment is reiterated preferably at least between 100 and 10000 times. The repeated iterations allow the determination ofthe probability to obtain the tested haplotype by chance. Assessment Of Statistical Association

To address the problem of false positives similar analysis may be performed with the same case-control populations in random genomic regions. Results in random regions and the candidate region are compared as described in a co-pending US Provisional Patent Application entitled "Methods, Software And Apparati For Identifying Genomic Regions Harboring A Gene Associated With A Detectable Trait," U.S. Serial Number 60/107,986, filed November 10, 1998, the contents of which are incoφorated herein by reference.

5) Evaluation Of Risk Factors

The association between a risk factor (in genetic epidemiology the risk factor is the presence or the absence of a certain allele or haplotype at marker loci) and a disease is measured by the odds ratio (OR) and by the relative risk (RR). If P(R ) is the probability of developing the disease for individuals with R and P(R^") is the probability for individuals without the risk factor, then the relative risk is simply the ratio ofthe two probabilities, that is: RR= P(R⁺)/P(R ) In case-control studies, direct measures ofthe relative risk cannot be obtained because ofthe sampling design. However, the odds ratio allows a good approximation ofthe relative risk for low- incidence diseases and can be calculated:

F⁺

OR =

1 - F⁺ (1 - E-)

ΟR= (F7(l-F⁺))/(F7(l-F-))

F⁺ is the frequency ofthe exposure to the risk factor in cases and F^' is the frequency ofthe exposure to the risk factor in controls. F⁺ and F^" are calculated using the allelic or haplotype frequencies ofthe study and further depend on the underlying genetic model (dominant, recessive, additive...).

One can further estimate the attributable risk (AR) which describes the proportion of individuals in a population exhibiting a trait due to a given risk factor. This measure is important in quantifying the role of a specific factor in disease etiology and in terms ofthe public health impact of a risk factor. The public health relevance of this measure lies in estimating the proportion of cases of disease in the population that could be prevented if the exposure of interest were absent. AR is determined as follows: AR = P_E (RR-1) / (P_E (RR-1)+1)

AR is the risk attributable to a biallelic marker allele or a biallelic marker haplotype. P_E is the frequency of exposure to an allele or a haplotype within the population at large; and RR is the relative risk which, is approximated with the odds ratio when the trait under study has a relatively low incidence in the general population.

Identification Of Biallelic Markers In Linkage Disequilibrium With The Biallelic Markers of the Invention

Once a first biallelic marker has been identified in a genomic region of interest, the practitioner of ordinary skill in the art, using the teachings ofthe present invention, can easily identify additional biallelic markers in linkage disequilibrium with this first marker. As mentioned before any marker in linkage disequilibrium with a first marker associated with a trait will be associated with the trait. Therefore, once an association has been demonstrated between a given biallelic marker and a trait, the discovery of additional biallelic markers associated with this trait is of great interest in order to increase the density of biallelic markers in this particular region. The causal gene or mutation will be found in the vicinity ofthe marker or set of markers showing the highest correlation with the trait.

Identification of additional markers in linkage disequilibrium with a given marker involves:

(a) amplifying a genomic fragment comprising a first biallelic marker from a plurality of individuals;

(b) identifying of second biallelic markers in the genomic region harboring said first biallelic marker; (c) conducting a linkage disequilibrium analysis between said first biallelic marker and second biallelic markers; and (d) selecting said second biallelic markers as being in linkage disequilibrium with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated.

Methods to identify biallelic markers and to conduct linkage disequilibrium analysis are described herein and can be carried out by the skilled person without undue experimentation. The present invention then also concerns biallelic markers which are in linkage disequilibrium with the specific biallelic markers Al to A32 and which are expected to present similar characteristics in terms of their respective association with a given trait. In a preferred embodiment, the invention concerns biallelic markers which are in linkage disequilibrium with the specific biallelic markers

Identification Of Functional Mutations Mutations in the HKLP gene which are responsible for a detectable phenotype or trait may be identified by comparing the sequences ofthe HKLP gene from trait positive and control individuals. Once a positive association is confirmed with a biallelic marker ofthe present invention, the identified locus can be scanned for mutations. In a preferred embodiment, functional regions such as exons and splice sites, promoters and other regulatory regions ofthe HKLP gene are scanned for mutations. In a preferred embodiment the sequence ofthe HKLP gene is compared in trait positive and control individuals. Preferably, trait positive individuals carry the haplotype shown to be associated with the trait and trait negative individuals do not carry the haplotype or allele associated with the trait. The detectable trait or phenotype may comprise a variety of manifestations of altered HKLP function.

The mutation detection procedure is essentially similar to that used for biallelic marker identification. The method used to detect such mutations generally comprises the following steps:

- amplification of a region ofthe HKLP gene comprising a biallelic marker or a group of biallelic markers associated with the trait from DNA samples of trait positive patients and trait- negative controls;

- sequencing ofthe amplified region; - comparison of DNA sequences from trait positive and control individuals;

- determination of mutations specific to trait-positive patients.

In one embodiment, said biallelic marker is selected from the group consisting of Al to A32, and the complements thereof. It is preferred that candidate polymoφhisms be then verified by screening a larger population of cases and controls by means of any genotyping procedure such as those described herein, preferably using a microsequencing technique in an individual test format. Polymoφhisms are considered as candidate mutations when present in cases and controls at frequencies compatible with the expected association results. Polymoφhisms are considered as candidate "trait-causing" mutations when they exhibit a statistically significant correlation with the detectable phenotype.

Recombinant Vectors

The term "vector" is used herein to designate either a circular or a linear DNA or RNA molecule, which is either double-stranded or single-stranded, and which comprise at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism. The present invention encompasses a family of recombinant vectors that comprise a regulatory polynucleotide derived from the HKLP genomic sequence, and/or a coding polynucleotide from either the HKLP genomic sequence or the cDNA sequence.

Generally, a recombinant vector ofthe invention may comprise any ofthe polynucleotides described herein, including regulatory sequences, coding sequences and polynucleotide constructs, as well as any HKLP primer or probe as defined above. More particularly, the recombinant vectors ofthe present invention can comprise any ofthe polynucleotides described in the "Genomic Sequences Of tThe HKLP Gene" section, the "HKLP cDNA Sequences" section, the "Coding

Regions" section, the "Polynucleotide constructs" section, and the "Oligonucleotide Probes And

Primers" section.

In a first preferred embodiment, a recombinant vector ofthe invention is used to amplify the inserted polynucleotide derived from a HKLP genomic sequence of SEQ ID Nos 1 and 2 or a HKLP cDNA, for example the cDNA of SEQ ID No 3 in a suitable cell host, this polynucleotide being amplified at every time that the recombinant vector replicates.

A second preferred embodiment ofthe recombinant vectors according to the invention consists of expression vectors comprising either a regulatory polynucleotide or a coding nucleic acid ofthe invention, or both. Within certain embodiments, expression vectors are employed to express the HKLP polypeptide which can be then purified and, for example be used in ligand screening assays or as an immunogen in order to raise specific antibodies directed against the HKLP protein.

In other embodiments, the expression vectors are used for constructing transgenic animals and also for gene therapy. Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression ofthe genes of interest in host cells. Dominant drug selection markers for establishing permanent, stable cell clones expressing the products are generally included in the expression vectors ofthe invention, as they are elements that link expression ofthe drug selection markers to expression ofthe polypeptide. More particularly, the present invention relates to expression vectors which include nucleic acids encoding a HKLP protein, preferably the HKLP protein ofthe amino acid sequence of SEQ ID

No 4 or variants or fragments thereof.

The invention also pertains to a recombinant expression vector useful for the expression of the HKLP coding sequence, wherein said vector comprises a nucleic acid of SEQ ID No 3. Recombinant vectors comprising a nucleic acid containing a HKZP-related biallelic marker is also part ofthe invention. In a preferred embodiment, said biallelic marker is selected from the group consisting of Al to A32, and the complements thereof.

Some ofthe elements which can be found in the vectors ofthe present invention are described in further detail in the following sections.

1. General features of the expression vectors of the invention

A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or even a linear DNA molecule which may consist of a chromosomal, non- chromosomal, semi-synthetic and synthetic DNA. Such a recombinant vector can comprise a transcriptional unit comprising an assembly of: (1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.

(2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and

(3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation ofthe host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion ofthe translated protein into the periplasmic space or the extracellular medium. In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5 '-flanking non-transcribed sequences. DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements. The in vivo expression of a HKLP polypeptide of SEQ ID No 4 or fragments or variants thereof may be useful in order to correct a genetic defect related to the expression ofthe native gene in a host organism or to the production of a biologically inactive HKLP protein.

Consequently, the present invention also deals with recombinant expression vectors mainly designed for the in vivo production ofthe HKLP polypeptide of SEQ ID No 4 or fragments or variants thereof by the introduction ofthe appropriate genetic material in the organism ofthe patient to be treated. This genetic material may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism, directly in vivo into the appropriate tissue. 2. Regulatory Elements

Promoters

The suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be expressed. The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression ofthe nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter. A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted. Promoter regions can be selected from any desired gene using, for example, CAT

(chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors. Preferred bacterial promoters are the Lad, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and tφ promoters (EP 0036776), the polyhedrin promoter, or the plO protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.

The choice of a promoter is well within the ability of a person skilled in the field of genetic egineering. For example, one may refer to the book of Sambrook et al.( 1989) or also to the procedures described by Fuller et al.(1996). Other regulatory elements

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation ofthe gene transcript. The nature ofthe polyadenylation signal is not believed to be crucial to the successful practice ofthe invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element ofthe expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

3. Selectable Markers Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRPl for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.

4. Preferred Vectors. Bacterial vectors

As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEM1 (Promega Biotec, Madison, WI, USA).

Large numbers of other suitable vectors are known to those of skill in the art, and commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).

Bacteriophage vectors

The PI bacteriophage vector may contain large inserts ranging from about 80 to about 100 kb. The construction of PI bacteriophage vectors such as pl58 or pl58/neo8 are notably described by Sternberg (1992, 1994). Recombinant PI clones comprising HKLP nucleotide sequences may be designed for inserting large polynucleotides of more than 40 kb (Linton et al., 1993). To generate PI DNA for transgenic experiments, a preferred protocol is the protocol described by McCormick et al.(1994). Briefly, E. coli (preferably strain NS3529) harboring the PI plasmid are grown overnight in a suitable broth medium containing 25 μg/ml of kanamycin. The PI DNA is prepared from the E. coli by alkaline lysis using the Qiagen Plasmid Maxi kit (Qiagen, Chatsworth, CA, USA), according to the manufacturer's instructions. The PI DNA is purified from the bacterial lysate on two Qiagen-tip 500 columns, using the washing and elution buffers contained in the kit. A phenol/chloroform extraction is then performed before precipitating the DNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), the concentration ofthe DNA is assessed by spectrophotometry.

When the goal is to express a PI clone comprising HKLP nucleotide sequences in a transgenic animal, typically in transgenic mice, it is desirable to remove vector sequences from the PI DNA fragment, for example by cleaving the PI DNA at rare-cutting sites within the PI polylinker (Sβl, Noil or Sail). The PI insert is then purified from vector sequences on a pulsed-field agarose gel, using methods similar using methods similar to those originally reported for the isolation of DNA from YACs (Schedl et al., 1993a; Peterson et al., 1993). At this stage, the resulting purified insert DNA can be concentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit (Millipore, Bedford, MA, USA - 30,000 molecular weight limit) and then dialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4; 250 μM EDTA) containing 100 mM NaCl, 30 μM spermine, 70 μM spermidine on a microdyalisis membrane (type VS, 0.025 μM from Millipore). The intactness ofthe purified PI DNA insert is assessed by electrophoresis on 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel and staining with ethidium bromide. Baculovirus vectors

A suitable vector for the expression ofthe HKLP polypeptide of SEQ ID No 3 or fragments or variants thereof is a baculovirus vector that can be propagated in insect cells and in insect cell lines. A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N°CRL 171 1) which is derived from Spodoptera frugiperda.

Other suitable vectors for the expression ofthe HKLP polypeptide of SEQ ID No 3 or fragments or variants thereof in a baculovirus expression system include those described by Chai et al.(1993), Vlasak et al.(1983) and Lenhard et al.(1996). Viral vectors

In one specific embodiment, the vector is derived from an adenovirus. Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996) or Ohno et al.(1994). Another preferred recombinant adenovirus according to this specific embodiment ofthe present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin ( French patent application N° FR-93.05954).

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo , particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA ofthe host. Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles ofthe present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus. Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728). Other preferred retroviral vectors are those described in Roth et al.(1996), PCT Application No WO 93/25234, PCT Application No WO 94/ 06920, Roux et al., 1989, Julan et al., 1992 and Neda et al., 1991. Yet another viral vector system that is contemplated by the invention consists in the adeno- associated virus (AAV). The adeno-associated vims is a naturally occurring defective virus that requires another virus, such as an adenovirus or a heφes vims, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., 1992). It is also one ofthe few viruses that

5 may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., 1992; Samulski et al., 1989; McLaughlin et al., 1989). One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells. BAC vectors The bacterial artificial chromosome (BAC) cloning system (Shizuya et al., 1992) has been 0 developed to stably maintain large fragments of genomic DNA (100-300 kb) in E. coli. A preferred BAC vector consists of pBeloBACl 1 vector that has been described by Kim et al.(1996). BAC libraries are prepared with this vector using size-selected genomic DNA that has been partially digested using enzymes that permit ligation into either the Bam HI or Hindϊll sites in the vector. Flanking these cloning sites are T7 and SP6 RNA polymerase transcription initiation sites that can 5 be used to generate end probes by either RNA transcription or PCR methods. After the construction of a BAC library in E. coli, BAC DNA is purified from the host cell as a supercoiled circle. Converting these circular molecules into a linear form precedes both size determination and introduction ofthe BACs into recipient cells. The cloning site is flanked by two Not I sites, permitting cloned segments to be excised from the vector by Not I digestion. Alternatively, the 0 DNA insert contained in the pBeloBAC 1 1 vector may be linearized by treatment ofthe BAC vector with the commercially available enzyme lambda terminase that leads to the cleavage at the unique cosN site, but this cleavage method results in a full length BAC clone containing both the insert DNA and the BAC sequences.

5. Delivery Of The Recombinant Vectors 5 In order to effect expression ofthe polynucleotides and polynucleotide constructs ofthe invention, these constructs must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain diseases states.

One mechanism is viral infection where the expression construct is encapsulated in an 0 infectious viral particle.

Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Graham et al., 1973; Chen et al., 1987;), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland et al., 35 1985), DNA-loaded liposomes (Nicolau et al., 1982; Fraley et al., 1979), and receptor-mediated transfection (Wu and Wu, 1987; 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome ofthe recipient cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior ofthe cell and has a physiological effect. This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.

Compositions for use in vitro and in vivo comprising a "naked" polynucleotide are described in PCT application N° WO 90/11092 (Vical Inc.) and also in PCT application No. WO 95/1 1307 (Institut Pasteur, INSERM, Universite d'Ottawa) as well as in the articles of Tacson et al.(1996) and of Huygen et al.(1996). In still another embodiment ofthe invention, the transfer of a naked polynucleotide ofthe invention, including a polynucleotide construct ofthe invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.(1987). In a further embodiment, the polynucleotide ofthe invention may be entrapped in a liposome (Ghosh and Bacchawat, 1991 ; Wong et al., 1980; Nicolau et al., 1987)

In a specific embodiment, the invention provides a composition for the in vivo production of the HKLP protein or polypeptide described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells ofthe tissue to express the said protein or polypeptide.

The amount of vector to be injected to the desired host organism varies according to the site of injection. As an indicative dose, it will be injected between 0,1 and 100 μg ofthe vector in an animal body, preferably a mammal body, for example a mouse body.

In another embodiment ofthe vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell. In a subsequent step, the cell that has been transformed with the vector coding for the desired HKLP polypeptide or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.

Cell Hosts

Another object ofthe invention consists of a host cell that has been transformed or transfected with one ofthe polynucleotides described herein, and in particular a polynucleotide either comprising a HKLP regulatory polynucleotide or the coding sequence ofthe HKLP polypeptide selected from the group consisting of SEQ ID Nos 1 -3 or a fragment or a variant thereof. Also included are host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as one of those described above. More particularly, the cell hosts ofthe present invention can comprise any ofthe polynucleotides described in the "Genomic Sequences Of tThe HKLP Gene" section, the "HKLP cDNA Sequences" section, the "Coding Regions" section, the "Polynucleotide constmcts" section, the "Oligonucleotide Probes And Primers" section and the "Recombinant Vectors" section.

A further recombinant cell host according to the invention comprises a polynucleotide containing a biallelic marker selected from the group consisting of Al to A32, and the complements thereof.

Preferred host cells used as recipients for the expression vectors ofthe invention are the following: a) Prokaryotic host cells: Escherichia coli strains (I.E.DH5-α strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streptomyces and

Staphylococcus. b) Eukaryotic host cells: HeLa cells (ATCC N°CCL2; N°CCL2.1; N°CCL2.2), Cv 1 cells (ATCC N°CCL70), COS cells (ATCC N°CRL1650; N°CRL1651), Sf-9 cells (ATCC N°CRL1711), C127 cells (ATCC N° CRL- 1804), 3T3 (ATCC N° CRL-6361), CHO (ATCC N° CCL-61), human kidney 293. (ATCC N° 45504; N° CRL-1573) and BHK (ECACC N° 84100501 ; N° 8411 1301). c) Other mammalian host cells.

The HKLP gene expression in mammalian, and typically human, cells may be rendered defective, or alternatively it may be proceeded with the insertion of a HKLP genomic or cDNA sequence with the replacement ofthe HKLP gene counteφart in the genome of an animal cell by a HKLP polynucleotide according to the invention. These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.

One kind of cell hosts that may be used are mammal zygotes, such as murine zygotes. For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml -for BAC inserts- 3 ng/μl -for PI bacteriophage inserts- in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and70 μM spermidine. When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al (1993b).

Anyone ofthe polynucleotides ofthe invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line. ES cell lines are derived from pluripotent, uncommitted cells ofthe inner cell mass of pre-implantation blastocysts. Preferred ES cell lines are the following: ES-E14TG2a (ATCC n° CRL- 1821), ES-D3 (ATCC n° CRL1934 and n° CRL-11632), YS001 (ATCC n° CRL-1 1776), 36.5 (ATCC n° CRL- 111 16). To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence. Preferred feeder cells consist of primary embryonic fibroblasts that are established from tissue of day 13- day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al.(1993) and are inhibited in growth by irradiation, such as described by Robertson (1987), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (1990).

The constmcts in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth ofthe host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in the expression of proteins can be dismpted by any convenient method, including freeze-thaw cycling, sonication, mechanical dismption, or use of cell lysing agents. Such methods are well known by the skill artisan.

Transgenic Animals

The terms "transgenic animals" or "host animals" are used herein designate animals that have their genome genetically and artificially manipulated so as to include one ofthe nucleic acids according to the invention. Preferred animals are non-human mammals and include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats) and Oryctogalus (e.g. rabbits) which have their genome artificially and genetically altered by the insertion of a nucleic acid according to the invention. In one embodiment, the invention encompasses non-human host mammals and animals comprising a recombinant vector ofthe invention or a HKLP gene disrupted by homologous recombination with a knock out vector. The transgenic animals ofthe invention all include within a plurality of their cells a cloned recombinant or synthetic DNA sequence, more specifically one ofthe purified or isolated nucleic acids comprising a HKLP coding sequence, a HKLP regulatory polynucleotide, a polynucleotide construct, or a DNA sequence encoding an antisense polynucleotide such as described in the present specification.

Generally, a transgenic animal according the present invention comprises any one ofthe

5 polynucleotides, the recombinant vectors and the cell hosts described in the present invention. More particularly, the transgenic animals ofthe present invention can comprise any ofthe polynucleotides described in the "Genomic Sequences Of tThe HKLP Gene" section, the " HKLP cDNA Sequences" section, the "Coding Regions" section, the "Polynucleotide constmcts" section, the "Oligonucleotide Probes And Primers" section, the "Recombinant Vectors" section and the "Cell Hosts" section.

10 A further transgenic animals according to the invention contains in their somatic cells and/or in their germ line cells a polynucleotide comprising a biallelic marker selected from the group consisting of Al to A32, and the complements thereof.

In a first preferred embodiment, these transgenic animals may be good experimental models in order to study the diverse pathologies related to cell differentiation, in particular concerning the

15 transgenic animals within the genome of which has been inserted one or several copies of a polynucleotide encoding a native HKLP protein, or alternatively a mutant HKLP protein.

In a second preferred embodiment, these transgenic animals may express a desired polypeptide of interest under the control ofthe regulatory polynucleotides ofthe HKLP gene, leading to good yields in the synthesis of this protein of interest, and eventually a tissue specific

20 expression of this protein of interest.

The design ofthe transgenic animals ofthe invention may be made according to the conventional techniques well known from the one skilled in the art. For more details regarding the production of transgenic animals, and specifically transgenic mice, it may be referred to US Patents Nos 4,873,191, issued Oct. 10, 1989; 5,464,764 issued Nov 7, 1995; and 5,789,215, issued Aug 4,

25 1998; these documents being herein incoφorated by reference to disclose methods producing transgenic mice.

Transgenic animals ofthe present invention are produced by the application of procedures which result in an animal with a genome that has incoφorated exogenous genetic material. The procedure involves obtaining the genetic material, or a portion thereof, which encodes either a

30 HKLP coding sequence, a HKLP regulatory polynucleotide or a DNA sequence encoding a HKLP antisense polynucleotide such as described in the present specification.

A recombinant polynucleotide ofthe invention is inserted into an embryonic or ES stem cell line. The insertion is preferably made using electroporation, such as described by Thomas et al.(1987). The cells subjected to electroporation are screened (e.g. by selection via selectable

35 markers, by PCR or by Southern blot analysis) to find positive cells which have integrated the exogenous recombinant polynucleotide into their genome, preferably via an homologous recombination event. An illustrative positive-negative selection procedure that may be used according to the invention is described by Mansour et al.(1988).

Then, the positive cells are isolated, cloned and injected into 3.5 days old blastocysts from mice, such as described by Bradley (1987). The blastocysts are then inserted into a female host animal and allowed to grow to term.

Alternatively, the positive ES cells are brought into contact with embryos at the 2.5 days old 8-16 cell stage (morulae) such as described by Wood et al.(1993) or by Nagy et al.(1993), the ES cells being internalized to colonize extensively the blastocyst including the cells which will give rise to the germ line. The offspring ofthe female host are tested to determine which animals are transgenic e.g. include the inserted exogenous DNA sequence and which are wild-type.

Thus, the present invention also concerns a transgenic animal containing a nucleic acid, a recombinant expression vector or a recombinant host cell according to the invention.

Recombinant Cell Lines Derived From The Transgenic Animals Of The Invention. A further object ofthe invention consists of recombinant host cells obtained from a transgenic animal described herein. In one embodiment the invention encompasses cells derived from non-human host mammals and animals comprising a recombinant vector ofthe invention or a HKLP gene disrupted by homologous recombination with a knock out vector.

Recombinant cell lines may be established in vitro from cells obtained from any tissue of a transgenic animal according to the invention, for example by transfection of primary cell cultures with vectors expressing owc-genes such as SV40 large T antigen, as described by Chou ( 1989) and Shay et al.( 1991).

Methods for screening substances interacting with a HKLP polypeptide

For the puφose ofthe present invention, a ligand means a molecule, such as a protein, a peptide, an antibody or any synthetic chemical compound capable of binding to the HKLP protein or one of its fragments or variants or to modulate the expression ofthe polynucleotide coding for HKLP or a fragment or variant thereof.

In the ligand screening method according to the present invention, a biological sample or a defined molecule to be tested as a putative ligand ofthe HKLP protein is brought into contact with the corresponding purified HKLP protein, for example the corresponding purified recombinant HKLP protein produced by a recombinant cell host as described hereinbefore, in order to form a complex between this protein and the putative ligand molecule to be tested.

As an illustrative example, to study the interaction ofthe HKLP protein, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 of the amino acid positions 1-478 ofthe SEQ ID No 4, with dmgs or small molecules, such as molecules generated through combinatorial chemistry approaches, the microdialysis coupled to HPLC method described by Wang et al. (1997) or the affinity capillary electrophoresis method described by Bush et al. (1997), the disclosures of which are incoφorated by reference, can be used. In further methods, peptides, drugs, fatty acids, lipoproteins, or small molecules which interact with the HKLP protein, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 of the amino acid positions 1-478 ofthe SEQ ID No 4 may be identified using assays such as the following. The molecule to be tested for binding is labeled with a detectable label, such as a fluorescent , radioactive, or enzymatic tag and placed in contact with immobilized HKLP protein, or a fragment thereof under conditions which permit specific binding to occur. After removal of non- specifically bound molecules, bound molecules are detected using appropriate means.

Another object ofthe present invention consists of methods and kits for the screening of candidate substances that interact with HKLP polypeptide.

The present invention pertains to methods for screening substances of interest that interact with a HKLP protein or one fragment or variant thereof. By their capacity to bind covalently or non-covalently to a HKLP protein or to a fragment or variant thereof, these substances or molecules may be advantageously used both in vitro and in vivo. In vitro, said interacting molecules may be used as detection means in order to identify the presence of a HKLP protein in a sample, preferably a biological sample.

A method for the screening of a candidate substance comprises the following steps : a) providing a polypeptide consisting of a HKLP protein or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 ofthe SEQ ID No 4 or a variant thereof; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; d) detecting the complexes formed between said polypeptide and said candidate substance.

The invention further concerns a kit for the screening of a candidate substance interacting with the HKLP polypeptide, wherein said kit comprises : a) a HKLP protein having an amino acid sequence selected from the group consisting ofthe amino acid sequences of SEQ ID No 4 or a peptide fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 ofthe SEQ ID No 4 or a variant thereof ; b) optionally means useful to detect the complex formed between the HKLP protein or a peptide fragment or a variant thereof and the candidate substance.

In a preferred embodiment ofthe kit described above, the detection means consist in monoclonal or polyclonal antibodies directed against the HKLP protein or a peptide fragment or a variant thereof.

Various candidate substances or molecules can be assayed for interaction with a HKLP polypeptide. These substances or molecules include, without being limited to, natural or synthetic organic compounds or molecules of biological origin such as polypeptides. When the candidate substance or molecule consists of a polypeptide, this polypeptide may be the resulting expression product of a phage clone belonging to a phage-based random peptide library, or alternatively the polypeptide may be the resulting expression product of a cDNA library cloned in a vector suitable for performing a two-hybrid screening assay.

The invention also pertains to kits useful for performing the hereinbefore described screening method. Preferably, such kits comprise a HKLP polypeptide or a fragment or a variant thereof, and optionally means useful to detect the complex formed between the HKLP polypeptide or its fragment or variant and the candidate substance. In a preferred embodiment the detection means consist in monoclonal or polyclonal antibodies directed against the corresponding HKLP polypeptide or a fragment or a variant thereof.

A. Candidate Iigands obtained from random peptide libraries In a particular embodiment ofthe screening method, the putative ligand is the expression product of a DNA insert contained in a phage vector (Parmley and Smith, 1988). Specifically, random peptide phages libraries are used. The random DNA inserts encode for peptides of 8 to 20 amino acids in length (Oldenburg K.R. et al., 1992; Valadon P., et al., 1996; Lucas A.H., 1994; Westerink M.A.J., 1995; Felici F. et al., 1991). According to this particular embodiment, the recombinant phages expressing a protein that binds to the immobilized HKLP protein is retained and the complex formed between the HKLP protein and the recombinant phage may be subsequently immunoprecipitated by a polyclonal or a monoclonal antibody directed against the HKLP protein.

Once the ligand library in recombinant phages has been constmcted, the phage population is brought into contact with the immobilized HKLP protein. Then the preparation of complexes is washed in order to remove the non-specifically bound recombinant phages. The phages that bind specifically to the HKLP protein are then eluted by a buffer (acid pH) or immunoprecipitated by the monoclonal antibody produced by the hybridoma anti-HKLP, and this phage population is subsequently amplified by an over-infection of bacteria (for example E. coli). The selection step may be repeated several times, preferably 2-4 times, in order to select the more specific recombinant phage clones. The last step consists in characterizing the peptide produced by the selected recombinant phage clones either by expression in infected bacteria and isolation, expressing the phage insert in another host-vector system, or sequencing the insert contained in the selected recombinant phages.

B. Candidate Iigands obtained by competition experiments.

Alternatively, peptides, drugs or small molecules which bind to the HKLP protein, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 of the SEQ ID No 4, may be identified in competition experiments. In such assays, the HKLP protein, or a fragment thereof, is immobilized to a surface, such as a plastic plate. Increasing amounts ofthe peptides, drugs or small molecules are placed in contact with the immobilized HKLP protein, or a fragment thereof, in the presence of a detectable labeled known HKLP protein ligand. For example, the HKLP ligand may be detectably labeled with a fluorescent, radioactive, or enzymatic tag. The ability of the test molecule to bind the HKLP protein, or a fragment thereof, is determined by measuring the amount of detectably labeled known ligand bound in the presence ofthe test molecule. A decrease in the amount of known ligand bound to the HKLP protein, or a fragment thereof, when the test molecule is present indicated that the test molecule is able to bind to the HKLP protein, or a fragment thereof.

C. Candidate Iigands obtained by affinity chromatography.

Proteins or other molecules interacting with the HKLP protein, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 of the SEQ ID No 4, can also be found using affinity columns which contain the HKLP protein, or a fragment thereof. The HKLP protein, or a fragment thereof, may be attached to the column using conventional techniques including chemical coupling to a suitable column matrix such as agarose, Affi Gel® , or other matrices familiar to those of skill in art. In some embodiments of this method, the affinity column contains chimeric proteins in which the HKLP protein, or a fragment thereof, is fused to glutathion S transferase (GST). A mixture of cellular proteins or pool of expressed proteins as described above is applied to the affinity column. Proteins or other molecules interacting with the HKLP protein, or a fragment thereof, attached to the column can then be isolated and analyzed on 2-D electrophoresis gel as described in Ramunsen et al. (1997), the disclosure of which is incoφorated by reference. Alternatively, the proteins retained on the affinity column can be purified by electrophoresis based methods and sequenced. The same method can be used to isolate antibodies, to screen phage display products, or to screen phage display human antibodies. D. Candidate Iigands obtained by optical biosensor methods

Proteins interacting with the HKLP protein, or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 ofthe SEQ ID No 4, can also be screened by using an Optical Biosensor as described in Edwards and Leatherbarrow (1997) and also in Szabo et al. (1995), the disclosure of which is incoφorated by reference. This technique permits the detection of interactions between molecules in real time, without the need of labeled molecules. This technique is based on the surface plasmon resonance (SPR) phenomenon. Briefly, the candidate ligand molecule to be tested is attached to a surface (such as a carboxymethyl dextran matrix). A light beam is directed towards the side ofthe surface that does not contain the sample to be tested and is reflected by said surface. The SPR phenomenon causes a decrease in the intensity of the reflected light with a specific association of angle and wavelength. The binding of candidate ligand molecules cause a change in the refraction index on the surface, which change is detected as a change in the SPR signal. For screening of candidate ligand molecules or substances that are able to interact with the HKLP protein, or a fragment thereof, the HKLP protein, or a fragment thereof, is immobilized onto a surface. This surface consists of one side of a cell through which flows the candidate molecule to be assayed. The binding ofthe candidate molecule on the HKLP protein, or a fragment thereof, is detected as a change ofthe SPR signal. The candidate molecules tested may be proteins, peptides, carbohydrates, lipids, or small molecules generated by combinatorial chemistry. This technique may also be performed by immobilizing eukaryotic or prokaryotic cells or lipid vesicles exhibiting an endogenous or a recombinantly expressed HKLP protein at their surface.

The main advantage ofthe method is that it allows the determination ofthe association rate between the HKLP protein and molecules interacting with the HKLP protein. It is thus possible to select specifically ligand molecules interacting with the HKLP protein, or a fragment thereof, through strong or conversely weak association constants.

E. Candidate Iigands obtained through a two-hybrid screening assay.

The yeast two-hybrid system is designed to study protein-protein interactions in vivo (Fields and Song, 1989), and relies upon the fusion of a bait protein to the DNA binding domain ofthe yeast Gal4 protein. This technique is also described in the US Patent N° US 5,667,973 and the US Patent N° 5,283,173 (Fields et al.) the technical teachings of both patents being herein incoφorated by reference.

The general procedure of library screening by the two-hybrid assay may be performed as described by Haφer et al. (1993) or as described by Cho et al. (1998) or also Fromont-Racine et al. (1997). The bait protein or polypeptide consists of a HKLP polypeptide or a fragment comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 ofthe SEQ ID No 4 or a variant thereof.

More precisely, the nucleotide sequence encoding the HKLP polypeptide or a fragment or variant thereof is fused to a polynucleotide encoding the DNA binding domain ofthe GAL4 protein, the fused nucleotide sequence being inserted in a suitable expression vector, for example pAS2 or pM3. Then, a human cDNA library is constructed in a specially designed vector, such that the human cDNA insert is fused to a nucleotide sequence in the vector that encodes the transcriptional domain ofthe GAL4 protein. Preferably, the vector used is the pACT vector. The polypeptides encoded by the nucleotide inserts ofthe human cDNA library are termed "pray" polypeptides.

A third vector contains a detectable marker gene, such as beta galactosidase gene or CAT gene that is placed under the control of a regulation sequence that is responsive to the binding of a complete Gal4 protein containing both the transcriptional activation domain and the DNA binding domain. For example, the vector pG5EC may be used.

Two different yeast strains are also used. As an illustrative but non limiting example the two different yeast strains may be the followings : - Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12, trpl-901, his3-D200, ade2-101, gal4Dgall80D URA3 GAL-LacZ, LYS GAL-HIS3, cyH); - Yl 87, the phenotype of which is (MATa gaU ga!80 his3 trpl-901 ade2-101 ura3-52 leu2-3, - 112 URA3 GAL-lacZmef), which is the opposite mating type of Y 190.

Briefly, 20 μg of pAS2/HKLP and 20 μg of pACT-cDNA library are co-transformed into yeast strain Y190. The transformants are selected for growth on minimal media lacking histidine, leucine and tryptophan, but containing the histidine synthesis inhibitor 3-AT (50 mM). Positive colonies are screened for beta galactosidase by filter lift assay. The double positive colonies (His^", beta-gat) are then grown on plates lacking histidine, leucine, but containing tryptophan and cycloheximide (10 mg/ml) to select for loss ofpAS2/HKLP plasmids bu retention of pACT-cDNA library plasmids. The resulting Y190 strains are mated with Y187 strains expressing HKLP or non- related control proteins; such as cyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Haφer et al. (1993) and by Bram et al. (1993), and screened for beta galactosidase by filter lift assay. Yeast clones that are beta gal- after mating with the control GaU fusions are considered false positives. In another embodiment ofthe two-hybrid method according to the invention, interaction between the HKLP or a fragment or variant thereof with cellular proteins may be assessed using the Matchmaker Two Hybrid System 2 (Catalog No. KI 604-1, Clontech). As described in the manual accompanying the Matchmaker Two Hybrid System 2 (Catalog No. K 1604- 1 , Clontech), the disclosure of which is incoφorated herein by reference, nucleic acids encoding the HKLP protein or a portion thereof, are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain ofthe yeast transcriptional activator GAL4. A desired cDNA, preferably human cDNA, is inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4. The two expression plasmids are transformed into yeast and the yeast are plated on selection medium which selects for expression of selectable markers on each ofthe expression vectors as well as GAL4 dependent expression ofthe HIS3 gene. Transformants capable of growing on medium lacking histidine are screened for GAL4 dependent lacZ expression. Those cells which are positive in both the histidine selection and the lacZ assay contain interaction between HKLP and the protein or peptide encoded by the initially selected cDNA insert.

Methods For Screening Substances Modulating The Activity Of The HKLP protein

The invention also concerns a method for screening new agents, or candidate substances which modulate the activity ofthe HKLP protein or a fragment thereof. Preferably, the HKLP protein or a fragment thereof is a polypeptide code comprising a contiguous span of at least 6 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1 ofthe amino acid positions 1-478 ofthe SEQ ID No 4. Preferably, the candidate substance is mixed with the HKLP protein and the activity ofthe HKLP protein is measured. Candidate substances include, without being limited to, natural or synthetic organic compounds or molecules of biological origin such as polypeptides. Various assays for biological activity of motor proteins are known (Sato-Yoshitake et al,

1992 and Scholey, 1993). In vitro motility assays to characterize specific KLPs, for example, include microtubule gliding assays demonstrating translocation of microtubules, organelle movement assays to visualize the movement of a cargo of interest, and single molecule motility assays (Howard 1989, Block 1990) to characterize structural elements. In short, microtubule gliding assays can be performed by applying a sample containing the

HKLP protein to a glass surface without any treatment and incubating with microtubules reconstituted from polymerized tubulin. Microtubule translocation activity and the direction of movement can be determined as in Nangaku 1994 by observing the movement of axonemes on the glass surface. Organelle movement assays can be performed by applying a composition containing the cargo of interest with a solution containing vesicles and the HKLP protein to a glass surface. Movement ofthe organelle can be observed, for example, by using a cargo-specific fluorescent probe to stain vesicles before incubation with the HKLP protein.

Methods For Inhibiting The Expression Of A HKLP Gene

Other therapeutic compositions according to the present invention comprise advantageously an oligonucleotide fragment ofthe nucleic sequence of HKLP as an antisense tool or a triple helix tool that inhibits the expression ofthe corresponding HKLP gene. A preferred fragment ofthe nucleic sequence of HKLP comprises an allele of at least one ofthe biallelic markers Al to A32.

Antisense Approach

Preferred methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al.(1995).

Preferably, the antisense tools are chosen among the polynucleotides (15-200 bp long) that are complementary to the 5'end ofthe HKLP mRNA. In another embodiment, a combination of different antisense polynucleotides complementary to different parts ofthe desired targeted gene are used. Preferred antisense polynucleotides according to the present invention are complementary to a sequence ofthe mRNAs of HKLP that contains either the translation initiation codon ATG or a splicing donor or acceptor site.

The antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression ofthe HKLP mRNA in the duplex. Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al., (1986) and Izant and Weintraub, (1984), the disclosures of which are incoφorated herein by reference.

In some strategies, antisense molecules are obtained by reversing the orientation ofthe HKLP coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed in the cell. The antisense molecules may be transcribed using in vitro transcription systems such as those which employ T7 or SP6 polymerase to generate the transcript. Another approach involves transcription of HKLP antisense nucleic acids in vivo by operably linking DNA containing the antisense sequence to a promoter in a suitable expression vector.

Alternatively, suitable antisense strategies are those described by Rossi et al.(1991), in the International Applications Nos. WO 94/23026, WO 95/04141 , WO 92/18522 and in the European Patent Application No. EP 0 572 287 A2

An alternative to the antisense technology that is used according to the present invention consists in using ribozymes that will bind to a target sequence via their complementary polynucleotide tail and that will cleave the corresponding RNA by hydrolyzing its target site (namely "hammerhead ribozymes"). Briefly, the simplified cycle of a hammerhead ribozyme consists of (1) sequence specific binding to the target RNA via complementary antisense sequences; (2) site-specific hydrolysis ofthe cleavable motif of the target strand; and (3) release of cleavage products, which gives rise to another catalytic cycle. Indeed, the use of long-chain antisense polynucleotide (at least 30 bases long) or ribozymes with long antisense arms are advantageous. A preferred delivery system for antisense ribozyme is achieved by covalently linking these antisense ribozymes to lipophilic groups or to use liposomes as a convenient vector. Preferred antisense ribozymes according to the present invention are prepared as described by Sczakiel et al.(1995), the specific preparation procedures being referred to in said article being herein incoφorated by reference.

Triple Helix Approach The HKLP genomic DNA may also be used to inhibit the expression ofthe HKLP gene based on intracellular triple helix formation.

Triple helix oligonucleotides are used to inhibit transcription from a genome. They are particularly useful for studying alterations in cell activity when it is associated with a particular gene. Similarly, a portion ofthe HKLP genomic DNA can be used to study the effect of inhibiting

HKLP transcription within a cell. Traditionally, homopurine sequences were considered the most useful for triple helix strategies. However, homopyrimidine sequences can also inhibit gene expression. Such homopyrimidine oligonucleotides bind to the major groove at homopurine:homopyrimidine sequences. Thus, both types of sequences from the HKLP genomic DNA are contemplated within the scope of this invention.

To carry out gene therapy strategies using the triple helix approach, the sequences ofthe HKLP genomic DNA are first scanned to identify 10-mer to 20-mer homopyrimidine or homopurine stretches which could be used in triple-helix based strategies for inhibiting HKLP expression. Following identification of candidate homopyrimidine or homopurine stretches, their efficiency in inhibiting HKLP expression is assessed by introducing varying amounts of oligonucleotides containing the candidate sequences into tissue culture cells which express the HKLP gene.

The oligonucleotides can be introduced into the cells using a variety of methods known to those skilled in the art, including but not limited to calcium phosphate precipitation, DEAE-Dextran, electroporation, liposome-mediated transfection or native uptake. Treated cells are monitored for altered cell function or reduced HKLP expression using techniques such as Northern blotting, RNase protection assays, or PCR based strategies to monitor the transcription levels ofthe HKLP gene in cells which have been treated with the oligonucleotide. The oligonucleotides which are effective in inhibiting gene expression in tissue culture cells may then be introduced in vivo using the techniques described above in the antisense approach at a dosage calculated based on the in vitro results, as described in antisense approach.

In some embodiments, the natural (beta) anomers ofthe oligonucleotide units can be replaced with alpha anomers to render the oligonucleotide more resistant to nucleases. Further, an intercalating agent such as ethidium bromide, or the like, can be attached to the 3' end ofthe alpha oligonucleotide to stabilize the triple helix. For information on the generation of oligonucleotides suitable for triple helix formation see Griffin et al.(l 989), which is hereby incoφorated by this reference. Computer-Related Embodiments

As used herein the term "nucleic acid codes ofthe invention" encompass the nucleotide sequences comprising, consisting essentially of, or consisting of any one ofthe following: a) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 5 1000 nucleotides of SEQ ID No 1, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 1 : 1-39624, 39705-40589, 40666-43629, 43710- 44203, 4431 1-45125, 45210-45440, 45622-45717, 45791-68580, 68675-70246, 70396-72421, 72601-73295, 73434-74648, 74898-83055, 83175-85192, 85279-85609, 85740-85906, 86070- 88304, 88396-90585, 90705-91767, 91824-94380, 94490-96296, 96364-97184, 97270-101 167,

10 101274-109465, 109581-1 10228, 1 10363-1 11819, 111882-113636, 113783-113945, 1 14186- 117002, 1 17075-1 19676, and 119677-121 162; b) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659-3829 and

15 8885-10884; c) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 3: 391-1619 and 6988-10682; d) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 , 2 or 3, or the complements

20 thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 nucleotide positions of any one ofthe following ranges of nucleotide positions of: (1) SEQ ID No 1 : 1-1000, 1001-2000, 2001- 3000, 3001-4000, 4001-5000, 5001-6000, 6001-7000, 7001-8000, 8001-9000, 9001-10000, 10001- 1 1000, 1 1001-12000, 12001-13000, 13001-14000, 14001-15000, 15001-16000, 16001-17000, 17001-18000, 18001-19000, 19001-20000, 20001-21000, 21001-22000, 22001-23000, 23001-

25 24000, 24001-25000, 25001-26000, 26001-27000, 27001-28000, 28001-29000, 29001-30000, 30001-31000, 31001-32000, 32001-33000, 33001-34000, 34001-35000, 35001-36000, 36001- 37000, 37001-38000, 38001-39000, 39001-39624, 39705-40589, 40666-43629, 43710-44203, 4431 1-45125, 45210-45440, 45622-45717, 45791-68580, 68675-70246, 70396-72421, 72601- 73295, 73434-74648, 74898-83055, 83175-85192, 85279-85609, 85740-85906, 86070-88304,

30 88396-90585, 90705-91767, 91824-94380, 94490-96296, 96364-97184, 97270-101 167, 101274- 109465, 109581-110228, 110363-111819, 111882-113636, 113783-113945, 114186-117002, 117075-1 19676, and 119677-121162; and (2) SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659- 3829 and 8885-10884; e) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein

35 said contiguous span comprises a G at position 7159 of SEQ ID No 1; f) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises a C either at 93 position 2551 or 4500 of SEQ ID No 2; g) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises a nucleotide selected in the group consisting of a C at position 5487, and a C at position 6265 of SEQ ID No 3; and, j) a nucleotide sequence 5 complementary to any one ofthe preceding nucleotide sequences.

The "nucleic acid codes ofthe invention" further encompass nucleotide sequences homologous to: a) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1 : 1-39624, 39705-40589,

10 40666-43629, 43710-44203, 4431 1-45125, 45210-45440, 45622-45717, 45791-68580, 68675- 70246, 70396-72421, 72601-73295, 73434-74648, 74898-83055, 83175-85192, 85279-85609, 85740-85906, 86070-88304, 88396-90585, 90705-91767, 91824-94380, 94490-96296, 96364- 97184, 97270-101167, 101274-109465, 109581-1 10228, 1 10363-1 1 1819, 1 1 1882-1 13636, 1 13783- 1 13945, 114186-117002, 1 17075-119676, and 1 19677-121162; b) a contiguous span of at least 12,

15 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659-3829 and 8885-10884 c) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said

20 contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 3: 391-1619 and 6988-10682; and d) sequences complementary to all ofthe preceding sequences. Homologous sequences refer to a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% homology to these contiguous spans. Homology may be determined using any method described herein, including BLAST2N with the default parameters or with any modified parameters.

25 Homologous sequences also may include RNA sequences in which uridines replace the thymines in the nucleic acid codes ofthe invention. It will be appreciated that the nucleic acid codes ofthe invention can be represented in the traditional single character format (See the inside back cover of Stryer, Lubert. Biochemistry, 3^rd edition. W. H Freeman & Co., New York.) or in any other format or code which records the identity ofthe nucleotides in a sequence.

30 As used herein the term "polypeptide codes ofthe invention" encompass the polypeptide sequences comprising a contiguous span of at least 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe amino acid positions 1-478 ofthe SEQ ID No 4. It will be appreciated that the polypeptide codes ofthe invention can be represented in the traditional single character format or three letter format (See the

35 inside back cover of Stryer, Lubert. Biochemistry, 3^rd edition. W. H Freeman & Co., New York.) or in any other format or code which records the identity ofthe polypeptides in a sequence. It will be appreciated by those skilled in the art that the nucleic acid codes ofthe invention and polypeptide codes ofthe invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. As used herein, the words "recorded" and "stored" refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on a computer readable medium to generate manufactures comprising one or more ofthe nucleic acid codes ofthe invention, or one or more ofthe polypeptide codes ofthe invention. Another aspect ofthe present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 nucleic acid codes ofthe invention. Another aspect ofthe present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 polypeptide codes ofthe invention.

Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.

Embodiments ofthe present invention include systems, particularly computer systems which store and manipulate the sequence information described herein. One example of a computer system 100 is illustrated in block diagram form in Figure 1. As used herein, "a computer system" refers to the hardware components, software components, and data storage components used to analyze the nucleotide sequences ofthe nucleic acid codes ofthe invention or the amino acid sequences ofthe polypeptide codes ofthe invention. In one embodiment, the computer system 100 is a Sun Enteφrise 1000 server (Sun Microsystems, Palo Alto, CA). The computer system 100 preferably includes a processor for processing, accessing and manipulating the sequence data. The processor 105 can be any well-known type of central processing unit, such as the Pentium III from Intel Coφoration, or similar processor from Sun, Motorola, Compaq or International Business Machines.

Preferably, the computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components. A skilled artisan can readily appreciate that any one ofthe currently available computer systems are suitable. In one particular embodiment, the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/or other computer readable media having data recorded thereon. In some embodiments, the computer system 100 further includes one or more data retrieving device 118 for reading the data stored on the internal data storage devices 110. The data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, etc. In some embodiments, the internal data storage device 1 10 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon. The computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.

The computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125a-c in a network or wide area network to provide centralized access to the computer system 100.

Software for accessing and processing the nucleotide sequences ofthe nucleic acid codes ofthe invention or the amino acid sequences ofthe polypeptide codes ofthe invention (such as search tools, compare tools, and modeling tools etc.) may reside in main memory 115 during execution. In some embodiments, the computer system 100 may further comprise a sequence comparer for comparing the above-described nucleic acid codes ofthe invention or the polypeptide codes ofthe invention stored on a computer readable medium to reference nucleotide or polypeptide sequences stored on a computer readable medium. A "sequence comparer" refers to one or more programs which are implemented on the computer system 100 to compare a nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences and/or compounds including but not limited to peptides, peptidomimetics, and chemicals stored within the data storage means. For example, the sequence comparer may compare the nucleotide sequences of nucleic acid codes ofthe invention or the amino acid sequences ofthe polypeptide codes ofthe invention stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies, motifs implicated in biological function, or stmctural motifs. The various sequence comparer programs identified elsewhere in this patent specification are particularly contemplated for use in this aspect ofthe invention.

Figure 2 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database. The database of sequences can be a private database stored within the computer system 100, or a public database such as GENBANK, PIR OR SWISSPROT that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100. As discussed above, the memory could be any type of memory, including RAM or .an internal storage device. The process 200 then moves to a state 204 wherein a database of sequences is opened for analysis and comparison. The process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer. A comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database. Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user ofthe computer system.

Once a comparison ofthe two sequences has been performed at the state 210, a determination is made at a decision state 210 whether the two sequences are the same. Of course, the term "same" is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as "same" in the process 200.

If a determination is made that the two sequences are the same, the process 200 moves to a state 214 wherein the name ofthe sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered. Once the name ofthe stored sequence is displayed to the user, the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220. However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database.

It should be noted that if a determination had been made at the decision state 212 that the sequences were not homologous, then the process 200 would move immediately to the decision state 218 in order to determine if any other sequences were available in the database for comparison. Accordingly, one aspect ofthe present invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid code ofthe invention or a polypeptide code ofthe invention, a data storage device having retrievably stored thereon reference nucleotide sequences or polypeptide sequences to be compared to the nucleic acid code ofthe invention or polypeptide code ofthe invention and a sequence comparer for conducting the comparison. The sequence comparer may indicate a homology level between the sequences compared or identify stmctural motifs in the nucleic acid code ofthe invention and polypeptide codes ofthe invention or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes. In some embodiments, the data storage device may have stored thereon the sequences of at least 2, 5, 10, 15, 20, 25, 30, or 50 of he nucleic acid codes ofthe invention or polypeptide codes ofthe invention. Another aspect ofthe present invention is a method for determining the level of homology between a nucleic acid code ofthe invention and a reference nucleotide sequence, comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through the use of a computer program which determines homology levels and determining homology between the nucleic acid code and the reference nucleotide sequence with the computer program. The computer program may be any of a number of computer programs for determining homology levels, including those specifically enumerated herein, including BLAST2N with the default parameters or with any modified parameters. The method may be implemented using the computer systems described above. The method may also be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 ofthe above described nucleic acid codes ofthe invention through the use ofthe computer program and determining homology between the nucleic acid codes and reference nucleotide sequences.

Figure 3 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous. The process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory. The second sequence to be compared is then stored to a memory at a state 256. The process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherein the first character ofthe second sequence is read. It should be understood that if the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U. If the sequence is a protein sequence, then it should be in the single letter amino acid code so that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the two characters are the same. If they are the same, then the process 250 moves to a state 268 wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process 250 continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process 250 moves to a decision state 274 to determine whether there are any more characters either sequence to read. If there aren't any more characters to read, then the process 250 moves to a state 276 wherein the level of homology between the first and second sequences is displayed to the user. The level of homology is determined by calculating the proportion of characters between the sequences that were the same out ofthe total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with a every character in a second sequence, the homology level would be 100%.

Alternatively, the computer program may be a computer program which compares the nucleotide sequences ofthe nucleic acid codes ofthe present invention, to reference nucleotide sequences in order to determine whether the nucleic acid code ofthe invention differs from a reference nucleic acid sequence at one or more positions. Optionally such a program records the length and identity of inserted, deleted or substituted nucleotides with respect to the sequence of either the reference polynucleotide or the nucleic acid code ofthe invention. In one embodiment, the computer program may be a program which determines whether the nucleotide sequences ofthe nucleic acid codes ofthe invention contain one or more single nucleotide polymoφhisms (SNP) with respect to a reference nucleotide sequence. These single nucleotide polymoφhisms may each comprise a single base substitution, insertion, or deletion.

Another aspect ofthe present invention is a method for determining the level of homology between a polypeptide code ofthe invention and a reference polypeptide sequence, comprising the steps of reading the polypeptide code ofthe invention and the reference polypeptide sequence through use of a computer program which determines homology levels and determining homology between the polypeptide code and the reference polypeptide sequence using the computer program.

Accordingly, another aspect ofthe present invention is a method for determining whether a nucleic acid code ofthe invention differs at one or more nucleotides from a reference nucleotide sequence comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through use of a computer program which identifies differences between nucleic acid sequences and identifying differences between the nucleic acid code and the reference nucleotide sequence with the computer program. In some embodiments, the computer program is a program which identifies single nucleotide polymoφhisms The method may be implemented by the computer systems described above and the method illustrated in Figure 3. The method may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 50 ofthe nucleic acid codes ofthe invention and the reference nucleotide sequences through the use ofthe computer program and identifying differences between the nucleic acid codes and the reference nucleotide sequences with the computer program. In other embodiments the computer based system may further comprise an identifier for identifying features within the nucleotide sequences ofthe nucleic acid codes ofthe invention or the amino acid sequences ofthe polypeptide codes ofthe invention.

An "identifier" refers to one or more programs which identifies certain features within the above-described nucleotide sequences ofthe nucleic acid codes ofthe invention or the amino acid sequences ofthe polypeptide codes ofthe invention. In one embodiment, the identifier may comprise a program which identifies an open reading frame in the cDNAs codes ofthe invention.

Figure 4 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence. The process 300 begins at a start state 302 and then moves to a state 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100. The process 300 then moves to a state 306 wherein a database of sequence features is opened. Such a database would include a list of each feature's attributes along with the name ofthe feature. For example, a feature name could be "Initiation Codon" and the attribute would be "ATG". Another example would be the feature name "TAATAA Box" and the feature attribute would be "TAATAA". An example of such a database is produced by the University of Wisconsin Genetics Computer Group (www.gcg.com).

Once the database of features is opened at the state 306, the process 300 moves to a state 308 wherein the first feature is read from the database. A comparison ofthe attribute ofthe first feature with the first sequence is then made at a state 310. A determination is then made at a decision state 316 whether the attribute ofthe feature was found in the first sequence. If the attribute was found, then the process 300 moves to a state 318 wherein the name ofthe found feature is displayed to the user. The process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324. However, if more features do exist in the database, then the process 300 reads the next sequence feature at a state 326 and loops back to the state 310 wherein the attribute ofthe next feature is compared against the first sequence.

It should be noted, that if the feature attribute is not found in the first sequence at the decision state 316, the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database.

In another embodiment, the identifier may comprise a molecular modeling program which determines the 3-dimensional stmcture ofthe polypeptides codes ofthe invention. In some embodiments, the molecular modeling program identifies target sequences that are most compatible with profiles representing the stmctural environments ofthe residues in known three-dimensional protein structures. (See, e.g., Eisenberg et al., U.S. Patent No. 5,436,850 issued July 25, 1995). In another technique, the known three-dimensional structures of proteins in a given family are superimposed to define the structurally conserved regions in that family. This protein modeling technique also uses the known three-dimensional structure of a homologous protein to approximate the stmcture ofthe polypeptide codes ofthe invention. (See e.g., Srinivasan, et al., U.S. Patent No. 5,557,535 issued September 17, 1996). Conventional homology modeling techniques have been used routinely to build models of proteases and antibodies. (Sowdhamini et al., 1997). Comparative approaches can also be used to develop three-dimensional protein models when the protein of interest has poor sequence identity to template proteins. In some cases, proteins fold into similar three-dimensional structures despite having very weak sequence identities. For example, the three- dimensional stmctures of a number of helical cytokines fold in similar three-dimensional topology in spite of weak sequence homology. The recent development of threading methods now enables the identification of likely folding patterns in a number of situations where the structural relatedness between target and template(s) is not detectable at the sequence level. Hybrid methods, in which fold recognition is performed using Multiple Sequence Threading (MST), stmctural equivalencies are deduced from the threading output using a distance geometry program DRAGON to construct a low resolution model, and a full-atom representation is constmcted using a molecular modeling package such as QUANTA.

According to this 3-step approach, candidate templates are first identified by using the novel fold recognition algorithm MST, which is capable of performing simultaneous threading of multiple aligned sequences onto one or more 3-D structures. In a second step, the structural equivalencies obtained from the MST output are converted into interresidue distance restraints and fed into the distance geometry program DRAGON, together with auxiliary information obtained from secondary structure predictions. The program combines the restraints in an unbiased manner and rapidly generates a large number of low resolution model confirmations. In a third step, these low resolution model confirmations are converted into full-atom models and subjected to energy minimization using the molecular modeling package QUANTA. (See e.g., Aszόdi et al., 1997). The results ofthe molecular modeling analysis may then be used in rational dmg design techniques to identify agents which modulate the activity ofthe polypeptide codes ofthe invention. Accordingly, another aspect ofthe present invention is a method of identifying a feature within the nucleic acid codes ofthe invention or the polypeptide codes ofthe invention comprising reading the nucleic acid code(s) or the polypeptide code(s) through the use of a computer program which identifies features therein and identifying features within the nucleic acid code(s) or polypeptide code(s) with the computer program. In one embodiment, computer program comprises a computer program which identifies open reading frames. In a further embodiment, the computer program identifies structural motifs in a polypeptide sequence. In another embodiment, the computer program comprises a molecular modeling program. The method may be performed by reading a single sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 ofthe nucleic acid codes ofthe invention or the polypeptide codes ofthe invention through the use ofthe computer program and identifying features within the nucleic acid codes or polypeptide codes with the computer program.

The nucleic acid codes ofthe invention or the polypeptide codes ofthe invention may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, they may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2,

SYBASE, or ORACLE. In addition, many computer programs and databases may be used as sequence comparers, identifiers, or sources of reference nucleotide or polypeptide sequences to be compared to the nucleic acid codes ofthe invention or the polypeptide codes ofthe invention. The following list is intended not to limit the invention but to provide guidance to programs and databases which are useful with the nucleic acid codes ofthe invention or the polypeptide codes ofthe invention. The programs and databases which may be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, 1990), FASTA (Pearson and Lipman, 1988), FASTDB (Bmtlag et al., 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the EMBL/Swissprotein database, the MDL Available Chemicals Directory database, the MDL Dmg Data Report data base, the Comprehensive Medicinal Chemistry database, Derwents's World Dmg Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure.

Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-tum-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion ofthe encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.

Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent specification referenced in this application are hereby incoφorated by reference into the present disclosure to more fully describe the sate ofthe art to which this invention pertains.

EXAMPLES

Example 1 Identification Of Biallelic Markers - DNA Extraction

Donors were unrelated and healthy. They presented a sufficient diversity for being representative of a French heterogeneous population. The DNA from 100 individuals was extracted and tested for the detection ofthe biallelic markers.

30 ml of peripheral venous blood were taken from each donor in the presence of EDTA. Cells (pellet) were collected after centrifugation for 10 minutes at 2000 φ . Red cells were lysed by a lysis solution (50 ml final volume: 10 mM Tris pH7.6; 5 mM MgCl₂; 10 mM NaCl). The solution was centrifuged (10 minutes, 2000 φm) as many times as necessary to eliminate the residual red cells present in the supernatant, after resuspension ofthe pellet in the lysis solution.

The pellet of white cells was lysed overnight at 42°C with 3.7 ml of lysis solution composed of:

- 3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM) / NaCl 0 4 M - 200 μl SDS 10%

- 500 μl K-proteinase (2 mg K-proteinase in TE 10-2 / NaCl 0.4 M).

For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) was added. After vigorous agitation, the solution was centrifuged for 20 minutes at 10000 φm. For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were added to the previous supernatant, and the solution was centrifuged for 30 minutes at 2000 φm. The DNA solution was rinsed three times with 70% ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 φm. The pellet was dried at 37°C, and resuspended in 1 ml TE 10-1 or 1 ml water. The DNA concentration was evaluated by measuring the OD at 260 nm (1 unit OD = 50 μg/ml DNA).

To determine the presence of proteins in the DNA solution, the OD 260 / OD 280 ratio was determined. Only DNA preparations having a OD 260 / OD 280 ratio between 1.8 and 2 were used in the subsequent examples described below.

The pool was constituted by mixing equivalent quantities of DNA from each individual.

Example 2

Identification Of Biallelic Markers: Amplification Of Genomic DNA By PCR

The amplification of specific genomic sequences ofthe DNA samples of example 1 was carried out on the pool of DNA obtained previously. In addition, 50 individual samples were similarly amplified. PCR assays were performed using the following protocol:

Final volume 25 μl

DNA 2 ng/μl

MgCl₂ 2 mM dNTP (each) 200 μM primer (each) 2.9 ng/μl

Ampli Taq Gold DNA polymerase 0.05 unit/μl

PCR buffer (lOx = 0.1 M TrisHCl pH8.3 0.5M KCl) lx

Each pair of first primers was designed using the sequence information ofthe HKLP gene disclosed herein and the OSP software (Hillier & Green, 1991). This first pair of primers was about 20 nucleotides in length and had the sequences disclosed in Table 1 in the columns labeled PU and RP.

Preferably, the primers contained a common oligonucleotide tail upstream ofthe specific bases targeted for amplification which was useful for sequencing. Primers PU contain the following additional PU 5' sequence:

TGTAAAACGACGGCCAGT; primers RP contain the following RP 5' sequence: CAGGAAACAGCTATGACC. The primer containing the additional PU 5' sequence is listed in SEQ ID No 9. The primer containing the additional RP 5' sequence is listed in SEQ ID No 10.

The synthesis of these primers was performed following the phosphoramidite method, on a GENSET UFPS 24.1 synthesizer. DNA amplification was performed on a Genius II thermocycler. After heating at 95°C for 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 95°C, 54°C for 1 min, and 30 sec at 72°C. For final elongation, 10 min at 72°C ended the amplification. The quantities ofthe amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as intercalant agent (Molecular Probes).

Table 1

104 Example 3

Identification Of Biallelic Markers - Sequencing Of Amplified Genomic DNA And

Identification Of Polymorphisms

The sequencing ofthe amplified DNA obtained in example 2 was carried out on ABI 377 5 sequencers. The sequences ofthe amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol. The products of the sequencing reactions were mn on sequencing gels and the sequences were determined using gel image analysis (ABI Prism DNA Sequencing Analysis software (2.1.2 version)).

The sequence data were further evaluated to detect the presence of biallelic markers within 0 the amplified fragments. The polymoφhism search was based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position as described previously.

In the 25 fragments of amplification, 32 biallelic markers were detected. The localization of these biallelic markers are as shown in Table 2.

5 Example 4

Validation Of The Polymorphisms Through Microsequencing

The biallelic markers identified in example 3 were further confirmed and their respective frequencies were determined through microsequencing. Microsequencing was carried out for each individual DNA sample described in Example 1. 0 Amplification from genomic DNA of individuals was performed by PCR as described above for the detection ofthe biallelic markers with the same set of PCR primers (Table 1).

The preferred primers used in microsequencing were about 19 nucleotides in length and hybridized just upstream ofthe considered polymoφhic base. According to the invention, the primers used in microsequencing are detailed in Table 4.

25 The microsequencing reaction was performed as follows :

After purification ofthe amplification products, the microsequencing reaction mixture was prepared by adding, in a 20μl final volume: 10 pmol microsequencing oligonucleotide, 1 U Thermosequenase (Amersham E79000G), 1.25 μl Thermosequenase buffer (260 mM Tris HCI pH 9.5, 65 mM MgCl₂), and the two appropriate fluorescent ddNTPs (Perkin Elmer, Dye Terminator Set

30 401095) complementary to the nucleotides at the polymoφhic site of each biallelic marker tested, following the manufacturer's recommendations. After 4 minutes at 94°C, 20 PCR cycles of 15 sec at 55°C, 5 sec at 72°C, and 10 sec at 94°C were carried out in a Tetrad PTC-225 thermocycler (MJ Research). The unincoφorated dye terminators were then removed by ethanol precipitation. Samples were finally resuspended in formamide-EDTA loading buffer and heated for 2 min at 95°C before being loaded on a polyacrylamide sequencing gel. The data were collected by an ABI PRISM 377 DNA sequencer and processed using the GENESCAN software (Perkin Elmer).

Following gel analysis, data were automatically processed with software that allows the determination ofthe alleles of biallelic markers present in each amplified fragment. The software evaluates such factors as whether the intensities ofthe signals resulting from the above microsequencing procedures are weak, normal, or saturated, or whether the signals are ambiguous. In addition, the software identifies significant peaks (according to shape and height criteria). Among the significant peaks, peaks corresponding to the targeted site are identified based on their position. When two significant peaks are detected for the same position, each sample is categorized classification as homozygous or heterozygous type based on the height ratio.

Table 2

BM refers to "biallelic marker" Alll and all2 refer respectively to a lele 1 and allele 2 of the biallelic marker. Table 3

Mis 1 and Mis 2 respectively refer to microsequencing primers which hybridized with the non-coding strand ofthe HKLP gene or with the coding strand ofthe HKLP gene. Example 5 Preparation of Antibody Compositions to the HKLP protein

Substantially pure protein or polypeptide is isolated from transfected or transformed cells containing an expression vector encoding the HKLP protein or a portion thereof. The concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:

A. Monoclonal Antibody Production bv Hvbridoma Fusion

Monoclonal antibody to epitopes in the HKLP protein or a portion thereof can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, C, ( 1975) or derivative methods thereof. Also see Harlow, E., and D. Lane. 1988.

Briefly, a mouse is repetitively inoculated with a few micrograms ofthe HKLP protein or a portion thereof over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells ofthe spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth ofthe system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots ofthe dilution placed in wells of a microtiter plate where growth ofthe culture is continued. Antibody- producing clones are identified by detection of antibody in the supernatant fluid ofthe wells by immunoassay procedures, such as ELISA, as originally described by Engvall, (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2.

B. Polyclonal Antibody Production bv Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes in the HKLP protein or a portion thereof can be prepared by immunizing suitable non-human animal with the HKLP protein or a portion thereof, which can be unmodified or modified to enhance immunogenicity. A suitable non- human animal is preferably a non-human mammal is selected, usually a mouse, rat, rabbit, goat, or horse. Alternatively, a crude preparation which has been enriched for HKLP concentration can be used to generate antibodies. Such proteins, fragments or preparations are introduced into the non- human mammal in the presence of an appropriate adjuvant (e.g. aluminum hydroxide, RIBI, etc.) which is known in the art. In addition the protein, fragment or preparation can be pretreated with an agent which will increase antigenicity, such agents are known in the art and include, for example, methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, and keyhole limpet hemocyanin (KLH). Serum from the immunized animal is collected, treated and tested according to known procedures. If the serum contains polyclonal antibodies to undesired epitopes, the polyclonal antibodies can be purified by immunoaffinity chromatography. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987). An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. (1971).

Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations ofthe antigen, begins to fall. See, for example, Ouchterlony, O. et al., (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 μM). Affinity ofthe antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., (1980).

Antibody preparations prepared according to either the monoclonal or the polyclonal protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels ofthe protein in the body.

While the preferred embodiment ofthe invention has been illustrated and described, it will be appreciated that various changes can be made therein by the one skilled in the art without departing from the spirit and scope ofthe invention.

FREE TEXT OF THE SEQUENCE LISTING

The following free text appears in the accompanying Sequence Listing : 3'regulatory region polymoφhic base or complement probe deletion of insertion of sequencing oligonucleotide Primer

Artificial Sequence REFERENCES

The following references cited herein are incoφorated herein by reference in their entireties Abbondanzo SJ et al., 1993, Methods in Enzymology, Academic Press, New York, pp. 803-

5 823 / Ajioka R.S. et al., Am. J. Hum. Genet., 60: 1439-1447, 1997 / Altschul et al., 1990, J. Mol. Biol. 215(3):403-410 / Altschul et al., 1993, Nature Genetics 3:266-272 / Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402 / Anton M. et al., 1995, J. Virol., 69 : 4600-4606 / Araki K et al. (1995) Vøc. Natl. Acad. Sci. USA. 92(l):160-4. / Aszόdi et al., Proteins: Structure, Function, and Genetics, Supplement 1 :38-42 (1997) / Baubonis W. (1991) Nucleic Acids Res.

10 21(9):2025-9. / Beaucage et al., Tetrahedron Lett 1981, 22: 1859-1862 / Block et al, 1990, Nature 348, 348-352 / Bradley A., 1987, Production and analysis of chimaeric mice. In: E.J. Robertson (Ed.), Teratocarcinomas and embryonic stem cells: A practical approach. IRL Press, Oxford, pp.l 13. / Bram RJ et al., 1993, Mol. Cell Biol., 13 : 4760-4769 / Brown EL, et al., Methods Enzymol 1979;68: 109-151 / Brutlag et al. Comp. App. Biosci. 6:237-245, 1990 / Bush et

15 al., 1997, J. Chromatogr., 777 : 31 1-328. / Chai H. et al. (1993) Biotechnol. Appl.

BiochemΛ 8:259-273. / Chee et al. (1996) Science. 274:610-614. / Chen et al. (1987) Mol. Cell. Biol. 7:2745-2752. / Chen et al. Proc. Natl. Acad. Sci. USA 94/20 10756-10761,1997 / Chen and Kwok Nucleic Acids Research 25:347-353 1997 / Cho RJ et al., 1998, Proc. Natl. Acad. Sci. USA, 95(7) : 3752-3757. / Chou J.Y., 1989, Mol. Endocrinol., 3: 1511-1514. / Clark A.G.

20 (1990) Mol. Biol. Evol. 7: 111-122. / Compton J. (1991) Nature. 350(6313):91-92. / Davis L.G., et al., Basic Methods in Molecular Biology, ed., Elsevier Press, NY, 1986 / Dempster et al., (1977) J. R. Stat. Soc, 39B:l-38. / Eckner R. et al. (\99\) EMBOJ. 10:3513-3522. / Edwards et Leatherbarrow, Analytical Biochemistry, 246, 1-6 (1997) / Engvall, E., Meth. Enzymol. 70:419 (1980) / Excoffier L. and Slatkin M. (1995) Mol. Biol. Evol, 12(5): 921-927.

25 / Feldman and Steg, 1996, Medecine/Sciences, synthese, 12:47-55 / Felici F., 1991, J. Mol. Biol., Vol. 222:301-310 / Fields and Song, 1989, Nature, 340 : 245-246 / Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980) / Flotte et al. (1992) Am. J. Respir. Cell Mol. Biol. 7:349-356. / Fodor et al. (1991) Science 251:767-777. / Fraley et al. (1979) Proc. Natl. Acad Sci. USA. 76:3348-3352.

30 / Fromont-Racine M. et al., 1997, Nature Genetics, 16(3) : 277-282. / Fuller S. A. et al. (1996) Immunology in Current Protocols in Molecular Biology, Ausubel et al.Eds, John Wiley & Sons, Inc., USA. / Furth P.A. et al. (1994) iVøc. Natl. Acad. Sci USA. 91 :9302-9306. / Geysen H. Mario et al. 1984. Proc. Natl. Acad. Sci. U.S.A. 81 :3998-4002 / Ghosh and Bacchawat, 1991, Targeting of liposomes to hepatocytes, IN: Liver Diseases, Targeted diagnosis and therapy using

35 specific rceptors and Iigands. Wu et al. Eds., Marcel Dekeker, New York, pp. 87-104. / Gonnet et al., 1992, Science 256: 1443-1445 / Gopal (1985) Mol. Cell. Biol, 5:1188-1 190. / Gossen 112 M. et al. (1992) Proc. Natl. Acad. Sci. USA. 89:5547-5551. / Gossen M. et al. (1995) Science. 268:1766-1769. / Graham et al. (1973) Virology 52:456-457. / Green et al., Ann. Rev. Biochem. 55:569-597 (1986) / Griffin et al. Science 245:967-971 (1989) / Grompe, M. (1993) Nαtwre Genetics. 5:111-117. / Grompe, M. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:5855-5892. 5 / Gu H. et al. (1993) Cell 73:1155-1164. / Gu H. et al. (1994) Science 265:103-106. / Guatelli J C et al. Proc. Natl. Acad. Sci. USA. 35:273-286. / Hacia JG, et al., Nat Genet 1996;14(4):441- 447 / HaffL. A. and Smirnov I. P. (1997) Genome Research, 7:378-388. / Hames B.D. and Higgins S.J. (1985) Nucleic Acid Hybridization: A Practical Approach. Hames and Higgins Ed., IRL Press, Oxford. / Harju L, et al., Clin Chem 1993;39(1 lPt l):2282-2287 / Harland et al.

10 (1985) J. Cell. Biol. 101 :1094-1095. / Harlow, E., and D. Lane. 1988. Antibodies A

Laboratory Manual. Cold Spring Harbor Laboratory, pp. 53-242 / Haφer JW et al., 1993, Cell, 75 : 805-816 / Hawley M.E. et al. (1994) Am. J. Phys. Anthropol. 18: 104. / Henikoff and Henikoff, 1993, Proteins 17:49-61 / Higgins et al., 1996, Methods Enzymol. 266:383-402 / Hillier L. and Green P. Methods Appl, 1991, 1: 124-8. / Hoess et al. (\9S6) Nucleic Acids Res.

15 14:2287-2300. / Howard et al (1989) Nature 342, 154-158 / Huang L. et al. (1996) Cancer Res 56(5): 1 137-1141. / Huygen et al. (1996) Nature Medicine. 2(8):893-898. / Izant JG, Weintraub H, Ce// 1984 Apr;36(4): 1007- 15 / Julan et al. (1992) J. Gen. Virol. 73:3251-3255. / Kanegae Y. et al., 1995 Nucl. Acids Res. 23:3816-3821. / Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268 / Khoury j. et al., Fundamentals of Genetic Epidemiology, Oxford

20 University Press, NY, 1993 / Kim U-J. et al. (1996) Genomics 34:213-218. / Klein et al. (1987) Nαtwre. 327:70-73. / Kohler, G. and Milstein, C, Nature 256:495 (1975) / Koller et al. (1992) Annu. Rev. Immunol. 10:705-730. / Kozal MJ, et al., Nat Med 1996;2(7):753-759 / Landegren U. et al. (1998) Genome Research, 8:769-776. / Lander and Schork, Science, 265, 2037-2048,

1994 / Lange K. (1997) Mathematical and Statistical Methods for Genetic Analysis. Springer, 25 New York. / Lenhard T. et al. (1996) Gene. 169:187-190. / Linton M.F. et al. (1993) J. Clin.

Invest. 92:3029-3037. / Liu Z. et al. (1994) Proc. Natl. Acad. Sci. USA. 91 : 4528-4262. / Livak KJ, Hainer JW, Hum Mutat 1994;3(4):379-385 / Livak et al., Nature Genetics, 9:341 -342,

1995 / Lucas A.H., 1994, In : Development and Clinical Uses of Haempophilus b Conjugate; / Mackey K, et al., 1998, Mol Biotechnol, 9(l):l-5 / Mansour S.L. et al. (1988) Nature. 336:348-

30 352. / Marshall R. L. et al. (1994) PCR Methods and Applications. 4:80-84. / McCormick et al. (1994) Genet. Anal. Tech. Appl. 11 :158-164. / McLaughlin B.A. et al. (1996) Am. J. Hum. Genet. 59:561-569. / Morton ^~N.E., Am.J. Hum.Genet., 7:277-318, 1955 / Muzyczka et al. (1992) Curr. Topics in Micro, and Immunol. 158:97-129. / Nada S. et al. (1993) Cell 73:1125- 1135. / Nagy A. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 8424-8428. / Nangaku M.

35 (1994) Cell 79, 1209- 1220 / Narang SA, et al., Methods Enzymol 1979;68:90-98 / Neda et al. (1991) J. Biol. Chem. 266: 14143-14146. / Newton et al. (1989) Nucleic Acids Res. 17:2503- 2516. / Nickerson D.A. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927. / Nicolau C. et al., 1987, Methods Enzymol., 149:157-76. / Nicolau et al. (1982) Biochim. Biophys. Acta. 721 :185-190. / Nyren P, Pettersson B, Uhlen M, Anal Biochem 1993;208(1):171-175 / O'Reilly et al. (1992) Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman and Co., New York. / Ohno et al. (1994) Science. 265:781-784. / Oldenburg K.R. et al., 1992, Proc. 5 Natl. Acad. Sci., 89:5393-5397. / Orita et al. (1989) Proc. Natl. Acad. Sci. US.A.86: 2776- 2770. / Ott J., Analysis of Human Genetic Linkage, John Hopkins University Press, Baltimore, 1991 / Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973) / Parmley and Smith, Gene, 1988, 73:305-318 / Pastinen et al., Genome Research 1997; 7:606-614 / Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 10 85(8):2444-2448 / Pease S. ans William R.S., 1990, Exp. Cell. Res., 190: 209-21 1. / Perlin et al. (1994) Am. J. Hum. Genet. 55:777-787. / Peterson et al., 1993, Proc. Natl. Acad. Sci. USA, 90 : 7593-7597. / Potter et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81(22):7161-7165. / Ramunsen et al., 1997, Electrophoresis, 18 : 588-598. / Reid L.H. et al. (1990) Proc. Natl. Acad. Sci. U A. 87:4299-4303. / Risch, N. and Merikangas, K. (Science, 273: 1516-1517, 1996 / 15 Robertson E., 1987, Embryo-derived stem cell lines. In: E.J. Robertson Ed. Teratocarcinomas and embrionic stem cells: a practical approach. IRL Press, Oxford, pp. 71. / Rossi et al., Pharmacol. Ther. 50:245-254, (1991) / Roth J.A. et al. (1996) Nαtwre Medicine. 2(9):985-991. / Roux et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:9079-9083. / Ruano et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:6296-6300. / Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory 20 Manual. 2ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. / Samson M, et al. (1996) Nαtwre, 382(6593):722-725. / Samulski et al. (1989) J. Virol. 63:3822-3828. / Sanchez-Pescador R. (1988) J. Clin. Microbiol. 26(10): 1934-1938. / Sarkar, G. and Sommer S.S. (1991) Biotechniques. I Sato-Yoshitake et al., J Biol Chem 1992 Νov 25;267(33):23930-6 / Sauer B. et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5166-5170. / Schedl A. et al., 1993a, 25 Nature, 362: 258-261. / Schedl et al., 1993b, Nucleic Acids Res., 21: 4783-4787. / Schneider et al.(1997) Arlequin: A Software For Population Genetics Data Analysis. University of Geneva. / Scholey, 1993, Motility assays for Motor Proteins, (San Diego, California : Academic Press) / Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation / Schaid D.J. et 30 a\., Genet. Epidemiol, 13:423-450, 1996 / Sczakiel G. et al. (1995) Trends Microbiol. 3(6):213- 217. / Shay J.W. et al., 1991, Biochem. Biophys. Acta, 1072: 1-7. / Sheffield, V.C. et al. (1991) Proc. Nαtl Acαd. Sci. U.S.A. 49:699-706. / Shizuya et al. (1992) Proc. Nαtl Acαd. Sci. U.S.A. 89:8794-8797. / Shoemaker DD, Lashkari DA, Morris D, Mittmann M, Davis RW, Nat Genet 1996;14(4):450-456 / Spielmann S. et al., Am. J. Hum. Genet., 52:506-516, 1993 / 35 Spielmann S. and Ewens W.J., Am. J. Hum. Genet., 62:450-458, 1998 / Smith (1957) Ann. Hum. Genet. 21 :254-276. / Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165. / Sowdhamini et al., Protein Engineering 10:207, 215 (1997) / Sternberg N.L. (1992) Trends Genet. 8:1-16. / Steinberg N.L. ( 1994) Mamm. Genome. 5:397-404. / Syvanen AC, Clin Chim Acta 1994;226(2):225-236 / Szabo A. et al. Curr Opin Struct Biol 5, 699-705 (1995) / Tacson et al. (1996) Nature Medicine. 2(8):888-892. / Te Riele et al. (1990) Nature. 348:649-651. / Terwilliger J.D. and Ott J., Handbook of Human Genetic Linkage, John Hopkins University Press, London, 1994 / Thomas K.R. et al. (1986) Cell. 44:419-428. / Thomas K.R. et al. (1987) Cell. 51 :503-512. / Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680 / Tur-Kaspa et al. (1986) Mol Cell Biol. 6:716-718. / Tyagi et al. (1998) Nature Biotechnology. 16:49-53. / Urdea M.S. (1988) Nucleic Acids Research. 11 :4937-4957. / Urdea M.S. et al.(1991) Nucleic Acids Symp. Ser. 24:197-200. / Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971) / Valadon P., et al., 1996, J. Mol. Biol., 261:11-22. / Van der Lugt et al. (1991) Ge«e. 105:263-267. / Vlasak R. et al. (1983) Eur. J. Biochem. 135:123-126. / Wabiko et al. (1986) Z)N .5(4):305-314. / Walker et al. (1996) Clin. Chem. 42:9-13. / Wang et al., 1997, Chromatographia, 44 : 205-208. / Weir, B.S. (1996) Genetic data Analysis II: Methods for Discrete population genetic Data, Sinauer Assoc, Inc., Sunderland, MA, U.S.A. I Westerink M.A.J., 1995, Proc. Natl. Acad. Sci., 92:4021-4025 / White, M.B. et al. (1992) Genomics. 12:301- 306. / White, M.B. et al. (1997) Genomics. 12:301-306. / Wong et al. (1980) Gene. 10:87-94. / Wood S.A. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 4582-4585. / Wu and Wu (1987) J. Biol. Chem. 262:4429-4432. / Wu and Wu (1988) Biochemistry. 27:887-892. / Wu et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:2757. / Yagi T. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:9918-9922. / Yonekawa Y. et al. (1998) J. Cell Biol. 141, 431-441 / Zhao et al., Am. J. Hum. Genet., 63:225-240, 1998 / Zou Y. R. et al. (1994) Curr. Biol. 4:1099-1103.

Claims

115 CLAIMS

1. An isolated, purified or recombinant polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No 1 or the complementary sequence thereof, wherein said contiguous span comprises either :

5 - at least 1 ofthe following nucleotide positions of SEQ ID No 1 : 1-39624, 39705-40589,

40666-43629, 43710-44203, 4431 1-45125, 45210-45440, 45622-45717, 45791-68580, 68675- 70246, 70396-72421, 72601-73295, 73434-74648, 74898-83055, 83175-85192, 85279-85609, 85740-85906, 86070-88304, 88396-90585, 90705-91767, 91824-94380, 94490-96296, 96364- 97184, 97270-101 167, 101274-109465, 109581-1 10228, 1 10363-111819, 1 1 1882-1 13636, 113783- 10 1 13945, 1 14186-1 17002, 1 17075-119676, and 119677-121162; or,

- a G at position 7159 of SEQ ID No 1.

2. An isolated, purified or recombinant polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No 2 or the complementary sequence thereof, wherein said

15 contiguous span comprises:

- at least 1 ofthe following nucleotide positions of SEQ ID No 2: 1-1600, 1751-2138, 2332- 2539, 2659-3829 and 8885-10884; or,

- a C either at position 2551 or 4500 of SEQ ID No 2.

20 3. An isolated, purified or recombinant polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No 3 or the complementary sequence thereof, wherein said contiguous span comprises either :

- at least 1 ofthe following nucleotide positions of SEQ ID No 3: 391-1619 and 6988- 10682; or,

25 - a C at position 5487, or a C at position 6265 of SEQ ID No 3.

4. An isolated, purified, or recombinant polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of SEQ ID Nos 1-3 and 5-8 or the complement thereof, wherein said span includes a HAZR-related biallelic marker in said sequence.

30

5. A polynucleotide according to claim 4, wherein said HAZR-related biallelic marker is selected from the group consisting of Al to A32, and the complements thereof.

6. A polynucleotide according to claim 4, wherein said HKZ -related biallelic marker is 35 selected from the group consisting of A 1 to A22 and A25 to A32, and the complements thereof.

7. A polynucleotide according to claim 4, wherein said HKL -related biallelic marker is selected from the group consisting of A23 and A24, and the complements thereof.

8. A polynucleotide according to any one of claims 4 to 7, wherein said contiguous span is 18 to 35 nucleotides in length and said biallelic marker is within 4 nucleotides ofthe center of said polynucleotide.

9. A polynucleotide according to claim 8, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide.

10. A polynucleotide according to claim 9, wherein said polynucleotide consists essentially of a sequence selected from the following sequences: PI to P30, and the complementary sequences thereto.

11. A polynucleotide according to any one of claims 4 to 7, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide and said biallelic marker is present at the 3' end of said polynucleotide.

12. A polynucleotide according to any one of claims 1 to 3, wherein the 3' end of said contiguous span is present at the 3' end of said polynucleotide.

13. A polynucleotide according to claim 12, wherein the 3' end of said polynucleotide is located within 20 nucleotides upstream of a H ZR-related biallelic marker in said sequence.

14. A polynucleotide according to claim 13, wherein the 3' end of said polynucleotide is located 1 nucleotide upstream of said HKLP-re\ateά biallelic marker in said sequence.

15. A polynucleotide according to claim 14, wherein said polynucleotide consists essentially of a sequence selected from the following sequences: Dl to D30, and El to E30.

16. An isolated, purified, or recombinant polynucleotide consisting essentially of a sequence selected from the following sequences: Bl to B19 and Cl to C25.

17. An isolated, purified, or recombinant polynucleotide which encodes a polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1 ofthe amino acid positions 1-478 ofthe SEQ ID No 4.

18. A polynucleotide according to any one of claims 1 to 17 attached to a solid support.

19. An array of polynucleotides comprising at least one polynucleotide according to claim 18.

20. An array according to claim 19, wherein said array is addressable.

21. A polynucleotide according to any one of claims 1 to 17 further comprising a label.

22. A recombinant vector comprising a polynucleotide according to any one of claims 1 to

17.

23. A host cell comprising a recombinant vector according to claim 22.

24. A non-human host animal or mammal comprising a recombinant vector according to claim 22.

25. A mammalian host cell comprising an HKLP gene disrupted by homologous recombination with a knock out vector, comprising a polynucleotide according to any one of claims 1 to 17.

26. A non-human host mammal comprising a HKLP gene disrupted by homologous recombination with a knock out vector, comprising a polynucleotide according to any one of claims 1 to 17.

27. A method of genotyping comprising determining the identity of a nucleotide at a HKLP- related biallelic marker or the complement thereof in a biological sample.

28. A method according to claim 27, wherein said biological sample is derived from a single subject.

29. A method according to claim 28, wherein the identity ofthe nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome.

30. A method according to claim 27, wherein said biological sample is derived from multiple subjects.

31. A method according to claim 27, further comprising amplifying a portion of said sequence comprising the biallelic marker prior to said determining step.

32. A method according to claim 31, wherein said amplifying is performed by PCR.

33. A method according to claim 27, wherein said determining is performed by a hybridization assay.

34. A method according to claim 27, wherein said determining is performed by a sequencing assay.

35. A method according to claim 27, wherein said determining is performed by a microsequencing assay.

36. A method according to claim 27, wherein said determining is performed by an enzyme- based mismatch detection assay.

37. A method of estimating the frequency of an allele of a HAZ -related biallelic marker in a population comprising: a) genotyping individuals from said population for said biallelic marker according to the method of claim 27; and b) determining the proportional representation of said biallelic marker in said population.

38. A method of detecting an association between a genotype and a trait, comprising the steps of: a) determining the frequency of at least one HAZ -related biallelic marker in trait positive population according to the method of claim 37; b) determining the frequency of said HKEP-related biallelic marker in a control population according to the method of claim 37; and c) determining whether a statistically significant association exists between said genotype and said trait.

39. A method of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising: a) genotyping at least one HKLP-related biallelic marker according to claim 29 for each individual in said population; b) genotyping a second biallelic marker by determining the identity ofthe nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome of each individual in said population; and c) applying a haplotype determination method to the identities ofthe nucleotides determined in steps a) and b) to obtain an estimate of said frequency.

40. A method according to claim 39, wherein said haplotype determination method is selected from the group consisting of asymmetric PCR amplifcation, double PCR amplification of specific alleles, the Clark method, or an expectation maximization algorithm.

41. A method of detecting an association between a haplotype and a trait, comprising the steps of: a) estimating the frequency of at least one haplotype in a trait positive population according to the method of claim 39; b) estimating the frequency of said haplotype in a control population according to the method of claim 39; and c) determining whether a statistically significant association exists between said haplotype and said trait.

42. A method according to claim 38, wherein said genotyping steps a) and b) are performed on a single pooled biological sample derived from each of said populations.

43. A method according to claim 38, wherein said genotyping steps a) and b) performed separately on biological samples derived from each individual in said populations.

44. A method according to either claim 38 or 41, wherein said control population is a trait negative population.

45. A method according to either claim 38 or 41, wherein said case control population is a random population.

46. An isolated, purified, or recombinant polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1 ofthe amino acid positions 1-478 ofthe SEQ ID No 4.

120 47. An isolated or purified antibody composition are capable of selectively binding to an epitope-containing fragment of a polypeptide according to claim 46, wherein said epitope comprises at least 1 ofthe amino acid positions 1-478 ofthe SEQ ID No 4.

48. A method according to any one of claims 27 to 45 wherein said H Z -related biallelic marker is selected from the group consisting of Al to A32, and the complements thereof.

49. A method according to any one of claims 27 to 45 wherein said HAEP-related biallelic marker is selected from the group consisting of Al to A22 and A25 to A32, and the complements thereof.

50. A method according to any one of claims 27 to 45 wherein said HAZ -related biallelic marker is selected from the group consisting of A23 and A24, and the complements thereof.

51. A diagnostic kit comprising a polynucleotide according to any one of claims 1 to 21.

52. Use of a polynucleotide comprising a contiguous span of at least 12 nucleotides of a sequence selected from the group consisting ofthe SEQ ID Nos 1-3 and 5-8 or the complementary sequence thereto for determining the identity ofthe nucleotide at a HKEP-related biallelic marker

53. Use according to claim 52 in a microsequencing assay, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide and wherein the 3' end of said polynucleotide is located 1 nucleotide upstream of said HKLP related biallelic marker in said sequence.

54. Use according to claim 52 in a hybridization assay, wherein said span includes said HKLP -related biallelic marker.

55. Use according to claim 52 in a specific amplification assay, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide and said biallelic marker is present at the 3' end of said polynucleotide.

56. Use according to claim 52 in a sequencing assay, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide.

57. Use according to any one of claims 52-56, wherein said HKLP -related biallelic is a biallelic marker selected in the group consisting ofthe biallelic markers Al to A32.

58. A computer readable medium having stored thereon a sequence selected from the group consisting of a nucleic acid code comprising one ofthe following: a) a contiguous span of at least 12 nucleotides of SEQ ID No 1, wherein said contiguous 5 span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 1 : 1-

39624, 39705-40589, 40666-43629, 43710-44203, 4431 1-45125, 45210-45440, 45622-45717, 45791-68580, 68675-70246, 70396-72421, 72601-73295, 73434-74648, 74898-83055, 83175- 85192, 85279-85609, 85740-85906, 86070-88304, 88396-90585, 90705-91767, 91824-94380, 94490-96296, 96364-97184, 97270-101 167, 101274-109465, 109581-1 10228, 1 10363-1 11819, 10 111882-113636, 113783-113945, 114186-117002, 1 17075-1 19676, and 119677-121162; b) a contiguous span of at least 12 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659-3829 and 8885-10884; c) a contiguous span of at least 12 nucleotides of SEQ ID No 1 or the complements thereof, 15 wherein said contiguous span comprises a G at position 7159 of SEQ ID No 1 ; d) a contiguous span of at least 12 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises a C either at position 2551 or 4500 of SEQ ID No 4; e) a contiguous span of at least 12 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide

20 positions of SEQ ID No 3: 391-1619 and 6988-10682; f) a contiguous span of at least 12 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises a nucleotide selected in the group consisting of a C at position 5487, and a C at position 6265 of SEQ ID No 3; and g) a nucleotide sequence complementary to any one ofthe contiguous spans of a), b), c), d), e), 25 or f).

59. A computer readable medium having stored thereon a sequence consisting of a polypeptide code comprising a contiguous span of at least 6 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1 ofthe amino acid positions 1-478 ofthe SEQ ID No 4.

30

60. A computer system comprising a processor and a data storage device wherein said data storage device a computer readable medium according to with claim 58 or 59.

61. A computer system according to claim 60, further comprising a sequence comparer and 35 a data storage device having reference sequences stored thereon.

62. A computer system of Claim 61 wherein said sequence comparer comprises a computer program which indicates polymoφhisms.

63. A computer system of Claim 60 further comprising an identifier which identifies features in said sequence.

64. A method for comparing a first sequence to a reference sequence, comprising the steps of: reading said first sequence and said reference sequence through use of a computer program which compares sequences; and determining differences between said first sequence and said reference sequence with said computer program, wherein said first sequence is selected from the group consisting of a nucleic acid code comprising one ofthe following: a) a contiguous span of at least 12 nucleotides of SEQ ID No 1 , wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 1 : 1-39624, 39705-40589, 40666-43629, 43710-44203, 4431 1-45125, 45210- 45440, 45622-45717, 45791-68580, 68675-70246, 70396-72421, 72601-73295, 73434- 74648, 74898-83055, 83175-85192, 85279-85609, 85740-85906, 86070-88304, 88396- 90585, 90705-91767, 91824-94380, 94490-96296, 96364-97184, 97270-101167, 101274-

109465, 109581-1 10228, 1 10363-1 11819, 1 1 1882-1 13636, 1 13783-1 13945, 114186- 1 17002, 1 17075-1 19676, and 1 19677-121162; b) a contiguous span of at least 12 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659-3829 and 8885-

10884; c) a contiguous span of at least 12 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises a G at position 7159 of SEQ ID No 1 ; d) a contiguous span of at least 12 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises a C either at position 2551 or 4500 of SEQ

ID No 4; e) a contiguous span of at least 12 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 3: 391-1619 and 6988-10682; f) a contiguous span of at least 12 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises a nucleotide selected in the group consisting of a C at position 5487, and a C at position 6265 of SEQ ID No 3; and g) a nucleotide sequence complementary to any one ofthe contiguous spans of a), b), c), d), e), or f); and a polypeptide code comprising a contiguous span of at least 6 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1 ofthe amino acid positions 1-478 ofthe SEQ ID No 4.

65. A method according to Claim 64, wherein said step of determining differences between the first sequence and the reference sequence comprises identifying at least one polymoφhism.

66. A method for identifying a feature in a sequence, comprising the steps of: reading said sequence through the use of a computer program which identifies features in sequences; and identifying features in said sequence with said computer program; wherein said sequence is selected from the group consisting of a nucleic acid code comprising one ofthe following: a) a contiguous span of at least 12 nucleotides of SEQ ID No 1, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 1: 1-39624, 39705-40589, 40666-43629, 43710-44203, 44311-45125, 45210- 45440, 45622-45717, 45791-68580, 68675-70246, 70396-72421, 72601-73295, 73434- 74648, 74898-83055, 83175-85192, 85279-85609, 85740-85906, 86070-88304, 88396-

90585, 90705-91767, 91824-94380, 94490-96296, 96364-97184, 97270-101 167, 101274- 109465, 109581-110228, 1 10363-11 1819, 1 1 1882-1 13636, 1 13783-1 13945, 1 14186- 1 17002, 117075-119676, and 119677- 121 162; b) a contiguous span of at least 12 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 2: 1-1600, 1751-2138, 2332-2539, 2659-3829 and 8885- 10884; c) a contiguous span of at least 12 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises a G at position 7159 of SEQ ID No 1 ; d) a contiguous span of at least 12 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises a C either at position 2551 or 4500 of SEQ ID No 4; e) a contiguous span of at least 12 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 ofthe following nucleotide positions of SEQ ID No 3: 391-1619 and 6988-10682; f) a contiguous span of at least 12 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises a nucleotide selected in the group consisting of a C at position 5487, and a C at position 6265 of SEQ ID No 3; and g) a nucleotide sequence complementary to any one ofthe contiguous spans of a), b), c), d), e), or f); and a polypeptide code comprising a contiguous span of at least 6 amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1 ofthe amino acid positions 1-478 ofthe SEQ ID No 4.

67. A method for the screening of a candidate substance interacting with a HKLP polyeptide comprising the following steps : a) providing a polypeptide consisting of a HKLP protein or a fragment comprising a contiguous span of at least 6 amino acids amino acids of SEQ ID No 4, wherein said contiguous span includes at least 1 ofthe amino acid positions 1-478 ofthe SEQ ID No 4 or a variant thereof; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; d) detecting the complexes formed between said polypeptide and said candidate substance.