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WO2001023403A1 - GENES DU CLADE ypga - Google Patents

GENES DU CLADE ypga Download PDF

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Publication number
WO2001023403A1
WO2001023403A1 PCT/US2000/026450 US0026450W WO0123403A1 WO 2001023403 A1 WO2001023403 A1 WO 2001023403A1 US 0026450 W US0026450 W US 0026450W WO 0123403 A1 WO0123403 A1 WO 0123403A1
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WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
ypga
group
isolated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US2000/026450
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English (en)
Inventor
James R. Brown
Alison F. Chalker
David J. Holmes
Edwina Imogen Wilding
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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Publication of WO2001023403A1 publication Critical patent/WO2001023403A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses.
  • the invention relates to polynucleotides and polypeptides of the ypgA clade family, as well as their variants, herein referred to as "ypgA clade genes," “ ypgA clade gene polynucleotide(s),” and “ypgA clade gene polypeptide(s),” as the case may be.
  • ypgA is found downstream of the gene encoding phosphomevalonate kinase and is part of the mevalonate kinase/decarboxylase operon. It is not found at this position in species of staphylococci, although a homologue is present in the genomes of the species examined. A ypgA homologue is also present in the ypgA clade in Borrelia b rgdoferi. No homologues were identified in eukaroytic organisms. Analysis of the amino-acid sequence for conserved motifs suggests that ypgA shows some distant homology to inosine 5'-monophosphate
  • ypgA is a member of the ypgA clade gene family.
  • the present invention relates to ypgA clade pathway genes, in particular ypgA polypeptides and polynucleotides, recombinant matenals and methods for their production
  • the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others
  • the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating m
  • the invention relates to ypgA clade gene polypeptides and polynucleotides as desc ⁇ bed in greater detail below
  • the invention relates to polypeptides and polynucleotides of ypgA clade genes that are related by amino acid sequence homology to ypgA clade polypeptides from other species
  • the invention relates especially to ypgA clade genes having a high degree of homology to the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NOs 1-16
  • the "ypgA clade gene family" of the invention means a set of genes encoding a set of polypeptides from Gram-positive bacteria falling within nodes A, B, C, D, E, F, and G of the phylogenetic tree depicted in Figure 1 comprising the genera Staphvlococcus, Streptococcus, and Enterococcus
  • ypgA clade gene(s) refers to a gene of the "ypgA clade pathway gene family", defined above "YpgA clade pathway gene polynucleot ⁇ de(s)” and “ypgA clade gene polypept ⁇ de(s)” means, respectively, a polynucleotide of the invention or polypeptide of the invention, as more particularly set forth elsewhere herein
  • the invention provides a set of genes encoding a set of polypeptides involved from Gram-positive bacteria falling within the clade defined by: node A of Figure 1 ; node B of Figure 1 ; node C of Figure 1 ; node D of Figure 1 ; node E of Figure 1 ; node F of Figure 1 ; and node G of the phylogenetic tree depicted in Figure 1.
  • the invention provides a set of genes encoding ypgA isolated from bacteria falling within the clade of Gram-positive bacteria of the phylogenetic tree depicted in Figure 1 comprising the species Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enter ococcus faecalis, and Bacillus subtilis.
  • sequences recited in the Sequence Listing below as "DNA” represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.
  • Staphylococcus epidermidis ypgA polynucleotide sequence [SEQ ID NO: l].
  • Steptococcus pyogenes ypgA polynucleotide sequence [SEQ ID NO: 13].
  • Bacillus subtilis ypgA polynucleotide sequence [SEQ ID NO: 15]. 5 ' - GTGACTCGAGCAGAACGAAAAAGACAACACATCAATCATGCCTTGTCCAT CGGCCAGAAGCGGGAAACAGGTCTTGATGATATTACGTTTGTTCACGTCA GTCTGCCCGATCTTGCATTAGAACAAGTAGATATTTCCACAAAAATCGGC GAACTTTCAAGCAGTTCGCCGATTTTTATCAATGCAATGACTGGCGGCGG CGGAAAACTTACATATGAGATTAATAAATCGCTTGCGCGAGCGGCTTC AGGCTGGAATTCCCCTTGCTGTGGGATCGCAAATGTCAGCATTAAAAGAT CCATCAGAGCGTCTTTCCTATGAAATTGTTCGAAAGGAAAACCCAAACGG GCTGATTTTTGCCAACCTGGGAAGCGAGGCAACGGCTGCTCAGGCAAAGG AAGCCGTTGAGATGATTGGAGCAACGGCTGCTCAGGCAAAGG AAGCCGTT
  • a deposit comprising a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland on 1 1 April 1996 and assigned deposit number 40794 The deposit was desc ⁇ bed as Streptococcus pneumoniae 0100993 on deposit
  • Streptococcus pneumoniae 0100993 DNA library in E coll was similarly deposited with the NCIMB and assigned deposit number 40800
  • the Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain"
  • the deposited strain comp ⁇ ses a full-length ypgA gene of the ypgA clade gene family
  • the sequence of the polynucleotides comp ⁇ sed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any desc ⁇ ption of sequences herein
  • Staphylococcus aureus WCUH 29 strain A deposit comp ⁇ smg a Staphylococcus aureus WCUH 29 strain has been deposited with the NCIMB), 23 St Machar Drive, Aberdeen AB2 1 RY, Scotland on 1 1 September 1995 and assigned NCIMB Deposit No 40771 , and referred to as Staphylococcus aureus WCUH29 on deposit
  • the Staphylococcus aureus strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain "
  • the deposited strain comp ⁇ ses a full-length ypgA gene of the ypgA clade gene family
  • the sequence of the polynucleotides comp ⁇ sed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any desc ⁇ ption of sequences herein
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure
  • the deposited strain will be irrevocably and without rest ⁇ ction or condition released to the public upon the issuance of a patent
  • the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C ⁇ 1 12
  • a license may be required to make, use or sell the deposited strain, and compounds de ⁇ ved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain, which polypeptide is comp ⁇ sed in the deposited strain
  • ypgA clade gene polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby.
  • mevalonate pathway gene polypeptide and polynucleotide sequences isolated from the deposited strain are also provided by the invention.
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is comprised in the deposited strain.
  • ypgA clade gene polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby.
  • Polypeptides Yp A clade gene polypeptides of the invention are substantially phylogenetically related to other proteins of the ypgA clade gene family.
  • Figure 1 shows the phylogenetic analysis of ypgA, a member of the ypgA clade gene family.
  • Phylogenetic trees are based on the neighbor-joining (NJ) method as implemented by the program NEIGHBOR of the PHYLIP 3.57c package (Felsenstein, J. 1993. Distributed by the author: http://evolution.genetics.washington. edu/phylip.html, Department of Genetics, University of Washington, Seattle.). The method used to create this phylogenetic tree is described in detail in Example 1.
  • polypeptides of ypgA clade genes referred to herein as "ypgA clade genes" and “ypgA clade gene polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • ypgA clade gene polypeptides encoded by naturally occurring alleles of a ypgA clade gene.
  • the present invention further provides for an isolated polypeptide that: (a) comprises or consists of an amino acid sequence that has at least 95% identity, most preferably at least 97- 99% or exact identity, to that of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16 over the entire length of said amino acid sequence; (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NOs: l, 3, 5, 7, 9, 1 1, 13, and 15 over the entire length of said polynucleotide sequence; (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 over the
  • polypeptides of the invention include the polypeptides of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of a ypgA clade gene, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] and also include portions of such polypeptides with such portion of the polypeptide generally comp ⁇ sing at least 30 amino acids and more preferably at least 50 amino acids
  • the invention also includes a polypeptide consisting of or comp ⁇ sing a polypeptide of the formula
  • a polypeptide of the invention is de ⁇ ved from a bacte ⁇ um of the ypgA clade gene family, however, it may preferably be obtained from other organisms of the same taxonomic genus
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order
  • a fragment is a va ⁇ ant polypeptide having an amino acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the invention
  • fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16], or of variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence.
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a bacterium of the ypgA clade gene family, are also preferred.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from such amino acid sequence.
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • the polynucleotide comprises a region encoding ypgA clade gene polypeptides comprising a sequence set out in Table 1 [SEQ ID NOs: l, 3, 5, 7, 9, 1 1, 13, and 15] that includes a full length gene, or a variant thereof.
  • a full-length gene from the ypgA clade gene family is essential to the growth and/or survival of an organism that possesses it, such as a bacterium from the ypgA clade gene family.
  • isolated nucleic acid molecules encoding and/or expressing ypgA clade gene polypeptides and polynucleotides, particularly ypgA clade gene polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs.
  • Further embodiments of the invention include biologically, diagnosttcally, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and va ⁇ ants thereof, and compositions comp ⁇ sing the same
  • Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a ypgA clade gene polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] and polynucleotides closely related thereto and va ⁇ ants thereof
  • a ypgA clade gene polypeptide from a bacterium of the ypgA clade gene family comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16], or a variant thereof
  • a polynucleotide of the invention encoding ypgA clade gene polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacte ⁇ a using cells from a bacte ⁇ um of the ypgA clade gene family as starting matenal, followed by obtaining a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15]
  • a library of clones of chromosomal DNA from a bacteria of the ypgA clade gene family in E coh or some other suitable host is probed with a radiolabeled o
  • each DNA sequence set out in Table 1 contains an open reading frame encoding a protein having about the number of ammo acid residues set forth m Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art
  • the present invention provides for an isolated polynucleotide comprising or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15 over the entire length of SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 over the entire length of said amino acid sequence
  • a polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than a bacte ⁇ um of the ypgA clade gene family, may be obtained by a process that comp ⁇ ses the steps of screening an approp ⁇ ate library under st ⁇
  • the invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15] Also provided by the invention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence
  • the polynucleotide of the invention may also comp ⁇ se at least one non-coding sequence, including for example, but not limited to, at least one non-coding 5' and 3' sequence, such as the transc ⁇ bed but non- translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ⁇ bosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadeny
  • the invention also includes a polynucleotide consisting of or comp ⁇ sing a polynucleotide of the formula X-(R ] ) m -(R 2 )-(R 3 ) n -Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of R ] and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide
  • a polynucleotide of the invention is de ⁇ ved from a bacte ⁇ um of the ypgA clade gene family, however, it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order
  • the term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of a ypgA clade gene having an amino acid sequence set out in Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16].
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may comprise coding and/or non-coding sequences.
  • the invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16]. Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
  • polynucleotides encoding ypgA clade gene variants that have the amino acid sequence of one of the ypgA clade gene polypeptides of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination.
  • ypgA clade gene polypeptide of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination.
  • silent substitutions, additions and deletions that do not alter the properties and activities of a ypgA clade gene polypeptide.
  • Preferred isolated polynucleotide embodiments also include polynucleotide fragments, such as a polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 1 1, 13, and 15, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, and 15.
  • polynucleotide fragments such as a polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, and 15.
  • polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding ypgA clade gene polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16], and polynucleotides that are complementary to such polynucleotides.
  • polynucleotides that comprise a region that is at least 95% are especially preferred.
  • those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred
  • Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, and 15]
  • polynucleotides that hyb ⁇ dize, particularly under st ⁇ ngent conditions, to ypgA clade gene polynucleotide sequences such as those polynucleotides in Table 1
  • the invention further relates to polynucleotides that hyb ⁇ dize to the polynucleotide sequences provided herein
  • the invention especially relates to polynucleotides that hyb ⁇ dize under stringent conditions to the polynucleotides desc ⁇ bed herein
  • the terms "st ⁇ ngent conditions” and “stnngent hyb ⁇ dization conditions” mean hyb ⁇ dization occur ⁇ ng only if there is at least 95% and preferably at least 97% identity between the sequences
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization
  • the invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15, or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully desc ⁇ bed elsewhere herein It is preferred that polynucleotides of the invention encoding ypgA be isolated from
  • Gram-positive bacteria of the phylogenetic tree depicted in Figure 1 It is more particularly preferred that such bacteria of the invention are bacteria of the genera Staphylococcus, Streptococcus, Enterococcus, or Bacillus It is most particularly preferred that such bacteria of the invention are bacteria of the species Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, or Bacillus subtilis
  • FIG. 1 For example, polynucleotides of the invention encoding ypgA falling within the clade defined by node A of Figure 1 , node B of Figure 1 , node C of Figure 1 , node C of Figure 1 , node D of Figure 1 , node E of Figure 1 , node F of Figure 1 , and node G of the phylogenetic tree of Figure 1
  • each is determined using the cladistical analyses disclosed herein, in Example 1
  • the polynucleotides of the invention may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding a ypgA clade gene and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a ypgA clade gene
  • Such probes generally will comp ⁇ se at least 15 nucleotide residues or base pairs
  • such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs
  • Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs
  • a coding region of a ypgA clade gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15] to synthesize an oligonucleotide probe
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hyb ⁇ dizes to
  • polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and mate ⁇ als for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relating to polynucleotide assays
  • the polynucleotides of the invention that are oligonucleotides derived from any polynucleotide or polypeptide sequence of Table 1 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained
  • the invention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids inte ⁇ or to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things As generally is the case in vivo, the additional amino acids may be processed away from a mature protein by cellular enzymes
  • a precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • the entire polypeptide encoded by an open reading frame is often not required for activity Accordingly, it has become routine in molecular biology to map the boundanes of the p ⁇ mary structure required for activity with N-terminal and C- terminal deletion expenments
  • expe ⁇ ments utilize exonuclease digestion or convenient rest ⁇ ction sites to cleave coding nucleic acid sequence
  • Promega (Madison, WI) sell an Erase-a-baseTM system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol available at www promega com)
  • the digested endpoints can be repaired (e g , by ligation to synthetic linkers) to the extent necessary to preserve an open reading frame
  • the nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity
  • a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotem, that is a precursor to a proprotein, having a leader sequence and one or more prosequences, that generally are removed dunng processing steps that produce active and mature forms of the polypeptide
  • the invention also relates to vectors that comp ⁇ se a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs de ⁇ ved from the DNA constructs of the invention
  • Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comp ⁇ sing expression systems Accordingly, in a further aspect, the present invention relates to expression systems that comp ⁇ se a polynucleotide or polynucleotides of the present invention, to host cells that are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques For recombinant production of the polypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY, ( 1986) and Sambrook, et al. , MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • Suitable hosts include, but are not limited to a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campy lobacter, Shigella, Legionella, Pseudomonas, Aeromona
  • vectors include, among others, chromosomal-, episomal- and virus-de ⁇ ved vectors, for example, vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those denved from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
  • the expression system constructs may comp ⁇ se control regions that regulate as well as engender expression
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard
  • the approp ⁇ ate DNA sequence may be inserted into the expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth in Sambrook, et al , MOLECULAR CLONING, A LABORA TOR Y MANUAL, supra
  • approp ⁇ ate secretion signals may be incorporated into the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • Polypeptides of the invention can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or pu ⁇ fication Diagnostic, Prognostic, Serotyping and Mutation Assays
  • This invention is also related to the use of ypgA clade gene polynucleotides and polypeptides of the invention for use as diagnostic reagents Detection of ypgA clade gene polynucleotides and/or polypeptides in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comp ⁇ sing the ypgA clade gene or protein, may be detected at the nucleic acid or ammo acid level by a va ⁇ ety of well known techniques as well as by methods provided herein Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials Polynucleotides from any of these sources,
  • an array of oligonucleotides probes comprising ypgA clade gene nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification
  • Array technology methods are well known and have general applicability and can be used to address a vanety of questions in molecular genetics including gene expression, genetic linkage, and genetic va ⁇ ability (see, for example, Chee, et al , Science, 274 610 (1996))
  • the present invention relates to a diagnostic kit that comprises
  • kits (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, or a fragment thereof, or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others
  • This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents Detection of a mutated form of a polynucleotide of the invention, preferably, SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, and 15, that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide
  • Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herein
  • the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by a bacte ⁇ um of the ypgA clade gene family, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15] Increased or decreased expression of a ypgA clade gene polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT- PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
  • a diagnostic assay in accordance with the invention for detecting over- expression of a ypgA clade gene polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techniques
  • Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical bra ⁇ es, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 ( 1991 )
  • Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases herein mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g , inhibit) the function of the polypeptide or polynucleotide
  • the present invention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the
  • the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comp ⁇ sing the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists, in the absence of an agonist or antagonist, by testing whether the candidate compound results in inhibition of activation of the polypeptide or
  • polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the invention also provides a method of screening compounds to identify those that enhance (agonist) or block (antagonist) the action of a ypgA clade gene polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bactencidal
  • the method of screening may involve high-throughput techniques
  • a synthetic reaction mix for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comp ⁇ sing a ypgA clade gene polypeptide and a labeled substrate or gand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a ypgA clade gene agonist or antagonist
  • the ability of the candidate molecule to agonize or antagonize the ypgA clade gene polypeptide is reflected in decreased binding of the labeled hgand or decreased production of product from such substrate Molecule
  • ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ * I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supematants, tissue extracts, bodily materials).
  • a source of the putative receptor e.g., cells, cell membranes, cell supematants, tissue extracts, bodily materials.
  • Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate.
  • Protein complexes such as formed by a ypgA clade gene polypeptide associating with another ypgA clade gene polypeptide or other polypeptide, labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein. It is preferred that this method be used to characterize small molecules that disrupt polypeptide complexes.
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of ypgA clade gene polypeptide dimers, trimers, tetramers or higher order structures, or structures formed by a ypgA clade gene polypeptide bound to another polypeptide.
  • a ypgA clade gene polypeptide can be labeled with both a donor and acceptor fluorophore. Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor. Compounds that block dimerization will inhibit fluorescence energy transfer.
  • Surface plasmon resonance can be used to monitor the effect of small molecules on a ypgA clade gene polypeptide self-association as well as an association of a ypgA clade gene polypeptide and another polypeptide or small molecule.
  • a ypgA clade gene polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric.
  • Solution protein can then passed over the ypgA clade gene polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index.
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for ypgA clade gene polypeptide self-association as well as an association of a ypgA clade gene polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to characterize the interaction between an association of a ypgA clade gene polypeptide with another ypgA clade gene polypeptide or a different polypeptide
  • a ypgA clade gene polypeptide can be coupled to a scintillation- filled bead Addition of radiolabeled ypgA clade gene polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid
  • signal is emitted upon a ypgA clade gene polypeptide binding and compounds that prevent a ypgA clade gene polypeptide self-association or an association of a ypgA clade gene polypeptide and another polypeptide or small molecule will diminish signal
  • inventions provide methods for identifying compounds that bind to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the invention compnsing contacting a polypeptide and/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and/or polynucleotide
  • an assay for ypgA clade gene agonists is a competitive assay that combines a ypgA clade gene and a potential agonist with ypgA clade gene-binding molecules, recombinant ypgA clade gene binding molecules, natural substrates or ligands, or substrate or hgand mimetics, under appropnate conditions for a competitive inhibition assay
  • a ypgA clade gene can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of ypgA clade gene molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure- based design of an agonist or antagonist of the polypeptide and/or polynucleotide, by (a) determining in the first
  • the present invention provides methods of treating abnormal conditions such as, for instance, a Disease, related to an excess of, an under-expression of, an elevated activity of, or a decreased activity of a ypgA clade gene polypeptide and/or polynucleotide
  • Typical examples of such competitors include fragments of a ypgA clade gene polypeptide and/or polypeptide
  • expression of a gene encoding an endogenous ypgA clade gene polypeptide can be inhibited using expression-blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation
  • An example of a known technique of this sort involves the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL ( 1988))
  • oligonucleotides that form triple helices with the gene can be supplied (see, for example, Lee, et al , Nucleic Acids Res ( 1979) 6 3073, Cooney, et al , Science (1988) 241 456, Dervan, et al , Science (1991) 251 1360)
  • These ohgomers can be administered per se or the relevant o
  • the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
  • the invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection.
  • the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial ypgA clade gene proteins that mediate tissue damage and/or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.
  • ypgA clade gene agonists and antagonists preferably bacteristatic or bactericidal agonists and antagonists.
  • the antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.
  • H. pylori Helicobacter pylori bacteria infect the stomachs of over one- third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France, http://www.uicc.ch/ecp/ecp 2904.htm). Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H. pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen.
  • Prefened antimicrobial compounds of the invention should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H. /ry/o ⁇ -induced cancers, such as gastrointestinal carcinoma. Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis.
  • Bodily material(s) means any material derived from an individual or from an organism infecting, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy materials.
  • Disease(s) means any disease caused by or related to infection by a bacteria, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as, for example, infection of cerebrospinal fluid, disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal
  • “Host cell(s)” is a cell that has been introduced (e.g., transformed or transfected) or is capable of introduction (e.g., transformation or transfection) by an exogenous polynucleotide sequence.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
  • Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs
  • Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, et al , J Molec Biol 215 403-410 (1990)
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894, Altschul, et al , J Mol Biol 215 403-410 (1990)
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NOs 1 , 3, 5, 7, 9, 11 , 13, and 15, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15, or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n
  • Alterations of a polynucleotide sequence encoding a polypeptide of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16
  • y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • “Individual(s)” means a multicellular eukaryote, including, but not limited to, a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human.
  • “Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living.
  • Organism(s) means a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campy lobacter, Shigella, Legionella, Pseudomonas, Aeromonas,
  • Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotide(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded, or triple-stranded regions, or a mixture of single- and double- stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • the term "polynucleotide(s)” also includes DNAs or RNAs as described above that comprise one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide(s) as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.
  • Polynucleotide(s) also embraces short polynucleotides often referred to as oligonucleotide(s).
  • Polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and ohgomers and to longer chains generally referred to as proteins. Polypeptides may comprise amino acids other than the 20 gene encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques.
  • Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • “Recombinant expression system(s)” refers to expression systems or portions thereof or polynucleotides of the invention introduced or transformed into a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the invention
  • Va ⁇ ant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any
  • Phylogenetic trees were constructed by neighbor-joining (N-J) and maximum parsimony (MP) methods for each set of alignments. N-J trees were based on pairwise distances between amino acid sequences using the programs NEIGHBOR and PROTDIST of the PHYLIP 3.57c package (Felsenstein, J. 1993. Distributed by the author: http devolution . genetics.washington.edu/phylip.html, Department of Genetics, University of Washington, Seattle.) The "Dayhoff ' program option was invoked in the latter program which estimates the expected amino acid replacements per position (EAARP) using a replacement model based on the Dayhoff 120 matrix.
  • EAARP expected amino acid replacements per position

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Abstract

La présente invention concerne des polypeptides et des polynucléotides du clade ypgA codant pour les polypeptides du clade ypgA et des méthodes de production de ces polypeptides par des techniques de recombinaison. Cette invention concerne aussi des méthodes permettant d'utiliser des polypeptides de clade ypgA dans la recherche de composés antibactériens.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006491A1 (fr) * 2000-07-18 2002-01-24 Center For Advanced Science And Technology Incubation, Ltd. Isopentenyl pyrophosphate isomerase

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus
WO1998018931A2 (fr) * 1996-10-31 1998-05-07 Human Genome Sciences, Inc. Polynucleotides et sequences de streptococcus pneumoniae

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus
WO1998018931A2 (fr) * 1996-10-31 1998-05-07 Human Genome Sciences, Inc. Polynucleotides et sequences de streptococcus pneumoniae

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KUNST ET AL.: "The complete genome sequence of the gram-positive bacterium bacillus subtilis", NATURE, vol. 390, no. 6657, 20 November 1997 (1997-11-20), pages 249 - 256, XP002936623 *
ROELS ET AL.: "Characterization of spoIVA, a sporulation gene involved in coat morphogenesis in bacillus subtilis", J. BACTERIOL., vol. 174, no. 2, January 1992 (1992-01-01), pages 575 - 585, XP002936626 *
SOROKIN ET AL.: "Sequence analysis of the bacillus subtilis chromosome region between the serA and kdg loci cloned in a yeast artificial chromosome", MICROBIOLOGY, vol. 142, no. PT. 8, 1996, pages 2005 - 2016, XP002936624 *
SOROKIN ET AL.: "The organization of the bacillus subtilis 168 chromosome region between the spoVA and serA genetic loci, based on seguence data", MOL. MICROBIOL., vol. 10, no. 2, 1993, pages 385 - 395, XP002936625 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006491A1 (fr) * 2000-07-18 2002-01-24 Center For Advanced Science And Technology Incubation, Ltd. Isopentenyl pyrophosphate isomerase

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