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WO2001007465A1 - Ga1R - Google Patents

Ga1R Download PDF

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Publication number
WO2001007465A1
WO2001007465A1 PCT/US2000/019529 US0019529W WO0107465A1 WO 2001007465 A1 WO2001007465 A1 WO 2001007465A1 US 0019529 W US0019529 W US 0019529W WO 0107465 A1 WO0107465 A1 WO 0107465A1
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Prior art keywords
polypeptide
seq
polynucleotide
sequence
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/US2000/019529
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English (en)
Inventor
Pan Fong Chan
Magdalena Zalacain
Michael A. Lonetto
David J. Holmes
Christopher M. Traini
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SmithKline Beecham Ltd
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Ltd
SmithKline Beecham Corp
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Publication of WO2001007465A1 publication Critical patent/WO2001007465A1/fr
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Classifications

    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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 polynucleoudes and polypeptides of the galR (repressor proteins) family, as well as their variants, herein referred to as "galR,” “galRpolynucleot ⁇ de(s),” and “galR polypept ⁇ de(s)” as the case may be
  • Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, 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 Since its isolation more than 100 years ago. Streptococcus pneumomae has been one of the more intensively studied microbes For example, much of our early understanding that DNA is.
  • Streptococcus pneumomae infections has risen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Streptococcus pneumomae strains that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism
  • the present invention relates to galR, in particular galR polypeptides and galR polynucleotides, recombinant materials and methods for their production
  • the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others
  • the mvention relates to methods for identifying agonists and antagomsts using the mate ⁇ als provided by the mvention, and for treating microbial infections and conditions associated with such infections with the identified agomst or antagonist compounds
  • the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting galR expression or activity
  • the invention relates to galR polypeptides and polynucleotides as described in greater detail below
  • the invention relates to polypeptides and polynucleotides of a galR of Streptococcus pneumomae, that is related by arruno acid sequence homology to Streptococcus thermophilus GalR polypeptide
  • the mvention relates especially to galR having a nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively
  • sequences recited m the Sequence Listing below as "DNA” represent an exemplification of the invention, smce those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides m general, including ⁇ bopolynucleotides
  • a deposit comprising a Streptococcus pneumomae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dnve, Aberdeen AB2 IRY, Scotland on 11 Apnl 1996 and assigned deposit number 40794
  • the deposit was described as Streptococcus pneumomae 0100993 on deposit On 17 Ap ⁇ l 1996 a Streptococcus pneumomae 0100993 DNA library in E co was similarly deposited with the NCEVIB and assigned deposit number 40800
  • the Streptococcus pneumomae strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited stram "
  • the deposited strain comprises a full length galR gene
  • the sequence of the polynucleotides comprised in the deposited strain, as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with
  • the deposit of the deposited stram 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 restriction or condition released to the public upon the issuance of a patent
  • the deposited stram is provided merely as convenience to those of skill m the art and is not an admission that a deposit is required for enablement such as that required under 35 U S C ⁇ 112
  • 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 pneumomae 0100993 strain, which polypeptide is comprised m the deposited stram
  • galR polynucleotide sequences in the deposited strain such as DNA and RNA
  • ammo acid sequences encoded thereby Also provided by the mvention are galR polypeptide and polynucleotide sequences isolated from the deposited stram
  • GalR polypeptide of the mvention is substantially phylogenetically related to other proteins of the galR (repressor proteins) family
  • galR polypeptides of Streptococcus pneumomae referred to herem as "galR” and “galR polypeptides” as well as biologically, diagnostically, prophylactically, climcally or therapeutically useful vanants thereof, and compositions comp ⁇ smg the same
  • the present mvention further provides for an isolated polypeptide that (a) composes or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide comprising or consistmg of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1 , (c) a polypeptide encoded by an isolated polynucleotide comp ⁇ smg or consistmg 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 ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • polypeptides of the mvention include a polypeptide of Table 1 [SEQ ID NO 2] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of galR, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ED NO 2] and also include portions of such polypeptides with such portion of the polypeptide generally comp ⁇ smg at least 30 ammo acids and more preferably at least 50 ammo acids
  • the mvention also mcludes a polypeptide consisting of or comp ⁇ smg a polypeptide of the formula X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the ammo terminus, X is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, R ⁇ and R3 are any ammo acid residue or modified ammo acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R 2 is an ammo acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R 2 is o ⁇ ented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy terminal ammo acid residue is at
  • a polypeptide of the mvention is de ⁇ ved from Streptococcus pneumomae, however, it may preferably be obtained from other organisms of the same taxonomic genus
  • a polypeptide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order
  • a fragment is a va ⁇ ant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention
  • fragments may be "free-standing,” or comp ⁇ sed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region in a single larger polypeptide
  • Prefe ⁇ ed fragments mclude, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of va ⁇ ants thereof, such as a continuous senes of residues that mcludes an amino- and/or carboxyl-terminal ammo acid sequence
  • Degradation forms of the polypeptides of the mvention produced by or in a host cell, particularly a Streptococcus pneumomae are also prefe ⁇ ed
  • fragments characterized by structural or functional attributes such as fragments that comp ⁇ se alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-formmg regions, coil and coil-formmg regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate bmdmg region, and high antigenic mdex regions
  • fragments mclude 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 NO:2, or an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2
  • Fragments of the polypeptides of the mvention may be employed for producing the co ⁇ esponding full-length polypeptide by peptide synthesis, therefore, these va ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the mvention Polynucleotides It is an object of the mvention to provide polynucleotides that encode galR polypeptides, particularly polynucleotides that encode a polypeptide herem designated galR
  • the polynucleotide comp ⁇ ses a region encodmg galR polypeptides comp ⁇ smg a sequence set out in Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a va ⁇ ant thereof
  • This mvention provides that this full length gene is essential to the growth and/or survival of an organism that possesses it, such as Streptococcus pneumomae
  • isolated nucleic acid molecules encoding and/or expressmg galR polypeptides and polynucleotides, particularly Streptococcus pneumomae galR polypeptides and polynucleotides, including, for example, unprocessed RNAs, ⁇ bozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides. and va ⁇ ants thereof, and compositions comp ⁇ smg the same
  • Another aspect of the mvention relates to isolated polynucleotides, including at least one full length gene, that encodes a galR polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and va ⁇ ants thereof
  • galR polypeptide from Streptococcus pneumomae comp ⁇ smg or consistmg of an ammo acid sequence of Table 1 [SEQ ID NO 2]. or a va ⁇ ant thereof Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO 2].
  • a polynucleotide of the mvention encodmg galR polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacte ⁇ a usmg Streptococcus pneumomae 0100993 cells as starting mate ⁇ al, followed by obtaining a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given m Table 1 [SEQ ID NO 1].
  • a library of clones of chromosomal DNA of Streptococcus pneumomae 0100993 m E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, de ⁇ ved from a partial sequence
  • Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent hyb ⁇ dization conditions
  • sequencmg the individual clones thus identified by hybndization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Convemently, such sequencmg is performed, for example, usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descnbed by Maniatis, T , F ⁇ tsch, E F and Sambrook et al , MOLE
  • the present mvention 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 NO 1 over the entire length of SEQ ID NO 1, or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (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 am o acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • a polynucleotide encodmg a polypeptide of the present mvention may be obtained by a process that comp ⁇ ses the steps of screening an approp ⁇ ate library under stringent hyb ⁇ dization conditions with a labeled or detectable probe consisting of or compnsing the sequence of SEQ ID NO 1 or a fragment thereof, and isolatmg a full-length gene and/or genomic clones compnsing said polynucleotide sequence
  • the mvention provides a polynucleotide sequence identical over its entire length to a codmg sequence (open readmg frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature polypeptide or a fragment in readmg frame with another codmg sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence
  • the polynucleotide of the mvention 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-mdependent termination signals), ⁇ bosome bmdmg sites.
  • the polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids.
  • a marker sequence that facilitates purification of a fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc. Natl. Acad.
  • Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression
  • a preferred embodiment of the invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 1006 set forth in SEQ ID NO:l of Table 1, both of that encode a galR polypeptide.
  • the invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:
  • R 1 X-(R 1 ) m -(R 2 )-(R 3 ) n -Y
  • X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond
  • Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond
  • each occurrence of R j 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
  • 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.
  • R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to R ⁇ and its 3' end nucleic acid residue is at the right, bound to R3.
  • Any stretch of nucleic acid residues denoted by either R j and/or R 2 , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • the polynucleotide of the above formula is a closed, circular polynucleotide, that can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary.
  • m and/or n is an integer between 1 and 1000.
  • Other prefe ⁇ ed embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polynucleotide of the invention is derived from Streptococcus pneumomae, 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.
  • polynucleotide encodmg a polypeptide encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention, particularly a bacte ⁇ al polypeptide and more particularly a polypeptide of the Streptococcus pneumomae galR havmg an ammo acid sequence set out in Table 1 [SEQ ID NO 2]
  • the term also encompasses polynucleotides that mclude a smgle 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 comp ⁇ se coding and/or non-coding sequences
  • the mvention further relates to vanants of the polynucleotides descnbed herem that encode vanants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention
  • prefe ⁇ ed embodiments are polynucleotides encoding galR va ⁇ ants, that have the ammo acid sequence of galR polypeptide of Table 1 [SEQ ID NO 2] m which several, a few, 5 to 10, 1 to 5. 1 to 3, 2, 1 or no ammo acid residues are substituted, modified, deleted and/or added, m any combination
  • Prefe ⁇ ed isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO:l, 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 NO: l
  • polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encodmg galR polypeptide havmg an ammo acid sequence set out in Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides
  • Most highly prefe ⁇ ed are polynucleotides that comp ⁇ se a region that is at least 95 % are especially prefe ⁇ ed
  • those with at least 97%> are highly prefe ⁇ ed 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% bemg the more prefe ⁇ ed
  • Prefe ⁇ ed embodiments are polynucleotides encodmg polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1]
  • the mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem
  • the mvention especially relates to polynucleotides that hyb ⁇ dize under stringent conditions to the polynucleotides descnbed herem
  • a specific example of stringent hybridization conditions is overnight mcubation at 42°C in a solution compnsing 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.
  • the mvention also provides a polynucleotide consisting of or compnsmg a polynucleotide sequence obtamed by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under st ⁇ ngent hybridization conditions with a probe havmg the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolatmg said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and pnmers fully descnbed elsewhere herein As discussed elsewhere herem regarding polynucleotide assays of the mvention, for instance, the polynucleotides of the mvention, may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encodmg galR and to
  • a codmg region of a galR gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to
  • polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relatmg to polynucleotide assays
  • polynucleotides of the invention that are ohgonucleotides derived from a sequence of Table
  • SEQ ID NOS 1 or 2 may be used in the processes herem as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or m part are transcnbed in bactena m infected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-terminal am o acids, or am o acids te ⁇ or to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance)
  • Such sequences may pla a role m processing of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other thmgs
  • the additional amm may pla a role
  • a precursor protem, havmg 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 As will be recognized, the entire polypeptide encoded by an open reading frame is often not required for activity Accordingly, it has become routme m molecular biology to map the boundanes of the primary structure required for activity with N-terminal and C-terminal deletion experiments These experiments utilize exonuclease digestion or convenient restnction sites to cleave codmg nucleic acid sequence For example, Promega (Madidi), Promega (Madi)
  • any contiguous fragment of SEQ ID NO 2 which retams at least 20%, preferably at least 50%, of an activity of the polypeptide encoded by the gene for SEQ ID NO 2 is withm the invention, as are corresponding fragment which are 70%, 80%, 90%, 95%,97%, 98% or 99% identical to such contiguous fragments
  • the contiguous fragment compnses at least 70% of the amino acid residues of SEQ ID NO 2, preferably at least 80%. 90% or 95% of the residues
  • a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (that may be refe ⁇ ed to as a preprotein), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem. or a preproprotem, that is a precursor to a proprotem, having a leader sequence and one or more prosequences. that generally are removed during processing steps that produce active and mature forms of the polypeptide
  • the mvention also relates to vectors that comp ⁇ se a polynucleotide or polynucleotides of the mvention, host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such protems usmg RNAs denved from the DNA constructs of the mvention
  • Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells comp ⁇ smg expression systems Accordingly, m a further aspect, the present mvention relates to expression systems that comp ⁇ se a polynucleotide or polynucleotides of the present mvention, to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention
  • Introduction of a polynucleotide mto the host cell can be effected by methods descnbed m 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, rmcromjection. cationic hpid-mediated transfection, electroporation, transduction. scrape loading, ballistic introduction and infection
  • bactenal cells such as cells of streptococci, staphylococci, enterococci E colt, streptomyces, cyanobacte ⁇ a, Bacillus subtihs, and Streptococcus pneumomae
  • fungal cells such as cells of a yeast, Kluveromyces . Saccharomyces. a basidiomycete.
  • Candida albwans and Aspergillus insect cells such as cells of Drosoph a S2 and Spodoptera Sf9, animal cells such as CHO. COS, HeLa, C127. 3T3. BHK. 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angiosperm
  • vectors include, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors de ⁇ ved from bactenal plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes.
  • the expression system constructs may compnse control regions that regulate as well as engender expression
  • any system or vector suitable to mamtam, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard
  • the appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routme techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABOR
  • appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectm chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication
  • This mvention is also related to the use of galR polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of galR polynucleotides and/or polypeptides m 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 orgamsm to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an orgamsm compnsmg the galR gene or protein, may be detected at the nucleic acid or ammo acid level by a vanety of well known techniques as well as by methods provided herem
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily matenals
  • Polynucleotides from any of these sources, particularly DNA or RNA may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique pnor to analysis RNA.
  • mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characterization of the species and strain of infectious or resident organism present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the orgamsm Deletions and insertions can be detected by a change m size of the amplified product in companson to a genotype of a reference sequence selected from a related orgamsm.
  • Po t mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled galR polynucleotide sequences Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences m melting temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected by alterations m the electrophoretic mobility of polynucleotide fragments m gels as compared to a reference sequence This may be earned out with or without denaturing agents Polynucleotide differences may also be detected by direct DNA or RNA sequencmg See.
  • Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase. VI and SI protection assay or a chemical cleavage method See, for example, Cotton et al . Proc Natl Acad Set , USA, 85 4397-4401 (1985)
  • an array of ohgonucleotides probes comp ⁇ smg galR 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 m molecular genetics including gene expression, genetic linkage, and genetic vanabihty (see, for example, Chee et al , Science, 274 610 (1996))
  • the present invention relates to a diagnostic kit that compnses (a) a polynucleotide of the present mvention, preferably the nucleotide sequence of SEQ ID NO 1, 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 NO 2 or a fragment thereof, or (d) an antibody to a polypeptide of
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1. that is associated with a disease or pathogemcity 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
  • Orgamsms, particularly infectious orgamsms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techmques, such as those descnbed elsewhere herem
  • a polynucleotide and/or polypeptide sequence between orgamsms possessmg a first phenotype and orgamsms possessmg a different, second different phenotype can also be determmed If a mutation is observed m some or all orgamsms possessmg the first phenotype but not m any organisms possessmg the second phenotype, then the mutation is likely to be the causative agent of the first phenotype
  • a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techmques. to allow for serotypmg, for example
  • RT-PCR can be used to detect mutations in the RNA It is particularly prefe ⁇ ed to use RT-PCR m conjunction with automated detection systems, such as. for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose.
  • PCR primers complementary to a polynucleotide encodmg galR polypeptide can be used to identify and analyze mutations
  • the mvention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end
  • These primers may be used for, among other thmgs, amphfying galR DNA and/or RNA isolated from a sample de ⁇ ved from an individual, such as a bodily mate ⁇ al
  • the primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to vanous techmques for elucidation of the polynucleotide sequence In this way. mutations m the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections caused by Streptococcus pneumomae, comp ⁇ smg determimng from a sample de ⁇ ved from an mdividual, such as a bodily matenal, an increased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1]
  • Increased or decreased expression of a galR polynucleotide can be measured using any on of the methods well known m the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybndization methods
  • a diagnostic assay m accordance with the mvention for detecting over-expression of galR polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to
  • Polypeptides and polynucleotides of the mvention may also be used to assess the bmdmg of small molecule substrates and hgands in, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures
  • substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Co gan et al , Current Protocols in Immunology 1 (2) Chapter 5 (1991)
  • Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, including many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agomze (e g , stimulate) or that antagonize (e g .inhibit) the function of the polypeptide or polynucleotide Accordmgly, m a further aspect, the present mvention provides for a method of screening compounds to identify those that agomze or that antagonize the function of a polypeptide or polynucleotide of the mvention, as well as related polypeptides and polynucleotides In general, agomsts or antagomsts (e g , inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agom
  • the screenmg methods may simply measure the bmdmg of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protem of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screenmg method may mvolve competition with a labeled competitor Further, these screemng methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide or polynucleotide, usmg detection systems appropnate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed m the presence of a
  • polypeptides and antibodies that bmd to and/or mteract with a polypeptide of the present mvention may also be used to configure screenmg methods for detectmg the effect of added compounds on the production of mRNA and/or polypeptide m cells
  • an ELISA assay may be constructed for measurmg secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agomst, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screemng compounds to identify those that enhance (agomst) or block (antagonist) the action of galR polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bacte ⁇ cidal
  • the method of screemng may mvolve high-throughput techmques For example, to screen for agomsts or antagomsts. a synthetic reaction mix.
  • a cellular compartment such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg galR polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a galR agomst or antagonist
  • the ability of the candidate molecule to agomze or antagonize the galR polypeptide is reflected in decreased bmdmg of the labeled hgand or decreased production of product from such substrate Molecules that bind gratuitously, i e , without mducmg the effects of galR polypeptide are most likely to be good antagomsts Molecules that b d well and, as the case may be, mcrease the rate of product production from substrate, mcrease signal transduction, or mcrease chemical channel activity are agomsts Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction.
  • a reporter system Reporter systems that may be useful in this regard mclude but are not limited to colo ⁇ metnc. labeled substrate converted mto product, a reporter gene that is responsive to changes in galR polynucleotide or polypeptide activity, and bmdmg assays known in the art Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known m the art These techmques mclude, but are not limited to, hgand bmdmg and crosshnking assays in which the polypeptide is labeled with a radioactive isotope (for instance.
  • ⁇ *I chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or punfication, and mcubated with a source of the putative receptor (e g . cells, cell membranes, cell supematants, tissue extracts, bodily matenals)
  • a source of the putative receptor e g . cells, cell membranes, cell supematants, tissue extracts, bodily matenals
  • Other methods include biophysical techmques such as surface plasmon resonance and spectroscopy
  • These screemng methods may also be used to identify agomsts and antagomsts of the polypeptide that compete with the bmdmg of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood m the art
  • the fluorescence polarization value for a fluorescenfly-tagged molecule depends on the rotational correlation time or tumbling rate
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of galR polypeptide dimers, tamers, tetramers or higher order structures, or structures formed by galR polypeptide bound to another polypeptide
  • GalR polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixmg 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 dime ⁇ zation will inhibit fluorescence energy transfer
  • GalR polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protem can then passed over the galR polypeptide -coated surface and specific bmdmg can be detected in real-time by momtonng the change m resonance angle caused by a change in local refractive mdex This technique can be used to charactenze the effect of small molecules on kinetic rates and equihbnum bmdmg constants for galR polypeptide self-association as well as an association of galR polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to charactenze the mteraction between an association of galR polypeptide with another galR polypeptide or a different polypeptide
  • GalR polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled galR polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid
  • signal is emitted upon galR polypeptide binding and compounds that prevent galR polypeptide self-association or an association of galR polypeptide and another polypeptide or small molecule will dimmish signal
  • identifying compounds that bind to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention comp ⁇ smg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably being associated with a second component capable of providing a detectable signal in response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determimng whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by
  • an assay for galR agomsts is a competitive assay that combmes GalR and a potential agomst with galR-binding molecules, recombinant galR bmdmg molecules, natural substrates or hgands.
  • GalR can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of galR molecules bound to a bmdmg molecule or converted to product can be determmed accurately to assess the effectiveness of the potential antagonist
  • a polypeptide and/or polynucleotide of the present mvention may also be used in a method for the structure-based design of an agomst or antagonist of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three- dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducmg the three-dimensional structure for the likely reactive s ⁇ te(s), bmdmg s ⁇ te(s) or mot ⁇ f(s) of an agomst or antagonist, (c) synthesizing candidate
  • testmg whether the candidate compounds are indeed agonists or antagonists It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps
  • the present mvention provides methods of treatmg abnormal conditions such as for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of. or a decreased activity of galR polypeptide and/or polynucleotide
  • expression of the gene encodmg endogenous galR polypeptide can be inhibited usmg expression blockmg techniques
  • This blocking may be targeted against any step in gene expression, but is preferably targeted agamst transcription and/or translation
  • An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988))
  • ohgonucleotides that form triple helices with the gene can be supplied (see.
  • each of the polynucleotide sequences provided herem may be used m the discovery and development of antibactenal compounds
  • the encoded protem upon expression, can be used as a target for the screemng of antibactenal drugs
  • the polynucleotide sequences encodmg the ammo terminal regions of the encoded protem 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 codmg sequence of mterest
  • the invention also provides the use of the polypeptide, polynucleotide, agomst or antagonist of the mvention 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 m the prevention of adhesion of bacteria, m particular gram positive and/or gram negative bacteria, to eukaryotic.
  • galR agomsts and antagomsts preferably bactenstatic or bactencidal agomsts and antagomsts
  • the antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases
  • Antagomsts of the mvention mclude, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the mvention and thereby inhibit or extinguish its activity or expression
  • Antagomsts also may be small organic molecules, a peptide.
  • a polypeptide such as a closely related protem or antibody that binds the same sites on a bmdmg molecule, such as a bmdmg molecule, without inducing galR-induced activities, thereby preventing the action or expression of galR polypeptides and/or polynucleotides by excluding galR polypeptides and/or polynucleotides from bmdmg
  • Antagomsts of the mvention also mclude a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventing bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small organic molecules.
  • Other antagomsts mclude antisense molecules (see Okano. J Neurochem 56 560 (1991). OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION.
  • antagomsts mclude compounds related to and va ⁇ ants of galR
  • polypeptide antagomsts mclude antibodies or, m some cases, ohgonucleotides or proteins that are closely related to the hgands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide. e g . a fragment of the hgands, substrates, receptors, enzymes, etc , or small molecules that bind to the polypeptide of the present mvention but do not ehcit a response, so that the activity of the polypeptide is prevented
  • Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons It is prefened that these small molecules are organic molecules
  • H pylori Helicobacter pylori bacteria infect the stomachs of over one-third of the world's population causmg 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/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastnc adenocarcmoma.
  • Prefened antmucrobial compounds of the invention (agomsts and antagomsts of galR polypeptides and/or polynucleotides) found using screens provided by the mvention, or known m the art, particularly narrow-spectrum antibiotics, should be useful m the treatment of H pylori mfection Such treatment should decrease the advent of H /ry/ ⁇ -induced cancers, such as gastromtestmal carcinoma Such treatment should also prevent, inhibit and/or cure gastnc ulcers and gastntis
  • Bodily mate ⁇ al(s) means any matenal denved from an mdividual or from an orgamsm infecting, infesting or inhabiting an mdividual, 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 mate ⁇ als
  • D ⁇ sease(s) means any disease caused by or related to infection by a bacte ⁇ a, 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
  • “Host cell(s)” is a cell that has been mtroduced (e g . transformed or transfected) or is capable of mtroduction (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 determmed 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 descnbed 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 mclude, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S F et al , J o/ec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al .
  • polynucleotides and polypeptides are provided in (1) and (2) below
  • Polynucleotide embodiments further mclude an isolated polynucleotide compnsmg a polvnucleotide sequence having at least a 95. 97 or 100% identity to the reference sequence of SEQ ID NO 1.
  • said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or msertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleotides m the reference sequence or m one or more contiguous groups withm the reference sequence, and wherem said number of nucleotide alterations is determmed by multiplying the total number of nucleotides m SEQ ID NO 1 by the integer definmg the percent identity divided by 100 and then subtractmg that product from said total number of nucleotides in SEQ ID NO 1. or
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides m SEQ ID NO 1
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator
  • any non-mteger product of x n and y is rounded down to the nearest mteger prior to subtractmg it from x n
  • Alterations of a polynucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Polypeptide embodiments further include an isolated polypeptide compnsing a polypeptide having at least a 95. 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain mteger number of ammo acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or msertion, and wherem said alterations may occur at the ammo- or carboxy-terrninal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups withm the reference sequence, and wherem said number of ammo acid alterations is determmed by multiplying the total number of ammo acids m SEQ ID NO 2 by the m
  • n a is the number of ammo acid alterations
  • x a is the total number of amino acids in SEQ ID NO 2
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest integer prior to subtractmg it from x a
  • Ind ⁇ v ⁇ dual(s) means a multicellular eukaryote, mcludmg, 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, / e , if it occurs m nature, it has been changed or removed from its o ⁇ ginal environment, or both
  • a polynucleotide or a polypeptide naturally present m a living orgamsm is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting mate ⁇ als of its natural state is “isolated”, as the term is employed herem
  • a polynucleotide or polypeptide that is introduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said orgamsm, which orgamsm may be living or non-living
  • Organ ⁇ sm(s) means a (l) prokaryote, mcludmg but not limited to, a member of the genus
  • Streptococcus Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Neissena, Haemoph ⁇ us, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersima, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschench a, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borreha, Leptospira, Spinllum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borreha andMycoplasma, and further mcludmg,
  • mcludmg but not limited to. a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lactis, or Candida albicans
  • Polynucleotide(s) generally refers to any polynbonucleotide or polydeoxy ⁇ bonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)” mclude. without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or single-, double- and tnple-stranded regions, smgle- and double-stranded RNA.
  • polynucleotide refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands m such regions may be from the same molecule or from different molecules The regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules One of the molecules of a t ⁇ ple-hehcal region often is an oligonucleotide As used herem, the term "polynucleotide(s)" also mcludes DNAs or RNAs as descnbed above that compnse one or more modified bases Thus, DNAs
  • amidation covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative. covalent attachment of a pid or hpid denvative. covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation.
  • GPI anchor formation hydroxylation, lodination, methylation, my ⁇ stoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins, such as argmylation.
  • Polypeptides may be branched or cychc. with or without branching Cyclic, branched and branched circular polypeptides may result from posttranslational 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 mvention introduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention
  • Va ⁇ ant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical va ⁇ ant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusion protems and truncations m the polypeptide encoded by the reference sequence, as discussed below
  • a typical vanant of a polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, m many regions, identical A vanant and reference polypeptide may differ m am
  • va ⁇ ants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combination
  • a vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic vanant, or it may be a vanant that is not known to occur naturally
  • Non-naturally occurnng vanants of polynucleotides and polypeptides may be made by mutagenesis techmques, by direct synthesis, and by other recombinant methods known to skilled artisans
  • Example 1 Strain selection, Library Production and Sequencing The polynucleotide having a DNA sequence given m Table 1 [SEQ ID NO 1] was obtamed from a library of clones of chromosomal DNA of Streptococcus pneumomae in E coli The sequencmg data from two or more clones compnsmg overlappmg Streptococcus pneumomae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared by routme methods, for example Methods 1 and 2 below
  • Total cellular DNA is isolated from Streptococcus pneumomae 0100993 according to standard procedures and size-fractionated by either of two methods
  • Method 1 Total cellular DNA is mechanically sheared by passage through a needle m order to size- fractionate accordmg to standard procedures
  • DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coh infected with the packaged library
  • the library is amplified by standard procedures
  • Total cellular DNA is partially hydrolyzed with a one or a combmation of restnction enzymes appropnate to generate a senes of fragments for cloning into library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coh infected with the packaged library
  • the library is amplified by standard procedures

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Abstract

L'invention concerne des polypeptides ga1R, des polynucléotides codant lesdits polypeptides ga1R et des méthodes de production de ces polypeptides par des techniques de recombinaison. L'invention concerne également des méthodes d'utilisation des polypeptides ga1R pour le criblage de composés antibactériens.
PCT/US2000/019529 1999-07-22 2000-07-18 Ga1R Ceased WO2001007465A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5096815A (en) * 1989-01-06 1992-03-17 Protein Engineering Corporation Generation and selection of novel dna-binding proteins and polypeptides
US5476929A (en) * 1991-02-15 1995-12-19 Uab Research Foundation Structural gene of pneumococcal protein
US5565204A (en) * 1994-08-24 1996-10-15 American Cyanamid Company Pneumococcal polysaccharide-recombinant pneumolysin conjugate vaccines for immunization against pneumococcal infections

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5096815A (en) * 1989-01-06 1992-03-17 Protein Engineering Corporation Generation and selection of novel dna-binding proteins and polypeptides
US5476929A (en) * 1991-02-15 1995-12-19 Uab Research Foundation Structural gene of pneumococcal protein
US5565204A (en) * 1994-08-24 1996-10-15 American Cyanamid Company Pneumococcal polysaccharide-recombinant pneumolysin conjugate vaccines for immunization against pneumococcal infections

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