WO2001008486A1 - POLYPEPTIDES ET POYLNUCLEOTIDES CoaA ET PROCEDES ASSOCIES - Google Patents

POLYPEPTIDES ET POYLNUCLEOTIDES CoaA ET PROCEDES ASSOCIES Download PDF

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Publication number
WO2001008486A1
WO2001008486A1 PCT/US2000/020744 US0020744W WO0108486A1 WO 2001008486 A1 WO2001008486 A1 WO 2001008486A1 US 0020744 W US0020744 W US 0020744W WO 0108486 A1 WO0108486 A1 WO 0108486A1
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Prior art keywords
polypeptide
seq
polynucleotide
sequence
isolated
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PCT/US2000/020744
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English (en)
Inventor
John P. Throup
Stephanie Van Horn
Richard L. Warren
David J. Holmes
Alison F. Chalker
Chi Young So
Karen A. Ingraham
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Smithkline Beecham Corporation
Smithkline Beecham Plc
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Publication of WO2001008486A1 publication Critical patent/WO2001008486A1/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
    • C07K14/315—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses
  • the invention relates to polynucleotides and polypeptides of the coaA family, as well as their vanants. herein referred to as "coaA.”
  • coaA polynucleot ⁇ de(s)," and “coaA polypept ⁇ de(s) as the case may be
  • Streptococci make up a medically important genera of microbes known to cause several tvpes of disease in humans, including, for example, otrtis 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 pneumoniae has been one of the more intensively studied microbes For example, much of our early understanding that DNA is.
  • the present invention relates to coaA, in particular coaA polypeptides and coaA 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 invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds.
  • the mvention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting coaA expression or activity.
  • the invention relates to coaA polypeptides and polynucleotides as described in greater detail below.
  • the invention relates to polypeptides and polynucleotides of a coaA of Streptococcus pneumoniae. that is related by amino acid sequence homology to coaA Streptomyces coelicolor polypeptide
  • the invention relates especially to coaA having a nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO:l and SEQ ID NO:2 respectively.
  • sequences recited in the Sequence Listing below as "DNA” represent an exemplification of the mvention. since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides
  • 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 IRY, Scotland on 11 April 1996 and assigned deposit number 40794. The deposit was described as Streptococcus pneumoniae 0100993 on deposit. On 17 April 1996 a Streptococcus pneumoniae 0100993 DNA library in E coli was similarly deposited with the NCIMB and assigned deposit number 40800 The Streptococcus pneumoniae strain deposit is referred to herein as "the deposited stram” or as "the DNA of the deposited strain "
  • the deposited strain compnses a full length coaA gene
  • the sequence of the polynucleotides compnsed in the deposited strain, as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling m the event of any conflict with any descnption of sequences herein
  • 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 stram will be irrevocably and without restnction or condition released to the public upon the issuance of a patent
  • the deposited stram 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 ⁇ 112
  • a license may be required to make, use or sell the deposited stram, and compounds denved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 stram, which polypeptide is compnsed in the deposited stram
  • coaA polynucleotide sequences m the deposited strain such as DNA and RNA
  • ammo acid sequences encoded thereby Also provided by the mvention are coaA polypeptide and polynucleotide sequences isolated from the deposited stram Polypeptides
  • CoaA polypeptide of the mvention is substantially phylogenetically related to other proteins of the coaA family
  • coaA polypeptides of Streptococcus pneumoniae referred to herem as "coaA” and “coaA polypeptides” as well as biologically, diagnostically.
  • the present mvention 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 NO 2 over the entire length of SEQ ID NO 2, (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 NO 1 over the entire length of SEQ ID NO 1.
  • 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 ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • polypeptides of the mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (m particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of coaA, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally compnsing at least 30 ammo acids and more preferably at least 50 ammo acids
  • the mvention also includes a polypeptide consisting of or compnsing a polypeptide of the formula
  • X-(Rl)m-(R2)-(R3)n-Y wherem, at the ammo termmus, X is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, and at the carboxyl termmus, Y is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, Ri 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 mteger between 1 and 1000 or zero, and R2 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, R2 is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to K ⁇ and its carboxy terminal ammo acid residue is at the nght, covalently bound to R3 Any stretch of ammo acid residues denoted by either R j or R3, where m and or
  • n is an mteger between 1 and 50, 100, or 500 It is most preferred that a polypeptide of the mvention is denved from Streptococcus pneumoniae 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 vanant 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-standmg,” or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle contmuous region m a smgle larger polypeptide
  • Preferred fragments m include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a contmuous senes of residues that includes an ammo- and/or carboxyl-terminal ammo acid sequence
  • Degradation forms of the polypeptides of the mvention produced by or m a host cell, particularly a Streptococcus pneumoniae are also preferred
  • fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-for mg regions, coil and coil-formmg regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic mdex 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 NO.2, or an isolated polypeptide comprising an amino acid sequence havmg at least 15,
  • Fragments of the polypeptides of the mvention may be employed for produc g the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the full-length polypeptides of the mvention Polynucleotides It is an object of the mvention to provide polynucleotides that encode coaA polypeptides, particularly polynucleotides that encode a polypeptide herem designated coaA
  • the polynucleotide compnses a region encoding coaA polypeptides compnsmg a sequence set out m 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 pneumoniae
  • isolated nucleic acid molecules encoding and/or expressmg coaA polypeptides and polynucleotides, particularly Streptococcus pneumoniae coaA polypeptides and polynucleotides, mcludmg, for example, unprocessed RNAs. nbozyme 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 vanants thereof, and compositions compnsmg the same
  • Another aspect of the mvention relates to isolated polynucleotides, mcludmg at least one full length gene, that encodes a coaA polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof
  • coaA polypeptide from Streptococcus pneumoniae comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof Usmg the information provided herem, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 2]
  • a polynucleotide of the mvention encoding coaA polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bactena usmg Streptococcus pneumoniae 0100993 cells as starting matenal, 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 pneumoniae 0100993 m E coh or some other smtable host is probed with a radiolabeled oligonucleotide, 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 hybndization conditions
  • a polynucleotide encoding a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than Streptococcus pneumoniae may be obtamed by a process that compnses the steps of screening an appropnate library under stringent hybndization conditions with a labeled or detectable probe consisting of or compnsmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones compnsmg said polynucleotide sequence
  • the mvention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading 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 coding sequence for a mature polypeptide or a fragment m 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 mvention may also compnse at least one non-coding sequence, mcludmg for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcnbed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termination signals), nbosome binding sites, Kozak sequences, sequences that stabilize mRNA, mtrons, and polyadeny
  • a preferred embodiment of the mvention is a polynucleotide of consisting of or compnsmg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 949 set forth m SEQ ID NO 1 of Table 1, both of that encode a coaA polypeptide
  • the mvention also mcludes a polynucleotide consisting of or compnsmg a polynucleotide of the formula
  • X-(R 1 ) m -(R 2 )-(R3) n -Y wherem, 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 Rj 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 In the polynucleotide formula above.
  • R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the ⁇ ght, bound to R3 Any stretch of nucleic acid residues denoted by either Rj and/or R 2 .
  • n 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 wherem 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 preferred embodiments of the mvention are provided where m is an mteger between 1 and 50. 100 or 500. and n is an mteger between 1 and 50, 100.
  • a polynucleotide of the mvention is de ⁇ ved from Streptococcus pneumoniae, however, it may preferably be obtamed from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtamed, for example, from organisms of the same taxonomic family or order
  • polynucleotide encoding 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 pneumoniae coaA havmg an ammo acid sequence set out m 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 havmg 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
  • polynucleotides encoding coaA vanants that have the ammo acid sequence of coaA polypeptide of Table 1 [SEQ ID NO 2] m which several, a few, 5 to 10, 1 to 5,
  • Preferred isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuchc acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO 1 , or an polynucleotide comprising a nucleic acid sequence havmg 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 ot SEQ ID NO 1
  • polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding coaA polypeptide havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides
  • polynucleotides that comp ⁇ se 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 NO 1]
  • polynucleotides that hyb ⁇ dize, particularly under stringent conditions, to coaA polynucleotide sequences such as those polynucleotides m Table 1
  • the mvention further relates to polynucleotides that hyb ⁇ dize to the polynucleotide sequences provided herem
  • the mvention especially relates to polynucleotides that hyb ⁇ dize under stringent conditions to the polynucleotides descnbed herem
  • strmgent hybridization conditions is overnight mcubation at 42°C m a solution compnsmg 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 support m 0 lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified m Sambrook, et al . Molecular Clomng A Laboratory Manual. Second Edition
  • the invention also provides a polynucleotide consisting of or compnsing a polynucleotide sequence obtained by screening an appropnate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under stringent hybridization conditions with a probe ing tin- sequence of said polynucleotide sequence set forth m SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully descnbed elsewhere herem
  • RNA may be used as a hybndization probe for RNA.
  • cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding coaA and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a coaA 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
  • Particularh prefened 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 coaA gene may be isolated by screening usmg a DNA sequence provided m
  • polynucleotides of the invention that are oligonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used m the processes herein as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herem 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 mvention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional ammo or carboxyl-terminal ammo acids, or ammo acids mte ⁇ or to a mature pohpep ⁇ de (when a mature form has more than one polypeptide cham, for instance) Such sequences may play a role m processmg 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 things As generally is the case in vivo, the additional ammo acids may be processed away from a mature protem by cellular enzymes For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to it It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary
  • a precursor protem. havmg a mature form of the polypeptide fused to one or more prosequences may be an mactive 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
  • 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
  • Nucleic acid sequences encoding such fragments of SEQ ID NO 2 and vanants thereof as descnbed herem are within the mvention, as are polypeptides so encoded
  • anv contiguous fragment of SEQ ID NO 2 which retains at least 20%, preferably at least 50%. of an activity of the polypeptide encoded by the gene for SEQ ID NO 2 is within the invention, as are correspondmg fragment which are 70%, 80%, 90%, 95% 97%, 98% or 99% identical to such contiguous fragments
  • the contiguous fragment comprises 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 refened to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotein, that is a precursor to a proprotem, havmg a leader sequence and one or more prosequences, that generally are removed du ⁇ ng processmg 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 engmeered with vectors of die mvention and die production of polypeptides of the mvention by recombmant techniques Cell-free translation systems can also be emploved to produce such protems usmg RNAs de ⁇ ved from the DNA constructs of the mvention
  • Recombmant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compnsmg 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 recombmant techmques
  • 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 .
  • bactenal cells such as cells of streptococci, staphylococci, enterococci E coh. streptomyces, cyanobacte ⁇ a.
  • Bacillus subtihs. and Streptococcus pneumoniae fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus, insect cells such as cells of Drosophila 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
  • a great va ⁇ ety of expression systems can be used to produce the polypeptides of die mvention
  • Such vectors mclude, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors de ⁇ ved from bacte ⁇ al plasmids, from bactenophage.
  • the expression system constructs may compnse control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, 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
  • a translated protem mto die lumen of the endoplasmic reticulum, mto the pe ⁇ plasmic space or mto the extracellular environment 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 recombmant cell cultures by well- known methods mcludmg 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 purification
  • Well known techmques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and or purification
  • This mvention is also related to the use of coaA polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of coaA 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 organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the coaA gene or protein, may be detected at the nucleic acid or ammo acid level by a vanety of well known techmques as well as by methods provided herem
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtamed 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 p ⁇ or to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characterization of the species and stram of infectious or resident organism present m an mdividual, may be made by an analysis of die genotype of a selected polynucleotide of the organism Deletions and insertions can be detected by a change in size of die amplified product m companson to a genotype of a reference sequence selected from a related organism preferably a different species of the same genus or a different stram of the same species Po
  • an anay of oligonucleotides probes comprising coaA nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification.
  • Anay technology methods are well known and have general apphcability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al., Science, 274: 610 (1996)).
  • the present invention relates to a diagnostic kit that comprises: (a) a polynucleotide of the present invention, 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 N0:2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2. 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, preferable. SEQ ID NO: 1. tiiat 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 variety of techniques, such as those described elsewhere herein.
  • the differences in a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined. If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype.
  • Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techmques, to allow for serotyping.
  • RT-PCR can be used to detect mutations in the RNA. It is particularly prefened to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan.
  • RNA, cDNA or genomic DNA may also be used for the same purpose, PCR.
  • PCR primers complementary to a polynucleotide encoding coaA polypeptide can be used to identify and analyze mutations.
  • the invention 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 things, amplifying coaA DNA and or RNA isolated from a sample derived from an individual, such as a bodily material.
  • 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 bacterial mfections, more preferably infections caused by Streptococcus pneumoniae, compnsmg determinmg from a sample denved from an individual, such as a bodily material, an increased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a coaA 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 mvention for detecting over-expression of coaA 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 determine levels of a coaA polypeptide.
  • Assay techmques that can be used to determine levels of a coaA polypeptide.
  • a sample denved from a host such as a bodily matenal
  • Such assay methods mclude radioimmunoassays, competitive-bmdmg assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays
  • Polypeptides and polynucleotides of the mvention may also be used to assess the bindmg of small molecule substrates and ligands m, for example, cells, cell-free preparations, chemical libra ⁇ es. and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g . Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
  • Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcludmg 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 tiiat antagonize
  • the present mvention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the mvention, as well as related polypeptides and polynucleotides
  • agonists or antagonists e g .
  • inhibitors may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned
  • Compounds may be identified from a va ⁇ etv of sources, for example, cells, cell-free preparations, chemical branes, and natural product mixtures
  • Such agonists and antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes etc , as the case may be. of coaA polypeptides and polynucleotides.
  • 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.
  • the screening method may involve competition with a labeled competitor.
  • 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 comprising 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 polynucleotide, as the case may be.
  • the screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide of the present invention, to form a mixture, measuring coaA polypeptide and/or polynucleotide activity in the mixture, and comparing the coaA polypeptide and/or polynucleotide activity of the mixture to a standard.
  • Fusion proteins such as those made from Fc portion and coaA polypeptide, as herein described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995): and K. Johanson et al, J Biol Chem, 270(16):9459-9471 (1995)).
  • 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 coaA polypeptides or polynucleotides, particularly those compounds that are bacteristatic and/or bactericidal.
  • the method of screening may involve high-throughput techniques. For example, to screen 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, comprising coaA polypeptide and a labeled substrate or ligand of such polypeptide is incubated in die absence or the presence of a candidate molecule that may be a coaA agonist or antagonist.
  • the ability of the candidate molecule to agonize or antagonize the coaA polypeptide is reflected m decreased bmdmg of the labeled hgand or decreased production of product from such substrate Molecules that bmd gratuitously, i e , without mducmg the effects of coaA polypeptide are most likely to be good antagonists Molecules that bmd 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, or chemical channel activity may be enhanced by usmg a reporter system Reporter systems that may be useful m this regard mclude but are not limited to colonmetnc, labeled substrate converted mto product, a reporter gene that is responsive to changes m coaA polynucleotide or polypeptide activity, and bmdmg
  • techmques mclude, but are not limited to, ligand binding and crosshnkmg 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.
  • a radioactive isotope for instance, ⁇ I
  • chemically modified for instance, biotinylated
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of coaA polypeptide dimers. t ⁇ mers. tetramers or higher order structures, or structures formed by coaA polypeptide bound to another polypeptide
  • CoaA 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 d me ⁇ zation will inhibit fluorescence energy transfer
  • CoaA polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monome ⁇ c Solution protem can then passed over the coaA polypeptide -coated surface and specific bmdmg can be detected in real-time by monitoring the change m resonance angle caused by a change m local refractive dex
  • This technique can be used to characterize the effect of small molecules on kmetic rates and equilibrium binding constants for coaA polypeptide self-association as well as an association of coaA polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to charactenze the interaction between an association of coaA polypeptide with another coaA polypeptide or a different polypeptide
  • CoaA polypeptide can be coupled to a scintillation-filled bead
  • Addition of radio-labeled coaA polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid
  • signal is emitted upon coaA polypeptide binding and compounds that prevent coaA polypeptide self-association or an association of coaA polypeptide and another polypeptide or small molecule will dimmish signal
  • bmdmg or mteraction preferably bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determinmg whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and
  • an assay for coaA agomsts is a competitive assay that combmes coaA and a potential agomst with coaA-binding molecules, recombmant coaA bmdmg molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay
  • CoaA can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of coaA molecules bound to a bmdmg 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 mvention may also be used m 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 polynucleot
  • the present mvention provides methods of treating 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 coaA polypeptide and/or polynucleotide
  • soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc in competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the coaA polypeptide and/or polypeptide
  • expression of the gene encoding endogenous coaA polypeptide can be inhibited using expression blocking techniques
  • This blocking may be targeted against any step m gene expression, but is preferably targeted against 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.
  • 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 o gomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • 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 encoding the ammo terminal regions of the encoded protem or Shme-Delgamo 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 mterest
  • the invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the mvention may be used m the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bactena, to eukaryotic, preferably mammalian, extracellular matrix proteins on m-dwellmg devices or to extracellular matnx proteins in wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial coaA proteins that mediate tissue damage and/or, to block the normal progression of pathogenesis in infections initiated other than by the implantation of m-dwellmg devices or by other surgical techniques
  • coaA agomsts and antagomsts coaA agomsts and antagoms
  • 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.
  • Antagomsts also may be small organic molecules a peptide a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule such as a bmdmg molecule, without mducmg coaA-induced activities, thereby preventing the action or expression of coaA polypeptides and/or polynucleotides by excluding coaA 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 preventmg bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small organic molecules peptides or peptide-hke molecules
  • Other antagomsts m clude antisense molecules (see Okano, J Neurochem 56 560 (1991), OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION CRC Press, Boca Raton, FL (1988), for a desc ⁇ ption of these molecules)
  • Preferred antagomsts m include compounds related to and vanants of coaA
  • polypeptide antagomsts include antibodies or, m some cases, oligonucleotides or proteins that are closely related to the ligands, substrates, receptor
  • Small molecules of the mvention 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
  • Hehcobacter pylori (herein "H pylori”) bactena mfect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Sch stosomes, Liver Flukes and Hehcobacter 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 gastric adenocarcinoma.
  • Preferred antimicrobial compounds of the mvention (agomsts and antagomsts of coaA polypeptides and/or polynucleotides) found using screens provided by the mvention, or known in the art, particularly narrow-spectrum antibiotics, should be useful m the treatment of H pylori infection Such treatment should decrease the advent of H pylori -induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis
  • Bodily mate ⁇ al(s) means any matenal denved from an mdividual or from an organism mfecting infesting or inhabiting an mdividual, mcludmg 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 matenals
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena, mcludmg , 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 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, mcludmg but not limited to those described in
  • 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, S . el al , J Mol Biol 215 403-410 (1990)
  • the well known Smith Waterman algorithm may also be used to determine identity Parameters for polypeptide sequence comparison mclude the following Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)
  • polynucleotide embodiments further mclude an isolated polynucleotide compnsmg a polynucleotide sequence havmg at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1 , wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, mcludmg 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
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in 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, and wherein any non-mteger 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 the polypeptide of SEQ ID NO 2 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 NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or:
  • n a is the number of amino 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 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 m 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 m 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, Corynebactenum, Mycobacte ⁇ um, Neissena, Haemophilia, Actmomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fanc sella, Pasturella, Moraxella, Acinetobacter, Erys ⁇ elothnx, Branhamella, Actinobacillus, Streptobacillus, L stena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamvdia, Borrelia and Mycoplasma, and further including
  • mcludmg but not limited to Archaehacter, and ( ) a unicellular or filamentous eukaryote, 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 lacts, or Candida albicans
  • Polynucleotide(s) generally refers to any poly ⁇ bonucleotide or polydeoxynbonucleotide, tiiat 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 smgle-, double- and t ⁇ ple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hyb ⁇ d molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or t ⁇ ple-stranded regions, or a mixture of smgle- and double-stranded regions
  • polynucleotide as used herem refers to t ⁇ ple-stranded
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleot ⁇ de(s)" as that term is intended herem
  • DNAs or RNAs compnsmg unusual bases, such as mosme. or modified bases, such as t ⁇ tylated bases, to name just two examples are polvnucleotidcs as the term is used herem
  • polynucleotide(s) as it is employed herem embraces such chemically, enzymatically or metabolically modified fonns of polynucleotides, as well as the chemical forms of DNA and RNA characte ⁇ stic of vinises and cells, mcludmg for example, simple and complex cells
  • Polynucleot ⁇ de(s) also embraces short polynucleotides often refened to
  • Modifications mclude. for example, acetylation. acylation, ADP- ⁇ bosylation. 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 lipid denvative, covalent attachment of phosphotidy nositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme.
  • Polypeptides may be branched or cyclic, witii 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
  • Recombmant expression system(s) refers to expression systems or portions tiiereof or polynucleotides of die mvention introduced or transformed mto a host cell or host cell lysate for die production of the polynucleotides and polypeptides of the mvention
  • Va ⁇ ant(s) " ' as die term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical vanant 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 amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusion protems 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 vanant are closely similar overall and, in many regions, identical
  • a vanant and reference polypeptide may differ in ammo acid
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombmant methods known to skilled artisans EXAMPLES
  • the polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E coh
  • the sequencmg data from two or more clones compnsing overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared routine methods, for example Methods 1 and 2 below
  • Total cellular DNA is mechanically sheared by passage through a needle m order to size- fractionate according to standard procedures
  • DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoR linkers added Fragments are gated 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
  • Method 2 Total cellular DNA is partially hydrolyzed with a one or a combmation of restriction enzymes appropnate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall. Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli mfected with the packaged library The library is amplified by standard procedures Example 2 CoaA Characterization
  • allelic replacement cassette was generated using PCR technology
  • the cassette consisted of a pair of 500bp chromosomal DNA fragments flanking an erythromycm resistance gene
  • the chromosomal DNA sequences are the 500bp preceding and followmg the DNA sequence encoding the NDP contained in Seq ID NO 1
  • the allelic replacement cassette was mtroduced mto S pneumoniae R6 by transformation Competent cells were prepared according to published protocols DNA was introduced into the cells by incubation of ng quantities of allelic replacement cassette with 106 cells at 30°C for 30 minutes The cells were transferred to 37°C for 90 minutes to allow expression of the erythromycm resistance gene Cells were plated in agar contammg lug erythromycm per ml Following incubation at 37°C for 36 hours, colonies are picked and grown overnight in Todd-Hewitt broth supplemented with 0 5% yeast extract Typically 1000 transformants contammg the appropriate allelic replacement are obtained If no transformants are obtamed in three separate transformation experiments as was the case for this gene coaA, then the gene is considered as being essential in vitro

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Abstract

La présente invention concerne des polypeptides coaA et des polynucléotides codant les polypeptides coaA, ainsi que des procédés permettant de produire les polypeptides précités au moyen de techniques de recombinaison. L'invention se rapporte enfin à des procédés d'utilisation des polypeptides coaA dans le criblage de composés antibactériens.
PCT/US2000/020744 1999-08-03 2000-07-31 POLYPEPTIDES ET POYLNUCLEOTIDES CoaA ET PROCEDES ASSOCIES WO2001008486A1 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1998043478A1 (fr) * 1997-04-01 1998-10-08 Merieux Oravax IDENTIFICATION DE POLYNUCLEOTIDES CODANT DE NOUVEAUX POLYPEPTIDES HELICOBACTER DANS LE GENOME $i(HELICOBACTER)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998043478A1 (fr) * 1997-04-01 1998-10-08 Merieux Oravax IDENTIFICATION DE POLYNUCLEOTIDES CODANT DE NOUVEAUX POLYPEPTIDES HELICOBACTER DANS LE GENOME $i(HELICOBACTER)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SONG ET AL.: "CoaA and rts are allelic and located ar kilobase 3532 on the escherichia coli physical map", JOURNAL OF BACTERIOLOGY, vol. 174, no. 5, March 1992 (1992-03-01), pages 1705 - 1706, XP002924295 *

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