WO2000058441A1 - STAPHYLOCOCCUS AUREUS ydjB APPARENTE A LA FAMILLE DE LUXS (SYNTHESE D'AUTOINDUCTEUR) - Google Patents

STAPHYLOCOCCUS AUREUS ydjB APPARENTE A LA FAMILLE DE LUXS (SYNTHESE D'AUTOINDUCTEUR) Download PDF

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Publication number
WO2000058441A1
WO2000058441A1 PCT/US2000/007597 US0007597W WO0058441A1 WO 2000058441 A1 WO2000058441 A1 WO 2000058441A1 US 0007597 W US0007597 W US 0007597W WO 0058441 A1 WO0058441 A1 WO 0058441A1
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Prior art keywords
polypeptide
seq
polynucleotide
sequence
amino acid
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PCT/US2000/007597
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English (en)
Inventor
John Peter Throup
Stephanie Van Horn
Richard L. Warren
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Smithkline Beecham Corporation
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Publication of WO2000058441A1 publication Critical patent/WO2000058441A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses.
  • the invention relates to polynucleotides and polypeptides of the luxS (autoinducer synthesis) family, as well as their variants, herein referred to as "ydjB,” “ydjB polynucleotide(s),” and “ydjB polypeptide(s)” as the case may be.
  • Staphylococcal genes and gene products are particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics.
  • the Staphylococci make up a medically important genera of microbes. They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues. S. aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.
  • Staphylococcus aureus 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 Staphylococcus aureus 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. Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics,” that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods.
  • polynucleotides and polypeptides such as the ydjB embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial activity.
  • Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease.
  • identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
  • the present invention relates to ydjB, in particular ydjB polypeptides and ydjB 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 invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting ydjB expression or activity.
  • the invention relates to ydjB polypeptides and polynucleotides as described in greater detail below.
  • the invention relates to polypeptides and polynucleotides of a ydjB of Staphylococcus aureus, that is related by amino acid sequence homology to HPO 105 Helicobacter pylori; ytjB Bacillus subtilis; YgaG Escherichia coli; YgaG Haemophilus influenzae; LuxS Vibrio harveyi; BB0377 Borrelia burgdorferi polypeptide.
  • the invention relates especially to ydjB 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 invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides .
  • NCIMB National Collections of Industrial and Marine Bacteria Ltd.
  • NCIMB National Collections of Industrial and Marine Bacteria Ltd.
  • the Staphylococcus aureus strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain.”
  • the deposited strain comprises a full length ydjB gene.
  • sequence of the polynucleotides comprised in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein.
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure.
  • the deposited strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent.
  • the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. ⁇ 112.
  • a license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is comprised in the deposited strain.
  • ydjB polynucleotide sequences in the deposited strain such as DNA and RNN and amino acid sequences encoded thereby.
  • ydjB polypeptide and polynucleotide sequences isolated from the deposited strain are also provided by the invention.
  • YdjB polypeptide of the invention is substantially phylogenetically related to other proteins of the luxS (autoinducer synthesis) family.
  • polypeptides o ⁇ Staphylococcus aureus referred to herein as "ydjB” and “ydjB polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • ydjB polypeptide encoded by naturally occurring alleles of a ydjB gene.
  • the present invention further provides for an isolated polypeptide that: (a) comprises or consists of an amino acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID ⁇ O: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; (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.
  • polypeptides of the invention 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 ydjB, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO:2] and also include portions of such polypeptides with such portion of the polypeptide generally comprising at least 30 amino acids and more preferably at least 50 amino acids.
  • the invention also includes a polypeptide consisting of or comprising a polypeptide of the formula:
  • X-(R ⁇ ) m -(R 2 )-(R 3 ) n -Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety described herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety described herein for modified polypeptides, Ri and R3 are any amino acid residue or modified amino 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 amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof.
  • R 2 is oriented so that its amino terminal amino acid residue is at the left, covalently bound to R ⁇ and its carboxy terminal amino acid residue is at the right, covalently bound to R3.
  • Any stretch of amino acid residues denoted by either R ⁇ or R3, where m and/or n is greater than 1, may be either a heteropolymer WO 00/58441 PCTtUSOO/07597
  • n is an integer between 1 and 50, 100, or 500.
  • a polypeptide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • a fragment is a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention
  • fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO 2], or of variants thereof, such as a continuous senes of residues that includes an amino- and/or carboxyl-terminal amino acid sequence Degradation forms of the polypeptides of the invention produced by or in a host cell, particularlv a Staphylococcus aureus.
  • 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-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions
  • fragments include an isolated polypeptide comprising an amino acid sequence havmg at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the ammo 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 l ention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the invention
  • polynucleotides that encode ydjB polypeptides particularly polynucleotides that encode a polypeptide herein designated ydjB
  • the polynucleotide compnses a region encoding polypeptides compnsing a sequence set out in Table 1 [SEQ ID NO 1] that includes a full length gene, or a ⁇ a ⁇ ant thereof The Applicants believe that this full length gene is essential to the growth and/or survival of an organism that possesses it.
  • isolated nucleic acid molecules encoding and/or expressing ydjB polypeptides and polynucleotides, particularly Staphylococcus aureus ydjB polypeptides and polynucleotides.
  • isolated nucleic acid molecules encoding and/or expressing ydjB polypeptides and polynucleotides, particularly Staphylococcus aureus ydjB polypeptides and polynucleotides.
  • isolated nucleic acid molecules encoding and/or expressing ydjB polypeptides and polynucleotides, particularly Staphylococcus aureus ydjB polypeptides and polynucleotides.
  • unprocessed RNAs nbozyme RNAs.
  • mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucle
  • Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a ydjB polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID N02] and polynucleotides closely related thereto and vanants thereof
  • Staphylococcus aureus comprising or consisting of an ammo acid sequence of Table 1 [SEQ ID NO.2], or a variant thereof
  • a polynucleotide of the invention encoding ydjB polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bactena using Staphylococcus aureus WCUH 29 cells as starting matenal, followed by obtaining a full length clone.
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1]
  • a library of clones of chromosomal DNA o ⁇ Staphylococcus aureus WCUH 29 ⁇ n £ colt or some other suitable host is probed with a radiolabeled oligonucleotide, preferabh a 17-mer or longer, de ⁇ ved from a partial sequence
  • Clones carrymg DNA identical to that of the probe can then be distinguished using strmgent hybridization conditions
  • sequencmg the individual clones thus identified b ⁇ hybridization with sequencmg primers designed from the o ⁇ gmal polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence
  • sequencmg is performed, for example, using denatured double stranded DNA prepared from a
  • each DNA sequence set out in Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art.
  • the present invention provides for an isolated polynucleotide comprising or consisting of: (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID 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 amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process that comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO:l or a fragment thereof; and isolating a full-length gene and/or genomic clones comprising said polynucleotide sequence.
  • the invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO: 1]. Also provided by the invention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence.
  • the polynucleotide of the invention may also comprise 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 transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNN introns, and polyadenylation signals.
  • the polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, 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. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al, Cell 37: 767 (1984), both of that may be useful in purifying polypeptide sequence fused to them.
  • 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
  • nucleotide immediately upstream of or including nucleotide 469 set forth in SEQ ID NO: 1 of Table 1, both of that encode a ydjB polypeptide.
  • the invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula: X-(R 1 ) m -(R 2 )-(R 3 ) n N 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 R j and R3 is mdependently any nucleic acid residue or modified nucleic acid residue, m is an mteger between 1 and 3000 or zero .
  • n is an mteger between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the mvention. particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof
  • R is o ⁇ ented 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 ] and/or R 2 , where m and/or n is greater than 1. may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, in a preferred embodiment.
  • 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 mteger between 1 and 1000.
  • m is an mteger between 1 and 50, 100 or 500, and n is an integer between 1 and 50. 100, or 500
  • a polynucleotide of the mvention is denved from Staphylococcus aureus, however, it may preferabh be obtained from other organisms of the same taxonomic genus
  • a polynucleotide of the mvention also be obtained, for example, from organisms of the same taxonomic family or order
  • polynucleotide encodmg a polypeptide encompasses polynucleotides that include a sequence encodmg a polypeptide of the mvention. particularly a bactena! polypeptide and more particularly a polypeptide of the Staphylococcus aureus ydjB havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2]
  • the term also encompasses pohmicleotides that mclude a single continuous region or discontmuous regions encodmg the polypeptide (for example, polynucleotides mterrupted bv mtegrated phage.
  • an mtegrated insertion sequence an mtegrated vector sequence, an mtegrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may compnse coding and/or non-coding sequences
  • the mvention further relates to vanants of the polynucleotides descnbed herem that encode vanants of a polypeptide a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the mvention ma ⁇ be used, for example, to s ⁇ thes ⁇ ze full-length polvnucleotides of the mvention
  • polynucleotides encodmg ⁇ djB vanants that have the ammo acid sequence of ⁇ djB pohpeptide 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, in any combination.
  • Prefened 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: l, 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 ' and/or 3 ' end of the polynucleotide sequence of SEQ ID
  • polynucleotides that are at least 95% or 97% identical over their entire length to a pohnucleotide encodmg ydjB polypeptide havmg an ammo acid sequence set out in Table 1 [SEQ ID NO 2]. and polynucleotides that are complementary to such polynucleotides.
  • polynucleotides that compnse a region that is at least 95% are especially preferred.
  • those with at least 97% are highly prefened among those with at least 95%. and among these those with at least 98% and at least 99% are particularly highly prefened, with at least 99% bemg the more prefened
  • Prefened 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].
  • polynucleotides that hybndize, particularly under stnngent conditions, to ydjB polynucleotide sequences, such as those pohnucleotides in Table 1
  • the mvention further relates to pohnucleotides that hvbndize to the polynucleotide sequences provided herem
  • the mvention especially relates to polynucleotides that hybndize under stnngent conditions to the polynucleotides descnbed herem
  • the terms "stnngent conditions” and “stnngent hvbndization conditions” mean hvbndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences
  • a specific example of stnngent hvbndrzation conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM
  • the mvention also provides a polynucleotide consisting of or compnsmg a polynucleotide sequence obtained by screening an appropnate library compnsmg a complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under stnngent hvbndization conditions with a probe having the 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 described elsewhere herem
  • the polynucleotides of the mvention may be used as a hvbndization probe for RNN cD ⁇ A and genomic D ⁇ A to isolate full-length cD ⁇ As and genomic clones encodmg ydjB and to isolate cD ⁇ A and genomic clones of other genes that have a high identity, particularly high sequence identity, to a ydjB gene.
  • Such probes generally will compnse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base parrs and may have at least 50 nucleotide residues or base pairs Particularly 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 ydjB gene may be isolated by screening usmg a D ⁇ A sequence provided in
  • Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe A labeled oligonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNN genomic D ⁇ A or mR ⁇ A to determine which members of the library the probe hybndizes to
  • primers designed to anneal withm the amplified product typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence
  • the products of this reaction can then be analvzed b ⁇ DNA sequencmg and a full-length DNA constructed either by joining the product directh to the existmg DNA to g ⁇ e a complete sequence, or earn ing out a separate full- length PCR using the new sequence information for the design of the 5' primer
  • the polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relating to polynucleotide assays.
  • polynucleotides of the invention that are oligonucleotides derived from a sequence of Table 1 [SEQ ID NOS: l or 2] may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
  • the invention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be processed away from a mature protein by cellular enzymes .
  • polynucleotide of the invention 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 protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide.
  • inactive precursors When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
  • the entire polypeptide encoded by an open reading frame is often not required for activity. Accordingly, it has become routine in molecular biology to map the boundaries of the primary structure required for activity with N-terminal and C-teiminal deletion experiments. These experiments utilize exonuclease digestion or convenient restriction sites to cleave coding nucleic acid sequence. For example, Promega (Madison, WI) sell an Erase-a-baseTM system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol available at www.promega.com). The digested endpoints can be repaired (e.g., by ligation to synthetic linkers) to the extent necessary to preserve an open reading frame.
  • nucleic acid of SEQ ID NO:l 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 variants thereof as described herein are within the invention, as are polypeptides so encoded.
  • a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, that is a precursor to a proprotein, 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.
  • a leader sequence which may be referred to as a preprotein
  • a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein or a preproprotein, that is a precursor to a proprotein, 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 invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems that comprise a polynucleotide or polynucleotides of the present invention, to host cells that are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techmques.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al, BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • bacterial cells such as cells of streptococci, staphylococci, enterococci E. coli, streptomyces, cyanobacteria, Bacillus subtilis, and Staphylococcus aureus
  • fungal cells such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus
  • insect cells such as cells o ⁇ Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells
  • plant cells such as cells of a gymnosperm or angiosperm.
  • vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses.
  • viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses.
  • the expression system constructs may compnse control regions that regulate as well as engender expression
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression m this regard
  • the appropnate DNA sequence may be inserted into the expression system by any of a vanetv of well-known and routme techmques. such as, for example, those set forth m Sambrook et al . MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
  • approp ⁇ ate 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 punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography. and lectin chromatography Most preferably, high performance hquid chromatography is employed for purification Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured dunng isolation and or purification
  • This mvention is also related to the use of ydjB polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of ⁇ djB pohnucleotides 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 ydjB gene or protein. mav be detected at the nucleic acid or ammo acid level by a vanetv 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 putativeh infected and/or infected individual's bodily matenals
  • Polynucleotides from any of these sources, particularh DNA or RNN may be used directh for detection or may be amplified enzvmatically by usmg PCR or other amplification technique pnor to anahsis R ⁇ N particularly mR ⁇ A.
  • cD ⁇ A and genomic D ⁇ A mav also be used m the same vva s Usmg amplification, characterization of the species and strain of infectious or resident organism present m an individual, mav be made bv an anahsis of the genotype of a selected pohnucleotide of the organism
  • 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 organism. preferably a different species of the same genus or a different strain of the same species.
  • Point mutations can be identified by hybndizmg amplified DNA to labeled ydjB polynucleotide sequences.
  • Polynucleotide sequence differences may also be detected by alterations in 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 arrav of oligonucleotides probes compnsmg ydjB 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 v anety of questions in molecular genetics including gene expression, genetic linkage, and genetic vanabihtv (see, for example, Chee et al . Science, 274 610 (1996))
  • the present mv ention 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 the present invention, preferably to the polypeptide of SEQ ID NO 2. It will be appreciated that m any such kit. (a) (b), (c) or (d) mav comprise a substantial component Such a kit will be of use in diagnosmg a disease or susceptibility to a Disease, among others
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polvnucleotide of the mvention, preferable.
  • SEQ ID NO 1. that is associated with a disease or pathogenicitv 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.
  • Organisms, particularly infectious organisms, carrying mutations m such polynucleotide may be detected at the polynucleotide level bv a vanetv of techniques such as those descnbed elsewhere herem
  • the differences in a polvnucleotide and or polvpeptide sequence between organisms possessing a first phenotvpe and organisms possessing a different, second different phenotvpe can also be determined If a mutation is observed in some or all organisms possessmg the first phenotvpe but not m 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 techniques, to allow for serotyping, for example.
  • 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.
  • RNN cD ⁇ A or genomic D ⁇ A may also be used for the same purpose, PCR.
  • PCR primers complementary to a polynucleotide encoding ydjB 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 ydjB D ⁇ A and/or R ⁇ A 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 various techniques for elucidation of the polynucleotide sequence. In this way, mutations in 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 invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID ⁇ O:l].
  • Increased or decreased expression of a ydjB polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
  • a diagnostic assay in accordance with the invention for detecting over-expression of ydjB polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example.
  • Assay techniques that can be used to determine levels of a ydjB polypeptide, in a sample derived from a host, such as a bodily material, are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.
  • Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et ⁇ /., Current Protocols in Immunology 1(2): Chapter 5 (1991). Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases herein mentioned.
  • the present invention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides and polynucleotides.
  • agonists or antagonists e.g. , inhibitors
  • Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. Such agonists and antagonists so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc., as the case may be, of ydjB polypeptides and polynucleotides; or may be structural or functional mimetics thereof (see Coligan et al, Current Protocols in Immunology l(2):Chapter 5 (1991)).
  • 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 ydjB polypeptide and/or polynucleotide activity in the mixture, and comparing the ydjB polypeptide and/or polynucleotide activity of the mixture to a standard.
  • Fusion proteins such as those made from Fc portion and ydjB 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, 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 m cells.
  • an ELISA assay may be constructed for measuring 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 agonist, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screening compounds to identify those that enhance (agonist) or block (antagonist) the action of ydjB polypeptides or polynucleotides, particularly those compounds that are bacte ⁇ static and/or bactencidal
  • the method of screening may mvolve high-throughput techniques. For example, to screen for agonists 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 ydjB polypeptide and a labeled substrate or kgand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a ydjB agonist or antagonist
  • the ability of the candidate molecule to agonize or antagonize the ydjB polypeptide is reflected m decreased binding of the labeled hgand or decreased production of product from such substrate Molecules that bmd gratuitously.
  • Reporter systems that may be useful m this regard mclude but are not limited to colo ⁇ metnc, labeled substrate converted mto product, a reporter gene that is responsive to changes m ydjB polynucleotide or polypeptide activity, and binding assays known m the art
  • Polypeptides of the invention mav be used to identify membrane bound or soluble receptors, if any. for such polypeptide. through standard receptor binding techmques known m the art These techmques mclude. but are not limited to. gand bmdmg and crosshnking assays m which the polypeptide is labeled with a radioactive isotope (for instance ⁇ 1), chemically modified (for instance, biotinvlated). or fused to a peptide sequence suitable for detection or punfication. and incubated with a source of the putative receptor (e g . cells, cell membranes, cell supernatants.
  • a source of the putative receptor e g . cells, cell membranes, cell supernatants.
  • tissue extracts, bodily matenals Other methods mclude biophysical techmques such as surface plasmon resonance and spectroscopv These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s). if anv Standard methods for conducting such assays are ell understood m the art
  • the fluorescence polarization v alue for a fiuorescenth -tagged molecule depends on the rotational conelation time or tumbling rate Protein complexes, such as formed by ydjB pohpeptide associating with another ydjB pohpeptide or other polypeptide. Labeled to comprise a fluorescently- labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein. It is preferred that this method be used to charactenze small molecules that disrupt polypeptide complexes
  • Fluorescence energy transfer may also be used charactenze small molecules that interfere with the formation of ydjB polypeptide dimers. tnmers. tetramers or higher order structures, or structures formed by ydjB polypeptide bound to another polypeptide YdjB 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 dimenzation will inhibit fluorescence energy transfer
  • YdjB polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protein can then passed over the ydjB polypeptide -coated surface and specific bmdmg can be detected m real-time by momtormg the change in 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 ydjB polypeptide self-association as well as an association of ydjB polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to charactenze the mteraction between an association of ydjB polypeptide with another v
  • identifying compounds that bmd to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polvnucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the pohpeptide and or polvnucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferabh bemg associated with a second component capable of providing a detectable signal in response to the bmdmg or mteraction of the pohpeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/
  • an assay for ydjB agonists is a competitive assay that combines ydjB and a potential agomst with ydjB-binding molecules, recombinant ydjB bmdmg molecules, natural substrates or hgands, or substrate or hgand mimetics, under appropnate conditions for a competitive inhibition assay
  • YdjB can be labeled, such as by radioactivity or a colo ⁇ metnc compound, such that the number of ydjB molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagomst
  • 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 antagomst of the polypeptide and/or polynucleotide, by (a) determining in
  • 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 ydjB polypeptide and/or polynucleotide
  • expression of the gene encodmg endogenous ydjB polypeptide can be inhibited usmg expression blocking techmques
  • This blocking may be targeted against any step m gene expression, but is preferably targeted against transcnption and/or translation
  • An examples of a known technique of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 m Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press.
  • oligonucleotides that form tnple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • polynucleotide sequences provided herem may be used in the discovery and development of antibacterial compounds
  • the encoded protem upon expression, can be used as a target for the screening of antibacterial drugs
  • the polynucleotide sequences encodmg the a mo 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 mvention also provides the use of the polypeptide, polynucleotide, agomst or antagomst of the mvention to mterfere 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 bactena, m particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matnx protems on m-dwellmg devices or to extracellular matnx protems m wounds, to block bactenal adhesion between eukaryotic, preferably mammalian, extracellular matnx protems and bactenal ydjB protems that mediate tissue damage and/or, to block the normal progression of pathogenesis m infections initiated other than by the implantation of m-dwellmg devices or
  • ydjB agonists and antagomsts preferably bactenstatic or bactencidal agonists and antagomsts
  • the antagonists and agonists of the mvention may be employed, for instance, to prevent, inhibit and or treat diseases
  • Hehcobacter pylori (herem "H pylon") 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) Schtstosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer.
  • 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 gastnc ulcers and gastntis
  • “Bodily matenal(s) means any matenal de ⁇ ved from an individual or from an organism infecting, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, u ⁇ ne, stool or autopsy mate ⁇ als
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena, including , for example, disease, such as, infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis. thyroiditis). lower respiratory (e g , empyema. lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g . secretory dianhoea. splenic absces. retropentoneal abscess). CNS (e g , cerebral abscess), eve (e g . blepha ⁇ tis. conjunctivitis, keratitis.
  • infections of the upper respiratory tract e g , otitis media, bactenal tracheitis, acute epiglottitis. thyroiditis.
  • lower respiratory e g , empyema. lung abscess
  • cardiac e
  • endophthalmitis preseptal and orbital celluhtis, darcryocystitis). kidnev and urinary tract (e g . epididvmitis. intrarenal and pennephnc absces, toxic shock syndrome), sk n (e g . impetigo, folhcuhtis, cutaneous abscesses, celluhtis, wound infection, bactenal myositis) bone and joint (e g , septic arthntis. osteomvehtis)
  • “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) bv an exogenous polynucleotide sequence
  • Identity as known in the art. is a relationship between two or more polypeptide sequences or two or more polvnucleotide sequences, as the case mav 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 bv the match between strings of such sequences "Identity” can be readilv calculated bv known methods, including but not limited to those described in (Computational Molecular Biology . Lesk. A M . ed Oxford University Press. New York, 1988, Biocomputmg Informatics and Genome Projects. Smith. D W . ed . Academic Press.
  • Computer program methods to determine identity between two sequences include, 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. Molec. Biol 215: 403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et 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 include the following: Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID 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, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the integer defining the percent identity divided by 100 and then subtracting that
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NOT
  • y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding 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 include up to a certain integer number of amino 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 ammo acid alterations
  • x a is the total number of ammo acids m 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 mteger 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, i e , if it occurs m nature, it has been changed or removed from its onginal 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 coexistmg matenals of its natural state is “isolated", as the term is employed herem
  • a polynucleotide or polypeptide that is introduced mto 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
  • 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, Yersinia, Fancisella, Pasturella, Morctxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibno, Proteus, Erwmia, Borrelia, Leptosptra, Spinllum, Campylobacter, Shigella, Legtonella
  • Polynucleot ⁇ de(s) generally refers to any polvnbonucleotide or polydeoxynbonucleotide, that mav be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)" mclude, without limitation.
  • DNN D ⁇ A that is a mixture of smgle- and double-stranded regions or single-, double- and t ⁇ ple-stranded regions, single- and double-stranded R ⁇ N and R ⁇ A that is mixture of single- and double-stranded regions, hybnd molecules compnsmg D ⁇ A and R ⁇ A that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions.
  • polynucleotide refers to tnple-stranded regions compnsing R ⁇ A or D ⁇ A or both R ⁇ A and D ⁇ A
  • 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 tnple-hehcal region often is an oligonucleotide
  • the term “polynucleotide(s)” also mcludes D ⁇ As or R ⁇ As as descnbed above that comp ⁇ se one or more modified bases
  • D ⁇ As or R ⁇ As with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herem
  • Polypept ⁇ de(s) refers to anv peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds
  • Polypeptide(s) refers to both short chains, commonlv refened to as peptides.
  • Polypeptide(s) mclude those modified either bv natural processes, such as processing and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a giv en pohpeptide Also, a given polypeptide mav compnse many types of modifications Modifications can occur anvwhere m a polypeptide.
  • mcludmg the peptide backbone the ammo acid side-chains, and the ammo or carboxvi termini Modifications mclude. for example acetviation. acvlation. ADP-nbosvlation. amidation. covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or hpid denvative, covalent attachment of phosphotidy nositol.
  • 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, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemrzation, 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 arginylation, and ubiquitination See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York (1993) and Wold, F , Posttrans
  • 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
  • 'Nar ⁇ ant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retams essential properties
  • a typical vanant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the vanant may or may not alter the ammo 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 variant 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 variant and reference polypeptide may
  • 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 recombinant methods known to skilled artisans.
  • the polynucleotide having a DNA sequence given in Table 1 [SEQ ID NOT] was obtained from a library of clones of chromosomal DNA o ⁇ Staphylococcus aureus in E. coli.
  • the sequencing data from two or more clones comprising overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NOT .
  • Libraries may be prepared by routine methods, for example: Methods 1 and 2 below.
  • Total cellular DNA is mechanically sheared by passage through a needle in 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 EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coli infected with the packaged library.
  • the library is amplified by standard procedures.
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures.
  • EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library.
  • the library is amplified by standard procedures.

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Abstract

L'invention porte sur des polypeptides ydjB et sur des polynucléotides les codant, ainsi que sur des procédés de production de ces polypeptides par des techniques de recombinaison. L'invention porte également sur des procédés d'utilisation des polypeptides ydjB pour rechercher des composés antibactériens.
PCT/US2000/007597 1999-03-26 2000-03-23 STAPHYLOCOCCUS AUREUS ydjB APPARENTE A LA FAMILLE DE LUXS (SYNTHESE D'AUTOINDUCTEUR) WO2000058441A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US27687299A 1999-03-26 1999-03-26
US09/276,872 1999-03-26

Publications (1)

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WO2000058441A1 true WO2000058441A1 (fr) 2000-10-05

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PCT/US2000/007597 WO2000058441A1 (fr) 1999-03-26 2000-03-23 STAPHYLOCOCCUS AUREUS ydjB APPARENTE A LA FAMILLE DE LUXS (SYNTHESE D'AUTOINDUCTEUR)

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Citations (1)

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

Patent Citations (1)

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

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