nrdG

Abstract: The invention provides nrdG polypeptides and polynucleotides encoding nrdG polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing nrdG polypeptides to screen for antibacterial compounds.

Description: nrdG FIELD OF THE INVENTION This invention relates to newly identified polynucleotides and pohpeptides. and their production and uses, as well as their variants, agonists and antagonists, and their uses hi particular, tlie invention relates to polynucleotides and polypeptides of tl e nrd family, as \\ ell as their variants, hereinafter referred to as "nrdG." "nrdG polynucleotιde(s)." and "nrdG polypeptιde(s) " BACKGROUND OF THE INVENTION It is particularly preferred to employ Staphylococcal genes and gene products as targets for tl e de\elopment of antibiotics Tl e Staphvlococci make up a medically important genera of microbes The\ are known to produce two types of disease, invasive and toxigenic I 'asive infections are characterized generalh by abscess fomiation effecting both skin surfaces and deep tissues S aureus is tlie second leading cause of bacteremia in cancer patients Osteoi e tis septic arthritis septic thrombophlebitis and acute bacterial endocarditis are also relatn eh common There are at least three clinical conditions resulting from tlie toxigenic properties of Staphvlococci Tlie 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 The frequency of Staphylococcu aureus infections has risen dramatically in tlie past few decades This has been attributed to tlie emergence of multiph antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of die standaid antibiotics Tins phenomenon has created an unmet medical need and demand for new ai ti-microbial agents, vaccines. drug screening methods, and diagnostic tests for tins organism Moreover, tlie drag discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics." that is. high throughput genome- or gene-based biologN Tins approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics relies heavily on tlie various tools of biourformatics to ldcnti gene sequences of potential interest from tlie many molecular biology databases now aλ/ailable as w ell as from otliei sources There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides. as targets for drag discover) Clearh. there exists a need for polynucleotides and pohpeptides. such as the nrdG embodiments of the invention, that have a present benefit of. among other tilings being useful to screen compounds for antibiotic activity Such factors are also useful to detennine their role in patliogenesis of mfection. dysfiinction and disease Tliere is also a need for identification and characterization of such factors and their antagonists and agonists to find \\a\s to prevent, ameliorate or correct such infection, dysfunction and disease Ceitain of the pohpeptides of tlie invention possess significant amino acid sequence homologλ to a known Phage T4 anaerobic nbonucleoside-tπphosphate reductase piotein see Sw issprot accession number P07075 SUMMARY OF THE INVENTION The present invention relates to nrdG in particulai nrdG poh peptides and nidG polynucleotides. recombinant materials and methods for their production In another aspect the invention relates to methods for using such pohpeptides and polynucleotides. including tlie treatment of microbial diseases, amongst others In a further aspect, 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 compounds In a still further aspect tlie lm ention relates to diagnostic assa\ s for detecting diseases associated with microbial infections and conditions associated with such infections such as assax s for detecting nrdG expression or activit} Vanous changes and modifications within tlie spirit and scope of tlie disclosed ιn\ ention w ill become readily apparent to those skilled in tlie art from reading tlie following descriptions and from reading the other parts of tlie present disclosure DESCRIPTION OF THE INVENTION The invention relates to nrdG pohpeptides and polynucleotides as descπbed in greater detail below In particular, tlie invention relates to polypeptides and polynucleotides of a nrdG of Staphylototcus aureus. winch is related by amino acid sequence homology to Phage T4 anaerobic nboiiucleoside-tπphosphate reductase polypeptide Tlie invention relates especially to nrdG having tlie nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively TABLE 1 NrdG Polynucleotide and Polypeptide Sequences . 9 . (A) Staphylococcus aureus nrdG polynucleotide sequence [SEQ ID NO 1] 5 ' -ATGACACTTTTAGACATTAAACAAGGACAAGGTTATATTGCTAAAATAGAATCAAATAGC TTTGTTGACGGTGAAGGAGTAAGATGCAGTGTTTATGTATCAGGATGTCCATTTAATTGT GTTGGATGTTATAACAAAGCCTCACAAAAGTTCAGATAT&&CGAGAA.ATAC^CT& TGAZ- ATATTAGCAGAAATATTAGATGATTGCGATCATGATTATATATCTGGCCTAΛGT^^ TT GGTGGCGAACCATTTTGTAATTTGGATATTACATTAAATCTTGTCAAAGCATTTCGAGCA CGTTTTGGAAATACAAAGACAATTTGGGTATGGACTGGATTTTTATATGAATATTTAGCA AATGATTGTACAGAACGTCGAGAGTTATTATCATACATTGACGTTTTAGTAGATGGTCTA TTTATACAACACTTATTCAAACCTGATTTACCATATAAAGGTTCTTTAAATCAACGCATT ATAGATGTACAACAATCACTCTCGCATGCGCGTATGATTGAATATATAGTTAGT-3 ' (B) Staphy lococcus aureus nrdG poh peptide sequence deduced from a poh nucleotide sequence in this table fSEQ ID NO 2] NH -MTLLDI KQGQGYIAKI ESNS FVDGEGVRCSVYVS GCPFNCVGCYNKASQKFRYGEKYTDE ILAEILDDCDHDYISGLSLLGGEPFCNLDITLNLVKAFRARFGNTKTIWVWTGF YEYLA NDCTERRELLSYIDVLVDGLFIQHLFKPDLPYKGSLNQRIIDVQQSLSHAI^MIEYIVS-COOH Deposited materials A deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with tlie National Collections of Industnal and Mamie Bactena Ltd (herein "NCIMB") 23 St Machai Drπ e Aberdeen AB2 IRY, Scotland on 11 September 1995 and assigned NCIMB Deposit No 40771 and refeired to as Staphylococcus aureus WCUH29 on deposit Tlie Staphylococcus aureus stram deposit is referred to herein as "the deposited stram" or as "tiie DNA of tlie deposited stram " Tlie deposited stram contains tlie full length nrdG gene Tlie sequence of the pohnucleotides contamed in tlie deposited stram. as well as the annno acid sequence of am pohpeptide encoded thereby, are controlling in tiie event of any conflict with am description of sequences herein Tlie deposit of the deposited strain has been made under tlie tenns of the Budapest Treats on tlie hitemational Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure Tlie strain will be irrevocably and without restnction or condition released to the public upon tlie issuance of a patent The deposited stram is provided merely as convenience to those of skill in tiie 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 tiie deposited strain and compounds derπed therefrom, and no such license is hereby granted In one aspect of tiie mvention there is provided an isolated nucleic acid molecule encoding a mature pohpeptide expressible by tlie Staphylococcus aureus WCUH 29 stram. winch polypeptide is contamed in tiie deposited strain Further provided by tlie mvention are nrdG polynucleotide sequences in tlie deposited strain, such as DNA and RNA. and ammo acid sequences encoded thereby .Also provided by the mvention are nrdG polypeptide and pohnucleotide sequences isolated from the deposited stram Polypeptides NrdG polypeptide of tlie mvention is structurally related to other proteins of tlie nrd fannh In one aspect of tlie mvention tliere are provided polypeptides of Staphylococcus aureus referred to herein as "nrdG" and "nrdG polypeptides" as well as biologicalh . diagnosticalh . proplrs lactical . clinicalh or tiierapeuticalh useful variants thereof, and compositions comprising tiie same Among tlie particularly preferred embodiments of tlie IΠΛ ention aic \ anants ot nidG polypeptide encoded by naturally occurring alleles of tiie nrdG gene Tlie present mvention further provides for an isolated polypeptide winch (a) comprises or consists of an ammo acid sequence which has at least 70% identity , preferably at least 80% identity, more preferably at least 90% identity, yet more preferably 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 which has at least 70% identity, preferably at least 80% identity . more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferabh 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 winch has at least 70% identity, preferabh at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% ldentitΛ even more preferably at least 97-99% or exact identity, to tlie annno acid sequence of SEQ ID NO 2. over the entire length of SEQ ID NO 2 Tlie polypeptides of tiie mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (in particular tiie mature polypeptide) as well as polypeptides and fragments, particularly tiiose winch have tiie biological activity of nrdG and also tiiose winch have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO l]or tiie relevant portion, preferabh at least 80% identity to a poh peptide of Table 1 [SEQ ID NO 2and more preferably at least 90% ιdentιt\ to a pohpeptide of Table 1 | SEQ ID NO 2] and still more preferably at least 95% identity to a pohpeptide of Table 1 |SEQ ID NO 2| and - 4 - also include portions of such polypeptides with such portion of tlie polypeptide general h containing at least 30 ammo acids and more preferably at least 50 annno acids The mvention also includes a polypeptide consisting of or compnsmg a polypeptide of tiie fom ula X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at tiie annno temnnus. X is hydrogen, a metal or am othei moιet\ described heiein for modified polypeptides. and at tiie carboxyl terminus. Y is lrsdrogen a metal oi am other moiets descnbed herein for modified polypeptides. R j and R3 are any annno acid residue or modified ammo acid residue, 111 is an mteger between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero. and R 2 is an annno acid sequence of tiie mvention. particularly an annno acid sequence selected from Table 1 or modified forms thereof hi tiie fomiula above. R 2 is oriented so that its annno terminal annno acid residue is at tiie left, covalently bound to R ] and its carbox terminal annno acid residue is at tiie nglit. covalently bound to R3 .Any stretch of annno acid residues denoted b\ eitliei R ] 01 R where m and/or n is greater than 1. may be either a heteropolymer or a homopohmer preferabh a heteropolymer Other preferred embodunents of tlie mvention are provided where m is an integer between 1 and 50, 100 or 500, and n is an mteger between 1 and 50. 100. or 500 It is most preferred that a polypeptide of tlie mvention is denved from Staphylococcus aureus. however, it may preferably be obtained from other organisms of tiie same taxonomic genus A polypeptide of tiie invention may also be obtained, for example, from organisms of the same taxonomic family or order A fragment is a variant polypeptide havmg an annno acid sequence that is entireh the same as part but not all of any annno acid sequence of any polypeptide of the invention As w ith nrdG polypeptides, fragments may be "free-standing." or composed within a larger polypeptide of which they form a part or region, most preferably as a single continuous region 111 a smgle larger polypeptide Preferred fragments mclude. for example, truncation polypeptides \ Ά\ ing a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2]. or of variants thereof, such as a continuous series of residues that mcludes an amino- and/or carboxyl-termnial annno acid sequence Degradation forms of the polypeptides of tlie mvention produced by or in a host cell, particularh a Staphylococcus aureus. are also preferred Further preferred are fragments charactenzed by structural or functional attnbutes such as fragments that compose alpha-helix and alpha-helix fomniig regions, beta-sheet and beta- sheet-formmg regions, turn and tum-fomnng regions, coil and coil-forming regions, hydroplnlic regions, hydrophobic regions, alpha amphipathic regions, beta amphipatlnc regions, flexible regions, surface-formmg regions, substrate binding region, and high antigcnic index regions Also preferred are biologically active fragments winch are those fragments tiiat mediate activities of nrdG. including tiiose with a smnlar activity or an improved activity , or with a decreased undesirable activity Also mcluded are tiiose fragments that are antigeinc or lmmunogenic ui an animal, especially in a human Particularly preferred are fragments compnsmg receptors or domains of enzymes that confer a function essential for viability of Staphylococcus aureus or tiie ability to initiate, or maintain cause Disease m an individual, particularly a human Fragments of tlie polypeptides of tiie mvention may be emplo\ed for producing tl e corresponding full-length polypeptide by peptide synthesis, therefore these \ aπants ma\ be emplo\ ed as mtennediates for producmg tiie full-length polypeptides of tlie invention In addition to the standard single and triple letter representations foi amino acids the term "X" or "Xaa" may also be used in describing certain polypeptides of the mvention "X" and "Xaa" mean that any of the twenty naturally occurring amino acids may appear at such a designated position m the polypeptide sequence Polynucleotides It is an object of tiie invention to provide polynucleotides that encode in dG pohpeptides particularly polynucleotides that encode tlie polypeptide herein designated nrdG hi a particularly preferred embodiment of the mvention tiie polynucleotide composes a region encoding nrdG polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1] winch includes a full length gene, or a variant thereof Tlie Applicants believe that tins full length gene is essential to the growth and/or survival of an organism winch possesses it. such as Staphylococcus aureus As a further aspect of the mvention tliere are provided isolated nucleic acid molecules encoding and/or expressing nrdG polypeptides and pohnucleotides. particularh Sttιph\ locaccιιs aureus nrdG polypeptides and polynucleotides. including for example, unprocessed RNAs nbozyme RNAs, n RNAs. cDNAs. genomic DNAs. B- and Z-DNAs Further embodiments of the mvention mclude biologically, diagiiostically, prophylactically. cluncally or tlierapeutically useful polynucleotides and polypeptides. and vanants thereof, and compositions compnsmg tiie same Another aspect of tlie mvention relates to isolated poly nucleotides including at least one full length gene, tiiat encodes a nrdG polypeptide having a deduced ammo acid sequence of Table 1 |SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof In another particularly preferred embodiment of the invention there is a nrdG polypeptide from Staphylococcus aureus comprising or consisting of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof Usmg tiie information provided hereui. such as a polynucleotide sequence set out ui Table 1 [SEQ ID NO 1J. a polynucleotide of the mvention encoding nrdG polypeptide ma\ be obtained us g standard clorung and screening methods, such as tiiose for cloning and sequencing chromosomal DNA fragments from bactena usmg Staphylococcus aureus WCUH 29 cells as starting matenal. followed by obtaunng a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a poh nucleotide sequence given in Table 1 [SEQ ID NO 1]. typically a libran of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E coh or some other suitable host is probed with a radiolabeled ohgonucleotide. preferabh a 17-mcr oi longei dei n ed from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions By sequencing the individual clones thus identified b\ hybridization with sequencing primers designed from the original polypeptide or poh nucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a frill length gene sequence Convenienth . such sequencing is perfoniied. for example using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Mamatis. T . Fntsch. E F and Sambrook et al MOLECULAR Cl ONING A LABORATORY MANUAL. 2nd Ed . Cold Spring Harbor Laboraton Pi ess Cold Spring Haiboi New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencing Denatured Doubie-Stranded DNA Templates 13 70) Direct genomic DNA sequencing ma\ also be performed to obtain a full length gene sequence Illustrative of the mvention. each polynucleotide set out in Table 1 [SEQ ID NO 1] was discovered ui a DNA library derived from Staphylococcus aureus WCUH 29 Moreover, each DNA sequence set out in Table I {SEQ ID NO 1 1 contains an open reading frame encoding a protein having about tlie number of annno acid residues set forth in TabL 1 |SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg annno acid residue molecular weight values well .known to those skilled ui tlie art Tlie polynucleotide of SEQ ID NO 1. between nucleotide number 1 and tlie stop codon winch beguis at nucleotide number 535 of SEQ ID NO 1. encodes tiie polypeptide of SEQ ID NO 2 hi a further aspect, tlie present mvention provides for an isolated pohnucleotide comprising or consisting of (a) a polynucleotide sequence which has at least 70% identity, preferabh at least 80% ιdentιt\ more preferably at least 90% identity, yet more preferably 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. (b) a polynucleotide sequence encodmg a pohpeptide which has at least 70% identity . preferabh at least 80% identity, more preferably at least 90% identity, yet more preferabh at least 95% identity, even more preferably at least 97-99% or 100% exact, to tiie am o acid sequence of SEQ ID NO 2. over the entire length of SEQ ID NO 2 A polynucleotide encodmg a polypeptide of tiie present mvention, uicluduig homologs and orthologs from species other than Staphylococcus aureus. may be obtamed by a process winch comprises tlie steps of screeinng an appropoate library under stringent hybodization conditions with a labeled or detectable probe consisting of or compnsmg tlie sequence of SEQ ID NO 1 oi a fragment thereof, and isolatmg a full-length gene and/or genomic clones containing said poh nucleotide sequence Tlie mvention provides a polynucleotide sequence identical o\ er its entire length to a coding sequence (open readmg frame) ui Table 1 [SEQ ID NO 1] Also provided by the lm ention 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 readmg frame with another codmg sequence, such as a sequence encodmg a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence Tlie polynucleotide of the mvention may also contain at least one non-codmg sequence including for example, but not lumted to at least one non-coding " and 3 * sequence, such as the transci ibed but non- translated sequences, termination signals (such as rho-dependent and iho-mdepcndent ternunation signals), nbosome buidmg sites. Kozak sequences, sequences that stabilize niRNA. nitrons, and polyadenylation signals The polynucleotide sequence may also compnse additional codmg sequence encodmg additional annno acids For example, a marker sequence that facilitates puπfication of tlie fused polypeptide can be encoded hi certaui embodunents of tlie mvention. the marker sequence is a hexa-lnstidme peptide. as provided in tiie pQE vector (Qiagen. Inc ) and described in Gcntz et al Proc Natl Acad S , USA 86 821 -824 ( 1989) or an HA peptide tag (λvilson L I al ( ell 3 " 767 (1984). both of which may be useful m punfymg polypeptide sequence fused to them Poly nucleotides of the mvention also mclude, but are not lumted to, polynucleotides compnsmg a stractural gene and its naturally associated sequences that control gene expression A preferred embodunent of the mvention is a polynucleotide of consisting of or compnsmg nucleotide 1 to tiie nucleotide immediately upstream of or uicluduig nucleotide 535 set forth in SEQ ID NO 1 of Table 1. both of winch encode the nrdG polypeptide The mvention also mcludes a polynucleotide consisting of or composing a poh nucleotide of the foπnula X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the 5' end of the molecule. X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule. Y is Irs drogen, a metal - 8 - or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of R ] and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero . n is an integer between 1 and 3000 or zero, and R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularh a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above. R is oriented so that its 5' end nucleic acid residue is at the left. bound to Rj 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 . where m and/or 11 is greater than 1. mas be either a heteropolymer or a homopolymer. preferablv a heteropohmcr Where in a preferred embodiment, X and Y together define a cos alent bond, the poh nucleotide of the abos e formula is a closed, circular polynucleotide. which can be a double-stranded poh nucleotide s heiein the formula shows a first strand to which the second strand is complementary In another pieferred embodiment m and/or n is an integer between 1 and 1000. Other preferred embodiments oftiie mvention are provided where 111 is an mteger between 1 and 50. 100 or 500. and 11 is an mteger between l and 50. 100. or 500 It is most preferred tiiat a polynucleotide of tiie invention is deπ ed from Stapln lococcus aureus. however, it may preferably be obtamed from other organisms oftiie same ta\onomιc genus A polynucleotide of tlie mvention may also be obtamed. for example, from organisms of tiie same taxonomic family or order The tenn "polynucleotide encodmg a polypeptide" as used hereui encompasses polynucleotides that mclude a sequence encodmg a polypeptide of tlie mvention. particularly a bacterial polypeptide and more particularly a polypeptide of tiie Staphylococcus aureus nrdG haλ ing an ammo acid sequence set out ui Table 1 [SEQ ID NO 2] The tenn also encompasses poh nucleotides that include a smgle continuous region or discontmuous regions encodmg the polypeptide (foi example polynucleotides mtemipted by mtegrated phage. an mtegrated uisertion 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 contain codmg and/or non-codmg sequences Tlie mvention further relates to vanants of tiie pohnucleotides described herem tiiat encode vanants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID N0 2| Fragments of a polynucleotides of tiie mvention may be used for example to ssnthesize full-length polynucleotides oftiie mvention Further particularly preferred embodunents are polynucleotides encodmg nrdG vanants. that have the annno acid sequence of nrdG polypeptide of Table 1 [SEQ ID NO 2J ui svlnch 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, ui any combination Especially preferred among these are silent substitutions, additions and deletions, that do not alter tiie properties and activities of nrdG polypeptide Further preferred embodiments of the mvention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encodmg nrdG poly peptide havmg an am o acid sequence set out ui Table 1 [SEQ ID NO 2], and polynucleotides tiiat are complementary to such polynucleotides Alternatively, most highly preferred are polynucleotides tiiat comprise a region tiiat is at least 80% identical over its entire length to a polynucleotide encodmg nrdG polypeptide and polynucleotides complementary thereto hi tins regard, polynucleotides at least 90% identical os'er their entire length to the same aie particularly preferred, and among these particularh prefened polynucleotides. tiiose with at least 95% are especially preferred Furthermore those ssith at least 97% are highly preferred among tiiose with at least 95%. and among these those s ith at least 98% and at least 99% are particularly highly preferred, with at least 99% be g tiie more preferred Preferred embodiments are polynucleotides encodmg polypeptides that retain substantialh the same biological function or activity as tiie mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1] hi accordance with certam preferred embodunents of tins mvention tliere aie provided polynucleotides that hybndize. particularly under strmgent conditions, to nrdG poh nucleotide sequences, such as tiiose polynucleotides ui Table 1 Tlie mvention further relates to polynucleotides tiiat hybndize to tiie polynucleotide sequences provided liereui hi tins regard, tlie invention especially relates to polynucleotides tiiat hybridize under strmgent conditions to tiie polynucleotides descnbed herein As herem used, tiie tenns "strmgent conditions" and "strmgent hybndization conditions" mean hybndization occurring onh if there is at least 95% and preferabh at least 97% identity between tlie sequences A specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate). 50 mM sodium phosphate (pH7.6). 5x Denhardt's solution, 10% dextran sulfate. and 20 micrograms/nil of denatured, sheared salmon speπn DNA. followed by washing the hybridization support m 0 lx SSC at about 65 °C Hs'bπdization and wash conditions are well known and exemplified in Sambrook. et al Molecular Cloning A Laboratory Manual. Second Edition. Cold Spring Harbor. N Y ( 1989) particularly Chapter 1 1 therein Solution hybridization may also be used ss ith the poh nucleotide sequences provided by the invention The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under stringent hybridization - 10 - conditions ith a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide mclude. for example, probes and primers fulls described elsewhere herein As discussed elsewhere liereui regarduig polynucleotide assays of tiie mvention. for uistance. tlie polynucleotides of tlie mvention. may be used as a hybndization probe for RNA, cDNA and genonnc DNA to isolate full-length cDNAs and genonuc clones encodmg nrdG and to isolate cDNA and genonnc clones of oilier genes that have a high identity, particularly high sequence identity, to tlie nrdG gene Such probes generally will comprise at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base pairs and mas has e at least 50 nucleotide residues or base pairs Particularh preferred piobes ss ill has e at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs A codmg region of a nrdG gene may be isolated by screenuig usmg a DNA sequence piovided in Table 1 [SEQ ID NO 1J to synthesize an ohgoiiucleotide probe A labeled oligonucleotide havuig a sequence complementary to tiiat of a gene of tiie invention is then used to screen a library of cDN A genonnc DNA or niRNA to deteninne which members oftiie library tlie probe hybndizes to There are several methods available and well known to those skilled in the art to obtain full-length DNAs, or extend short DNAs. for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see. for example. Frohman. et al . PNAS USA 85. 8998- 9002. 1988) Recent modifications ofthe technique, exemplified by the Marathon™ teclniology (Clontech Laboratories Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technolog . cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence hgated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within 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 known gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design ofthe 5' primer The polynucleotides and polypeptides of tiie invention may be employ ed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relatuig to polynucleotide assays - 1 1 - The polynucleotides of the invention that are ohgonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herein as described, but preferabh for PCR. to determine whether or not the polynucleotides identified herein in sshole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences w ill also has e utility in diagnosis ofthe stage of infection and type of infection the pathogen has attained The mvention also provides polynucleotides that encode a polypeptide that is the mature prote plus additional annno or carboxyl-tennuial ammo acids, or ammo acids uitenor to tiie mature polypeptide (when tlie mature fonn has more than one polypeptide chain, for uistance) Such sequences may plav a role m processuig of a protein from precursor to a mature form, mas alloss protein transport mas lengtiien or shorten protein half-life or mas facilitate manipulation of a protein for assay or production, among other tilings As generalls is tiie case in vivo the additional am o acids may be processed away from tlie mature protem by cellular enzymes For each and every polynucleotide of tlie mvention tliere is pro ided a polynucleotide complementary to it It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide with winch they are complementary A precursor protem. having a mature fonn of tiie pohpeptide fused to one or more prosequences may be an inactive fonn of tiie polypeptide When prosequcnces aie remov d such mactive precursors generally are activated Some or all of tlie prosequences may be remov ed before activation Generally, such precursors are called proprotems In addition to the standard A, G, C. T/U representations for nucleotides. the term "N" may also be used in describing certain polynucleotides of the invention "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position m the DNA or RNA sequence except it is preferred that N is not a nucleic acid that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame would have the effect of generating a premature termination codon in such reading frame In sum, a polynucleotide ofthe mvention may encode a mature protein, a matuie protem plus a leader sequence (winch may be referred to as a preprotem). a precursor of a mature protem having one or more prosequences that are not tiie leader sequences of a preprotem. or a preproprotem. winch is a precursor to a proprotem. having a leader sequence and one or more prosequences. winch generally are removed duπng processuig steps that produce active and mature fonns oftiie polypeptide Vectors, Host Cells, Expression Systems The mvention also relates to vectors tiiat compnse a polynucleotide or poly nucleotides of tlie mvention. host cells that are genetically engineered with vectors oftiie mvention and tiie production of 12 polypeptides of tlie mvention by recombinant techniques Cell-free translation systems can also be employed to produce such protems usmg RNAs denved from tlie DNA constructs oftiie mvention Recombinant polypeptides oftiie present mvention may be prepared by processes well known ui tiiose skilled in tlie art from genetically enguieered host cells comprising expression sv stems Accorduigly. ui a further aspect, tlie present mvention relates to expression sv stems which comprise a polynucleotide or polynucleotides ofthe present mvention. to host cells winch are genetically engmeered with such expression systems, and to tlie production of polypeptides oftiie invention by recombuiant techniques For recombuiant production of tiie polypeptides of tiie invention, host cells can be genetically engmeered to incorporate expression sv stems or portions thereof or polynucleotides of the inv ention Introduction of a polynucleotide mto the host cell can be effected by methods described in mam standard laboratory manuals, such as Davis, et al . BASIC METHODS IN MOLECULAR BIOLOG) (1986) and Sambrook. et al . MOLECULAR CLONING A LABORATORY MAX UAL 2nd Ed Cold Spring Harbor Laboratory Press. Cold Spnng Harbor. N Y (1989). such as. calcium phosphate transfection. DEAE-dextran mediated transfection. transvection. nncrouijection. cationic pid-mediated transfection. electroporation. transduction. scrape loadmg. ballistic introduction and mfection Representative examples of appropnate hosts mclude bacterial cells, such as cells of streptococci, staphylococci. enterococci E coh. streptomy ces. cy anobacteπa. Bacillus sublihs and Staphylococcus aureus. fungal cells, such as cells of a yeast. Kluveromyces Saccharomyces. a basidiomycete, Candida albicans and Aspergillus. sect cells such as cells of Drosophila S2 and Spodoptera Sf9. animal cells such as CHO. COS. HeLa. C127. 3T3. BHK. 293. CVJ and Bowes melanoma cells, and plant cells, such as cells of a gynmospenn or angiospenn A great vanety of expression sy stems can be used to produce the polypeptides of tiie mvention Such vectors include, among others, chromosomal-, episomal- and v iras-denved vectors for example, vectors denved from bacterial plasnnds. from bactenophage from transposons. from yeast episomes. from insertion elements, from yeast chromosomal elements, from v iruses such as baculoviruses, papova viruses, such as SV40. vaccuua virases. adenoviruses. fowl pox virases pseudorabies virases. picomavirases and retrovirases. and vectors denved from combu ations thereof such as tiiose denved from plasnnd and bactenophage genetic elements, such as cosnuds and phagenuds Tlie expression system constnicts may contaui control regions that regulate as well as engender expression Generally, any svstem or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression in this regard The appropnate DNA sequence may be mserted mto tiie expression system by any of a variety of well- known and routme techniques, such as. for example, tiiose set forth m Sambrook et al . MOLECULAR CLONING A LABORATORY MANUAL, (supra) In recombinant expression systems in eukaryotes. for secretion of a translated protem mto tlie lumen of tiie endoplasmic reticulum. mto tlie penplasnnc space or into tiie extracellular environment, appropnate secretion signals may be uicorporated mto tlie expressed poly peptide These signals may be endogenous to tlie polypeptide or they may be heterologous signals Polypeptides ofthe mvention can be recovered and punfied from recombuiant cell cultures by well-known methods uicluduig ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography. phosphocellulose chromatography. hydrophobic uiteraction cliromatographv affinity chromatography hvdroxylapatite chromatography and lectin cliromatography Most preferabh'. high perfonnance liquid chromatography is employed foi puπfication Well known techniques for refolduig protein mav be employed to regenerate active confonnation when tlie polypeptide is denatured duruig isolation and or purification Diagnostic, Prognostic, Serotyping and Mutation Assays Tins mvention is also related to tlie use of nrdG poly/nucleotides and polypeptides of tiie mvention for use as diagnostic reagents Detection of nrdG polynucleotides and/or polypeptides ui a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staguig of disease or response of an infectious orgamsm to drags Eukarvotes particularly mammals, and especially humans, particularly tiiose mfected or suspected to be infected with an orgamsm compnsmg tlie nrdG gene or protem, may be detected at tlie nucleic acid or ammo acid level by a vanety of well known techniques as well as by methods provided liereui Polypeptides and polynucleotides for prognosis, diagnosis or other analv sis may be obtamed from a putatively mfected and/or mfected individual's bodily matenals Polynucleotides from any of these sources, particularly DNA or RNA. may be used directly for detection or mav be amplified enzymatically by usmg PCR or εmy other amplification technique pnor to analv sis RNA particularly mRNA, cDNA and genonnc DNA may also be used ui tiie same way s Using amplification charactenzation of tlie species and stram of mfectious or resident orgamsm present in an individual may be made by an analysis of tiie genotype of a selected polynucleotide of tiie orgamsm Deletions and insertions can be detected by a change ui size oftiie amplified product in companson to a genotype of a reference sequence selected from a related organism, preferably a different species of tiie same genus or a different stram of tiie same species Point mutations can be identified bv hv bπdizuig amplified DNA to labeled nrdG polynucleotide sequences Perfectly or significantly matched sequences can be distinguished from unperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively . or by detectuig differences m melting temperatures - 14 - or renaturation kinetics Polynucleotide sequence differences may also be detected by alterations m tlie electrophoretic mobility of polynucleotide fragments ui gels as compared to a reference sequence Tins may be earned out with or without denaturuig agents Polynucleotide differences may also be detected by direct DNA or RNA sequencing See. for example. Myers et al . Science, 230 1242 (1985) Sequence changes at specific locations also may be revealed by nuclease protection assay s. 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) hi another embodunent. an array of ohgonucleotides probes compnsmg nrdG nucleotide sequence or fragments thereof can be constracted to conduct efficient screenmg 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 variety of questions m molecular genetics lncluduig gene expression, genetic linkage, and genetic variability (see for example Chee et al . Science, 274 610 (\996)) Thus in another aspect, the present invention relates to a diagnostic kit which comprises (a) a poly nucleotide ofthe 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 ofthe present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibody to a polypeptide ofthe 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 Tins invention also relates to tlie use of polynucleotides oftiie piesent invention as diagnostic reagents Detection of a mutated fonn of a polynucleotide ofthe invention, preferable SEQ ID NO 1 which is associated with a disease or pathogemcity will provide a diagnostic tool tiiat can add to or define, a diagnosis of a disease, a prognosis of a course of disease, a deteπmnation of a stage of disease, or a susceptibility to a disease, winch results from under-expression. over-expression or altered expression oftiie polynucleotide Org∑unsms. particularly mfectious organisms, carryuig mutations ui such polynucleotide may be detected at tiie polynucleotide level by a variety of techniques, such as those desenbed elsewhere hereui Tlie nucleotide sequences oftiie present invention are also valuable for organism chromosome identification The sequence is specifically targeted to. and can hybndize w itii, a particular location on - 15 - an organism's chromosome, particularly to a Staphylococcus aureus chromosome Tlie mapping of relevant sequences to chromosomes accorduig to tlie present mvention may be an important step u correlating tiiose sequences with pathogenic potential and/or an ecological niche of an organism and/or drag resistance of an orgamsm. as well as tiie essentiality oftiie gene to tiie orgamsm Once a sequence has been mapped to a precise chromosomal location, tiie physical position ofthe sequence on tiie chromosome can be correlated with genetic map data Such data may be found on-line in a sequence database Tlie relationship between genes and diseases that hav e been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (coinhentance of physically adjacent genes) or matmg studies The differences in a polynucleotide and/or polypeptide sequence betvv een organisms possessing a first phenoty pe and organisms possessing a different, second different phenotv pe 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 phenotv pe then the mutation is likelv to be the causative agent of the first phenotype Cells from an orgamsm carryuig mutations or polymorphisms (alle c variations) in a polynucleotide and/or polypeptide of the invention may also be detected at tiie polynucleotide or polypeptide level by a variety of techmques. to allow for serotypuig. for example For example. RT- PCR can be used to detect mutations ui tlie RNA It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as. for example. GeneScan RNA cDNA or genonnc DNA may also be used for tiie same purpose. PCR As an example PCR primers complementary to a polynucleotide encodmg nrdG polypeptide can be used to identify and analyze mutations These pruners may be used for. among other things, amplifying nrdG DNA and or RNA isolated from a sample denved from an individual, such as a bodily mateπal The pruners may be used to amplify a polynucleotide isolated from an mfected individual, such that tlie polynucleotide may then be subject to various techmques for elucidation oftiie polynucleotide sequence In tins vvav mutations m tlie polynucleotide sequence may be detected and used to diagnose and/or prognosc the infection or its stage or course, or to serotype and/or classify tlie mfectious agent Tlie mvention further provides a process for diagnosing, disease, preferabh bacteπal 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 NO 1] Increased or decreased expression of a nrdG 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 - 16 PCR. RT-PCR, RNase protection. Northern blotting, spectrometry and other hybridization methods hi addition, a diagnostic assay ui accordance with the invention for detecting over-expression of nrdG polypeptide compared to normal control tissue samples mav be used to detect tlie piesence of an infection, for example Assay techniques tiiat can be used to determine les els or a nidG polypeptide. ui a sample denved from a host, such as a bodily material, are ell -blown to those of skill m the art. Such assay methods mclude radiounniunoassays. competitive-bmdmg assays. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays Differential Expression Tlie polynucleotides and polynucleotides of tlie mvention mav be used as reagents for differential screening methods Tliere are many differential screening and differential display methods known m tiie art ui winch the polynucleotides and polypeptides of tiie mvention mav be used Foi example, the differential display technique is described by Chuang et al . J Bactenol 175 2026-2036 (1993) This method identifies those genes which are expressed m an orgamsm by identifying niRNA present using randomly-primed RT-PCR By comparing pre-mfection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF "unknowns " In Vivo Expression Technology (IVET) is desciibed by Cannlli e i al Proc NaT! Acad Sci USA 91 2634-2638 (1994) IVET identifies genes up-regulated during infection when compared to laboratory cultivation, implying an important role in infection ORFs identified by this technique are implied to have a significant role m infection establishment and/or maintenance In this technique random chromosomal fragments of target orgamsm are cloned upstream of a promoter-less recombinase gene a plasmid vector This construct is introduced into the target orgamsm which carries an antibiotic resistance gene flanked bv resolvase sites Growth m the presence of the antibiotic removes from the population those fragments cloned into the plasmid vector capable of supporting transcription of the recombinase gene and therefore have caused loss of antibiotic resistance The resistant pool is introduced into a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance The chromosomal fragment carried by each antibiotic sensitiv e bacterium should carry a promoter or portion of a gene normally upregulated during infection Sequencing upstream of the recombinase gene allows identification of the up regulated gene RT-PCR may also be used to analyze gene expression patterns For RT PCR using the polynucleotides of the invention, messenger RNA is isolated from bacterial mfected tissue e g 48 hour murme lung infections, and the amount of each niRNA species assessed bv reverse - 17 - transcription of the RNA sample primed with random hexanucleotides followed bv PCR with gene specific primer pairs The determination of the presence and amount of a particular mR A species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed in the infected tissue Analysis of gene transcription can be carried out at different times of infection to gain a detailed knowledge of gene regulation m bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacteπals Because of the gene specific nature of the PCR primers employed it should be understood that the bacterial mRNA preparation need not be free of mammalian RNA This allows the investigator to carry out a simple and quick RNA preparation from mfected tissue to obtain bacterial mRNA species which are very short lived in the bacterium (in the order of 2 minute halfhves) Optimally the bacterial mRNA is prepared from infected muπne lung tissue by mechanical dismption m the presence of TRlzole (GIBCO-BRL) for very short periods of time, subsequent processing atøøording to the manufacturers of TRlzole reagent and DNAase treatment to remove contaminating DNA Preferably the process is optimized by finding those conditions which give a maximum amount of Staphylococcus aureus 16S πbosomal RNA as detected by probing Northerns with a suitably labeled sequence specific oligonucleotide probe Typically a 5' dye labeled primer is used in each PCR primer pair in a PCR reaction which is terminated optimally between 8 and 25 cycles The PCR products are separated on 6% polyacrylannde gels with detection and quantification using GeneScanner (manufactured by ABI) Antibodies Tlie polypeptides and polynucleotides oftiie invention or vanants thereof, or cells expressing tiie same can be used as uiimunogens to produce antibodies lmmunospccific for such po peptides or polynucleotides respectively hi certaui preferred embodunents of the mvention tliere are prov ided antibodies against nrdG polypeptides or polynucleotides Antibodies generated agauist tlie polypeptides or polynucleotides of tlie mvention can be obtamed by administering tiie polypeptides and/or polynucleotides of tlie mvention. or epitope-beanng fragments of either or both, analogues of either or both, or cells expressmg either or both, to an animal. preferably a nonhuman. usmg routuie protocols For preparation of monoclonal antibodies, any technique known ui tiie art that provides antibodies produced by continuous cell line cultuies can be used Examples mclude vanous techmques. such as tiiose in Koliler. G and Milstem. C . Nature 256 495-497 (1975). Kozbor et al . Immunology Today 4 72 (1983). Cole et al , pg 77-96 ui MONOCLONAL ANTIBODIES AND CANCER THERAPY. Alan R Liss. Inc (1985) Techniques for tlie production of single chain antibodies (U S Patent No 4 946 778) can be adapted to produce suigle chaui antibodies to polypeptides or polynucleotides of this ins ention Also transgemc nnce. or otiier orgamsms such as otiier mammals, may be used to express humanized antibodies unmunospecific to tiie polypeptides or polynucleotides oftiie mvention Alternatively, phage display technology may be utilized to select antibody genes with binding activities tosvards a pol peptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-nrdG or from naive libraries (McCafferty. et al . ( 1990). Nature 348. 552-554. Marks et al . ( 1992) Biotechnology 10. 779- 783) The affinity of these antibodies can also be improved bv . for example chain shuffling (Clackson et al . (199 \) Nature 352 628) Tlie above-descπbed antibodies may be employed to isolate or to identify' clones expressing tiie polypeptides or polynucleotides of tlie invention to punfy tlie polypeptides or polynucleotides by. for example, affinity chromatography Thus among others, antibodies against nrdG-pohpeptide or nrdG-polv nucleotide mav be employed to treat uifections. particulai ly bacterial mfections Polypeptide variants include antigenically. epitopically or mimunologicallv equiv alent variants fomi a particular aspect of this invention A polypeptide or polynucleotide of the invention, such as an antigenically or immunologically equivalent derivative or a fusion protem of the polypeptide is used as an antigen to immunize a mouse or other animal such as a rat or chicken The fusion protem may provide stability to the polypeptide The antigen may be associated, for example bv conjugation with an immunogenic carrier protein for example bovme serani albumin, keyhole limpet haemocv anm oi tetanus toxoid Alternatively, a multiple antigeinc polypeptide comprising multiple copies of the polypeptide. or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigeinc to improve lmmunogenicity so as to obviate the use of a carrier Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual For example if the individual is human the antibody mav most preferably be "humanized." where the comphmentaπty determining region or regions of the hv biidoma-deπv ed antibody has been transplanted mto a human monoclonal antibody for example as described Jones et al (1986). Nature 321. 522-525 or Tempest et al . (1991 ) Biotechnology 9, 266-273 In accordance with an aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization Among tiie particularly preferred embodunents oftiie invention are naturally occurruig allehc vanants of nrdG polynucleotides and polypeptides encoded thereby - 19 - The use of a polynucleotide of the invention m genetic immunization w ill preferabh employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al Hum Mol Genet (1992) 1 363. Manthorpe et al , Hum Gene Ther (1983) 4 419). delivers of DNA complexed with specific protem carriers (Wu et al , J Biol Chem (1989) 264 16985) coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef. PNAS USA (1986) 83 9551) encapsulation of DNA in various forms of hposomes (kaneda et al Science (1989) 243 375). particle bombardment (Tang et al Nature ( 1992) 356 152 Eisenbraun et al DNA Cell B ol (1993) 12 791) and in vivo infection usmg cloned retrov iral v ectois (Seeger c l al . PNAS USA (1984) 81 5849) Antagonists and Agonists - Assays and Molecules Pohpeptides and polynucleotides of tlie mvention may also be used to assess the bmduig of small molecule substrates and ligands ui. 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 stractural or functional mrmetics See. e g Cohgan et al Current Protocols in Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides oftiie present mvention are responsible for many biological functions, mcluduig many disease states, ui particular tlie Diseases hereinbefore mentioned It is therefore desirable to devise screemng methods to identify compounds which stunulate or winch uihibit tiie function oftiie polypeptide or polynucleotide Accorduigly, in a further aspect, tiie present mvention provides for a method of screemng compounds to identify tiiose which stimulate or winch inhibit tlie function of a polypeptide or polynucleotide oftiie invention as well as related po peptides and polynucleotides hi general, agonists or antagonists may be employ ed for therapeutic and prophy lactic purposes for such Diseases as nereuibefore mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures Such agonists, antagomsts or uilnbitors so-identified may be natural or modified substrates, ligands. receptors, enzymes, etc . as the case may be. of nrdG polypeptides and polynucleotides, or may be structural or functional mmietics thereof (see Cohgan et al Current Protocols in Immunology 1(2) Chapter 5 (1991)) The screening methods may simply measure the bmduig of a candidate compound to the polypeptide or polynucleotide. or to cells or membranes bearing the polypeptide or polynucleotide. or a fusion protem ofthe polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition vv ith a labeled competitor Further, these screening methods mav test w hether the candidate compound results m a signal generated by activ ation or inhibition of the po peptide or - 20 - polynucleotide. using detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a know n 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 poly peptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation ofthe polypeptide or polynucleotide. as the case may be Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide ofthe present invention, to fonn a mixture. measuring nrdG polypeptide and/or polynucleotide activity in the mixture, and comparing the nrdG polypeptide and/or poly nucleotide activity of the mixture to a standard Fusion proteins such as those made from Fc portion and nrdG pohpeptide as heieinbefore desci ibed can also be used for high-throughput screening assay s to identify antagonists of the po peptide of the piesent invention, as well as of phylogenetically and and/or functionally related poly peptides (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)) Tlie polynucleotides. pohpeptides 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/oi poh peptide in cells For example, an ELISA assay may be constracted 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 which 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 screemng compounds to identity those which enhance (agomst) or block (antagomst) tlie action of nrdG pohpeptides or poh nucleotides paiticulaih those compounds that are bactenostatic and/or bacteriocidal Tlie method of scieening may inv olv e 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 am thereof, compnsmg nrdG polypeptide and a labeled substrate or ligand of such polypeptide is mcubated in the absence or the presence of a candidate molecule that may be a nrdG agomst or antagonist Tlie ability of tiie candidate molecule to agonize or antagonize the nrdG po peptide is reflected in decreased binding of tiie labeled ligand or decreased production of product from such substrate Molecules that bind gratuitously, / e . without mducmg tiie effects of nrdG polypeptide are most likely to be good antagomsts Molecules that bmd well and, as tlie case may be. uicrease tiie rate of product production - 21 - from substrate, uicrease signal transduction. or uicrease chemical channel activity are agonists Detection of tlie rate or level of. as tiie case may be. production of product from substrate, signal transduction. or chemical channel activity mav be enhanced bv using a reporter sy stem Reportei systems that may be useful ui tins regard clude but are not lumted to coloπmetπc labeled substrate converted mto product, a reporter gene that is responsive to changes ui nrdG polynucleotide 01 polypeptide activity, and bmduig assays known in tiie art Polypeptides ofthe invention may be used to identify membrane bound or soluble receptors, if any . for such poly peptide. through standard receptor binding techmques known in the art These techniques include, but are not limited to. ligand binding and crosshnking assay s m which the polypeptide is labeled with a radioactive isotope (for instance. ' ^I). chemically modified (for instance, biotmylated). or fxiscd to a peptide sequence suitable for detection oi purification, and incubated with a source of the putative receptor (e g . cells, cell membranes, cell supeniatants. tissue extracts, bodily materials) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopv These screening methods may also be used to identify agonists and antagonists ofthe polypeptide which compete with the binding of the polypeptide to its receptor(s). if any Standard methods for conducting such assays are well understood in the art hi otiier emboduiients of tiie mvention there are provided methods for identify ing compounds which bmd to or otherwise interact with and uilnbit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsmg contactuig a polypeptide and/or polynucleotide oftiie mvention with a compound to be screened under conditions to pemnt bmduig to or otiier interaction between tiie compound and the polypeptide and/or polynucleotide to assess tiie bmduig to or other mteraction with the compound, such bmduig or interaction preferabh being associated with a second component capable of providmg a detectable signal ui response to the bmduig or mteraction of tiie polypeptide and/or polynucleotide with tiie compound, and determunng whetiier the compound bmds to or otherwise mteracts with and activates or mhtbits an activity or expiession of the polypeptide and/or polynucleotide by detectmg tlie presence or absence of a signal generated from the binding or mteraction oftiie compound with tiie polypeptide and/or polynucleotide Another example of an assay for nrdG agomsts is a competitive assay that combuies nrdG and a potential agomst with nrdG-binduig molecules, recombuiant nrdG binding molecules natural substrates or ligands, or substrate or ligand mimetics. under appropriate conditions foi a competitive inhibition assay NrdG can be labeled, such as bv radioactiv ity oi a colonmetiic compound such that tlie number of nrdG molecules bound to a bmduig molecule or converted to product can be detennined accurately to assess tiie effectiveness oftiie potential antagomst - 22 - Potential antagonists mclude. among others, small organic molecules, peptides. polypeptides and antibodies that bind to a polynucleotide and/or poly peptide of the invention and thereby inhibit or extmguish its activity or expression Potential antagonists also may be small organic molecules, a peptide. a polypeptide such as a closely related protem or antibody that binds tiie same sites on a bmduig molecule, such as a bmduig molecule, without uiducing nrdG-mduced activities, tiiereby preventing tiie action or expression of nrdG polypeptides and/or polynucleotides by excluduig nrdG polypeptides and/or polynucleotides from binduig Potential antagonists mclude a small molecule tiiat buids to and occupies the binding site of tiie polypeptide thereby prevent g bmduig to cellular binding molecules such that noimal biological activity is prevented Examples of small molecules mclude but are not limited to small organic molecules, peptides or peptide-like molecules Otiier potential antagomsts clude antisense molecules (see Okano. ./ Neurochem 56 560 (1991). OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION CRC Press. Boca Raton. FL (1988). for a description of these molecules) Preferred potential antagomsts clude compounds related to and vanants of nrdG Otiier examples of potential polypeptide antagonists include antibodies or some cases, ohgonucleotides or protems winch are closely related to the ligands. substrates, receptors cnzsmes etc . as the case may be. oftiie polypeptide, e g . a fragment oftiie ligands. substrates, receptors enzymes, etc , or small molecules which bmd to tlie polypeptide oftiie present mvention but do not elicit a response, so that tlie activity oftiie polypeptide is prevented Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands receptors, substrates, enzymes, etc for a po peptide and or polynucleotide of the present invention, or compounds which decrease oi enhance the production of such polypeptides and/or polynucleotides . which composes (a) a polypeptide and/or a polynucleotide ofthe present invention. (b) a recombinant cell expressing a polypeptide and/or polynucleotide ofthe present invention. (c) a cell membrane expressing a polypeptide and/or polynucleotide ofthe present invention, or (d) antibody to a polypeptide and/or polynucleotide ofthe present invention. which poly peptide is preferably that of SEQ ID NO 2. and which polynucleotide is preferabh that of SEQ ID NO 1 It will be appreciated that in any such kit. (a), (b). (c) or (d) may comprise a substantial component It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide ofthe present invention may also be used in a method for the stnicture-based design of an agonist, antagonist or inhibitor of the polypeptide and/or polynucleotide. by - 23 - (a) determining m the first instance the three-dimensional stnicture of the poh peptide and/oi polynucleotide. or complexes thereof, (b) deducing the three-dimensional stracture for the likely reactive sιte(s), binding sιte(s) or motιf(s) of an agonist, antagomst or inhibitor. (c) synthesizing candidate compounds that are predicted to bind to or react ith the deduced binding sιte(s). reactive sιte(s). and/or motifrs). and (d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors It will be further appreciated that this will noπnally be an iterative process, and this iterative process may be perfoπned using automated and computer-controlled steps In a further aspect, tiie present mvention provides methods of treatmg abnonnal 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 nrdG polypeptide and/or polynucleotide If tiie expression and/or activity ofthe pohpeptide and/or polynucleotide is in excess several approaches are available One approach comprises administering to an u div ldual need thereof an inhibitor compound (antagomst) as liereui descnbed. optionally in combination ss itli a phanuaceuticallv acceptable earner, m an amount effective to mhibit tl e function and/or expression oftiie po peptide and/or polynucleotide. such as. for example, by blockuig tiie bmduig of ligands. substrates, receptors, enzymes, etc . or by uilnbituig a second signal, and tiiereby alleviatmg tlie abnonnal condition In another approach, soluble fonns ofthe polypeptides still capable of binding the ligand. substrate. enzymes, receptors, etc m competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the nrdG pohpeptide and/or polypeptide In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions ofthe constant regions of heavy or light chains of lmmimoglobuhns of various subclasses (IgG. IgM. IgA. IgE) Preferred as an lmmunoglobulin is the constant part of the heavy chain of human IgG. particularly IgGl. where fusion takes place at the hinge region In a particular embodiment, the Fc part can be remov ed simply bv incorporation of a cleav age sequence which can be cleaved with blood clotting factor Xa Furthermore, this mv ention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drag screening, diagnosis and therapy A further aspect of the inv ention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914 - 24 In still another approach, expression ofthe gene encoding endogenous nrdG polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation Ai examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see. for example. O ' Connor J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression CRC Press Boca Raton. FL (1988)) Alternatively, oligonucleotides which form tuple helices ith 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 Each of the polynucleotide sequences provided herein may be used m the discov ers and development of antibacterial compounds The encoded protein upon expression can be used as a target for the screening of antibacterial drags Additionally , the po nucleotide sequences encodmg the annno terminal regions ofthe encoded protein or Shine-Delgarno or otliei translation facilitating sequences of the respective mRNA can be used to constnict antisense sequences to control the expression ofthe coding sequence of interest The invention also provides the use of the polypeptide. polynucleotide. agonist or antagonist of the mvention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic. preferabh mammalian, host responsible for sequelae ot infection In particular, the molecules of the invention may be used in the prevention ot adhesion ot bactei ia in particular gram positive and/or gram negative bacteria, to eukary otic preferabh mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix protems in wounds, to block nrdG protein-mediated mammalian cell invasion by. for example, initiating phosphors lation of mammalian tyrosine kinases (Rosenshine et al . Infect Immun 60 221 1 (1992). to block bacterial adhesion between eukaryotic. preferabh mammalian extracellular matrix proteins and bacterial nrdG proteins that mediate tissue damage and/or to block the normal progression of pathogenesis in infections initiated other than by the implantation of indwelling devices or by other surgical techniques hi accordance with yet another aspect oftiie invention, tliere are provided nrdG agonists and antagomsts. preferably bacteπostatic or bactenocidal agonists and antagomsts The antagomsts and agonists of tiie invention may be employed, for instance, to prevent mhibit and/or treat diseases Hehcobacter pylori (herein "H pylori") bacteria infect the stomachs of ov er one-thud ot the world's population causing stomach cancer, ulcers, and gastritis (International Agency foi - 25 - Research on Cancer (1994) Schistosυmes. Liver Flukes and Hehcυbacter Pylori (International Agency for Research on Cancer, Lyon. France, http //www uicc clι/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and- effect relationship between H pylori and gastric adenocarcinoma. classify ing the bacteπum as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of nrdG polypeptides and/or polynucleotides) found using screens provided by the invention, or known m the art. particularly narrow-spectram antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H pylon-mduceά cancers such as gastrointestinal carcinoma Such treatment should also prev ent. inhibit and/or cure gastric ulcers and gastritis Vaccines Tliere are provided by tlie mvention. products, compositions and methods for assessing nrdG expression, treatmg disease, assay ing genetic vanation. and administering a nrdG poly peptide and/or polynucleotide to an orgamsm to raise an unmunological response against a bactena. especially a Staphylococcus aureus bacteria Another aspect of the invention relates to a method for inducing an miniunological response m an individual, particularly a mammal which comprises inoculating the indiv idual with nrdG polynucleotide and/or polypeptide. or a fragment or variant thereof adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus infection Also provided are methods whereby such unmunological response slows bacterial replication Yet another aspect of the invention relates to a method of inducing lmmunological response in an individual which comprises deliv enng to such individual a nucleic acid vector, sequence or πbozv me to direct expression of nrdG polynucleotide and/or poly peptide. or a fragment or a v aπant thereof for expressing mdG polynucleotide and/or polypeptide. or a fragment oi a v ariant theieof //-? vivo in order to induce an lmmunological response, such as. to produce antibody and/ or T cell immune response, including, for example, cytokine-producmg T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not One example of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise Such nucleic acid vector may comprise DNA. RNA a πbozyme. a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA- protein complex A further aspect of the invention relates to an lmmunological composition that when introduced into an individual, preferably a human, capable of having induced within it an - 26 - lmmunological response induces an lmmunological response in such individual to a nrdG polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant nrdG polynucleotide and/or poh peptide encoded therefrom and/or composes DNA and or RNA which encodes and expresses an antigen of said nrdG poly nucleotide poh peptide encoded therefrom, or other polypeptide of the invention The lmmunological response mav be used tlierapeutically or prophylactically and may take the fonn of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells A nrdG polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not bv itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which ss ill hav e antigenie and/or immunogenic properties, and preferably protective properties Thus fused recombinant protem preferably further composes an antigeinc co-protein, such as hpoprotcin D from Hemophilus influenzae. Glutathione-S-transferase (GST) or beta-galactosidase. or any other relatively large co-protein which solubihzes the protein and facilitates production and purification thereof Moreover, the co-protein mav act as an adjuvant in the sense of providing a geneiahzed stimulation of the immune system of the orgamsm receiv ing the protein The co-protein mav be attached to either the ammo- or carboxv -teoninus of the first protein Provided by this invention are compositions, particularly v accine compositions and methods comprising the polypeptides and/or polynucleotides of the invention and lmiminostiniulatory DNA sequences, such as those described in Sato. Y et al Science 273 352 (1996) Also, provided by this invention are methods using the described poly nucleotide or particular fragments thereof, which have been shown to encode non-v ariable regions of bacterial cell surface proteins, in polynucleotide constnicts used in such genetic immunization expei intents in animal models of infection with Staphylococcus aureus Such experiments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value derived from the requisite organ of the animal successfully resisting oi clearing infection tor the development of prophy lactic agents or therapeutic treatments of bacterial infection paiticulai h Si phx lυcotcus am ens infection in mammals, particularly humans A polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue Examples of tissue damage include wounds m skin or - 27 - connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage oi by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth throat, mammary glands, urethra or vagina The invention also includes a vaccine foniiulation which composes an immunogenic recombinant polypeptide and/or polynucleotide of the invention together w itli a suitable carrier, such as a pharmaceutically acceptable carrier Since the polypeptides and polynucleotides may be broken down m the stomach, each is preferabh administered paienteralls including foi example, administration that is subcutaneous intramuscular, intiav enous oi mtradennal Foonulations suitable for parenteral administration include aqueous and non-aqueous stenle mjection solutions which may contain anti-oxidants. buffers, bactenostatic compounds and solutes which render the foonulation isotonic with the bodily fluid, preferabh the blood, of the individual, and aqueous and non-aqueous sterile suspensions which mav include suspending agents or thickening agents The formulations mav be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and mav be stored in a freeze-di led condition requiring only the addition of the sterile liquid carrier immediately poor to use The vaccine foonulation may also include adjuvant systems for enhancing the lmmunogenicit of the formulation, such as oil-m water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation While the invention has been described with reference to certain nrdG po peptides and polynucleotides. it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides. and similar polypeptides and poly nucleotides w ith additions deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant polypeptides or polynucleotides Compositions, kits and administration In a further aspect of tiie mvention tliere are provided compositions composing a nrdG polynucleotide and/or a nrdG polypeptide for adnumstration to a cell or to a multicellular organism Tlie mvention also relates to compositions composing a polynucleotide and/or a poh peptides discussed herein or their agonists or antagomsts The polypeptides and polynucleotides ofthe invention may be employed m combmation with a non-stenle or stenle earner or carriers for use ith cells. tissues or orgamsms. such as a pharmaceutical earner suitable for administration to an individual Such compositions comprise, for instance, a media additive or a tlierapeutically effective amount of a pohpeptide and or polynucleotide of tlie invention and a pharmaceutically acceptable carrier or excipient Such earners may include, but are not lumted to. saline, buffeied saline dextrose svatei - 28 - glycerol. ethanol and combuiations tiiereof Tlie formulation should suit the mode of administration The mvention further relates to diagnostic and phannaceutical packs and kits compnsmg one or more containers filled vv itli one or more of tlie ingredients of tlie aforementioned compositions of tiie mvention Polypeptides. polynucleotides and otiier compounds of tiie mvention may be employ ed alone or m conjunction with otiier compounds, such as therapeutic compounds The pharmaceutical compositions may be administered in any effective, convenient mannei uicluduig, for uistance. administration by topical, oral. anal, vaguial. intrav enous, lntrapeπtoneal. mtramuscular. subcutaneous, uitranasal or uitradennal routes among others In therapy or as a prophylactic, the active agent may be administered to an individual as an liijectable composition, for example as a sterile aqueous dispersion, preferably lsotonic Alternatively the composition may be fonnulated for topical application for example m the form of ointments, creams lotions ev e ointments ev diops eai drops mouthwash. impregnated dressings and sutures and aerosols, and mav contain appropriate conventional additives, including, for example, preservatives, solvents to assist diug penetration and emollients m ointments and creams Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oley l alcohol for lotions Such carriers may constitute from about 1 % to about 98% bv ss eight of the formulation more usually they will constitute up to about 80% by weight of the formulation hi a further aspect, tlie present invention provides for pharmaceutical compositions comprising a tlierapeutically effective amount of a polypeptide and or polynucleotide. such as the soluble foim of a polypeptide and/or polynucleotide of tlie present mvention. agomst or antagomst peptide or small molecule compound, ui combmation with a phannaceutically acceptable carrier or excipient Such earners mclude. but are not lumted to, saluie. buffered salme. dextrose, water, glycerol. ethanol. and combuiations tiiereof Tlie invention further relates to phannaceutical packs and kits compnsmg one or more contauiers filled ith one or more oftiie uigredients oftiie aforementioned compositions of the mvention Polypeptides, polynucleotides and otiier compounds of the present inv ention mav be employed alone or ui conjunction with otiier compounds, such as therapeutic compounds Tlie composition will be adapted to tlie route of adnninstration. for instance by a sv stennc or an oral route Preferred fonns of systemic administration mclude injection, typically by intravenous mjection Other uηection routes, such as subcutaneous, mtramuscular. or uitrapeπtoneal. can be used Alternative means for systemic administration mclude transmucosal and transdennal administration usmg penetrants such as bile salts or ftisidic acids or otiier detergents In addition if a pohpeptide or other compounds of tlie present invention can be fonnulated ui an enteoc or an encapsulated - 29 - formulation, oral admunstration may also be possible Admmistration of these compounds mav also be topical and or localized, m tlie fonn of salves, pastes, gels, and tlie like For administration to mammals, and particularly humans it is expected that the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg t piealls around i mg/kg The physician in any event will detennine the actual dosage which w ill be most suitable tor an individual and will vary with the age. weight and response of the particular lndiv ldual The above dosages are exemplary of the average case There can. of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention In-dwelling devices include surgical implants, prosthetic devices and catheters, i e dev ices that are introduced to the body of an individual and remain in position for an extended time Such devices include, for example, artificial joints, heart valves pacemakers v asculai grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters The composition of the invention mav be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device Treatment may be continued after surgery during the ln-bodv time of the device In addition, the composition could also be used to broaden peooperative cover for anv surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections Many orthopedic surgeons consider that humans with prosthetic |omts should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation In addition to the therapy described above, the compositions of this invention mav be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to or in conjunction with, antibiotic prophylaxis Alternatively, the composition ofthe invention may be used to bathe an indwelling device immediately before insertion The active agent will preferably be present at a concentration of 1 μg/ml to 1 Omg/ml for bathing of wounds or indwelling devices A vaccine composition is conveniently in ιn|cctablc form Conv entional adjuv ants mav be employed to enhance the immune response A suitable unit dose tor v accination is 0 ^ microgram/kg of antigen, and such dose is preferably administered 1 -3 times and sv ith an interval of 1-3 weeks With the indicated dose range, no adverse toxicological effects will be observed - 30 - with the compounds of the invention which would preclude their administration to suitable individuals Sequence Databases, Sequences in a Tangible Medium, and Algorithms Polynucleotide and polypeptide sequences fonn a valuable urfonnation resource with svluch to detemnne their 2- and 3-duιιensιonal structures as well as to identify further sequences of sumlar homology These approaches are most easily facilitated by storing tiie sequence ui a computer readable medium and then us g tlie stored data m a known macromolecular stracture program or to search a sequence database usmg well known searclnng tools, such as GCC The polynucleotide and polypeptide sequences of the invention are particularly useful as components in databases useful for search analy ses as ssell as in sequence analy is algorithms As used in this section entitled "Sequence Databases Sequences in α Tangible Medium and Algorithms." and in claims related to this section, the terms "polynucleotide of the inv ention" and "poly nucleotide sequence of the invention" mean any detectable chemical or physical characteristic of a polynucleotide of the invention that is or may be reduced to or stored m a tangible medium, preferably a computer readable foon For example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases and mass spcctrographic data As used in this section entitled Databases and Algorithms and in claims related thereto the terms "polypeptide of the invention" and "polypeptide sequence of the inv ention" mean am detectable chemical or physical characteristic of a polypeptide of the invention that is or may be reduced to or stored in a tangible medium, preferably a computer readable form For example, chromatographic scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data Tlie mvention provides a computer readable medium hav ing stored theieon pols peptide sequences of the invention and/or poly nucleotide sequences of the ins ention Foi example a computer readable medium is provided composing and having stored thereon a member selected from the group consisting of a polynucleotide composing the sequence of a poly nucleotide of the invention, a polypeptide comp sing the sequence of a polypeptide sequence ofthe invention, a set of polynucleotide sequences wherein at least one of the sequences comprises the sequence of a polynucleotide sequence of the invention, a set of polypeptide sequences wherein at least one of the sequences comprises the sequence of a pohpeptide sequence ot the ins ention a data set representing a polynucleotide sequence comprising the sequence of poly nucleotide sequence of the invention, a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention, a polynucleotide composing the sequence of a polynucleotide sequence of the invention, a polypeptide composing the sequence of a polypeptide sequence of the inv ention a set ot poly nucleotide sequences ss herein at least one of the sequences comprises the sequence of a pols nucleotide sequence of the ins ention α set of polypeptide sequences wherein at least one of said sequences composes the sequence ot a polypeptide sequence of the invention, a data set representing a polynucleotide sequence comprising the sequence of a polynucleotide sequence of the invention, a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention The computer readable medium can be any composition of matter used to store information or data, including for example commercially av ailable floppy disks, tapes, chips, hard drives, compact disks, and video disks Also provided by the invention are methods for the analy sis of character sequences or strings, particularly genetic sequences or encoded genetic sequences Preferred methods of sequence analysis mclude. for example, methods of sequence homology analv sis. such as identity and similarity analy sis. RNA structure anal vsis sequence assembly cladistic analy sis sequence motif analysis open readmg frame determination nucleic acid base calling nucleic acid base trimming, and sequencing chromatogram peak analv sis A computer based method is provided for perfooinng homology identification This method comprises the steps of providing a polynucleotide sequence comprising the sequence a polynucleotide of the invention m a computer readable medium, and comparing said polynucleotide sequence to at least one polvnucleotide or polypeptide sequence to identify homology A computer based method is also provided for perfoomng homology identification said method composing the steps of providing a polypeptide sequence comprising the sequence of a polypeptide of the invention m a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology A computer based method is still further provided for polynucleotide assembly , said method composing the steps of providing a first polynucleotide sequence composing the sequence of a polynucleotide of the invention in a computer readable medium and screening for at least one overlapping region between said first polvnucleotide sequence and a second polynucleotide sequence A further embodiment ofthe invention provides a computer based method for perfooinng homology identification, said method composing the steps of providing a polvnucleotide sequence composing the sequence of a poh nucleotide of the invention in a computer readable medium and comparing said polvnucleotide sequence to at least one polvnucleotide oi polypeptide sequence to identify homology - 32 - A further embodiment ofthe invention provides a computer based method for perfoiming homology identification, said method comprising the steps of providing a polypeptide sequence composing the sequence of a polypeptide of the invention in a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology A further embodiment of the invention provides a computer based method for polynucleotide assembly , said method composing the steps of prov iding a first polv nucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium, and screening for at least one overlapping region between said first pol nucleotide sequence and a second polynucleotide sequence In another preferred embodiment of tiie mvention tliere is provided a computei readable medium having stored thereon a member selected from the group consisting of a poh nucleotide comprising the sequence of SEQ ID NO 1. a polypeptide comprising the sequence of SEQ ID NO 2. a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 1. a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2. a data set representing a polynucleotide sequence composing the sequence of SEQ ID NO 1. a data set representing a polynucleotide sequence encoding a polypeptide sequence composing the sequence of SEQ ID NO 2, a polynucleotide composing the sequence of SEQ ID NO 1. a polypeptide comp sing the sequence of SEQ ID NO 2. a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 1. a set of polypeptide sequences wherein at least one ot said sequences comprises the sequence of SEQ ID NO 2. a data set representing a polv nucleotide sequence comp sing the sequence of SEQ ID NO 1. a data set representing a polynucleotide sequence encodmg a polypeptide sequence composing the sequence of SEQ ID NO 2 A further preferred embodiment of the invention provides a computer based method for perfonning homology identification, said method comprising the steps of providing a polynucleotide sequence composing the sequence of SEQ ID NO 1 m a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology A still further preferred embodiment of the invention provides a computer based method for perfooinng homology identification, said method composing the steps of providing a polypeptide sequence composing the sequence of SEQ ID NO 2 in a computer readable medium and comparing said polypeptide sequence to at least one polv nucleotide oi pohpeptide sequence to identify homology A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence composing the sequence of SEQ ID NO 1 in a computer readable medium, and screening for at least one overlapping region between said first polv nucleotide sequence and a second poly nucleotide sequence A further embodiment ofthe invention provides a computer based method for performing homology identification, said method composing the steps of providing a polynucleotide sequence composing the sequence of SEQ ID NO 1 in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology A further embodiment of the inv ention provides a computer based method foi pci foi ming homology identification, said method comprising the steps of prov iding α poh peptide sequence composing the sequence of SEQ ID NO 2 in a computer readable medium, and compaung said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method composing the steps of providing a first polynucleotide sequence composing the sequence of SEQ ID NO 1 in a computer readable medium and screening for at least one overlapping region between said first polv nucleotide sequence and a second polynucleotide sequence All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference m their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated bv reference herein as being fully set forth Any patent application to which this application claims priority is also incorporated by reference herein in its entirety GLOSSARY Tlie following definitions are provided to facilitate understanding of certain terms used frequently herein ' Antιbody(ιes) ' as used herein includes polyclonal and monoclonal antibodies, chimeπc single chain, and humamzed antibodies, as well as Fab fragments, including the products of an Fab or other lnimunoglobuhn expression library "Antigenically equivalent deovatιve(s)" as used herein encompasses a poh peptide polynucleotide. or the equivalent of either which will be specifically recognized by certain antibodies which, when raised to the protem. polypeptide or polynucleotide according to the - 34 - invention, interferes vv ith the immediate phv sical interaction between pathogen and mammalian host "Bispecific antιbody(ιes)" means an antibody comprising at least two antigen binding domains, each domain directed against a different epitope "Bodily mateπal(s) means any matenal denved from an individual or from an organism uifectmg. mfestuig or uiliabitmg an uidividual. uicluduig but not lumted to. cells, tissues and waste such as. bone, blood, seram. cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, uruie. stool or autopsy materials "Dιsease(s)" means any disease caused by or related to mfection by a bacteria, uicluduig for example, disease, such as. infections oftiie upper respiratory tract (e g . otitis media bacterial tracheitis. acute epiglottitis. tliyroiditis). lower respiratory (e g . empyema. lung abscess) cardiac (e g mfective endocarditis), gastrouitestmal (e g . secretory diarrhoea, splenic absces reti opeπtoneal abscess). CNS (e g . cerebral abscess), eve (e g . blepharitis, conjunctivitis, keratitis. endophthalnutis preseptal and orbital celluhtis. darcryocystitis). kidney and uπnary tract (e g . epididvmrtis. uitiarenal and pennephπc absces. toxic shock syndrome), skin (e g . mipetigo. folhculitis. cutaneous abscesses, celluhtis. wound mfection. bactenal myositis) bone and joint (e g . septic arthritis osteomv elitis) "Fusion protem(s)" refers to a protem encoded by two often unrelated fused genes oi fragments thereof In one example. EP-A-0464 discloses fusion proteins composing v anous portions of constant region of lmmunoglobulm molecules together with another human protein or part thereof In many cases, employing an lnimunoglobulin Fc region as a part of a fusion protem is advantageous for use in therapy and diagnosis resulting in. for example, improved pharmacokinetic properties [see. e g . EP-A 0232262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed. detected and purified "Host cell(s)" is a cell which has been transfoπned or transfected or is capable of transfoonation or transfection by an exogenous polynucleotide sequence "Identity," as known in tiie art. is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as tlie case may be. as detennined by coniparuig tlie sequences hi tiie art. "identit " also means the degree of sequence relatedness between poly peptide or polynucleotide sequences, as the case may be. as detennined bv the match bet een strings of such sequences "Identity" can be readily calculated bv known methods including but not limited to those described in (Computational Molecular Biology. Lesk A M . ed Oxford Univ ersity Press. New York. 1988. Biocomputing Informatics and Genome Projects. Smith. D W . ed Academic Press. New York. 1993. Computer Analysis of Sequence Data. Part I. Griffin. A M . and Griffin. H G . eds . Humana Press. New Jersey. 1994. Sequence Analysis in Molecular Biology, von Heinje. G . Academic Press. 1987. and Sequence Analysis Primer Gπbskov M and Devereux, J , eds . M Stockton Press. New York. 1991. and Ca llo. H and Lipman. D SIAM J Applied Math , 48 1073 (1988) Methods to deteonme identity are designed to give the largest match between the sequences tested Moreover, methods to detemime identity are codified in publicly available computer programs Computer program methods to determine identity between two sequences mclude. but are not limited to. the GCG program package (Devereux J . et al . Nucleic Acids Research 12(1) 387 ( 1984)) BLASTP BLASTN and FΛSTΛ (Atschul S F et al . ./ Molec Biol 215 403-410 ( 1990) The BLAST X program is publicly av ailable from NCBI and other sources (BLAST Manual. Altschul. S . el al NCBI NLM NIH Bcthesdu MD 20894. Altschul. S . et al . ./ Mol Biol 215 403-410 (1990) The well known Smith Watennan algorithm may also be used to detennme identity Parameters for polypeptide sequence compaoson include the follow mg 1) Algorithm Needlenian and Wunscli. J Mol Biol 48 443-453 ( 1970) Compaoson 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) Parameters for polynucleotide comparison include the following 1) Algorithm Needlenian and Wunsch. J Mol Biol 48 443-453 ( 1970) Comparison matrix, matches = +10. mismatch = 0 Gap Penalty 50 Gap Length Penalty 3 Available as The "gap" program from Genetics Computer Group. Madison WI These are the default parameters for nucleic acid comparisons A preferred meaning for "identity " for polynucleotides and poly peptides as the case mav be. are provided in (1) and (2) below ( 1 ) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50. 60. 70. 80. 85. 90. 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 mteger - 36 - number of nucleotide alterations as compared to the reference sequence ss herein 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' teniimal positions ofthe reference nucleotide sequence or anywhere bet een those tenninal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence, and herein said number of nucleotide alterations is detennined by multiplying the total number of nucleotides m SEQ ID NO 1 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO 1. or n n < x n - (x n • y). wherein n n is the numbei of nucleotide alterations. \ n is the total number of nucleotides in SEQ ID NO 1. y is 0 50 for 50%. 0 60 for 60%. 0 70 for 70% 0 80 for 80% 0 for 85% 0 90 foi 90%. 0 95 for 95%. 0 97 for 97% or 1 00 for 100%. and • 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 frameslnft mutations in this coding sequence and thereby alter the polypeptide encoded by the poly nucleotide following such alterations By way of example, a polynucleotide sequence of the present inv ention mav be identical to the reference sequence of SEQ ID NO 2. that is it may be 100% identical or it mav include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion. or insertion, and wherein said alterations may occur at the 5' or 3' tenninal positions of the reference polynucleotide sequence or anywhere between those terminal positions lnteispersed either individually among the nucleic acids m the reference sequence or in one or moie contiguous groups within the reference sequence The number of nucleic acid alterations for a given percent identity is deteonined by multiplying the total number of ammo acids m SEQ ID NO 2 by the integer defining the percent identity divided bv 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2. or n n ≤ x n " ( x n • y)- wherein n n is the number of annno acid alterations, x n is the total number of ammo acids in SEQ ID NO 2, y is. for instance 0 70 for 70%. 0 80 for 80%. 0 85 for 85% etc . • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded dosvn to the nearest mteger poor to subtracting it from x n (2) Polypeptide embodiments further include an isolated po peptid compnsmg a polypeptide having at least a 50.60. 70. 80. 85. 90. 95. 97 or 100% identity to a po peptide 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 ammo acid deletion, substitution including conservative and non- conservative substitution or insertion, and wherein said alterations mas occur at the amino- or carboxy-teoiiinal positions ofthe reference polypeptide sequence oi anvwhcie between those tenninal positions, interspersed either individually among the ammo 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 detennined by multiplying the total number of annno 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 ammo acids in SEQ ID NO 2 oi n a < x a - (x a • y), wherein n a is the number of ammo acid alterations. x a is the total number of ammo acids in SEQ ID NO 2 y is 0 50 for 50%. 0 60 for 60%. 0 70 for 70%. 0 80 for 80%. 0 85 for 85% 0 90 for 90%. 0 95 foi 95%. 0 97 for 97% or 1 00 for 100%. and • 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 By way of example, a polypeptide sequence ofthe present invention mav be identical to the reference sequence of SEQ ID NO 2. that is it may be 100% identical, or it may include up to a certain integer number of annno acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consisting of at least one annno acid deletion substitution including conserv ativ e and non- conservative substitution, or insertion, and wherein said alterations mav occur at the ammo- or carboxy-teπninal positions ofthe 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 The number of amino acid alterations for a given % identity is detennined bv multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity div ided bv 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2. or n a ≤ a " ( a • )> wherein n a is the number of ammo acid alterations. x a is the total number of amino acids in SEQ ID NO 2 y is for instance 0 70 for 70% 0 80 for 80%. 0 85 for 85% etc . and • is the sy mbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded dossn to the nearest integer prior to subtracting it from x a "Immunologically equivalent deovatιve(s)" as used herein encompasses a poh peptide. poly nucleotide. or the equivalent of either which when used in a suitable formulation to raise antibodies m a vertebrate, the antibodies act to interfere with the immediate physical interaction bet een pathogen and mammalian host Jmmunospecific " means tiiat characteostic of -an antibody whereby it possesses substantially greater affinity for tiie polypeptides oftiie invention or tlie polynucleotides ofthe mvention than its affinity for otiier related polypeptides or polynucleotides respectively . particularly tiiose polypeptides and polynucleotides m the poor art "hιdιvιdual(s)" means a multicellular eukar ote. including, but not lumted to a metazoan. a mammal, an ovid. a bovid. a simian, a pπniate. and a human "Isolated" means altered "by tlie hand of man" from its natural state. / e . if it occurs in nature, it has been changed or removed from its ooguial environment, or both For example a polvnucleotide or a polypeptide naturally present ui a hvuig orgamsm is not "isolated. " but the same polvnucleotide or polypeptide separated from tlie coexistmg mateoals of its natural state is "isolated" as the tenn is employed liereui Moreover, a polynucleotide or polypeptide that is uitroduced into an orgamsm bv transfonnation. genetic mampulation or by any otiier recombmant metiiod is "isolated" even if it is still present ui said orgamsm. winch organism may be vuig or non-living "Organιsm(s)" means a (l) prokaryote. uicluduig but not limited to. a member of the genus Streptococcus Staphylococcus, Bordetel/a Corynebactenum, Mycobactenum Neissena Haemophilus, Actino ycetes, Streptomycetes Nocardia Enterobacier Yers/nia fancisella Pasturella, Moraxella, Acinetobacter, Erysipelothnx Branhame/la Actmobacillus Strepiobacillus Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium Treponema Escherichia Salmonella, Kleibsiella, Vibrio, Proteus Erwinia Borreha, Leptospira, Spirillum Campy/obacter - 39 - Shigella. Legionella, Pseudomonas. Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma. and further including, but not limited to, a member of the species or group. Group A Streptococcus. Group B Streptococcus, Group C Streptococcus, Group D Streptococcus. Group G Streptococcus. Streptococcus pneumoniac. Streptococcus pyogenes. Streptococcus aga/acuae. Streptococcus faecalis, Streptococcus faecium. Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacierium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis. Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes. Bυrdetella pertusis. Bordatella parapertusis, Bordetella bronchi septica, Escherichia co/i. Shigella dysenteriae. Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae. Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis. Proteus vulgaris. Yersinia peslis, Kleibsiella pneumoniac, Serratia marcessens, Serratia liquefaciens, Vibrio cholera. Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis. Brucella abυrtis. Bacillus anthracis. Bacillus cereus. Clostridium perfringens. Clostridium tetani. Clostridium botulinum. Treponema pallidum, Rickettsia rickcttsii and Chlamydia trachomitis. (ii) an archaeon. uicluding but not lumted to Achaebacter. and (iii) a unicellular or filamentous eukaryote. including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces. Kluveromyces, or Candida, .and a member of tiie species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans, "Polynucleotide(s)" generally refers to any polyriboiiucleotide or polyJeoxribonucleotide. which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA. DNA tiiat is a mixture of single- and double- stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA. and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions, hi addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. Tlie strands in such regions may be from tlie same molecule or from different molecules. Tlie regions may include all of one or more of the molecules, but more typically uivolve only a region of some of the molecules. One of tiie molecules of a triple-helical region often is an oligonucleotide. As used herein, tlie term "polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus. DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein. Moreover. DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are - 40 - polynucleotides as tiie tenn is used liereui It will be appreciated that a great v aπetv of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in tiie art The tenn "polynucleotιde(s)" as it is employed herem embraces such chemically, enzymatically or metabolically modified fomis of polynucleotides. as well as tlie chemical fomis of DNA and RNA charactenstic of virases and cells, uicluduig. for example, sunple and complex cells "Pohnucleotιde(s)" also embraces short polynucleotides often refened to as olιgonucleotιde(s) "Polypeptιde(s)" refers to any peptide or protein compnsmg two oi more annno acids joined to each otiier by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains commonly referred to as peptides. o gopeptides and ohgomers and to longei chains generally leterred to as protems Polypeptides may contaui annno acids other than tiie 20 gene encoded annno acids "Polypeptιde(s)" mclude tiiose modified either by natural processes, such as processing and other post- translational modifications, but also bv chemical modification techmques Such modifications are well descnbed in basic texts and in moie detailed monographs, as well as in a v oluminous research literature, and they are well known to tiiose of skill in tiie art It ill be appreciated that the same tv pe of modification may be present m tiie same or varying degree at several sites in a given pohpeptide Also, a given polypeptide may contaui many types of modifications Modifications can occur anywhere ui a polypeptide. uicluduig tlie peptide backbone, tlie annno acid side-chains, and tiie ammo or carboxyl teninni Modifications clude. for example, acetylation. acylation. ADP-nbosv lation. anndation. covalent attachment of flavin, covalent attaclnnent of a heme moiety, covalent attaclnnent of a nucleotide or nucleotide denvative. covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidyluiositol. cross-luikuig. cychzation disulfide bond fonnation. demcthv lation fonnation of covalent cross-hnks. fonnation of cvstetnc. formation of pv roglutamate foi mv lation gamma-carboxylation. glycosylation. GPI anchor formation, hy droxvlation lodination methv lation mynstoylation. oxidation, proteolytic processuig. phosphorylation. preny lation. racemization. glycosylation. lipid attaclnnent. sulfation. gamma-carboxylation of glutannc acid residues, hydroxylation and ADP-πbosylation. selenoylation, sulfation. transfer-RNA mediated addition of ammo acids to protems. such as arguiylation. and ubiquituiation See. for uistaiice. PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES. 2nd Ed . T E Creighton W H Freeman and Company, New York (1993) and Wold. F . Posttranslational Prote Modifications Perspectiv es and Prospects, pgs 1-12 ui POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS. B C Johnson. Ed . Academic Press. New York (1983), Seifter et al , Meth Enzymol 182 626-646 (1990) and Rattan et al . Protein Synthesis Posttranslational Modifications and Aging. Ann N Y Acad Sci 663 48-62 (1992) Polypeptides mav be branched or cyclic, with or without branching 41 Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well "Recombuiant expression system(s)" refers to expression systems or portions tiiereof or polynucleotides of the mvention introduced or transfonned mto a host cell or host cell lysate for tiie production oftiie polynucleotides and polypeptides oftiie mvention 'Naoant(s) " as the teon is used herein, is a polynucleotide or poh peptide that differs from a reference polynucleotide or polypeptide respectively but retains essential properties A typical variant of a polynucleotide differs m nucleotide sequence from another reference polynucleotide Changes in the nucleotide sequence ofthe variant 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 proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below A ty pical variant of a polypeptide differs in amino acid sequence from anothei refeicnce po peptide Generally differences are limited so that the sequences of the reference poh peptide and the v ai lant are closely similar overall and. in many regions, identical A variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code Tlie present invention also includes mclude variants of each of tiie polypeptides of the mvention. that is polypeptides that van' from the referents bv conservativ e annno acid substitutions whereby a residue is substituted by anotiier with like characteristics Typical such substitutions aie among Aa. Val. Leu and He. among Ser and Thr. among tlie acidic residues Asp and Glu. among Asn and Gin, and among the basic residues Lys and Arg. or aromatic residues Phe and Tyr Particularly preferred are vanants ui winch several. 5-10. 1-5. 1-3. 1-2 or 1 annno acids are substituted, deleted, or added ui any combmation A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it mav be a variant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and po peptides mav be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods kno n to skilled artisans EXAMPLES Tlie examples below are earned out using standard techmques. which are well known and routuie to tiiose of skill m tlie art. except where otherwise descnbed ui detail Tlie examples are illustrative, but do not lu t tiie invention Example 1 Strain selection, Library Production and Sequencing - 42 - The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO 1] was obtamed from a library of clones of chromosomal DNA of Staphylococcus aureus in E coll The sequencing data from two or more clones containing ovei lapping Staphylococcus aureus DNAs was used to constnict the contiguous DNA sequence in SEQ ID NO 1 Libiaπes mav be prepared by routine methods, for example Methods 1 and 2 below Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures DNA fragments of up to 1 lkbp in size aie rendered blunt by treatment with exonuclease and DNA polynierase. and EcoRI linkers added Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI. the library packaged bv standard procedures and E cυh infected with the packaged library The library is amplified by standard procedures Method 2 Total cellular DNA is partially hydrolvzed w ith a one or a combination of lestπction enzymes appropriate to generate a series of fragments for cloning into library v ectors (e g Rsal Pall. Alul. Bshl235I). and such fragments are size-fractionated according to standaid 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 coll infected with the packaged library The hbrarv is amplified by standard procedures Example 2 NrdG Characterization Nucleoside reductases (obonucleoside diphosphate reductases) products of the nrd genes, are responsible for catalysing the reduction of the 2 hy droxyl group of the ribose ring of πbonucleosides to the corresponding deoxyribonucleosides This is the first step in the synthesis of a DNA from a corresponding RNA and the nrd gene products are therefore ubiquitous in nature There are 3 classes of Nrd. one of which (class-I) is subdivided into la (NrdA/B) and lb (NrdE/F) In classes I and III. the enzymes are of alpha2-beta2 quateoiary structure The redox state of the alpha-subumt thiols is modulated by the action of the b subumt whose role is generation of redox-active radicals NrdA/B enzymes are widely distributed in nature but NrdE/F and NrdD/G may be more closely associated with bacteria Inhibition of cellular activ ltv of the bacterial NrdD/G enzyme can compromise the viability of the bactenal cell and the enzyme therefore presents as a molecular target against which antibacterial compounds can be directed - 43 -

Claims: What is claimed is: 1 Ai isolated poly peptide selected from the group consisting of (i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least (a) 70% identity. (b) 80% identity . (c) 90% identity, or (d) 95% identity to the ammo acid sequence of SEQ ID NO 2 ov er the entire length of SEQ I D NO 2. (n) an isolated poly peptide comprising the amino acid sequence of SEQ ID NO 2 oi (in) an isolated polypeptide which is the annno acid sequence of SEQ ID NO 2 2 Ai isolated poly nucleotide selected from tlie group consistuig of (i) an isolated polynucleotide comprising a nucleotide sequence encoding a pols peptide that has at least (a) 70% identity . (b) 80% identity, (c) 90% identity, or (d) 95% identity. to tiie annno acid sequence of SEQ ID NO 2. over the entire length of SEQ ID NO 2 (u) an isolated polv nucleotide compnsmg a nucleotide sequence that has at least (a) 70% identity (b) 80% identity. (c) 90% identity, or (d) 95% identity. over its entire length to a nucleotide sequence encodmg tlie polypeptide of SEQ ID NO 2. (in) an isolated polynucleotide compnsmg a nucleotide sequence hich has at least (a) 70% identity. (b) 80% identity. (c) 90% identity, or (d) 95% identity. - 44 - to that of SEQ ID NO 1 os er the entne length of SEQ ID NO 1 (IV) an isolated polynucleotide compnsmg a nucleotide sequence encoding the pohpeptide of SEQ ID NO 2. (vi) an isolated polynucleotide which is the polynucleotide of SEQ ID NO 1. or (vi) an isolated polynucleotide obtainable by screemng an appropnate library under strmgent hy bπdization conditions with a labeled probe having tiie sequence of SEQ ID NO 1 or a fragment thereof. (vn) an isolated polynucleotide encoding tiie same mature poh peptide expressed bv the nrdG gene contamed ui tlie Staphylococcus aureus or a nucleotide sequence complementary to said isolated polvnucleotide 3 Ai antibody lmmunospecific for the polypeptide of claim 1 4 A method for the treatment of an individual (I) in need of enhanced activity or expression of the poly peptide of claim 1 compnsmg (a) administering to the individual a tlierapeutically effectiv e amount of an agomst to said polypeptide. and/or (b) providing to the individual an isolated poly nucleotide composmg a nucleotide sequence encodmg said polypeptide ui a form so as to effect production of said polypeptide activity in vivo . or (n) having need to inhibit activity or expression of the polypeptide of claim 1 comprising (a) administering to the individual a tlierapeutically effectiv e amount of an antagonist to said polypeptide. and/or (b) administering to the individual a nucleic acid molecule that inhibits the expression of a nucleotide sequence encodmg said polypeptide. and/or (c) administering to the individual a tlierapeutically effectiv e amount of a polypeptide that competes with said poh peptide for its ligand substrate or receptor 5 A process for diagnosing a disease or a susceptibility to a disease in an individual related to expression or activity of the polypeptide of claim 1 ui an uidividual comprising 45 (a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in the genome of said individual, and/or (b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said individual 6 A method for screening to identify compounds tiiat activ ate and/or that inhibit tiie function ofthe polypeptide of claun 1 winch composes a metiiod selected from tiie group consisting of (a) measuring the bmduig of a candidate compound to the poh peptide (or to the cells oi membranes bearing the polypeptide) or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound. (b) measuring the buiding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor. (c) testing whether the candidate compound results in a signal generated bv activ ation oi inhibition of the polypeptide. using detection sy stems appropriate to the cells or cell membranes bearing the polypeptide. (d) mixing a candidate compound with a solution containing a polypeptide of claim 1. to form a mixture, measuring activity ofthe polypeptide in the mixture, and comparing the activity of the mixture to a standard. (e) detecting the effect of a candidate compound on the production of niRN A encodmg said polypeptide and said polypeptide in cells using foi instance an LLIS A assαv oi (f) (1) contacting a composition compnsmg tiie polypeptide with the compound to be screened under conditions to pemnt mteraction between tlie compound and tlie polypeptide to assess tiie mteraction of a compound, such mteraction beuig associated with a second component capable of providmg a detectable signal m response to tiie mteraction of the polypeptide with tlie compound, and (2) detennunng whetiier tiie compound uiteracts w ith and activ ates oi inhibits an activity oftiie polypeptide by detecting the presence or absence of a signal geneiated from the mteraction oftiie compound with tlie polypeptide 7 An agonist or an antagonist ofthe activity or expression polypeptide of claim 1 8 Ai expression system compnsmg a polvnucleotide capable of producing α po peptide of claim I when said expression system is present in a compatible host cell - 46 - 9 A host cell composing the expression system of claim 8 or a membrane thereof expressing the polypeptide of claim 1 10 A process for producing a polypeptide of claim 1 compnsmg cultui mg a host cell of claim 9 under conditions sufficient for the production of said poly peptide 1 1 A process for producing a host cell as defined in claim 9 composing transfoomng or transfectmg a cell with the expression system of claim 8 such the host cell, under appropriate culture conditions produces a pohpeptide of claim 1 12 A host cell produced bv the process of claim 1 I oi a membiane theieot expi essmg α polypeptide of claim 1 13 A computer readable medium having stored thereon a member selected from the group consisting of a poly nucleotide composing the sequence of SEQ ID NO 1. a polypeptide composing the sequence of SEQ ID NO 2. a set of pol nucleotide sequences wherein at least one of said sequences composes the sequence of SEQ ID NO 1. a set of poh peptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2 a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1. a data set representing a polynucleotide sequence encodmg a polypeptide sequence comprising the sequence of SEQ ID NO 2. a polynucleotide composing the sequence of SEQ ID NO 1. a polypeptide composmg the sequence of SEQ ID NO 2. a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 1 a set of poly peptide sequences wherein at least one of said sequences composes the sequence of SEQ I D NO 2 a data set representing a polynucleotide sequence composing the sequence of SEQ ID NO 1 a data set representing a polynucleotide sequence encodmg a polypeptide sequence composing the sequence of SEQ ID NO 2 14 A computer based method for perforating homology identification, said method comprising the steps of providing a polynucleotide sequence composing the sequence of SEQ ID NO 1 in a computer readable medium, and conipaπng said polv nucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology - 47 15 A further embodiment of the invention provides a computer based method for polynucleotide assembly said method composing the steps of providing a first poly nucleotide sequence comp sing the sequence of SEQ ID NO 1 in a computer ieadable medium, and screening for at least one overlapping region between said first polv nucleotide sequence and a second polynucleotide sequence - 48

Inventors: BLACK MICHAEL T, BLACK, MICHAEL, T.
Applicants: SMITHKLINE BEECHAM CORP, SMITHKLINE BEECHAM CORPORATION
Application year: 1999
Main classification: A61K35
IPC codes: A61K38, A61K39, A61K45, A61K48, A61P31, C07K14, C07K16, C12N1, C12N5, C12N9, C12N15, C12P21, C12Q1, G01N33