EP1087792A1 - Ribonucleotidtriphosphatreduktase-vermittelte anaerobie - Google Patents

Ribonucleotidtriphosphatreduktase-vermittelte anaerobie

Info

Publication number
EP1087792A1
EP1087792A1 EP99957023A EP99957023A EP1087792A1 EP 1087792 A1 EP1087792 A1 EP 1087792A1 EP 99957023 A EP99957023 A EP 99957023A EP 99957023 A EP99957023 A EP 99957023A EP 1087792 A1 EP1087792 A1 EP 1087792A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
sequence
polynucleotide
seq
identity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99957023A
Other languages
English (en)
French (fr)
Inventor
Edwina I. Wilding
Michael T. Black
Christopher M. Traini
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Publication of EP1087792A1 publication Critical patent/EP1087792A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0093—Oxidoreductases (1.) acting on CH or CH2 groups (1.17)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y117/00—Oxidoreductases acting on CH or CH2 groups (1.17)
    • C12Y117/04—Oxidoreductases acting on CH or CH2 groups (1.17) with a disulfide as acceptor (1.17.3)
    • C12Y117/04002—Ribonucleoside-triphosphate reductase (1.17.4.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses
  • the invention relates to polynucleotides and polypeptides of the nrdD (anaerobic ⁇ bonucleotide t ⁇ phosphate reductase) family, as well as their variants, hereinafter referred to as "nrdD,” “nrdD polynucleot ⁇ de(s),” and “nrdD polypept ⁇ de(s)" as the case may be
  • Staphylococcal genes and gene products are particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics
  • the Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigenic Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common.
  • Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common
  • There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock
  • Staphylococcus aureus infections has ⁇ sen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism
  • nrdD embodiments of the invention that have a present benefit of, among other things, bemg useful to screen compounds for antibiotic activity
  • Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease
  • identification and characte ⁇ zation of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease
  • polypeptides of the invention possess significant amino acid sequence homology to a known nrdD protein
  • the present invention relates to nrdD, m particular nrdD polypeptides and nrdD polynucleotides. recombinant materials and methods for their production
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of microbial diseases, amongst others
  • the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified compounds
  • the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting nrdD expression or activity
  • the invention relates to nrdD polypeptides and polynucleotides as desc ⁇ bed in greater detail below
  • the invention relates to polypeptides and polynucleotides of a nrdD of Staphylococcus aureus. which is related by arruno acid sequence homology to nrdD polypeptide
  • the invention relates especially to nrdD having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively
  • a deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dnve, Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCIMB Deposit No 40771, and referred to as Staphylococcus aureus WCUH29 on deposit
  • NCIMB National Collections of Industrial and Marine Bactena Ltd
  • the strain will be irrevocably and without restnction or condition released to the public upon the issuance of a patent
  • the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C ⁇ 112
  • a license may be required to make, use or sell the deposited strain, and compounds de ⁇ ved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is contained in the deposited strain
  • nrdD polynucleotide sequences in the deposited strain such as DNA and RNA.
  • amino acid sequences encoded thereby Also provided by the invention are nrdD polypeptide and polynucleotide sequences isolated from the deposited stram Polypeptides
  • NrdD polypeptide of the invention is substantially phylogenetically related to other proteins of the nrdD (anaerobic ⁇ bonucleotide t ⁇ phosphate reductase) family
  • nrdD Staphylococcus aureus referred to herein as "nrdD” and “nrdD polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comp ⁇ smg the same
  • the present mvention further provides for an isolated polypeptide which (a) comprises or consists of an ammo acid sequence which has at least 70% identit , 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,
  • polypeptides of the mvention include a polypeptide of Table 1 [SEQ ID NO 2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of nrdD. and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO l]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO 2] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids
  • the mvention also mcludes a polypeptide consistmg of or comp ⁇ smg a polypeptide of the formula
  • X is hydrogen, a metal or any other moiety described herem for modified polypeptides. and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, Ri and R3 are any ammo acid residue or modified ammo acid residue, m is an mteger between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R 2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof.
  • R 2 is oriented so that its amino terminal amino acid residue is at the left, covalently bound to R ] and its carboxy terminal amino acid residue is at the right, covalently bound to R3.
  • Any stretch of arnino acid residues denoted by either Ri or R3, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polypeptide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • a fragment is a variant polypeptide having an arnino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention.
  • fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or of variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence.
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Staphylococcus aureus, are also preferred.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-fo ⁇ riing regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-foiming regions, substrate binding region, and high antigenic index regions.
  • X or "Xaa” may also be used in describing certain polypeptides of the invention "X” and “Xaa” mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the polypeptide sequence
  • nrdD polypeptides
  • the polynucleotide comprises a region encoding nrdD polypeptides comp ⁇ smg a sequence set out m Table 1 [SEQ ID NO 1] which mcludes a full length gene, or a variant thereof The Applicants believe that this full length gene is essential to the growth and/or survival of an organism which possesses it, such as Staphylococcus aureus
  • isolated nucleic acid molecules encoding and or expressmg nrdD polypeptides and polynucleotides, particularly Staphylococcus aureus nrdD polypeptides and polynucleotides, including, for example, unprocessed RNAs, ⁇ bozyme RNAs.
  • Another aspect of the mvention relates to isolated polynucleotides. including at least one full length gene, that encodes a nrdD polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and va ⁇ ants thereof
  • nrdD polypeptide from Staphylococcus aureus comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2 or a variant thereof
  • a polynucleotide of the mvention encoding nrdD polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacte ⁇ a usmg Staphylococcus aureus WCUH 29 cells as startmg mate ⁇ al, followed by obtairung a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1].
  • a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 m E colt or some other suitable host is probed with a radiolabeled o gonucleotide, preferably a 17-mer or longer, derived from a partial sequence
  • Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent hybridization conditions
  • sequencmg primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techmques are described by Maniatis, T .
  • each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having about the number of ammo acid residues set forth m Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled in the art
  • the present mvention provides for an isolated polynucleotide comp ⁇ smg or consistmg of
  • polynucleotide sequence may also contain at least one non-codmg sequence, mcludmg for example, but not limited to at least one non-codmg 5' and 3' sequence, such as the transc ⁇ bed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ⁇ bosome binding sites, Kozak sequences, sequences that stabilize mRNA, rntrons, and polyadenylation signals
  • the polynucleotide sequence may also comp ⁇ se additional coding sequence encoding additional
  • a prefe ⁇ ed embodiment of the mvention is a polynucleotide of consistmg of or comp ⁇ smg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 1849 set forth m SEQ ID NO 1 of Table 1, both of which encode the nrdD polypeptide
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof
  • R is oriented so that its 5' end nucleic acid residue is at the left, bound to R ⁇ and its 3' end nucleic acid residue is at the ⁇ ght, bound to R3
  • Any stretch of nucleic acid residues denoted by either R ⁇ and/or R 2 , where m and or n is greater than 1 may be either a heteropolymer or a homopolymer, preferably a heteropolymer
  • X and Y together define a covalent bond
  • the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second
  • polynucleotide of the mvention is de ⁇ ved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus nrdD having an ammo acid sequence set out m Table 1
  • the mvention further relates to va ⁇ ants of the polynucleotides descnbed herem that encode va ⁇ ants of a polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of a polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention
  • the mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem
  • the mvention especially relates to polynucleotides that hybndize under strmgent conditions to the polynucleotides desc ⁇ bed herem
  • strmgent conditions and “stringent hybndization conditions” mean hybndrzation occumng only if there is at least 95% and preferably at least 97% identity between the sequences
  • strmgent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6).
  • the invention also provides a polynucleotide consistmg 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 conditions with a probe havmg the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fra
  • the polynucleotides of the mvention may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding nrdD and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the nrdD gene
  • Such probes generally will comp ⁇ se at least 15 nucleotide residues or base pairs
  • such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs
  • Particularly prefe ⁇ ed probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs
  • a coding region of a nrdD gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe
  • a labeled oligonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or rnRNA to determine which members of the library the probe hybndizes to
  • [SEQ ID NOS 1 or 2] may be used m the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein m whole or m part are transc ⁇ bed in bacteria in infected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is the mature protem plus additional ammo or carboxyl-teiminal ammo acids, or ammo acids mtenor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance)
  • Such sequences may play a role in processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half- life or may facilitate manipulation of a protem for assay or production, among other things
  • the additional am o acids may be processed away from the mature protem
  • a precursor protem, havmg a mature form of the polypeptide fused to one or more prosequences may be an mactive form of the polypeptide When prosequences are removed such mactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • 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 m combination with adjacent nucleotide positions, when read m the correct reading frame, would have the effect of generating a premature termination codon in such reading frame
  • the mvention also relates to vectors that comp ⁇ se a polynucleotide or polynucleotides of the mvention, host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs de ⁇ ved from the DNA constructs of the mvention
  • Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells comp ⁇ smg expression systems Accordingly, in a further aspect, the present mvention relates to expression systems which compnse a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techmques
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention
  • Introduction of a polynucleotide mto the host cell can be effected by methods descnbed m many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press Cold Spring Harbor, N Y (1989).
  • calcium phosphate transfection DEAE-dextran mediated transfection.
  • bacte ⁇ al cells such as cells of streptococci, staphylococci. enterococci E coh, streptomyces. cyanobactena. Bacillus subtths, and Staphylococcus aureus fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus.
  • insect cells such as cells of Drosoph ⁇ a S2 and Spodoptera Sf9
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells
  • plant cells such as cells of a gvmnosperm or angiosperm
  • vectors include, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bacte ⁇ al plasmids, from bacte ⁇ ophage. from transposons, from yeast episomes.
  • the expression system constructs may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard
  • the approp ⁇ ate DNA sequence may be inserted mto the expression system by any of a va ⁇ ety of well-known and routme techmques.
  • Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectm chromatography Most preferably, high performance liquid chromatography is employed for purification
  • Well known techmques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected mdividual's bodily mate ⁇ als
  • Polynucleotides from any of these sources, particularly DNA or RNA may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique p ⁇ or to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characte ⁇ zation of the species and stram of infectious or resident orgamsm present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the orgamsm Deletions and insertions can be detected by a change m size of the amphfied product m compa ⁇ son to a genotype of a reference sequence selected from a related orgamsm, preferably a different species of the
  • Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and SI protection assay or a chemical cleavage method See, for example, Cotton et al , Proc Natl Acad Set , USA, 85 4397-4401 (1985)
  • an a ⁇ ay of oligonucleotides probes compnsmg nrdD nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification
  • Anay technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic va ⁇ abihty (see, for example. Chee et al , Science, 274 610 (1996))
  • the present mvention relates to a diagnostic kit which compnses
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or
  • kits an antibod ⁇ to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO 2
  • a kit will be of use in diagnosmg a disease or susceptibility to a Disease, among others
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to.
  • Organisms particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techmques, such as those descnbed elsewhere herem
  • the nucleotide sequences of the present mvention are also valuable for orgamsm chromosome identification
  • the sequence is specifically targeted to, and can hybndize with, a particular location on an organism's chromosome, particularly to a Staphylococcus aureus chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention may be an important step m co ⁇ elating those sequences with pathogenic potential and or an ecological mche of an orgamsm and or drug resistance of an organism, as ell as the essentiality of the gene to the orgamsm
  • the physical position of the sequence on the chromosome can be co ⁇ elated with genetic map data Such data may be found on-lme m a sequence database
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (coinhe ⁇ tance of physically adjacent genes) or
  • the mvention also includes p ⁇ mers of the formula
  • X is hydrogen, a metal or a modified nucleotide residue, and at the 3' end of the molecule.
  • Y is hydrogen, a metal or a modified nucleotide residue
  • Rj and R3 are any nucleic acid residue or modified nucleotide residue
  • m is an mteger between 1 and 20 or zero
  • n is an mteger between 1 and 20 or zero
  • R is a pnmer sequence of the mvention, particularly a p ⁇ mer sequence selected from Table 2
  • R is o ⁇ ented so that its 5' end nucleotide residue is at the left, bound to Ri and its 3' end nucleotide residue is at the nght, bound to R3
  • Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer bemg complementary to a region of a polynucleotide of Table 1
  • m and/or n is
  • the mvention further provides these primers with 1, 2. 3 or 4 nucleotides removed from the 5' and/or the 3' end
  • These p ⁇ mers may be used for among other things, amplifying nrdD DNA and/or RNA isolated from a sample de ⁇ ved from an individual, such as a bodily matenal
  • the primers may be used to amplify a polynucleotide isolated from an infected mdividual, such that the polynucleotide may then be subject to vanous techmques for elucidation of the polynucleotide sequence In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus, compnsmg determining from a sample denved from an individual, such as a bodily material, an increased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a nrdD polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides.
  • a diagnostic assay in accordance with the mvention for detectmg over-expression of mdD polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a mdD polypeptide, m a sample de ⁇ ved from a host, such as a bodily mate ⁇ al, are well-known to those of skill m the art
  • Assay techmques that can be used to determine levels of a mdD polypeptide, m a sample de ⁇ ved from a host, such as a bodily mate ⁇ al, are well-known to those of skill m the art
  • Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays Differential Expression
  • RT-PCR may also be used to analyze gene expression patterns
  • messenger RNA is isolated from bacterial infected tissue, e g , 48 hour munne lung infections, and the amount of each mRNA species assessed by reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs
  • the determination of the presence and amount of a particular mRNA 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 gam a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacterials
  • 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 infected tissue to obtain bacterial mRNA
  • the polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high density arrays or grids These high density arrays are particularly useful for diagnostic and prognostic purposes
  • a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention may be used for probing, such as usmg hybridization or nucleic acid amplification, using a probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence m an mdividual
  • Such a presence may indicate the presence of a pathogen, particularly Staphylococcus aureus, and may be useful in diagnosing and/or prognosmg disease or a course of disease
  • a grid compnsing a number of variants of the polynucleotide sequence of SEQ ID NO 1 are preferred Also preferred is a comprising a number of variants of
  • Antibodies The polypeptides and polynucleotides of the mvention or vanants thereof, or cells expressmg the same can be used as lmmunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively
  • antibodies against nrdD polypeptides or polynucleotides Antibodies generated against the polypeptides or polynucleotides of the mvention can be obtained by admmistenng the polypeptides and or polynucleotides of the mvention, or epitope-bea ⁇ ng fragments of either or both, analogues of either or both, or cells expressmg either or both, to an animal, preferably a nonhuman, usmg routme protocols
  • any technique known in the art that provides antibodies produced by continuous cell lme cultures can be used Examples mclude va ⁇ ous techniques, such as those m Kohler.
  • phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-nrdD or from naive hbranes (McCafferty, et al , (1990). Nature 348, 552-554. Marks, et al , (1992) Biotechnology 10, 779-783) The affimty of these antibodies can also be improved by. for example, chain shuffling (Clackson et al , (1991) Nature 352
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify' clones expressmg the polypeptides or polynucleotides of the mvention to punfy the polypeptides or polynucleotides by, for example, affinity chromatography
  • antibodies agamst nrdD-polypeptide or mdD-polynucleotide may be employed to treat infections, particularly bacte ⁇ al infections
  • the antibody or variant thereof is modified to make it less lmmunogemc in the individual
  • the antibody may most preferably be "humanized,” where the comphmentanty determining region or regions of the hybridoma-de ⁇ ved antibody has been transplanted into a human monoclonal antibody, for example as described m Jones et al (1986), Nature
  • Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and ligands m. for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g .
  • the present mvention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the mvention, as well as related polypeptides and polynucleotides
  • agomsts or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned
  • Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agomsts, antagonists or inhibitors so-identified may be natural or modified
  • these screening methods may test whether the candidate compound results m a signal generated b> activation or inhibition of the polypeptide or polynucleotide.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, m the absence of an agonist or inhibitor, by testing whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide, as the case may be
  • the screemng methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring nrdD polypeptide and/or polynucleotide activity
  • Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide. through standard receptor binding techniques known in the art These techniques mclude. but are not limited to, hgand binding and crosshnking assays m which the polypeptide is labeled with a radioactive isotope (for instance, 1 * "I), chemically modified (for instance, biot ylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g .
  • a radioactive isotope for instance, 1 * "I
  • chemically modified for instance, biot ylated
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of nrdD polypeptide d mers, turners, tetramers or higher order structures, or structures formed by nrdD polypeptide bound to another polypeptide NrdD polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dime ⁇ zation will inhibit fluorescence energy transfer
  • an assay for nrdD agomsts is a competitive assay that combines mdD and a potential agomst with nrdD-binding molecules, recombinant nrdD bmdmg molecules, natural substrates or ligands. or substrate or hgand mimetics, under approp ⁇ ate conditions for a competitive inhibition assay mdD can be labeled, such as by radioactivity or a colo ⁇ met ⁇ c compound, such that the number of mdD molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • Potential antagonists mclude. among others, small orgamc molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide and or polypeptide of the mvention and thereby inhibit or extinguish its activity or expression
  • Potential antagonists also may be small orgamc molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule, without inducing nrdD-rnduced activities, thereby preventmg the action or expression of mdD polypeptides and or polynucleotides by excluding mdD polypeptides and or polynucleotides from bmdmg
  • Potential antagonists m include a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small orgamc molecules, peptides or peptide-hke molecules
  • Other potential antagonists m include antisense molecules (see Okano, J Neurochem 56 560 (1991) OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press.
  • Preferred potential antagonists m include compounds related to and vanants of mdD
  • Other examples of potential polypeptide antagonists m include antibodies or, m some cases, ohgonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be. of the polypeptide, e g .
  • Certam of the polypeptides of the mvention are biomimetics.
  • functional mimetics of the natural mdD polypeptide These functional mimetics may be used for. among other things, antagonizing the activity of mdD polypeptide or as a antigen or lmmunogen m a manner desc ⁇ bed elsewhere herem
  • Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides For example, preferred functional mimetics mclude.
  • Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefe ⁇ ed that these cassettes compnse 5' and 3' rest ⁇ ction sites to allow for a convement means to hgate the cassettes together when desired It is further prefe ⁇ ed that these cassettes compnse gene expression signals known in the art or desc ⁇ bed elsewhere herem
  • the present invention relates to a screening kit for identifying agomsts, antagonists, ligands receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present invention, or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises
  • polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide and/or polynucleotide, by
  • the present mvention provides methods of treatmg abnormal conditions such as, for instance, a Disease, related to either an excess of. an under-expression of, an elevated activity of. or a decreased activity of mdD polypeptide and or polynucleotide
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present mvention, or a fragment thereof, and vanous portions of the constant regions of heavy or light chains of immunoglobulms of various subclasses (IgG, IgM, IgA, IgE) Preferred as an lmmunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hmge region
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa
  • this invention relates to processes for the preparation of these fusion protems by genetic engineering, and to the use thereof for drug screenmg. diagnosis and therapy
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion prote technology can be found in International Patent Application Nos W094/29458 and W094/2
  • expression of the gene encoding endogenous mdD polypeptide can be inhibited using expression blocking techniques
  • This blocking may be targeted against any step m gene expression, but is preferably targeted against transcription and/or translation
  • An examples of a known technique of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example. O'Connor, J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press. Boca Raton, FL (1988))
  • ohgonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073. Cooney et al Science (1988) 241 456, Dervan et al Science (1991) 251 1360)
  • These o gomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds
  • the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs
  • polynucleotide sequences encodmg the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the codmg sequence of interest
  • the invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the invention may be used in the prevention of adhesion of bacteria, m particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds, to block mdD protem-mediated mammalian cell invasion by.
  • mdD agomsts and antagonists preferably bacte ⁇ static or bactencidal agomsts and antagonists
  • Hehcobacter pylori (herein "H pylori”) bactena infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer. L ⁇ on. France, http //www uicc ch ecp/ecp2904 htm)
  • the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcinoma.
  • Preferred antimicrobial compounds of the invention agonists and antagonists of nrdD polypeptides and or polynucleotides found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H pylori -induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis Vaccines
  • mvention products, compositions and methods for assessmg mdD expression, treatmg disease, assaying genetic vanation, and administering a nrdD polypeptide and/or polynucleotide to an orgamsm to raise an lmmunological response against a bactena. especially a Staphylococcus aureus bactena
  • Another aspect of the mvention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with mdD polynucleotide and or polypeptide.
  • a further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, mduces an immunological response in such individual to a mdD polynucleotide and or polypeptide encoded therefrom, wherein the composition comprises a recombinant mdD polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said mdD polynucleotide.
  • polynucleotide or particular fragments thereof which have been shown to encode non-va ⁇ able regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Staphylococcus aureus
  • Such experiments will be particularly useful for identifying protem 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, de ⁇ ved from the requisite organ of the animal successfully resisting or clearmg infection, for the development of prophylactic agents or therapeutic treatments of bacterial mfection, particularly Staphylococcus aureus infection, in mammals, particularly humans
  • a polypeptide of the invention may be used as an antigen for vaccmation of a host to produce specific antibodies which protect against invasion of bactena. for example by blocking adherence of bacteria to damaged tissue
  • tissue damage mclude wounds in skm or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or 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 formulation which comprises an lmmunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable earner Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, mcludmg, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal
  • mcludmg for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants.
  • the formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampoules and vials and may be stored m a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid carrier immediately prior to use
  • the vaccine formulation may also include adjuvant systems for enhancing the lmmunoge city of the formulation, such as oil-m water systems and other systems known m the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation
  • compositions for purposes of compositions, kits and administration
  • compositions comp ⁇ smg a mdD polynucleotide and/or a mdD polypeptide for administration to a cell or to a multicellular orgamsm
  • the mvention also relates to compositions compnsrng a polynucleotide and/or a polypeptides discussed herem or their agomsts or antagonists
  • the polypeptides and polynucleotides of the mvention may be employed m combination with a non-ste ⁇ le or stenle earner or earners for use with cells, tissues or orgamsms, such as a pharmaceutical earner suitable for administration to an mdividual Such compositions compnse.
  • earners may mclude, but are not limited to, salme, buffered salme. dextrose, water, glycerol. ethanol and combmations thereof
  • the formulation should suit the mode of administration
  • the mvention further relates to diagnostic and pharmaceutical packs and kits compnsrng one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention
  • Polypeptides, polynucleotides and other compounds of the mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
  • compositions may be admmistered in any effective, convement manner mcludmg, for instance, admmistration by topical, oral, anal, vaginal, mtravenous, mtrape ⁇ toneal. intramuscular, subcutaneous, mtranasal or lntradermal routes among others
  • the active agent may be administered to an mdividual as an mjectable composition, for example as a sterile aqueous dispersion, preferably isotomc
  • composition may be formulated for topical application for example m the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in omtments and creams
  • topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions
  • Such carriers may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation
  • the present mvention provides for pharmaceutical compositions compnsrng a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and or polynucleotide of the present mvention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient
  • a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, salme. buffered salme.
  • the mvention further relates to pharmaceutical packs and kits compnsrng one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention
  • Polypeptides, polynucleotides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • the composition will be adapted to the route of admmistration, for instance by a systemic or an oral route
  • Prefe ⁇ ed forms of systemic admmistration m clude injection, typically by mtravenous injection
  • Other injection routes such as subcutaneous, intramuscular, or mtrape ⁇ toneal
  • Alternative means for systemic admmistration m include transmucosal and transdermal admmistration usmg penetrants such as bile salts or fusidic acids or other detergents
  • a polypeptide or other compounds of the present mvention can be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • the composition will
  • the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg
  • the physician in any event will determine the actual dosage which will be most suitable for an mdividual and will vary with the age, weight and response of the particular individual
  • 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 mvention
  • In-dwellmg devices include surgical implants, prosthetic devices and catheters, l e , devices that are introduced to the body of an individual and remain in position for an extended time
  • Such devices mclude. for example, artificial jomts, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospmal fluid shunts, urinary catheters, continuous ambulatory pentoneal dialysis (CAPD) catheters
  • composition of the mvention may be administered by injection to achieve a systemic effect agamst relevant bacteria shortly before insertion of an in-dwelling device Treatment may be continued after surgery during the m-body time of the device
  • composition could also be used to broaden pe ⁇ operative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix protems exposed in wound tissue and for prophylactic use m dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis
  • a vaccine composition is conveniently in mjectable form
  • Conventional adjuvants may be employed to enhance the immune response
  • a suitable unit dose for vaccination is 0 5-5 microgram kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks With the mdicated dose range, no adverse toxicological effects will be observed 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 form a valuable information resource with which to determme their 2- and 3-d ⁇ mens ⁇ onal structures as well as to identify further sequences of similar homology
  • These approaches are most easily facilitated by storing the sequence in a computer readable medium and then usmg the stored data m a known macromolecular structure program or to search a sequence database usmg well known searching tools, such as GCC
  • polynucleotide and polypeptide sequences of the invention are particularly useful as components in databases useful for search analyses as well as in sequence analysis algonthms
  • sequence databases Sequences in a Tangible Medium, and Algorithms
  • polynucleotide of the invention and polynucleotide 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 in a tangible medium, preferably a computer readable form For example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data
  • polypeptide of the invention and polypeptide sequence of the invention mean any detectable chemical or physical characteristic of a polypeptide of the mvention that is or may be reduced to or
  • a computer based method for performing homology identification This method comprises the steps of providing a polynucleotide sequence comprising the sequence a polynucleotide of the invention in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology
  • a computer based method is also provided for performing homology identification, said method comprising the steps of providing a polypeptide sequence comprising the sequence of a polypeptide of the mvention in 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 comprising the steps of providing a first polynucleotide 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 polynucleotide sequence and a second polynucleotide sequence
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polypeptide sequence compnsrng the sequence of a polypeptide of the mvention 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 comprising the steps of providing a first polynucleotide 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 polynucleotide sequence and a second polynucleotide sequence
  • a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 a data set representing a polynucleotide sequence encoding a polypeptide sequence compnsrng the sequence of SEQ ID NO 2
  • a polynucleotide comprising the sequence of SEQ ID NO 1 a polypeptide compnsrng 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 further preferred embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising 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 further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of SEQ ID NO 1 in a computer readable medium, and screening for at least one overlappmg region between said first polynucleotide sequence and a second polynucleotide sequence
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising 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 mvention provides a computer based method for performing homology identification, said method comprising the steps of providing a polypeptide sequence compnsrng the sequence of SEQ ID NO 2 in a computer readable medium, and comparing said polypeptide sequence to at least one polyn
  • 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 comprising the sequence of SEQ ID NO 1 in a computer readable medium, and screemng for at least one overlappmg region between said first polynucleotide sequence and a second polynucleotide sequence
  • ant ⁇ body( ⁇ es) as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c. single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulin expression library
  • Antigemcally equivalent de ⁇ vat ⁇ ve(s) as used herem encompasses a pohpeptide, polynucleotide. or the equivalent of either which will be specifically recognized b ⁇ certain antibodies which, when raised to the protem, polypeptide or polynucleotide according to the mvention. interferes with the immediate physical interaction between pathogen and mammalian host
  • Bispecific antibody( ⁇ es) means an antibody comprising at least two antigen bmdmg domains, each domain directed against a different epitope
  • Bodily mate ⁇ al(s) means any matenal denved from an mdividual or from an orgamsm infecting, infesting oi inhabiting an mdividual.
  • mcludmg but not limited to. cells, tissues and waste, such as. bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy mate ⁇ als
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena. mcludmg , for example, disease, such as.
  • “Host cell(s)” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences
  • Identity can be readily calculated by known methods, including but not limited to those described m (Computational Molecular Biology Lesk. A M . ed . Oxford University Press. New York, 1988, Biocomputtng Informatics and Genome Projects Smith, D W ed Academic Press, New York. 1993. Computer Analysis of Sequence Data. Part I.
  • Parameters for polypeptide sequence companson include the following
  • Parameters for polynucleotide companson include the following
  • Polynucleotide embodiments further mclude 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 ma ⁇ mclude up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion.
  • alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides SEQ ID NO: 1
  • n n is the number of nucleotide alterations.
  • x 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 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtractmg it from x n
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and alter the polypeptide encoded by the polynucleotide following such alterations
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 1. that is it may be 100% identical, or it may include up to a certain mteger number of nucleic 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 w herem said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleic acids in the reference sequence or m one or more contiguous groups withm the reference sequence
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids m SEQ ID NO 1. or
  • n n is the number of nucleic acid alterations
  • x n is the total number of nucleic acids m SEQ ID NO 1
  • 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-mteger product of x n and y is rounded down to the nearest mteger prior to subtracting it from x n
  • Polypeptide embodiments further include an isolated polypeptide compnsrng a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtracting that product from said
  • n a is the number of ammo acid alterations
  • x a is the total number of amino acids in SEQ ID NO 2.
  • y is 0 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 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtractmg it from x a
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may mclude up to a certain integer number of amino 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 consistmg of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence
  • the number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids m SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2, or
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO 2
  • y is. for instance 0 70 for 70%. 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest mteger prior to subtracting it from x a
  • “Immunologically equivalent de ⁇ vat ⁇ ve(s)” as used herein encompasses a polypeptide, poh nucleotide or the equivalent of either which when used in a suitable formulation to raise antibodies in a ⁇ ertebrate.
  • Immunospecific means that charactenstic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the mvention or the polynucleotides of the mvention than its affimty for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the p ⁇ or art "Ind ⁇ v ⁇ dual(s)” means a multicellular eukaryote. mcludmg. but not limited to a metazoan, a mammal, an ovid, a bovid. a simian, a primate, and a human
  • Isolated means altered “by the hand of man” from its natural state, / , if it occurs m nature, it has been changed or removed from its onginal environment, or both
  • a polynucleotide or a polypeptide naturally present m a Irving orgamsm is not “isolated,” but the same polynucleotide or polypeptide separated from the coexistmg matenals of its natural state is “isolated", as the term is employed herem
  • a polynucleotide or polypeptide that is mtroduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said orgamsm, which orgamsm may be living or non-living
  • Organ ⁇ sm(s) means a (I) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus Staphylococcus, Bordetella, Corynebactenum, Mycobacterium, Neissena, Haemophilus, Actinomycetes Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acmetobacter Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschench a, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas
  • mclud g but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenv seae, Kluveromyces lactis, or Candida albicans
  • Polynucleot ⁇ de(s) generally refers to any polynbonucleoti.de or polydeoxynbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)” mclude, without limitation, smgle- and double-stranded DNA.
  • DNA that is a mixture of single- and double-stranded regions or single-, double- and t ⁇ ple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of s gle- and double-stranded regions
  • hybnd molecules comp ⁇ smg DNA and RNA that may be single-stranded or, more typically, double-stranded, or t ⁇ ple-stranded regions, or a mixture of smgle- and double-stranded regions
  • polynucleotide refers to tnple-stranded regions comp ⁇ smg RNA or DNA or both RNA and DNA
  • the strands m such regions may be from the same molecule or from different molecules
  • the regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules
  • One of the molecules of a tnple-he cal region often is an oligonucleotide As used herem,
  • Modifications mclude, for example, acetylation, acylation. ADP- nbosylation. amidation. covalent attachment of flavrn, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide de ⁇ vative, covalent attachment of a pid or lipid denvative, covalent attachment of phosphotidy nositol cross-linking, cvchzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme. formation of pyroglutamate. formylation.
  • Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention
  • “Subtraction set” is one or more, but preferably less than 100, polynucleotides compnsrng at least one polynucleotide of the invention
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions m any combination
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code
  • the present invention also includes mclude va ⁇ ants of each of the polypeptides of the mvention. that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted b ⁇ another with like charactenstics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu.
  • Example 1 Strain selection, Library Production and Sequencing
  • the polynucleotide having a DNA sequence given m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E cob
  • the sequencing data from two or more clones containmg overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may 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 l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI.
  • the library is amplified by standard procedures Method 2 Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning mto library vectors (e g . Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library
  • the library is amplified by standard procedures

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
EP99957023A 1998-06-17 1999-06-09 Ribonucleotidtriphosphatreduktase-vermittelte anaerobie Withdrawn EP1087792A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US9867898A 1998-06-17 1998-06-17
US98678 1998-06-17
PCT/US1999/012975 WO1999065530A1 (en) 1998-06-17 1999-06-09 NrdD

Publications (1)

Publication Number Publication Date
EP1087792A1 true EP1087792A1 (de) 2001-04-04

Family

ID=22270435

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99957023A Withdrawn EP1087792A1 (de) 1998-06-17 1999-06-09 Ribonucleotidtriphosphatreduktase-vermittelte anaerobie

Country Status (3)

Country Link
EP (1) EP1087792A1 (de)
JP (1) JP2002517993A (de)
WO (1) WO1999065530A1 (de)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6737248B2 (en) * 1996-01-05 2004-05-18 Human Genome Sciences, Inc. Staphylococcus aureus polynucleotides and sequences
US6107071A (en) * 1996-09-24 2000-08-22 Smithkline Beecham Corporation Histidinol dehydrogenase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9965530A1 *

Also Published As

Publication number Publication date
JP2002517993A (ja) 2002-06-25
WO1999065530A1 (en) 1999-12-23

Similar Documents

Publication Publication Date Title
WO1999062527A1 (en) nrdE
US6346397B1 (en) GyrA
EP0967990A1 (de) Udp-n-acetylmuramoyl-l-alanin:d-glutamat ligase (murd) aus staphylococcus aureus
WO2000029547A1 (en) YfiI PSEUDOURIDINE SYNTHASE
WO1999053020A1 (en) 6-phosphogluconate dehydrogenase of streptococcus pneumoniae
WO1999026970A1 (en) NOVEL pth
US6555338B1 (en) NrdF from Staphylococcus aureus
EP0961778A1 (de) 3-hydroxyacyl-coa-dehydrogenase von staphylococcus aureus
US6287804B1 (en) nrdG
WO2000030662A1 (en) Fabz
WO1999055729A1 (en) STAPHYLOCOCCUS AUREUS pyrH POLYPEPTIDES AND POLYNUCLEOTIDES
WO1999017795A1 (en) RIBOSOME RECYCLING FACTOR (FRR) OF $i(STAPHYLOCOCCUS AUREUS)
EP1114145A1 (de) Topa
EP1084141A1 (de) nrdF
WO2000028819A1 (en) Yfil pseudouridine synthase
EP0961781A1 (de) AMA-Polypeptide
EP1109566A1 (de) Gcp
EP1077983A2 (de) Pria
WO2000028820A1 (en) DnaB
WO2000023467A1 (en) birA
EP1105411A1 (de) nrdE
WO1999065530A1 (en) NrdD
EP1107977A1 (de) Ratc
WO2000039286A1 (en) DnaE
WO2000023611A1 (en) FabG

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010117

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): BE CH DE DK FR GB IT LI NL

RIN1 Information on inventor provided before grant (corrected)

Inventor name: TRAINI,CHRISTOPHER,M., SMITHKLINE BEECHAM CORP.

Inventor name: BLACK, MICHAEL, T.

Inventor name: WILDING, EDWINA, I.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 20020207