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WO2000039286A1 - DnaE - Google Patents

DnaE Download PDF

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Publication number
WO2000039286A1
WO2000039286A1 PCT/US1999/028970 US9928970W WO0039286A1 WO 2000039286 A1 WO2000039286 A1 WO 2000039286A1 US 9928970 W US9928970 W US 9928970W WO 0039286 A1 WO0039286 A1 WO 0039286A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
sequence
seq
identity
Prior art date
Application number
PCT/US1999/028970
Other languages
French (fr)
Inventor
Earl W. May
Andrew P. Fosberry
Diana Hoult
Michelle Williams
Original Assignee
Smithkline Beecham Corporation
Smithkline Beecham, Plc
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 Corporation, Smithkline Beecham, Plc filed Critical Smithkline Beecham Corporation
Publication of WO2000039286A1 publication Critical patent/WO2000039286A1/en

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    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses
  • the invention relates to polynucleotides and polypeptides of the DNA Polymerase family, as well as their va ⁇ ants, hereinafter referred to as "DnaE,” “DnaE polynucleot ⁇ de(s),” and “DnaE polypept ⁇ de(s)” as the case may be
  • Staphylococcal genes and gene products 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 Staphylococcus aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bactenal endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome
  • polynucleotides and polypeptides such as the DnaE embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial activity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
  • the present invention relates to DnaE, in particular DnaE polypeptides and DnaE polynucleotides, recombinant materials and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others.
  • the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting DnaE expression or activity.
  • the invention relates to DnaE polypeptides and polynucleotides as described in greater detail below.
  • the invention relates to polypeptides and polynucleotides of a DnaE of Staphylococcus aureus, which is related by amino acid sequence homology to DnaE polypeptide.
  • the invention relates especially to DnaE having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 and SEQ ID NO:2 respectively.
  • sequences recited in the Sequence Listing below as "DNA” represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.
  • a deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the
  • NCIMB National Collections of Industrial and Marine Bacteria Ltd.
  • the Staphylococcus aureus strain deposit is refe ⁇ ed to herein as "the deposited strain” or as "the DNA of the deposited strain.”
  • the deposited strain contains a full length DnaE gene.
  • the sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein.
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the
  • the deposited strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent.
  • the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. ⁇ 112.
  • a license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is contained in the deposited strain.
  • DnaE polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby.
  • DnaE polypeptide and polynucleotide sequences isolated from the deposited strain are also provided by the invention.
  • DnaE polypeptide of the invention is substantially phylogenetically related to other proteins of the DNA Polymerase family.
  • polypeptides of Staphylococcus aureus refe ⁇ ed to herein as "DnaE” and “DnaE polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • the present invention further provides for an isolated polypeptide which: (a) comprises or consists of an amino 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 preferably at least 97-99% or exact identity to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; (c) a polypeptide encoded by an isolated polynucleotide
  • polypeptides of the invention 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 DnaE, 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 amino acids and more preferably at least 50 amino acids.
  • the invention also includes a polypeptide consisting of or comprising a polypeptide of the formula:
  • R 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
  • Ri and its carboxy terminal amino acid residue is at the right, covalently bound to R3.
  • Any stretch of amino 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 prefe ⁇ ed embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polypeptide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • a fragment is a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention.
  • fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
  • Prefe ⁇ ed 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 prefe ⁇ ed. Further prefe ⁇ ed are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • prefe ⁇ ed fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2. Also prefe ⁇ ed are biologically active fragments that are those fragments that mediate activities of
  • DnaE including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly prefe ⁇ ed are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Staphylococcus aureus or the ability to initiate, or maintain cause Disease in an individual, particularly a human.
  • Fragments of the polypeptides of the invention may be employed for producing the co ⁇ esponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • 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.
  • the polynucleotide comprises a region encoding DnaE polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:l] which includes a full length gene, or a variant thereof.
  • SEQ ID NO:l a sequence set out in Table 1 [SEQ ID NO:l] which includes 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 that possesses it, such as Staphylococcus aureus.
  • isolated nucleic acid molecules encoding and or expressing DnaE polypeptides and polynucleotides, particularly Staphylococcus aureus DnaE polypeptides and polynucleotides, including, for example, unprocessed RNAs, ⁇ bozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • DnaE polypeptides and polynucleotides particularly Staphylococcus aureus DnaE polypeptides and polynucleotides, including, for example, unprocessed RNAs, ⁇ bozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and va ⁇ ants thereof, and compositions comp ⁇ sing the same
  • Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a DnaE polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and va ⁇ ants thereof
  • a DnaE polypeptide from
  • Staphylococcus aureus comp ⁇ smg or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof
  • a polynucleotide of the invention encoding DnaE polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacte ⁇ a using Staphylococcus aureus WCUH 29 cells as starting mate ⁇ al, followed by obtaining a full length clone
  • a polynucleotide sequence of the mvention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1]
  • typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E colt or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, denved from a partial sequence Clones carrying DNA identical to that of the
  • each DNA sequence set out in Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled m the art
  • the present mvention provides for an isolated polynucleotide comprising or consisting of (a) 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 preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encoding a polypeptide 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 preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which compnses the steps of screening an approp ⁇ ate library under stringent hybndization conditions with a labeled or detectable probe consisting of or compnsing the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones containing said polynucleotide sequence
  • the invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO 1]
  • a coding sequence for a mature polypeptide or a fragment thereof by itself as well as a codmg sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence
  • the polynucleotide of the mvention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcnbed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termination signals), nbosome bmdmg sites, Kozak sequences, sequences that stabilize mRNA.
  • the polynucleotide sequence may also compnse additional coding sequence encoding additional ammo acids
  • a marker sequence that facilitates pu ⁇ fication of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed in Gentz et al , Proc Natl Acad Set , USA 86 821-824 (1989), or an HA peptide tag (W ⁇ son et al , Cell 37 767 (1984), both of which may be useful in pu ⁇ fying polypeptide sequence fused to them
  • Polynucleotides of the mvention also mclude, but are not limited to, polynucleotides compnsing a structural gene and its naturally associated sequences that control gene expression
  • a prefe ⁇ ed embodiment of the invention is a polynucleotides compnsing a structural gene and its naturally associated sequence
  • the invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:
  • Ri and R3 is independently any nucleic acid residue or modified nucleic acid residue
  • m is an integer between 1 and 3000 or zero
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof.
  • R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to R ⁇ and its 3' end nucleic acid residue is at the right, bound to R3.
  • Any stretch of nucleic acid residues denoted by either R ⁇ and/or R 2 , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary.
  • m and/or n is an integer between 1 and 1000.
  • Other prefe ⁇ ed embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polynucleotide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Staphylococcus aureus DnaE having an amino acid sequence set out in Table 1 [SEQ ID NO:2],
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.
  • the mvention further relates to vanants of the polynucleotides descnbed herein that encode vanants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention
  • Prefe ⁇ ed embodiments are polynucleotides encoding DnaE vanants, that have the ammo acid sequence of DnaE polypeptide of Table 1 [SEQ ID NO 2] m which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no am o acid residues are substituted, modified, deleted and/or added, m any combination Especially prefe ⁇ ed among these are silent substitutions, additions and deletions, that do not alter the properties and activities of DnaE polypeptide
  • Prefe ⁇ ed embodiments of the mvention are polynucleo
  • polynucleotides that hybndize, particularly under stringent conditions, to DnaE polynucleotide sequences such as those polynucleotides in Table 1
  • the mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herein
  • the mvention especially relates to polynucleotides that hybndize under stringent conditions to the polynucleotides descnbed herem
  • the terms "stringent conditions” and “stringent hybndization conditions” mean hybndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences
  • a specific example of stringent hybndization conditions is overnight mcubation at 42°C m a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM t
  • the mvention also provides a polynucleotide consisting of or compnsmg a polynucleotide sequence obtained by screening an appropriate library contammg the complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under st ⁇ ngent hybndization conditions with 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 include, for example, probes and primers fully descnbed elsewhere herem
  • 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 DnaE and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the DnaE gene
  • Such probes generally will compnse 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 pa rs
  • a coding region of a DnaE gene may be isolated by screening usmg a DNA sequence provided in Table 1 [SEQ JD NO 1] to synthesize an oligonucleotide probe
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to
  • polynucleotides of the mvention that are ohgonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herein as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herein m whole or m part are transcribed in bactena m infected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is the mature protem plus additional ammo or carboxyl-terminal am o 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 As generally is the case in vivo, the additional ammo acids may be processed away from the mature protem by cellular enzymes
  • a precursor protein, havmg a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • N may also be used in desc ⁇ bmg certain polynucleotides of the mvention "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken in combmation with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such readmg frame
  • a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (which may be refe ⁇ ed to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotein, which is a precursor to a proprotem, havmg a leader sequence and one or more prosequences, which generally are removed du ⁇ ng processmg steps that produce active and mature forms of the polypeptide
  • the invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems.
  • the present invention relates to expression systems that comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al, BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • bacterial cells such as cells of streptococci, staphylococci, enterococci, E. coli, streptomyces, cyanobacteria, Bacillus subtilis, and Staphylococcus aureus
  • fungal cells such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus
  • insect cells such as cells of Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells
  • plant cells such as cells of a gymnosperm or angiosperm.
  • vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculovirases, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the expression system by any of a va ⁇ ety of well-known and routine techmques, such as, for example, those set forth in Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
  • appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification
  • Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and or punfication
  • This mvention is also related to the use of DnaE polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of DnaE polynucleotides and/or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the DnaE gene or protein, may be detected at the nucleic acid or ammo acid level by a vanety of well known techniques as well as by methods provided herem
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected mdividual's bodily matenals
  • Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification techmque pnor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characterization of the species and strain of infectious or resident organism present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism Deletions and insertions can be detected by a change m size of the amplified product m companson to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species Pomt mutations can be
  • 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 assays, such as RNase, VI and SI protection assay or a chemical cleavage method. See, for example, Cotton et al. , Proc. Natl. Acad. Sci. , USA, 85: 4397-4401 (1985).
  • an a ⁇ ay of oligonucleotides probes comprising DnaE nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al, Science, 274: 610 (1996)).
  • the present invention relates to a diagnostic kit which comprises: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2.
  • kits may comprise a substantial component.
  • a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others.
  • This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents.
  • Detection of a mutated form of a polynucleotide of the invention will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide.
  • Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.
  • the nucleotide sequences of the present invention are also valuable for organism chromosome identification.
  • the sequence is specifically targeted to, and can hybridize 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 invention may be an important step in co ⁇ elating those sequences with pathogenic potential and/or an ecological niche of an organism and/or drug resistance of an organism, as well as the essentiality of the gene to the organism.
  • 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 (coinheritance of physically adjacent genes) or mating studies, such as by conjugation
  • the differences in a polynucleotide and or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined. If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype.
  • Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example.
  • RT-PCR can be used to detect mutations in the RNA. It is particularly prefe ⁇ ed to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan.
  • RNA, cDNA or genomic DNA may also be used for the same purpose, PCR.
  • PCR primers complementary to a polynucleotide encoding DnaE polypeptide can be used to identify and analyze mutations. Examples of representative primers are shown below in Table 2.
  • the invention also includes primers of the formula: 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, and at the 3' end of the molecule, Y is hydrogen, a metal or a modified nucleotide residue, R ⁇ and R3 are any nucleic acid residue or modified nucleotide residue, m is an integer between 1 and 20 or zero , n is an integer between 1 and 20 or zero, and R 2 is a primer sequence of the invention, particularly a primer sequence selected from Table 2.
  • R is oriented so that its 5' end nucleotide residue is at the left, bound to Ri and its 3' end nucleotide residue is at the right, 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 being complementary to a region of a polynucleotide of Table 1.
  • m and/or n is an integer between 1 and 10.
  • the mvention further provides these pnmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end
  • These pnmers may be used for, among other things, amplifying DnaE DNA and/or RNA isolated from a sample denved from an mdividual, such as a bodily mate ⁇ al
  • the pnmers may be used to amplify a polynucleotide isolated from an infected mdividual, such that the polynucleotide may then be subject to vanous techniques for elucidation of the polynucleotide sequence In this way, mutations m the polynucleotide sequence may be detected and used to diagnose andor prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections caused by Staphylococcus aureus, compnsmg detenmmng from a sample derived from an mdividual, 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 DnaE polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
  • a diagnostic assay m accordance with the mvention for detectmg over-expression of DnaE 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 DnaE polypeptide
  • the polynucleotides and polynucleotides of the mvention may be used as reagents for differential screening methods
  • differential screening and differential display methods There are many differential screening and differential display methods known in the art in which the polynucleotides and polypeptides of the mvention may be used
  • the differential display technique is descnbed by Chuang et al , J Bacterwl 175 2026-2036 (1993) This method identifies those genes which are expressed in an organism by identifying mRNA present usmg randomly-primed RT-PCR By comparing pre-infection 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 described by Camilh et al , Proc Nat'l Acad Sci
  • IVET identifies genes up-regulated during infection when compared to laboratory cultivation, implying an important role m infection ORFs identified by this techmque are implied to have a significant role m infection establishment and/or maintenance
  • random chromosomal fragments of target organism are cloned upstream of a promoter-less recombmase gene in a plasmid vector
  • This construct is introduced mto the target organism which ca ⁇ ies an antibiotic resistance gene flanked by resolvase sites Growth in 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 sensitive bacterium should carry a promoter or portion of a gene normally upregulated during infection Sequencing upstream of the recombin
  • RT-PCR may also be used to analyze gene expression patterns
  • messenger RNA is isolated from bactenal mfected tissue, e g , 48 hour mu ⁇ ne 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 pnmer 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 transcnbed in the mfected tissue Analysis of gene transcnption can be earned out at different times of mfection to gam a detailed knowledge of gene regulation m bactenal pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacte ⁇ als Because of the gene specific nature of the PCR primers employed it should be understood that the bactenal mRNA preparation need not be free of mammalian RNA This allows the investigator
  • polynucleotides of the invention may be used as components of polynucleotide a ⁇ ays, preferably high density a ⁇ ays or grids These high density a ⁇ ays are particularly useful for diagnostic and prognostic purposes
  • a set of spots each comprismg a different gene, and further comprising a polynucleotide or polynucleotides of the invention may be used for probing, such as using hybridization or nucleic acid amplification, using a probes obtamed or denved from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual
  • Such a presence may indicate the presence of a pathogen, particularly Staphylococcus aureus, and may be useful in diagnosmg and/or prognosmg disease or a course of disease
  • a grid comprismg a number of variants of the polynucleotide sequence of SEQ ID NO 1 are preferred Also prefe ⁇
  • polypeptides and polynucleotides of the mvention or vanants thereof, or cells expressmg the same can be used as lmmunogens to produce antibodies lmmunospecific for such polypeptides or polynucleotides respectively
  • Antibodies generated against the polypeptides or polynucleotides of the mvention can be obtamed by administering the polypeptides and/or polynucleotides of the 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 routme protocols
  • any techmque known m the art that provides antibodies produced by contmuous cell lme cultures can be used Examples mclude vanous techmques, such as those in Kohler, G and Milstem, C , Nature 256 495-497 (1975), Kozbor et al , Immunology Today 4 72 (1983), Cole et al , pg 77-96 m MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R L ⁇ ss, Inc (1985)
  • smgle chain antibodies can be adapted to produce s gle chain antibodies to polypeptides or polynucleotides of this mvention
  • transgenic mice, or other organisms such as other rnammals may be used to express humanized antibodies lmmunospecific to the polypeptides or polynucleotides of the mvention
  • phage display technology may be utilized to select antibody genes with bmdmg activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-DnaE or from naive libraries (McCafferty, et al , (1990).
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptides or polynucleotides of the mvention to purify the polypeptides or polynucleotides by, for example, affinity chromatography
  • antibodies against DnaE-polypeptide or DnaE-polynucleotide may be employed to treat infections, particularly bactenal infections
  • Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form 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 protein may provide stability to the polypeptide
  • the antigen may be associated, for example by conjugation, with an lmmunogemc carrier protein for example bovme serum albumin, keyhole limpet haemocyanin or tetanus toxoid
  • an lmmunogemc carrier protein for example bovme serum albumin, keyhole limpet haemocyanin or tetanus toxoid
  • a multiple antigenic polypeptide comprismg multiple copies of the polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogenicity so as to obviate the use of a earner
  • the antibody or vanant thereof is modified to make it less immunogemc in the individual
  • the antibody may most preferably be "humanized,” where the comphmentanty determimng region or regions of the hybndoma-denved antibody has been transplanted into a human monoclonal antibody, for example as descnbed in lones et al (1986), Nature
  • a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization
  • particularly prefe ⁇ ed embodiments of the mvention are naturally occurring allelic va ⁇ ants of DnaE polynucleotides and polypeptides encoded thereby
  • a polynucleotide of the invention in genetic immunization will preferably 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), delivery of DNA complexed with specific protem earners (Wu et al , J Biol Chem (1989) 264 16985), coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef, RN-4S USA, (1986) 83 9551), encapsulation of D ⁇ A m various forms of liposomes (Kaneda et al , Science (1989) 243 375), particle bombardment (Tang et al Nature (1992) 356 152, Eisenbraun et al , DNA Cell Biol (1993) 12 791) and in vivo infection using cloned retroviral vectors (See
  • Polypeptides and polynucleotides of the mvention may also be used to assess the bmdmg of small molecule substrates and ligands m, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
  • Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, including many disease states, in particular the Diseases herembefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide Accordingly, m a further aspect, 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 poly
  • Such agonists, antagonists or inhibitors so- ldentified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of DnaE polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Coligan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991))
  • the screenmg methods may simply measure the bmdmg of a candidate compound to the polypeptide or polynucleotide. or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screenmg method may involve competition with a labeled competitor
  • these screenmg methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide
  • usmg detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testmg whether the candidate compound results in inhibition of activation of the 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 of the present invention, to form a mixture, measuring DnaE polypeptide and/or polynucleotide activity in the
  • Fusion proteins such as those made from Fc portion and DnaE polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al., I Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16):9459-9471 (1995)).
  • polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells.
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents 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 screening compounds to identify those which enhance
  • a synthetic reaction mix a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising DnaE polypeptide and a labeled substrate or Ugand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a DnaE agonist or antagonist.
  • the ability of the candidate molecule to agonize or antagonize the DnaE polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate.
  • Molecules that bind gratuitously, i.e., without inducing the effects of DnaE polypeptide are most likely to be good antagonists.
  • Molecules that bind well and, as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists. Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system.
  • Reporter systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in DnaE polynucleotide or polypeptide activity, and binding assays known in the art.
  • Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 1), chemically modified (for instance biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily matenals) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the bindmg of the polypeptide to its receptor(s), if any Standard methods for conductmg such assays are well understood in the art
  • the fluorescence polanzation value for a fluorescently-tagged molecule depends on the rotational co ⁇ elation time or tumbling rate Protem complexes, such as formed by DnaE polypeptide associatmg with another DnaE polypeptide or other polypeptide, labeled to compnse a fluorescently- labeled molecule will have higher polanzation values than a fluorescently labeled monomenc protem It is preferred that this method be used to charactenze small molecules that disrupt polypeptide complexes
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of DnaE polypeptide dimers, t ⁇ mers, tetramers or higher order structures, or structures formed by DnaE polypeptide bound to another polypeptide
  • DnaE polypeptide can be labeled with both a donor and acceptor fluorophore
  • fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dime ⁇ zation will inhibit fluorescence energy transfer
  • Surface plasmon resonance can be used to monitor the effect of small molecules on DnaE polypeptide self-association as well as an association of DnaE polypeptide and another polypeptide or small molecule
  • DnaE polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protem can then passed over the DnaE polypeptide -coated surface and specific binding can be detected m real-time by monitoring
  • a scintillation proximity assay may be used to charactenze the interaction between an association of DnaE polypeptide with another DnaE polypeptide or a different polypeptide DnaE polypeptide can be coupled to a scmtillation-filled bead Addition of radio-labeled DnaE polypeptide results in binding where the radioactive source molecule is m close proximity to the scintillation fluid Thus, signal is emitted upon DnaE polypeptide bmdmg and compounds that prevent DnaE polypeptide self-association or an association of DnaE polypeptide and another polypeptide or small molecule will dimmish signal
  • ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidin- facilitated ion channels m suspended membrane bilayers and hence to a measurable change m the admittance (similar to impedance) of the biosensor This approach is lmear over six decades of admittance change and is ideally suited for large scale, high through-put screening of small molecule combinatorial hbranes
  • identifying compounds which bmd to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably bemg associated with a second component capable of providmg a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleo
  • Potential antagonists include, among others, small organic 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 organic molecules, a peptide, a polypeptide such as a closely related protem or antibody that binds the same sites on a bmdmg molecule, such as a bmdmg molecule, without mducmg DnaE-mduced activities, thereby preventing the action or expression of DnaE polypeptides and/or polynucleotides by excluding DnaE 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 binding to cellular bmdmg molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules
  • Other potential antagomsts m clude antisense molecules (see Okano, J Neurochem 56 560 (1991), OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a descnption of these molecules)
  • Prefe ⁇ ed potential antagomsts mclude compounds related to and vanants of DnaE
  • Other examples of potential polypeptide antagomsts include antibodies or, m some cases, ohgonucleotides or proteins which are closely related to the ligands, substrates, receptors,
  • the present invention relates to a screenmg kit for identifying agonists, 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 compnses (a) a polypeptide and/or a polynucleotide of the present invention, (b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention, (c) a cell membrane expressing a polypeptide and/or polynucleotide of the present mvention, or (d) antibody to a polypeptide and/or polynucleotide of the present invention, which polypeptide is preferably that of SEQ ID NO 2, and which polynucleotide is preferably that of SEQ ID NO 1
  • m any such kit (a) a polypeptide and/or
  • a 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 (a) determining m the first instance the three-dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive s ⁇ te(s), bmdmg s ⁇ te(s) or mot ⁇ f(s) of an agonist, antagonist or inhibitor, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced bmdmg s ⁇ te(s), reactive s ⁇ te(s), and/or mot ⁇ f(s), and (d) testing whether the candidate compounds are mdeed agomsts, antagonists or inhibitors
  • the present mvention provides methods of treating abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of DnaE polypeptide and/or polynucleotide
  • the present invention relates to genetically engmeered soluble fusion proteins comprising a polypeptide of the present mvention, or a fragment thereof, and various portions of the constant regions of heavy or light chams of lmmunoglobulms of vanous subclasses (IgG, IgM.
  • IgA, IgE Preferred as an lmmunoglobulm is the constant part of the heavy cham of human IgG, particularly IgGl , where fusion takes place at the hinge 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 proteins 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 protein technology can be found m International Patent Application Nos W094/29458 and W094/22914
  • expression of the gene encoding endogenous DnaE polypeptide can be inhibited usmg expression blockmg techmques
  • This blockmg may be targeted against any step m gene expression, but is preferably targeted agamst transcnption and/or translation
  • An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 m Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988))
  • ohgonucleotides which form tnple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360)
  • These ohgomers can be administered per se or the relevant
  • 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 antibactenal drugs
  • the polynucleotide sequences encoding the ammo terminal regions of the encoded protein or Shme-Delgarno or other translation facihtatmg sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest
  • the invention also provides the use of the polypeptide, polynucleotide, agomst or antagonist of the invention to interfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the invention may be used m the prevention of adhesion of bactena, in particular gram positive and/or gram negative bactena, to eukaryotic, preferably mammalian, extracellular matnx proteins on m-dwellmg devices or to extracellular matrix protems in wounds, to block bacterial adhesion between eukaryotic.
  • DnaE agomsts and antagomsts preferably bacte ⁇ static or bactencidal agomsts and antagomsts
  • the antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases
  • Hehcobacter pylori herem "H pylori" bacteria infect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, Lyon, 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 adenocarcmoma, classifying the bacterium as a Group I (definite) carcinogen
  • Preferred antimicrobial compounds of the invention agonists and antagonists of DnaE polypeptides and/or polynucleotides found usmg screens provided by the invention, or known m the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent
  • Another aspect of the invention relates to a method for inducing an immunological response m an individual, particularly a mammal which comprises inoculating the mdividual with DnaE 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 immunological response slows bacterial replication
  • Yet another aspect of the invention relates to a method of inducing immunological response m an individual which comprises dehvenng to such mdividual a nucleic acid vector, sequence or nbozyme to direct expression of DnaE polynucleotide and/or polypeptide, or a fragment or a vanant thereof, for expressmg DnaE polynucleotide and/or polypeptide, or a fragment or a vanant thereof in vivo in order to mduce an immunological response
  • a further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having mduced within it an immunological response, induces an immunological response in such mdividual to a DnaE polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant DnaE polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said DnaE polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention
  • the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity ansmg from CTL or CD4+ T cells
  • a DnaE polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or lmmunogenic properties, and preferably protective properties
  • fused recombinant protein preferably further compnses an antigenic co-protem, such as hpoprotein 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
  • the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protem
  • the co-protem may be attached to either the ammo- or carboxy-terminus of the first protem
  • compositions particularly vaccme compositions, and methods compnsmg the polypeptides and/or polynucleotides of the mvention and lmmunostimulatory DNA sequences, such as those described in Sato, Y et al Science 273 352 (1996)
  • this mvention are methods using the descnbed polynucleotide or particular fragments thereof, which have been shown to encode non-variable regions of bactenal 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, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Staphylococcus aureus 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 agamst invasion of bactena, for example by blocking adherence of bacteria to damaged tissue Examples of tissue damage mclude wounds in skin or connective tissue caused, for example, by mechanical,
  • the invention also includes a vaccine formulation which compnses an immunogemc recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal
  • aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacte ⁇ static compounds and solutes which render the formulation lsotonic with the bodily fluid, preferably the blood, of the mdividual, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents
  • the formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze- d ⁇ ed condition
  • compositions for purposes of compositions, kits and administration
  • compositions compnsmg a DnaE polynucleotide and/or a DnaE polypeptide for admmistration to a cell or to a multicellular organism
  • the mvention also relates to compositions compnsmg a polynucleotide and/or a polypeptides discussed herem or their agomsts or antagomsts
  • the polypeptides and polynucleotides of the mvention may be employed m combination with a non-sterile or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for admmistration to an mdividual
  • Such compositions compnse for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the mvention and a pharmaceutically acceptable earner or excipient
  • Such earners may mclude, but are not limited to, sahne, buffered salme, dextrose, water, glycerol, ethanol and combmations thereof
  • the formulation should suit the mode of admmistration
  • the mvention further relates
  • 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 administered m any effective, convement manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, lntrapentoneal, intramuscular, subcutaneous, lntranasal or mtradermal routes among others
  • the active agent may be administered to an mdividual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotomc
  • compositions may be formulated for topical application for example in the form of omtments, creams, lotions, eye omtments, 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 m ointments and creams
  • topical formulations may also contain compatible conventional carriers, for example cream or omtment bases, and ethanol or oleyl alcohol for lotions
  • Such carriers may constitute from about 1% to about 98% by weight of the fonnulation, more usually they will constitute up to about 80% by weight of the formulation
  • the present mvention provides for pharmaceutical compositions compnsmg 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
  • the mvention further relates to pharmaceutical packs and kits compnsmg 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 m conjunction with other compounds such as therapeutic compounds
  • 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 include injection, typically by mtravenous injection
  • Other injection routes such as subcutaneous, intramuscular, or intrapentoneal
  • Alternative means for systemic admmistration m include transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents
  • oral administration may also be possible
  • Administration of these compounds may also be topical and/or localized, m the form of salves, pastes, gels, and the like
  • 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 individual and will vary with the age, weight and response of the particular individual
  • the above dosages are exemplary of the average case
  • In-dwelling devices mclude surgical implants, prosthetic devices and catheters, I e , devices that are introduced to the body of an mdividual and remain in position for an extended time
  • Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, and contmuous ambulatory pentoneal dialysis (CAPD) catheters
  • composition of the mvention may be administered by injection to achieve a systemic effect agamst relevant bactena shortly before insertion of an in-dwelling device Treatment may be contmued after surgery during the m-body time of the device
  • composition could also be used to broaden preoperative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections
  • compositions of this mvention may be used generally as a wound treatment agent to prevent adhesion of bactena to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or m conjunction with, antibiotic prophylaxis
  • composition of the mvention may be used to bathe an indwelling device immediateh before insertion
  • the active agent will preferably be present at a concentration of 1 ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices
  • 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 indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their admmistration to suitable individuals
  • Polynucleotide and polypeptide sequences form a valuable information resource with which to determine 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 m 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 m sequence analysis algorithms
  • 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 mvention and “polypeptide sequence of the invention” mean any detectable chemical or physical charactenstic of a polypeptide of the invention that is or may be reduced to
  • the mvention provides a computer readable medium having stored thereon polypeptide sequences of the invention and/or polynucleotide sequences of the invention
  • a computer readable medium comprising and having stored thereon a member selected from the group consisting of a polynucleotide comprismg the sequence of a polynucleotide of the invention, a polypeptide comprising the sequence of a polypeptide sequence of the 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 polypeptide sequence of the invention, a data set representing a polynucleotide sequence comprising the sequence of polynucleotide sequence of the invention, a data set representmg a polynucleotide sequence encoding a polypeptide sequence compnsmg the sequence of a poly
  • sequence analysis includes, for example, methods of sequence homology analysis, such as identity and simila ⁇ ty analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open readmg frame determmation, nucleic acid base callmg, nucleic acid base tnmmmg, and sequencmg chromatogram peak analysis
  • a computer based method for performing homology identification This method comprises the steps of providmg a first polynucleotide sequence comprising the sequence a polynucleotide of the invention in a computer readable medium; and comparing said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
  • a computer based method for performing homology identification, said method comprising the steps of: providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; and comparing said first polypeptide sequence to at least one second 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 at least one second polynucleotide or polypeptide sequence.
  • a computer based method is still further provided for polynucleotide assembly, said method comprising the steps of: providing a first polypeptide sequence comprising a polypeptide of the invention in a computer readable medium; and screening for at least one overlapping region between said first polypeptide sequence and at least one second polynucleotide or polypeptide sequence.
  • Preferred embodiments of the assemble methods of the invention use the assembly method set forth in United States Patent Number 5,618,672.
  • a computer readable medium having stored thereon a member selected from the group consisting of: a polynucleotide comprising the sequence of SEQ ID NO:l; 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 comprising the sequence of SEQ ID NO: l; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO:2; a polynucleotide comprising the sequence of SEQ ID NO:l; a polypeptide comprising the sequence of SEQ ID NO:2; a set of polynucleotide sequences wherein at least
  • 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:l in 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 mvention provides a computer based method for performing homology identification, said method comprising the steps of providing a polypeptide sequence comprising the sequence of SEQ ID NO 2 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 compnsmg the steps of providing a first polynucleotide sequence compnsmg the sequence of SEQ ID NO 1 in a computer readable medium, and screenmg for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence
  • a further embodiment of the mvention provides a computer based method for performing homology identification, said method compnsmg the steps of providmg 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
  • “Ant ⁇ body( ⁇ es)” as used herem includes polyclonal and monoclonal antibodies, chimenc, smgle chain, and humamzed antibodies, as well as Fab fragments, mcludmg the products of an Fab or other lmmunoglobulm expression library
  • Antigenically equivalent de ⁇ vative(s) encompasses a polypeptide, polynucleotide, or the equivalent of either which will be specifically recognized by certain antibodies which, when raised to the protein, polypeptide or polynucleotide according to the invention, interferes with the immediate physical interaction between pathogen and mammalian host
  • Bispecific ant ⁇ body( ⁇ es) means an antibody comprising at least two antigen binding domains, each domain directed agamst a different epitope
  • Bodily matenal(s) means any matenal denved from an mdividual or from an organism infecting, infesting or inhabiting an mdividual, mcludmg but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy matenals
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena, mcludmg , for example, disease, such as, infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g , empyema, lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g . secretory dia ⁇ hoea, splemc absces. retrope ⁇ toneal abscess), CNS (e g .
  • infections of the upper respiratory tract e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis
  • lower respiratory e g , empyema, lung abscess
  • cardiac e g , infective endocarditis
  • gastrointestinal e g . secretory dia ⁇ h
  • Fusion prote ⁇ n(s) refers to a protem encoded by two, often unrelated, fused genes or fragments thereof
  • EP-A-0464 discloses fusion proteins comprising various portions of constant region of lmmunoglobulm molecules together with another human protein or part thereof
  • employmg an lmmunoglobulm Fc region as a part of a fusion protem is advantageous for use in therapy and diagnosis resultmg m, for example, improved pharmacokmetic properties [see, e g , EP-A 0232262]
  • “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,” as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity” can be readily calculated by known methods, mcludmg but not limited to those described m (Computational Molecular Biology, Lesk, A M , ed , Oxford University 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, Gnffin, A M , and Gnffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Primer, Gnbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Carillo, H , and Lip
  • Polynucleotide embodiments further include an isolated polynucleotide compnsmg 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 integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either mdividually among the nucleotides in the reference sequence or m 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
  • 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 pnor to subtracting it from x n
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or ftameshift mutations m this codmg sequence and thereby 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 integer 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 wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either mdividually among the nucleic acids in the reference sequence or in one or more contiguous groups within 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 in 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-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n
  • Polypeptide embodiments further include an isolated polypeptide comprising 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 amino 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, mcludmg 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 ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from
  • 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 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-mteger product of x a and y is rounded down to the nearest integer pnor to subtracting
  • a polypeptide sequence of the present mvention may 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 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 consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either mdividually among the amino acids m 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 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 ammo 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 wherein any non-mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • Imunologically equivalent denvat ⁇ ve(s) encompasses a polypeptide, polynucleotide, 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 mteraction between pathogen and mammalian host
  • 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 affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the pnor 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, . e , if it occurs m nature, it has been changed or removed from its onginal environment, or both
  • a polynucleotide or a polypeptide naturally present m a living orgamsm is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting 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
  • Organism(s) means a (I) prokaryote, mcludmg but not limited to, a member of the genus
  • Streptococcus Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Neissena, Haemoph lus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus L stena, Calymmatobacterium, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further mcludmg
  • Polynucleotide(s) generally refers to any polynbonucleotide or polydeoxynbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleot ⁇ de(s)" mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or smgle-, double- and tnple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions
  • polynucleotide as used herem refers to tnple-stranded regions compnsmg RNA
  • Polypept ⁇ de(s) refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds
  • Polypept ⁇ de(s) refers to both short chains, commonly refe ⁇ ed to as peptides, o gopeptides and ohgomers and to longer chains generally refe ⁇ ed to as proteins
  • Polypeptides may contain ammo acids other than the 20 gene encoded amino acids
  • Polypeptide(s)” mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and in more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications
  • Polypeptides may be branched or cyclic, with or without branchmg Cyclic, branched and branched circular polypeptides may result from posttranslational natural processes and may be
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides of the mvention introduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention "Subtraction set" is one or more, but preferably less than 100, polynucleotides comprising at least one polynucleotide of the invention
  • 'Na ⁇ ant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical vanant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the 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 m the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in ammo acid sequence from another reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitution
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis and bv other recombinant methods known to skilled artisans
  • EXAMPLES The examples below are earned out usmg standard techmques, which are well known and routme to those of skill m the art, except where otherwise descnbed m detail The examples are illustrative, but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing
  • the polynucleotide havmg a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus m E colt
  • the sequencmg data from two or more clones containing overlappmg 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 accordmg to standard procedures
  • DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coli infected with the packaged library
  • the library is amplified by standard procedures
  • 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 into library vectors (e g . Rsal. Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coll infected with the packaged library The library is amplified by standard procedures Example 2 DnaE Characterization
  • TheStaphylococcus aureusDnaE gene was PCR amplified from Staphylococcus aureus WCUH29 chromosomal DNA usmg the following pnmers
  • Staphylococcus aureusDnaE gene from strain WCUH29 was cloned as a Ndel/Xhol fragment into pBluePet and pET20BHC [ both of these constructs are derived from the Novagen pET vectors]
  • Expression studies were performed m Ecoh BL21DE3 and a overexpressed protem band of correct size for Staphylococcus aureusDnaE( ⁇ l 17kDa) was clearly visible after Coomassie stamed SDSPAGE

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Abstract

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

Description

DnaE
FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses In particular, the invention relates to polynucleotides and polypeptides of the DNA Polymerase family, as well as their vaπants, hereinafter referred to as "DnaE," "DnaE polynucleotιde(s)," and "DnaE polypeptιde(s)" as the case may be
BACKGROUND OF THE INVENTION It is 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 Staphylococcus aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bactenal endocarditis are also relatively common There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome
The frequency of Staphylococcus aureus infections has πsen dramatically in the past few decades This has been attπbuted to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Staphylococcus aureus strains that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism Moreover, the drug discovery process is cuπently undergoing a fundamental revolution as it embraces "functional genomics," that is, high throughput genome- or gene-based biology This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics relies heavily on the vaπous tools of bioinfoπriatics to identify gene sequences of potential interest from the manv molecular biology databases now available as well as from other 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 drug discovery
Clearly, there exists a need for polynucleotides and polypeptides, such as the DnaE embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial activity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
SUMMARY OF THE INVENTION The present invention relates to DnaE, in particular DnaE polypeptides and DnaE polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including 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 agonist or antagonist compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting DnaE expression or activity.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The invention relates to DnaE polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a DnaE of Staphylococcus aureus, which is related by amino acid sequence homology to DnaE polypeptide. The invention relates especially to DnaE having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 and SEQ ID NO:2 respectively. Note that sequences recited in the Sequence Listing below as "DNA" represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.
TABLE 1 DnaE Polynucleotide and Polypeptide Sequences
(A) Staphylococcus aureus DnaE polynucleotide sequence [SEQ ID NO:l]. 5 ' - atgtcttttgttcacctgcaagtgcatagcgggtatagcctgctaaacagcgccgcggctgtggaagaactcgtc agtga agctgacaggctcggatatgcgtctttggcgctgacagatgatcatgtcatgtacggagctat caattttataa agctt gcaaggcgagagggatcaacccaataatcggtttgacggcttctgtttttacagatgacagcgagcttgaagcat acccg c cg cctgctggcaaaatcgaatacaggctatcaaaacctgctgaaaatcagcagcgttctgcaatctaaatca aaagg cggactgaagccgaaatggcttcacag ta cgagaagggattatcgcaataacgcccggcgaaaaaggatatat tgaaa cgcttcttgaaggagggctgttcgaacaggctgctcaagcctcgctggaatttcagtctatattcggaaagggag ctttt tatttctcctatcagccttttaaagggaatcaagtcttgtctgagcagattctaaagctgtcagaagaaacgggt atccc tgttacggccacaggcgatgtgcattacataaggaaggaagataaggctgcctaccgttgtttgaaagcgattaa agcgg gggaaaaactgacggatgcacccgctgaagatttgcctgatctcgatttgaaaccactcgaagaaatgcaaaaca tttat cgagagcatcccgaggcactgcaagcttctgtagagatcgcggaacaatgccgggttgacgtcagtctcgggcag acccg tctaccgtcttttccgaccccagacggaacgtccgctgatgattatttaacggatatctgtatggagggactccg cagcc gtttcggcaagcctgatgaacgataccttcgccgtcttcaa at-gagcttgatgtcatcaagcggat-gaagt-tca gcgat tactttctgatcgtgtgggattttatgaagcacgcccatgaaaaagggattgttacaggaccgggaaggggttct gcggc gggttctcttgtggcatatgtgctgtatatcacagatgttgacccgattaagcatcaccttctgtttgaaagatt tttaa atcccgaacgcgtcagcatgccggatattgatattgactttcccgatactagaagggatgaagtcattcaatatg ttcag cagaaatacggtgcaatgcacgtcgcacagat a cacctttggaacacttgcggcaaaggcggcgctccgggac gtcgg cagagtattcggcgtcagcccgaaggaggcagatcagcttgcaaaactcattccttcaagaccgggaatgacgct ggatg aagcgagacagcagtcaccgcagctggataagcggcttcgggagtcaagcctgcttcagcaagtctattcaattg cccgg aaaatagaagggctgccaaggcatgcatcaacccacgcagcgggcgttgtcctgagtgaagaaccgttaacagat gttgt tccgcttcaagaagggcatgaagggatctatttaacgcaatacgcgatggaccatcttgaagacttgggcctttt aaaaa tggatttcttaggtttgaggaatctgacacttattgaatccatcacttcaatgattgaaaaagaagaaaatatta aaata gacctttcaagtatttcctacagcgatgacaaaacgttctccttgctgtcaaaaggggatacgacaggcattttc cagct tgaatctgcagggatgagaagcgtcttgaaacggctgaaaccttcgggcctagaagatattgtggcggtcaatgc actgt accggccgggtccgatggaaaatattccgtta atcgaccgtaaacacggtcgggcgcctgttcactatcctc atgaa gat taaggagcattttggaagatacatatggcg ca gtgtatcaggaacaaattatgatgattgcttcccgt atggc ggggttttctttaggcgaagcggacctattgagaagggcggtcagcaaaaagaaaaaagaaatcctggacagaga gcgaa gccattttgttgaagggtgcttaaaaaaggagtattctgtagacactgcaaatgaggtctacgatttaatcgtca aattc gcaaactatggctttaacagaagccatgcggtggcatacagcatgatcgggtgccagcttgcgtatttgaaagct catta tccgttatattttatgtgcggattgctgacaagcgtcattggcaatgaggataaaatctctcaatatctttacga agcaa aggggagcgggattcgtatccttcctcc tcagtgaataaaagcagttttc g ttacagttgaaaacggatctg tcaga tacagcctccgcgcgataaaaagtgtaggcgtctcagcagtcaaagatatatataaagcaagaaaagagaaacca tttga agatcttttcgatttctgctttcgcgtgccgtcaaaaagcgtaaatcgcaaaatgcttgaagcacttatcttttc tggtg cgatggacgaattcggccaaaaccgggccacgctgctcgcatccattgatgttgctttggaacatgctgagc at tcgcc gcggacgatgaccaaatgggattgtttttagatgaatcgttttcaattaagccgaagtatgtggagacggaggag cttcc tctggtggatttacttgcgtttgaaaaggaaacgcttggcatttatttttcaaatcatcccctctccgccttccg aaaac agctcactgcgcaaggagcagtctccattctgcaggctcagcgggcagtcaaaaggcagctttcactaggcgttc tgctg tcgaagatcaaaacgattcggacgaaaacaggtcaaaacatggcgtttttaacgctcagtgatgaaaccggcgag atgga agccgtagtcttccctgaacaattcagacagctttctcccgttttaagagaaggcgctct-tttgttcactgcggg gaaat gtgaagttagacaagataagatccaattca catgtcccgggcagaa tat agaagatatggatgcagaaaaag cgcca tcggtttatataaaaatagaaagcagtcagcacagccaggagatcctggcaaagattaagcgcattttgctggag cataa aggggaaacaggcgttta ctctat atgaaaggcaaaaacagacgattaagctacctgagtcgttccatatcaa tgcag atcaccaggtgttatatcgcttaaaagagctgttgggtcaaaaaaacgtcgttttaaaacagtggtaa-3 '
(B) Staphylococcus aureus DnaE polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO:2],
NH2-
MSFVH QVHSGYSLLNSAAAVEELVSEADRLGYASLALTDDHVMYGAIQFYKACKARGINPIIGLTASVFTDDSE EAY LVLLAKSNTGYQNLLKISSVLQSKSKGG KPK LHSYREGIIAITPGEKGYIETLLEGGLFEQAAQASLEFQSIF
GKGAF
YFSYQPFKGNQVLSEQILKLSEETGIPVTATGDVHYIRKEDKAAYRCLKAIKAGEKLTDAPAEDLPD DLKPLEE
MQNIY
REHPEALQASVEIAEQCRVDVS GQTR PSFPTPDGTSADDYLTDICMEGLRSRFGKPDERYLRRLQYELDVIKR MKFSD
YFLIV DFMKHAHEKGIVTGPGRGSAAGSLVAYVLYITDVDPIKHHL FERFLNPERVSMPDIDIDFPDTRRDEV
IQYVQ
QKYGAMHVAQIITFGTLAAKAALRDVGRVFGVSPKEADQI-AKLIPSRPGMTLDEARQQSPQLDKRLRESSLLQQV
YSIAR KIEGLPRHASTHAAGWLSEEPLTDWPLQEGHEGIYLTQYAMDH EDLGLLKMDFLG RNLTLIESITSMIEKE
ENIKI
DLSSISYSDDKTFSLLSKGDTTGIFQLESAGMRSVLKRLKPSGLEDIVAVNALYRPGPMENIPLFIDRKHGRAPV
HYPHE
DLRSI EDTYGVIVYQEQIMMIASRMAGFSLGEADLLRRAVSKKKKEILDRERSHFVEGCLKKEYSVDTANEVYD LIVKF
ANYGFNRSHAVAYSMIGCQLAYLKAHYPLYFMCGLLTSVIGNEDKISQY YEAKGSGIRILPPSVNKSSFPFTVE
NGSVR
YSLRAIKSVGVSAVKDIYKARKEKPFEDLFDFCFRVPSKSVNRK LEALIFSGAMDEFGQNRATLLASIDVALEH
AE FA ADDDQ G FLDESFSIKPKYVETEELPLVDLLAFEKET GIYFSNHPLSAFRKQLTAQGAVSILQAQRAVKRQLS
LGVLL
SKIKTIRTKTGQN AFLT SDETGEMEAWFPEQFRQLSPV REGALLFTAGKCEVRQDKIQFIMSRAELLEDMD
AEKAP
SVYIKIESSQHSQEILAKIKRILLEHKGETGVYLYYERQKQTIKLPESFHINADHQVLYRLKELLGQKNWLKQ -COOH Deposited materials
A deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the
National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive,
Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCIMB Deposit No. 40771, and refeπed to as Staphylococcus aureus WCUH29 on deposit. . The Staphylococcus aureus strain deposit is refeπed to herein as "the deposited strain" or as "the DNA of the deposited strain."
The deposited strain contains a full length DnaE gene. The sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein. The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The deposited strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. §112. A license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
In one aspect of the invention there is provided 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. Further provided by the invention are DnaE polynucleotide sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby. Also provided by the invention are DnaE polypeptide and polynucleotide sequences isolated from the deposited strain.
Polypeptides
DnaE polypeptide of the invention is substantially phylogenetically related to other proteins of the DNA Polymerase family. In one aspect of the invention there are provided polypeptides of Staphylococcus aureus refeπed to herein as "DnaE" and "DnaE polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
Among the particularly prefeπed embodiments of the invention are variants of DnaE polypeptide encoded by naturally occurring alleles of the DnaE gene. The present invention further provides for an isolated polypeptide which: (a) comprises or consists of an amino 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 preferably at least 97-99% or exact identity to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide 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 preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.
The polypeptides of the invention 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 DnaE, 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 amino acids and more preferably at least 50 amino acids.
The invention also includes a polypeptide consisting of or comprising a polypeptide of the formula:
X-(Rι )m-(R2)-(R3)n-Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety described herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety described herein for modified polypeptides, Ri and R3 are any amino acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof. In the formula above, R2 is oriented so that its amino terminal amino acid residue is at the left, covalently bound to
Ri and its carboxy terminal amino acid residue is at the right, covalently bound to R3. Any stretch of amino 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 prefeπed 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.
It is most prefeπed that a polypeptide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
A fragment is a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention. As with DnaE polypeptides, 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. Prefeπed 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 prefeπed. Further prefeπed are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Further prefeπed fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2. Also prefeπed are biologically active fragments that are those fragments that mediate activities of
DnaE, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly prefeπed are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Staphylococcus aureus or the ability to initiate, or maintain cause Disease in an individual, particularly a human.
Fragments of the polypeptides of the invention may be employed for producing the coπesponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
In addition to the standard single and triple letter representations for amino acids, the term "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.
Polynucleotides
It is an object of the invention to provide polynucleotides that encode DnaE polypeptides, particularly polynucleotides that encode the polypeptide herein designated DnaE.
In a particularly prefeπed embodiment of the invention the polynucleotide comprises a region encoding DnaE polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:l] which includes 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 that possesses it, such as Staphylococcus aureus. As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and or expressing DnaE polypeptides and polynucleotides, particularly Staphylococcus aureus DnaE polypeptides and polynucleotides, including, for example, unprocessed RNAs, πbozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vaπants thereof, and compositions compπsing the same
Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a DnaE polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vaπants thereof In another particularly prefeπed embodiment of the mvention there is a DnaE polypeptide from
Staphylococcus aureus compπsmg or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof
Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO 1] a polynucleotide of the invention encoding DnaE polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteπa using Staphylococcus aureus WCUH 29 cells as starting mateπal, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the mvention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1], typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E colt or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, denved from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent hybπdization conditions By sequencing the individual clones thus identified by hybridization with sequencmg primers designed from the oπgmal polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence Conveniently, such sequencmg is performed, for example, usmg denatured double stranded DNA prepared from a plasrmd clone Suitable techmques are described by Maniatis, T , Fπtsch, E F and Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (see in particular Screemng By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencmg may also be performed to obtain a full length gene sequence Illustrative of the mvention, each polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered in a DNA library denved from Staphylococcus aureus WCUH 29
Moreover, each DNA sequence set out in Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled m the art The polynucleotide of SEQ ID NO 1. between nucleotide number 1 and the stop codon which begins at nucleotide number 3346 of SEQ ID NO 1, encodes the polypeptide of SEQ ID NO 2
In a further aspect, the present mvention provides for an isolated polynucleotide comprising or consisting of (a) 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 preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encoding a polypeptide 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 preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than Staphylococcus aureus, may be obtained by a process which compnses the steps of screening an appropπate library under stringent hybndization conditions with a labeled or detectable probe consisting of or compnsing the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones containing said polynucleotide sequence
The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO 1] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence The polynucleotide of the mvention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcnbed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termination signals), nbosome bmdmg sites, Kozak sequences, sequences that stabilize mRNA. lntrons, and polyadenylation signals The polynucleotide sequence may also compnse additional coding sequence encoding additional ammo acids For example, a marker sequence that facilitates puπfication of the fused polypeptide can be encoded In certain embodiments of the mvention, the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed in Gentz et al , Proc Natl Acad Set , USA 86 821-824 (1989), or an HA peptide tag (Wύson et al , Cell 37 767 (1984), both of which may be useful in puπfying polypeptide sequence fused to them Polynucleotides of the mvention also mclude, but are not limited to, polynucleotides compnsing a structural gene and its naturally associated sequences that control gene expression A prefeπed embodiment of the invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 3346 set forth in SEQ ID NO: 1 of Table 1, both of which encode the DnaE polypeptide.
The invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:
X-(Rι)m-(R2)-(R3)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 hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of Ri 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 R2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof. In the polynucleotide formula above, R2 is oriented so that its 5' end nucleic acid residue is at the left, bound to R\ and its 3' end nucleic acid residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either R\ and/or R2, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where, in a preferred embodiment, 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 strand is complementary. In another preferred embodiment m and/or n is an integer between 1 and 1000. Other prefeπed 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.
It is most prefeπed that a polynucleotide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Staphylococcus aureus DnaE having an amino acid sequence set out in Table 1 [SEQ ID NO:2], The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences. The mvention further relates to vanants of the polynucleotides descnbed herein that encode vanants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention Further particularly prefeπed embodiments are polynucleotides encoding DnaE vanants, that have the ammo acid sequence of DnaE polypeptide of Table 1 [SEQ ID NO 2] m which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no am o acid residues are substituted, modified, deleted and/or added, m any combination Especially prefeπed among these are silent substitutions, additions and deletions, that do not alter the properties and activities of DnaE polypeptide Further prefeπed embodiments of the mvention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding DnaE polypeptide having an ammo acid sequence set out in Table 1 [SEQ ID NO 2] and polynucleotides that are complementary to such polynucleotides Alternatively most highly prefeπed are polynucleotides that compπse a region that is at least 80% identical over its entire length to a polynucleotide encoding DnaE polypeptide and polynucleotides complementary thereto In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly prefeπed, and among these particularly prefeπed polynucleotides, those with at least 95% are especially prefeπed Furthermore, those with at least 97% are highly prefeπed among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly prefeπed, with at least 99% being the more prefeπed Prefeπed embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1]
In accordance with certain prefeπed embodiments of this mvention there are provided polynucleotides that hybndize, particularly under stringent conditions, to DnaE polynucleotide sequences, such as those polynucleotides in Table 1 The mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herein In this regard, the mvention especially relates to polynucleotides that hybndize under stringent conditions to the polynucleotides descnbed herem As herein used, the terms "stringent conditions" and "stringent hybndization conditions" mean hybndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of stringent hybndization conditions is overnight mcubation at 42°C m a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM tπsodmm citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support m 0 lx SSC at about 65°C Hybndization and wash conditions are well known and exemplified m Sambrook, et al , Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor, N Y , (1989), particularly Chapter 11 therein Solution hybndization may also be used with the polynucleotide sequences provided by the invention
The mvention also provides a polynucleotide consisting of or compnsmg a polynucleotide sequence obtained by screening an appropriate library contammg the complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under stπngent hybndization conditions with 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 include, for example, probes and primers fully descnbed elsewhere herem
As discussed elsewhere herem regarding polynucleotide assays of the mvention, for instance, the polynucleotides of the mvention, may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding DnaE and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the DnaE gene Such probes generally will compnse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs Particularly prefeπed probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pa rs
A coding region of a DnaE gene may be isolated by screening usmg a DNA sequence provided in Table 1 [SEQ JD NO 1] to synthesize an oligonucleotide probe A labeled oligonucleotide having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to
There are several methods available and well known to those skilled m 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 of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, 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 earned out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide pnmers The PCR reaction is then repeated using "nested" primers, that is, pnmers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' m the adaptor sequence and a gene specific pnmer that anneals further 5' in the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencmg 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 usmg the new sequence information for the design of the 5' pnmer The polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relating to polynucleotide assays
The polynucleotides of the mvention that are ohgonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herein as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herein m whole or m part are transcribed in bactena m infected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained
The mvention also provides polynucleotides that encode a polypeptide that is the mature protem plus additional ammo or carboxyl-terminal am o 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 As generally is the case in vivo, the additional ammo acids may be processed away from the mature protem by cellular enzymes
For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to it It is prefeπed that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary
A precursor protein, havmg a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
In addition to the standard A, G, C, T/U representations for nucleotides, the term "N" may also be used in descπbmg certain polynucleotides of the mvention "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken in combmation with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such readmg frame
In sum, a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (which may be refeπed to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotein, which is a precursor to a proprotem, havmg a leader sequence and one or more prosequences, which generally are removed duπng processmg steps that produce active and mature forms of the polypeptide
Vectors, Host Cells, Expression Systems The invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention. Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems that comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques. For recombinant production of the polypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al, BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as cells of streptococci, staphylococci, enterococci, E. coli, streptomyces, cyanobacteria, Bacillus subtilis, and Staphylococcus aureus; fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insect cells such as cells of Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells, such as cells of a gymnosperm or angiosperm.
A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculovirases, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. 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 in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a vaπety of well-known and routine techmques, such as, for example, those set forth in Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
In recombinant expression systems in eukaryotes. for secretion of a translated protem into the lumen of the endoplasmic reticulum, into the penplasmic space or into the extracellular environment, appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and or punfication
Diagnostic, Prognostic, Serotyping and Mutation Assays This mvention is also related to the use of DnaE polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of DnaE polynucleotides and/or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the DnaE gene or protein, may be detected at the nucleic acid or ammo acid level by a vanety of well known techniques as well as by methods provided herem
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected mdividual's bodily matenals Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification techmque pnor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characterization of the species and strain of infectious or resident organism present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism Deletions and insertions can be detected by a change m size of the amplified product m companson to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species Pomt mutations can be identified by hybndizing amplified DNA to labeled DnaE polynucleotide sequences Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried out with or without denaturing 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 assays, such as RNase, VI and SI protection assay or a chemical cleavage method. See, for example, Cotton et al. , Proc. Natl. Acad. Sci. , USA, 85: 4397-4401 (1985).
In another embodiment, an aπay of oligonucleotides probes comprising DnaE nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al, Science, 274: 610 (1996)).
Thus in another aspect, the present invention relates to a diagnostic kit which comprises: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others. This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide of the invention, preferable, SEQ ID NO: 1, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.
The nucleotide sequences of the present invention are also valuable for organism chromosome identification. The sequence is specifically targeted to, and can hybridize 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 invention may be an important step in coπelating those sequences with pathogenic potential and/or an ecological niche of an organism and/or drug resistance of an organism, as well as the essentiality of the gene to the organism. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be coπelated with genetic map data. Such data may be found on-line in 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 (coinheritance of physically adjacent genes) or mating studies, such as by conjugation.
The differences in a polynucleotide and or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined. If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype.
Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations in the RNA. It is particularly prefeπed to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose, PCR. As an example, PCR primers complementary to a polynucleotide encoding DnaE polypeptide can be used to identify and analyze mutations. Examples of representative primers are shown below in Table 2.
Table 2 Primers for amplification of DnaE polynucleotides SEQ ID NO PRIMER SEQUENCE
3 5 ' -CCATTCAATCATATGGTGGCATATTTAAATATTC-3 '
4 5 ' -CACAACACTCGAGTTAGCATTCCCCTTTCGTAGC-3 '
The invention also includes primers of the formula: X-(R1)m-(R2)-(R3)n-Y wherein, at the 5' end of the molecule, 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, R\ and R3 are any nucleic acid residue or modified nucleotide residue, m is an integer between 1 and 20 or zero , n is an integer between 1 and 20 or zero, and R2 is a primer sequence of the invention, particularly a primer sequence selected from Table 2. In the polynucleotide formula above R is oriented so that its 5' end nucleotide residue is at the left, bound to Ri and its 3' end nucleotide residue is at the right, 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 being complementary to a region of a polynucleotide of Table 1. In a prefeπed embodiment m and/or n is an integer between 1 and 10. The mvention further provides these pnmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These pnmers may be used for, among other things, amplifying DnaE DNA and/or RNA isolated from a sample denved from an mdividual, such as a bodily mateπal The pnmers may be used to amplify a polynucleotide isolated from an infected mdividual, such that the polynucleotide may then be subject to vanous techniques for elucidation of the polynucleotide sequence In this way, mutations m the polynucleotide sequence may be detected and used to diagnose andor prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
The mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections caused by Staphylococcus aureus, compnsmg detenmmng from a sample derived from an mdividual, 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 DnaE polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods In addition, a diagnostic assay m accordance with the mvention for detectmg over-expression of DnaE 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 DnaE polypeptide, m a sample denved from a host, such as a bodily matenal, 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
The polynucleotides and polynucleotides of the mvention may be used as reagents for differential screening methods There are many differential screening and differential display methods known in the art in which the polynucleotides and polypeptides of the mvention may be used For example, the differential display technique is descnbed by Chuang et al , J Bacterwl 175 2026-2036 (1993) This method identifies those genes which are expressed in an organism by identifying mRNA present usmg randomly-primed RT-PCR By comparing pre-infection 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 described by Camilh et al , Proc Nat'l Acad Sci
USA 91 2634-2638 (1994) IVET identifies genes up-regulated during infection when compared to laboratory cultivation, implying an important role m infection ORFs identified by this techmque are implied to have a significant role m infection establishment and/or maintenance In this technique random chromosomal fragments of target organism are cloned upstream of a promoter-less recombmase gene in a plasmid vector This construct is introduced mto the target organism which caπies an antibiotic resistance gene flanked by resolvase sites Growth in 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 sensitive 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 mvention, messenger RNA is isolated from bactenal mfected tissue, e g , 48 hour muπne 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 pnmer 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 transcnbed in the mfected tissue Analysis of gene transcnption can be earned out at different times of mfection to gam a detailed knowledge of gene regulation m bactenal 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 bactenal 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 species which are very short lived m the bacterium (in the order of 2 mmute halflives) Optimally the bactenal mRNA is prepared from mfected munne lung tissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL) for very short periods of time, subsequent processmg according to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA Preferably the process is optimized by fmdmg those conditions which give a maximum amount of Staphylococcus aureus 16S nbosomal RNA as detected by probmg Northerns with a suitably labeled sequence specific oligonucleotide probe Typically a 5' dye labeled pnmer is used m each PCR primer pair m a PCR reaction which is terminated optimally between 8 and 25 cycles The PCR products are separated on 6% polyacrylamide gels with detection and quantification using GeneScanner (manufactured by ABI) Gridding and Polynucleotide Subtraction
Methods have been described for obtaining information about gene expression and identity using so called "high density DNA arrays" or gnds See, e g , M Chee et al , Science, 274 610-614 (1996) and other references cited therein Such gridding assays have been employed to identify certain novel gene sequences, referred to as Expressed Sequence Tags (EST) (Adams et a , Science, 252 1651-1656 (1991)) A vanety of techniques have also been descnbed for identifying particular gene sequences on the basis of their gene products For example, see International Patent Application No WO91/07087, published May 30, 1991 In addition, methods have been descnbed for the amplification of desired sequences For example, see International Patent Application No W091/17271, published November 14, 1991
The polynucleotides of the invention may be used as components of polynucleotide aπays, preferably high density aπays or grids These high density aπays are particularly useful for diagnostic and prognostic purposes For example, a set of spots each comprismg a different gene, and further comprising a polynucleotide or polynucleotides of the invention, may be used for probing, such as using hybridization or nucleic acid amplification, using a probes obtamed or denved from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual Such a presence may indicate the presence of a pathogen, particularly Staphylococcus aureus, and may be useful in diagnosmg and/or prognosmg disease or a course of disease A grid comprismg a number of variants of the polynucleotide sequence of SEQ ID NO 1 are preferred Also prefeπed is a comprismg a number of vanants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO 2
Antibodies
The polypeptides and polynucleotides of the mvention or vanants thereof, or cells expressmg the same can be used as lmmunogens to produce antibodies lmmunospecific for such polypeptides or polynucleotides respectively
In certain prefeπed embodiments of the mvention there are provided antibodies against DnaE polypeptides or polynucleotides
Antibodies generated against the polypeptides or polynucleotides of the mvention can be obtamed by administering the polypeptides and/or polynucleotides of the 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 routme protocols For preparation of monoclonal antibodies, any techmque known m the art that provides antibodies produced by contmuous cell lme cultures can be used Examples mclude vanous techmques, such as those in Kohler, G and Milstem, C , Nature 256 495-497 (1975), Kozbor et al , Immunology Today 4 72 (1983), Cole et al , pg 77-96 m MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Lιss, Inc (1985)
Techmques for the production of smgle chain antibodies (U S Patent No 4,946,778) can be adapted to produce s gle chain antibodies to polypeptides or polynucleotides of this mvention Also, transgenic mice, or other organisms such as other rnammals, may be used to express humanized antibodies lmmunospecific to the polypeptides or polynucleotides of the mvention Alternatively, phage display technology may be utilized to select antibody genes with bmdmg activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-DnaE 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 by, for example, chain shuffling (Clackson et al , (1991) Nature 352 628)
The above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptides or polynucleotides of the mvention to purify the polypeptides or polynucleotides by, for example, affinity chromatography Thus, among others, antibodies against DnaE-polypeptide or DnaE-polynucleotide may be employed to treat infections, particularly bactenal infections
Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form 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 protein may provide stability to the polypeptide The antigen may be associated, for example by conjugation, with an lmmunogemc carrier protein for example bovme serum albumin, keyhole limpet haemocyanin or tetanus toxoid Alternatively, a multiple antigenic polypeptide comprismg multiple copies of the polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogenicity so as to obviate the use of a earner
Preferably, the antibody or vanant thereof is modified to make it less immunogemc in the individual For example, if the mdividual is human the antibody may most preferably be "humanized," where the comphmentanty determimng region or regions of the hybndoma-denved antibody has been transplanted into a human monoclonal antibody, for example as descnbed in lones 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 the particularly prefeπed embodiments of the mvention are naturally occurring allelic vaπants of DnaE polynucleotides and polypeptides encoded thereby
The use of a polynucleotide of the invention in genetic immunization will preferably 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), delivery of DNA complexed with specific protem earners (Wu et al , J Biol Chem (1989) 264 16985), coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef, RN-4S USA, (1986) 83 9551), encapsulation of DΝA m various forms of liposomes (Kaneda et al , Science (1989) 243 375), particle bombardment (Tang et al Nature (1992) 356 152, Eisenbraun et al , DNA Cell Biol (1993) 12 791) and in vivo infection using cloned retroviral vectors (Seeger et al , PNAS USA (1984) 81 5849) Antagonists and Agonists - Assays and Molecules
Polypeptides and polynucleotides of the mvention may also be used to assess the bmdmg 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 , Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, including many disease states, in particular the Diseases herembefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide Accordingly, m a further aspect, 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 In general, agonists 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 agonists, antagonists or inhibitors so- ldentified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of DnaE polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Coligan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991))
The screenmg methods may simply measure the bmdmg of a candidate compound to the polypeptide or polynucleotide. or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screenmg method may involve competition with a labeled competitor
Further, these screenmg methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, usmg detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testmg whether the candidate compound results in inhibition of activation of the 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 of the present invention, to form a mixture, measuring DnaE polypeptide and/or polynucleotide activity in the mixture, and comparing the DnaE polypeptide and/or polynucleotide activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and DnaE polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al., I Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16):9459-9471 (1995)).
The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents 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 screening compounds to identify those which enhance
(agonist) or block (antagonist) the action of DnaE polypeptides or polynucleotides, particularly those compounds that are bacteristatic and/or bactericidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising DnaE polypeptide and a labeled substrate or Ugand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a DnaE agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the DnaE polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i.e., without inducing the effects of DnaE polypeptide are most likely to be good antagonists. Molecules that bind well and, as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists. Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in DnaE polynucleotide or polypeptide activity, and binding assays known in the art.
Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^1), chemically modified (for instance biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily matenals) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the bindmg of the polypeptide to its receptor(s), if any Standard methods for conductmg such assays are well understood in the art
The fluorescence polanzation value for a fluorescently-tagged molecule depends on the rotational coπelation time or tumbling rate Protem complexes, such as formed by DnaE polypeptide associatmg with another DnaE polypeptide or other polypeptide, labeled to compnse a fluorescently- labeled molecule will have higher polanzation values than a fluorescently labeled monomenc protem It is preferred that this method be used to charactenze small molecules that disrupt polypeptide complexes
Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of DnaE polypeptide dimers, tπmers, tetramers or higher order structures, or structures formed by DnaE polypeptide bound to another polypeptide DnaE 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 dimeπzation will inhibit fluorescence energy transfer Surface plasmon resonance can be used to monitor the effect of small molecules on DnaE polypeptide self-association as well as an association of DnaE polypeptide and another polypeptide or small molecule DnaE polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protem can then passed over the DnaE polypeptide -coated surface and specific binding can be detected m real-time by monitoring the change in resonance angle caused by a change in local refractive index This techmque can be used to characterize the effect of small molecules on kinetic rates and equihbnum bmdmg constants for DnaE polypeptide self-association as well as an association of DnaE polypeptide and another polypeptide or small molecule
A scintillation proximity assay may be used to charactenze the interaction between an association of DnaE polypeptide with another DnaE polypeptide or a different polypeptide DnaE polypeptide can be coupled to a scmtillation-filled bead Addition of radio-labeled DnaE polypeptide results in binding where the radioactive source molecule is m close proximity to the scintillation fluid Thus, signal is emitted upon DnaE polypeptide bmdmg and compounds that prevent DnaE polypeptide self-association or an association of DnaE polypeptide and another polypeptide or small molecule will dimmish signal
ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidin- facilitated ion channels m suspended membrane bilayers and hence to a measurable change m the admittance (similar to impedance) of the biosensor This approach is lmear over six decades of admittance change and is ideally suited for large scale, high through-put screening of small molecule combinatorial hbranes
In other embodiments of the mvention there are provided methods for identifying compounds which bmd to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably bemg associated with a second component capable of providmg a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the bmdmg or mteraction of the compound with the polypeptide and/or polynucleotide Another example of an assay for DnaE agonists is a competitive assay that combmes DnaE and a potential agonist with DnaE-binding molecules, recombinant DnaE bmdmg molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay DnaE can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of DnaE molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
Potential antagonists include, among others, small organic 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 organic molecules, a peptide, a polypeptide such as a closely related protem or antibody that binds the same sites on a bmdmg molecule, such as a bmdmg molecule, without mducmg DnaE-mduced activities, thereby preventing the action or expression of DnaE polypeptides and/or polynucleotides by excluding DnaE polypeptides and or polynucleotides from bmdmg
Potential antagonists mclude a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventmg binding to cellular bmdmg molecules, such that normal biological activity is prevented Examples of small molecules mclude but are not limited to small organic molecules, peptides or peptide-like molecules Other potential antagomsts mclude antisense molecules (see Okano, J Neurochem 56 560 (1991), OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a descnption of these molecules) Prefeπed potential antagomsts mclude compounds related to and vanants of DnaE Other examples of potential polypeptide antagomsts mclude 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 , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented Certain of the polypeptides of the mvention are biomimetics, functional mimetics of the natural DnaE polypeptide These functional mimetics may be used for, among other things, antagonizing the activity of DnaE polypeptide or as a antigen or lmmunogen m a manner descnbed elsewhere herem Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides For example, prefeπed functional mimetics mclude, a polypeptide compnsing the polypeptide sequence set forth m SEQ ID NO 2 lacking 20, 30, 40, 50, 60, 70 or 80 ammo- or carboxy-terminal ammo acid residues, including fusion proteins compnsing one or more of these truncated sequences Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefeπed that these cassettes compnse 5' and 3' restπction sites to allow for a convement means to ligate the cassettes together when desired It is further prefeπed that these cassettes compnse gene expression signals known m the art or descnbed elsewhere herem
Thus, in another aspect, the present invention relates to a screenmg kit for identifying agonists, 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 compnses (a) a polypeptide and/or a polynucleotide of the present invention, (b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention, (c) a cell membrane expressing a polypeptide and/or polynucleotide of the present mvention, or (d) antibody to a polypeptide and/or polynucleotide of the present invention, which polypeptide is preferably that of SEQ ID NO 2, and which polynucleotide is preferably that of SEQ ID NO 1 It will be appreciated that m any such kit, (a), (b), (c) or (d) may compnse a substantial component
It will be readily appreciated by the skilled artisan that a 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 (a) determining m the first instance the three-dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive sιte(s), bmdmg sιte(s) or motιf(s) of an agonist, antagonist or inhibitor, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced bmdmg sιte(s), reactive sιte(s), and/or motιf(s), and (d) testing whether the candidate compounds are mdeed agomsts, antagonists or inhibitors
It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed usmg automated and computer-controlled steps
In a further aspect, the present mvention provides methods of treating abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of DnaE polypeptide and/or polynucleotide
If the expression and/or activity of the polypeptide and/or polynucleotide is in excess, several approaches are available One approach compnses administering to an mdividual m need thereof an inhibitor compound (antagonist) as herem descnbed, optionally in combmation with a pharmaceutically acceptable earner m an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide. such as, for example, by blockmg the bmdmg of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of bmdmg the ligand, substrate, enzymes, receptors, etc in competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the DnaE polypeptide and/or polypeptide In a further aspect, the present invention relates to genetically engmeered soluble fusion proteins comprising a polypeptide of the present mvention, or a fragment thereof, and various portions of the constant regions of heavy or light chams of lmmunoglobulms of vanous subclasses (IgG, IgM. IgA, IgE) Preferred as an lmmunoglobulm is the constant part of the heavy cham of human IgG, particularly IgGl , where fusion takes place at the hinge region In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion proteins 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 protein technology can be found m International Patent Application Nos W094/29458 and W094/22914
In still another approach, expression of the gene encoding endogenous DnaE polypeptide can be inhibited usmg expression blockmg techmques This blockmg may be targeted against any step m gene expression, but is preferably targeted agamst transcnption and/or translation An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 m Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, ohgonucleotides which form tnple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed m vivo
Each of the 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 antibactenal drugs Additionally, the polynucleotide sequences encoding the ammo terminal regions of the encoded protein or Shme-Delgarno or other translation facihtatmg sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest
The invention also provides the use of the polypeptide, polynucleotide, agomst or antagonist of the invention to interfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection In particular, the molecules of the invention may be used m the prevention of adhesion of bactena, in particular gram positive and/or gram negative bactena, to eukaryotic, preferably mammalian, extracellular matnx proteins on m-dwellmg devices or to extracellular matrix protems in wounds, to block bacterial adhesion between eukaryotic. preferably mammalian, extracellular matnx proteins and bactenal DnaE proteins that mediate tissue damage and/or, to block the normal progression of pathogenesis m infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques
In accordance with yet another aspect of the mvention, there are provided DnaE agomsts and antagomsts, preferably bacteπstatic or bactencidal agomsts and antagomsts
The antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases
Hehcobacter pylori (herem "H pylori") bacteria infect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcmoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of DnaE polypeptides and/or polynucleotides) found usmg screens provided by the invention, or known m 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 gastrointestmal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastntis Vaccines
There are provided by the mvention, products, compositions and methods for assessmg DnaE expression, treating disease, assaying genetic vanation, and administering a DnaE polypeptide and or polynucleotide to an organism to raise an immunological response against a bactena, especially a Staphylococcus aureus bactena
Another aspect of the invention relates to a method for inducing an immunological response m an individual, particularly a mammal which comprises inoculating the mdividual with DnaE 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 immunological response slows bacterial replication Yet another aspect of the invention relates to a method of inducing immunological response m an individual which comprises dehvenng to such mdividual a nucleic acid vector, sequence or nbozyme to direct expression of DnaE polynucleotide and/or polypeptide, or a fragment or a vanant thereof, for expressmg DnaE polynucleotide and/or polypeptide, or a fragment or a vanant thereof in vivo in order to mduce an immunological response, such as, to produce antibody and/ or T cell immune response, mcludmg, for example, cytokine- producmg T cells or cytotoxic T cells, to protect said mdividual, preferably a human, from disease, whether that disease is already established within the individual or not One example of administering the gene is by acceleratmg it mto the desired cells as a coatmg on particles or otherwise Such nucleic acid vector may comprise DNA, RNA, a nbozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protem complex or an RNA-protein complex
A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having mduced within it an immunological response, induces an immunological response in such mdividual to a DnaE polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant DnaE polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said DnaE polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity ansmg from CTL or CD4+ T cells
A DnaE polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or lmmunogenic properties, and preferably protective properties Thus fused recombinant protein, preferably further compnses an antigenic co-protem, such as hpoprotein 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 may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protem The co-protem may be attached to either the ammo- or carboxy-terminus of the first protem
Provided by this invention are compositions, particularly vaccme compositions, and methods compnsmg the polypeptides and/or polynucleotides of the mvention and lmmunostimulatory DNA sequences, such as those described in Sato, Y et al Science 273 352 (1996)
Also, provided by this mvention are methods using the descnbed polynucleotide or particular fragments thereof, which have been shown to encode non-variable regions of bactenal 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, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Staphylococcus aureus 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 agamst invasion of bactena, for example by blocking adherence of bacteria to damaged tissue Examples of tissue damage mclude wounds in skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds m the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagma
The invention also includes a vaccine formulation which compnses an immunogemc recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteπstatic compounds and solutes which render the formulation lsotonic with the bodily fluid, preferably the blood, of the mdividual, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents The formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze- dπed condition requiring only the addition of the stenle liquid earner immediately pnor to use The vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-in 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
While the invention has been descnbed with reference to certain DnaE polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the lmmunogenic properties of the recombinant polypeptides or polynucleotides
Compositions, kits and administration
In a further aspect of the mvention there are provided compositions compnsmg a DnaE polynucleotide and/or a DnaE polypeptide for admmistration to a cell or to a multicellular organism
The mvention also relates to compositions compnsmg a polynucleotide and/or a polypeptides discussed herem or their agomsts or antagomsts The polypeptides and polynucleotides of the mvention may be employed m combination with a non-sterile or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for admmistration to an mdividual Such compositions compnse for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the mvention and a pharmaceutically acceptable earner or excipient Such earners may mclude, but are not limited to, sahne, buffered salme, dextrose, water, glycerol, ethanol and combmations thereof The formulation should suit the mode of admmistration The mvention further relates to diagnostic and pharmaceutical packs and kits compnsmg 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
The pharmaceutical compositions may be administered m any effective, convement manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, lntrapentoneal, intramuscular, subcutaneous, lntranasal or mtradermal routes among others
In therapy or as a prophylactic, the active agent may be administered to an mdividual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotomc
Alternatively the composition may be formulated for topical application for example in the form of omtments, creams, lotions, eye omtments, 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 m ointments and creams Such topical formulations may also contain compatible conventional carriers, for example cream or omtment bases, and ethanol or oleyl alcohol for lotions Such carriers may constitute from about 1% to about 98% by weight of the fonnulation, more usually they will constitute up to about 80% by weight of the formulation In a further aspect, the present mvention provides for pharmaceutical compositions compnsmg 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, m combination with a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, salme, buffered salme. dextrose, water, glycerol, ethanol, and combmations thereof The mvention further relates to pharmaceutical packs and kits compnsmg 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 m 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 Prefeπed forms of systemic admmistration mclude injection, typically by mtravenous injection Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used Alternative means for systemic admmistration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if a polypeptide or other compounds of the present mvention can be formulated m an entenc or an encapsulated formulation, oral administration may also be possible Administration of these compounds may also be topical and/or localized, m the form of salves, pastes, gels, and the 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, typically around 1 mg/kg The physician in any event will determine the actual dosage which will be most suitable for an individual 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 mdividual instances where higher or lower dosage ranges are merited, and such are withm the scope of this mvention
In-dwelling devices mclude surgical implants, prosthetic devices and catheters, I e , devices that are introduced to the body of an mdividual and remain in position for an extended time Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, and contmuous ambulatory pentoneal dialysis (CAPD) catheters
The composition of the mvention may be administered by injection to achieve a systemic effect agamst relevant bactena shortly before insertion of an in-dwelling device Treatment may be contmued after surgery during the m-body time of the device In addition, the composition could also be used to broaden preoperative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections
Many orthopedic surgeons consider that humans with prosthetic joints 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 jomt 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 descnbed above, the compositions of this mvention may be used generally as a wound treatment agent to prevent adhesion of bactena to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or m conjunction with, antibiotic prophylaxis
Alternatively, the composition of the mvention may be used to bathe an indwelling device immediateh before insertion The active agent will preferably be present at a concentration of 1 μg/ml to lOmg/ml for bathing of wounds or indwelling devices
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 indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their admmistration to suitable individuals
Sequence Databases, Sequences in a Tangible Medium, and Algorithms
Polynucleotide and polypeptide sequences form a valuable information resource with which to determine their 2- and 3-dιmensιonal structures as well as to identify further sequences of similar homology These approaches are most easily facilitated by storing the sequence m 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
The polynucleotide and polypeptide sequences of the invention are particularly useful as components in databases useful for search analyses as well as m sequence analysis algorithms As used m this section entitled "Sequence Databases, Sequences in a Tangible Medium, and Algorithms," and in claims related to this section, the terms "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 As used m this section entitled Databases and Algorithms and in claims related thereto, the terms "polypeptide of the mvention" and "polypeptide sequence of the invention" mean any detectable chemical or physical charactenstic 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, chromatograpmc scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data
The mvention provides a computer readable medium having stored thereon polypeptide sequences of the invention and/or polynucleotide sequences of the invention For example, a computer readable medium is provided comprising and having stored thereon a member selected from the group consisting of a polynucleotide comprismg the sequence of a polynucleotide of the invention, a polypeptide comprising the sequence of a polypeptide sequence of the 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 polypeptide sequence of the invention, a data set representing a polynucleotide sequence comprising the sequence of polynucleotide sequence of the invention, a data set representmg a polynucleotide sequence encoding a polypeptide sequence compnsmg the sequence of a polypeptide sequence of the invention, a polynucleotide compnsmg the sequence of a polynucleotide sequence of the invention, a polypeptide compnsmg the sequence of a polypeptide sequence of the invention, a set of polynucleotide sequences wherein at least one of the sequences comprises the sequence of a polynucleotide sequence of the mvention, a set of polypeptide sequences wherein at least one of said sequences compnses the sequence of 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 available floppy disks, tapes, chips, hard drives, compact disks, and video disks
Also provided by the invention are methods for the analysis of character sequences or strings, particularly genetic sequences or encoded genetic sequences Prefeπed methods of sequence analysis mclude, for example, methods of sequence homology analysis, such as identity and similaπty analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open readmg frame determmation, nucleic acid base callmg, nucleic acid base tnmmmg, and sequencmg chromatogram peak analysis
A computer based method is provided for performing homology identification This method comprises the steps of providmg a first polynucleotide sequence comprising the sequence a polynucleotide of the invention in a computer readable medium; and comparing said first polynucleotide sequence to at least one second 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 first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; and comparing said first polypeptide sequence to at least one second 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 at least one second polynucleotide or polypeptide sequence.
A computer based method is still further provided for polynucleotide assembly, said method comprising the steps of: providing a first polypeptide sequence comprising a polypeptide of the invention in a computer readable medium; and screening for at least one overlapping region between said first polypeptide sequence and at least one second polynucleotide or polypeptide sequence.
Preferred embodiments of the assemble methods of the invention use the assembly method set forth in United States Patent Number 5,618,672.
In another prefeπed embodiment of the invention there is provided a computer readable medium having stored thereon a member selected from the group consisting of: a polynucleotide comprising the sequence of SEQ ID NO:l; 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 comprising the sequence of SEQ ID NO: l; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO:2; a polynucleotide comprising the sequence of SEQ ID NO:l; 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: l; 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 comprising the sequence of SEQ ID NO:l; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising 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:l in 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 mvention provides a computer based method for performing homology identification, said method comprising the steps of providing a polypeptide sequence comprising the sequence of SEQ ID NO 2 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 compnsmg the steps of providing a first polynucleotide sequence compnsmg the sequence of SEQ ID NO 1 in a computer readable medium, and screenmg for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence
A further embodiment of the mvention provides a computer based method for performing homology identification, said method compnsmg the steps of providmg 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
All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth Any patent application to which this application claims pπoπty is also incorporated by reference herein in its entirety m the manner described above for publications and references
GLOSSARY
The following definitions are provided to facilitate understanding of certain terms used frequently herem
"Antιbody(ιes)" as used herem includes polyclonal and monoclonal antibodies, chimenc, smgle chain, and humamzed antibodies, as well as Fab fragments, mcludmg the products of an Fab or other lmmunoglobulm expression library
"Antigenically equivalent deπvative(s)" as used herein encompasses a polypeptide, polynucleotide, or the equivalent of either which will be specifically recognized by certain antibodies which, when raised to the protein, polypeptide or polynucleotide according to the invention, interferes with the immediate physical interaction between pathogen and mammalian host
"Bispecific antιbody(ιes)" means an antibody comprising at least two antigen binding domains, each domain directed agamst a different epitope
"Bodily matenal(s) means any matenal denved from an mdividual or from an organism infecting, infesting or inhabiting an mdividual, mcludmg but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy matenals
"Dιsease(s)" means any disease caused by or related to infection by a bactena, mcludmg , for example, disease, such as, infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g , empyema, lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g . secretory diaπhoea, splemc absces. retropeπtoneal abscess), CNS (e g . cerebral abscess), eye (e g , blephantis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e g , epididymitis, lntrarenal and peπnephnc absces, toxic shock syndrome), skin (e g , impetigo, folhcuhtis, cutaneous abscesses, cellulitis, wound infection, bactenal myositis) bone and jomt (e g , septic arthntis, osteomyelitis)
"Fusion proteιn(s)" refers to a protem encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0464 discloses fusion proteins comprising various portions of constant region of lmmunoglobulm molecules together with another human protein or part thereof In many cases, employmg an lmmunoglobulm Fc region as a part of a fusion protem is advantageous for use in therapy and diagnosis resultmg m, for example, improved pharmacokmetic 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 protem has been expressed, detected and purified
"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," as known m the art, 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 In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, mcludmg but not limited to those described m (Computational Molecular Biology, Lesk, A M , ed , Oxford University 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, Gnffin, A M , and Gnffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Primer, Gnbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Carillo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S F et al , J Molec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990) The well known Smith Waterman algorithm may also be used to determine identity Parameters for polypeptide sequence comparison include the following Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)
Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4
A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps)
Parameters for polynucleotide companson include the following Algorithm Needleman 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 polypeptides, as the case may be, are provided m (1) and (2) below
(1) Polynucleotide embodiments further include an isolated polynucleotide compnsmg 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 integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either mdividually among the nucleotides in the reference sequence or m 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 m SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtractmg that product from said total number of nucleotides m SEQ ID NO 1, or nn ≤ xn " (xn • y)>
wherein nn is the number of nucleotide alterations, xn 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%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer pnor to subtracting it from xn Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or ftameshift mutations m this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
By way of example, 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 integer 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 wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either mdividually among the nucleic acids in the reference sequence or in one or more contiguous groups within 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
nn < xn - (xn • y),
wherein nn is the number of nucleic acid alterations, xn is the total number of nucleic acids in 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-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn
(2) Polypeptide embodiments further include an isolated polypeptide comprising 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 amino 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, mcludmg 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 ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO 2. or
na < xa - (xa • y),
wherein na is the number of ammo acid alterations, xa 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 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of xa and y is rounded down to the nearest integer pnor to subtracting
By way of example, a polypeptide sequence of the present mvention may 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 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 consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either mdividually among the amino acids m 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 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 ammo acids in SEQ ID NO 2, or
na < xa - (xa • y),
wherein na is the number of amino acid alterations, xa 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 symbol for the multiplication operator, and wherein any non-mteger product of xa and y is rounded down to the nearest integer prior to subtracting it from xa
"Immunologically equivalent denvatιve(s)" as used herein encompasses a polypeptide, polynucleotide, 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 mteraction between pathogen and mammalian host
"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 affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the pnor art
"Indιvιdual(s)" means a multicellular eukaryote, mcludmg, but not limited to a metazoan, a mammal, an ovid. a bovid, a simian, a primate, and a human
"Isolated" means altered "by the hand of man" from its natural state, . e , if it occurs m nature, it has been changed or removed from its onginal environment, or both For example, a polynucleotide or a polypeptide naturally present m a living orgamsm is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is "isolated", as the term is employed herem Moreover, 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 "Organism(s)" means a (I) prokaryote, mcludmg but not limited to, a member of the genus
Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Neissena, Haemoph lus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus L stena, Calymmatobacterium, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further mcludmg, 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 pneumomae, Streptococcus pyogenes Streptococcus agalactiae, Streptococcus faecahs, Streptococcus faecium, Streptococcus durans, Neissena gonorrheae, Neissena meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vaginalis, Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actmomyctes israeln, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coh, Shigella dysentenae, Haemophilus nfluenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella Salmonella typhi Citrobacter freundu Proteus mirabilis, Proteus vulgans, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia hquefaciens, Vibno cholera, Shigella dysentern, Shigella flexnen, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis Bacillus anthracis, Bacillus cereus, Clostndium perfnngens, Clostndium tetani Clostndium botulmum Treponema palhdum Rickettsia rickettsn and Chlamydia trachomitis, (n) an archaeon, mcludmg but not limited to Archaebacter, and (111) a unicellular or filamentous eukaryote, mcludmg but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lacts, or Candida albicans
"Polynucleotide(s)" generally refers to any polynbonucleotide or polydeoxynbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or smgle-, double- and tnple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions In addition "polynucleotide" as used herem refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands m such regions may be from the same molecule or from different molecules The regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules One of the molecules of a tπple-hehcal region often is an oligonucleotide As used herem, the term "polynucleotide(s)" also mcludes DNAs or RNAs as descnbed above that contain one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is intended herem Moreover, DNAs or RNAs compnsmg unusual bases, such as inosine, or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill m the art The term "polynucleotide(s)" as it is employed herem embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, mcludmg, for example, simple and complex cells "Polynucleotide(s)" also embraces short polynucleotides often refeπed to as ohgonucleotιde(s)
"Polypeptιde(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly refeπed to as peptides, o gopeptides and ohgomers and to longer chains generally refeπed to as proteins Polypeptides may contain ammo acids other than the 20 gene encoded amino acids "Polypeptide(s)" mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and in more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation ADP-nbosylation, amidation covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or lipid deπvative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, lodination, methylation, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- nbosylation, selenoylation. sulfation, transfer-RNA mediated addition of ammo acids to proteins, such as arginylation, and ubiquitination See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York (1993) and Wold, F , Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 in 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 may be branched or cyclic, with or without branchmg Cyclic, branched and branched circular polypeptides may result from posttranslational natural processes and may be made by entirely synthetic methods, as well
"Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the mvention introduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention "Subtraction set" is one or more, but preferably less than 100, polynucleotides comprising at least one polynucleotide of the invention
'Naπant(s)" as the term is used herem, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical vanant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the 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 m the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in ammo acid sequence from another reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical A variant and reference polypeptide may differ in amino 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 The present mvention also includes mclude vanants 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 by another with like charactenstics Typical such substitutions are among Ala, Val, Leu and lie, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin. and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly prefeπed are vanants in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combmation A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis and bv other recombinant methods known to skilled artisans EXAMPLES The examples below are earned out usmg standard techmques, which are well known and routme to those of skill m the art, except where otherwise descnbed m detail The examples are illustrative, but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing
The polynucleotide havmg a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus m E colt The sequencmg data from two or more clones containing overlappmg 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 accordmg to standard procedures DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coli infected with the packaged library The library is amplified by standard procedures
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 into library vectors (e g . Rsal. Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coll infected with the packaged library The library is amplified by standard procedures Example 2 DnaE Characterization
TheStaphylococcus aureusDnaE gene was PCR amplified from Staphylococcus aureus WCUH29 chromosomal DNA usmg the following pnmers
DnaE Forward
Ndel
5 ' CCATTCAATCATATGGTGGCATATTTAAATATTC 3 ' [SEQ ID NO 5]
DnaE Reverse
Xhol
5 ' CACAACACTCGAGTTAGCATTCCCCTTTCGTAGC 3 ' [SEQ ID NO 6]
The Staphylococcus aureusDnaE gene from strain WCUH29 was cloned as a Ndel/Xhol fragment into pBluePet and pET20BHC [ both of these constructs are derived from the Novagen pET vectors] Expression studies were performed m Ecoh BL21DE3 and a overexpressed protem band of correct size for Staphylococcus aureusDnaE(~l 17kDa) was clearly visible after Coomassie stamed SDSPAGE

Claims

What is claimed is: 1 An isolated polypeptide selected from the group consisting of
(1) an isolated polypeptide comprising an ammo acid 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 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide compnsmg the amino acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombmant polynucleotide compnsmg the polynucleotide sequence of SEQ ID NO 1
2 An isolated polynucleotide selected from the group consisting of
(l) an isolated polynucleotide compnsmg a polynucleotide sequence encodmg a polypeptide that has 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, over the entire length of SEQ ID NO 2, (n) an isolated polynucleotide compnsmg a polynucleotide sequence that has at least
(a) 70% identity
(b) 80% identity,
(c) 90% identity, or
(d) 95% identity, over its entire length to a polynucleotide sequence encodmg the polypeptide of SEQ ID
NO 2,
(m) an isolated polynucleotide compnsmg a nucleotide sequence which has at least
(a) 70% identity,
(b) 80% identity,
(c) 90% identity, or
(d) 95% identity, to that of SEQ ID NO 1 over the entire length of SEQ ID NO 1 ,
(iv) an isolated polynucleotide compnsmg a nucleotide sequence encodmg the polypeptide of SEQ ID NO 2,
(v) an isolated polynucleotide which is the polynucleotide of SEQ ID NO 1 , (vi) an isolated polynucleotide obtainable by screenmg an appropnate library under stringent hybndization conditions with a probe havmg the sequence of SEQ ID NO 1 or a fragment thereof, (vn) an isolated polynucleotide encodmg a mature polypeptide expressed by the DnaE gene contamed m the Staphylococcus aureus, and
(vin) a polynucleotide sequence complementary to said isolated polynucleotide of (I), (u), (in), (iv), (v), (vi) or (vn)
3 An antibody antigenic to or 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 polypeptide of claim 1 comprising the step of
(a) administering to the individual a therapeutically effective amount of an agomst to said polypeptide, or
(b) providing to the mdividual an isolated polynucleotide compnsmg a polynucleotide sequence encodmg said polypeptide m a form so as to effect production of said polypeptide activity in vivo, or
(n) havmg need to inhibit activity or expression of the polypeptide of claim 1 comprising
(a) administering to the individual a therapeutically effective amount of an antagonist to said polypeptide, or
(b) administering to the individual a nucleic acid molecule that inhibits the expression of a polynucleotide sequence encoding said polypeptide, or
(c) administering to the mdividual a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate , or receptor
5 A process for diagnosmg or prognosmg a disease or a susceptibility to a disease m an individual related to expression or activity of the polypeptide of claim 1 m an mdividual comprising the step of
(a) determining the presence or absence of a mutation m the nucleotide sequence encodmg said polypeptide m the genome of said mdividual, 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 that activate or that inhibit the function of the polypeptide of claim 1 which compnses a method selected from the group consisting of
(a) measuring the bmdmg of a candidate compound to the polypeptide or to the cells or membranes bearmg the polypeptide or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound,
(b) measuring the bmdmg of a candidate compound to the polypeptide or to the cells or membranes bearmg the polypeptide or a fusion protem thereof m the presence of a labeled competitor,
(c) testing whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, using detection systems 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, measurmg activity of the 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 mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay, or
(f) (1) contacting a composition compnsmg the polypeptide with the compound to be screened under conditions to permit mteraction between the compound and the polypeptide to assess the mteraction of a compound, such mteraction bemg associated with a second component capable of providmg a detectable signal m response to the mteraction of the polypeptide with the compound, and
(2) deteimining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the mteraction of the compound with the polypeptide
7 An agonist or an antagonist of the activity or expression polypeptide of claim 1
8 An expression system compnsmg a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell
9 A host cell compnsmg the expression system of claim 8 or a membrane thereof expressing a polypeptide selected from the group consistmg of (1) an isolated polypeptide compnsmg an ammo acid sequence selected from the group havmg at least
(a) 70% identity,
(b) 80% identity,
(c) 90%) identity, or
(d) 95% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide compnsmg the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1
10 A process for producing a polypeptide selected from the group consisting of
(I) an isolated polypeptide compnsmg an ammo 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 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1. compnsmg the step of cultunng a host cell of claim 9 under conditions sufficient for the production of said polypeptide
11 A process for producmg a host cell compnsmg the expression system of claim 8 or a membrane thereof expressmg a polypeptide selected from the group consisting of
(I) an isolated polypeptide compnsmg an ammo acid sequence selected from the group havmg 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 over the entire length of SEQ ID NO 2, (u) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombmant polynucleotide compnsing the polynucleotide sequence of SEQ ID NO 1, said process compnsmg the step of transforming or transfectmg a cell with an expression system compnsmg a polynucleotide capable of producmg said polypeptide of (I), (ii), (m) or (iv) when said expression system is present in a compatible host cell such the host cell, under appropriate culture conditions produces said polypeptide of (1) (n), (in) or (iv)
12 A host cell produced by the process of claim 11 or a membrane thereof expressmg a polypeptide selected from the group consisting of
(1) an isolated polypeptide compnsmg 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 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2, (m) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombmant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1
13 A computer readable medium having stored thereon a member selected from the group consisting of a polynucleotide compnsmg 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 representmg a polynucleotide sequence comprising the sequence of SEQ ID NO 1 , a data set representmg a polynucleotide sequence encoding a polypeptide sequence compnsmg the sequence of SEQ ID NO 2, a polynucleotide compnsmg the sequence of SEQ ID NO 1, a polypeptide compnsmg the sequence of SEQ ID NO 2, a set of polynucleotide sequences wherem at least one of said sequences compnses the sequence of SEQ ID NO 1. a set of polypeptide sequences wherem 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 representmg a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO 2
14 A computer based method for performing homology identification, said method compnsing the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 in a computer readable medium, and companng said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology
15 A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method compnsmg the steps of providmg a first polynucleotide sequence compnsmg the sequence of SEQ ID NO 1 in a computer readable medium, and screenmg for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence
16 A polynucleotide consistmg of a polynucleotide of the formula
X-(Rι )m-(R2)-(R3)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 hydrogen, a metal, 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 mteger between 1 and 3000 or zero , n is an mteger between 1 and 3000 or zero, and R is a nucleic acid sequence or modified nucleic acid sequence set forth m SEQ ID NO 1
PCT/US1999/028970 1998-12-23 1999-12-08 DnaE WO2000039286A1 (en)

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US09/219,034 1998-12-23

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KIM ET AL.: "In vivo assembly of overproduced DNA polymerase III. Overproduction, purification and characterization of the alpha, alpha-epsilon and alpha-epsilon-theta subunits", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 34, August 1996 (1996-08-01), pages 20681 - 20689, XP002924885 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1404865A2 (en) * 2001-05-14 2004-04-07 Replidyne, Inc. System for discovery of agents that block yersinia pestis and pseudomonas aeruginosa dna replication
EP1404865A4 (en) * 2001-05-14 2005-06-01 Replidyne Inc System for discovery of agents that block yersinia pestis and pseudomonas aeruginosa dna replication
JP2008271967A (en) * 2001-05-14 2008-11-13 Replidyne Inc System for developing curative agent blocking yersinia pestis and pseudomonas aeruginosa dna replication

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