+

WO2001011970A1 - yloV - Google Patents

yloV Download PDF

Info

Publication number
WO2001011970A1
WO2001011970A1 PCT/US2000/021869 US0021869W WO0111970A1 WO 2001011970 A1 WO2001011970 A1 WO 2001011970A1 US 0021869 W US0021869 W US 0021869W WO 0111970 A1 WO0111970 A1 WO 0111970A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
sequence
ofthe
Prior art date
Application number
PCT/US2000/021869
Other languages
French (fr)
Inventor
Martin K. R. Burnham
Magdalena Zalacain
Sanjoy Biswas
Patrick V. Warren
John D. Holmes
Alison F. Chalker
Stephanie Van Horn
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 WO2001011970A1 publication Critical patent/WO2001011970A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)

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 ofthe unknown function family, as well as their vanants, herein referred to as "yloV,” “yloV polynucleot ⁇ de(s),” and “yloV polypept ⁇ de(s)” as the case may be
  • Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia. meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one ofthe more intensively studied microbes For example, much of our early understanding that DNA is, in fact, the genetic matenal was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe Despite the vast amount of research with S pneumoniae, many questions concerning the virulence of this microbe remain It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics
  • polynucleotides and polypeptides such as the yloV 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 m pathogenesis of infection, dysfunction and disease
  • identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease
  • the present invention relates to yloV, in particular yloV polypeptides and yloV polynucleotides, recombinant matenals 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 yloV expression or activity
  • the invention relates to yloV polypeptides and polynucleotides as descnbed in greater detail below
  • the invention relates to polypeptides and polynucleotides of a yloV of Streptococcus pneumoniae, that is related by amino acid sequence homology to YloV protein [Bacillus subtihs]
  • the invention relates especially to yloV having a 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 ofthe invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ⁇ bopolynucleotides
  • a deposit comprising a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 IRY, Scotland on 11 April 1996 and assigned deposit number 40794. The deposit was described as Streptococcus pneumoniae 0100993 on deposit.
  • NCIMB National Collections of Industrial and Marine Bacteria Ltd.
  • Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly deposited with the NCIMB and assigned deposit number 40800.
  • the Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain” or as "the DNA ofthe deposited strain.”
  • the deposited strain compnses a full length yloV gene
  • the sequence of the polynucleotides comprised in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descnption 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 restnction or condition released to the public upon the issuance of a patent
  • the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C ⁇ 112
  • a license may be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain, which polypeptide is compnsed in the deposited strain
  • yloV polynucleotide sequences in the deposited strain such as DNA and RNA.
  • amino acid sequences encoded thereby Also provided by the invention are yloV polypeptide and polynucleotide sequences isolated from the deposited strain
  • YloV polypeptide of the invention is substantially phylogenetically related to other proteins of the unknown function family
  • polypeptides of Streptococcus pneumoniae referred to herein as “yloV” and “yloV polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same
  • the present invention further provides for an isolated polypeptide that (a) compnses or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • polypeptides ofthe invention include a polypeptide of Table 1 [SEQ ID NO 2] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of yloV, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also include portions of such polypeptides with such portion of the polypeptide generally comprising at least 30 am o acids and more preferably at least 50 amino acids
  • the mvention also mcludes a polypeptide consisting of or compnsmg a polypeptide ofthe formula X-(R ⁇ ) m -(R 2 )-(R 3 ) n -Y wherein, at the ammo terminus, X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, R ⁇ and R3 are any ammo acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R 2 is an amino acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R 2 is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy terminal ammo acid residue is at the nght, co
  • a polypeptide of the mvention is denved from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms ofthe same taxonomic genus
  • a polypeptide of the mvention may also be obtained, for example, from organisms ofthe same taxonomic family or order
  • a fragment is a variant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention
  • fragments may be "free-standing,” or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle contmuous region m a smgle larger polypeptide
  • Preferred fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a contmuous se ⁇ es of residues that mcludes an ammo- and/or carboxyl-termmal am o acid sequence
  • Degradation forms ofthe polypeptides of the mvention produced by or in a host cell, particularly a Streptococcus pneumoniae are also preferred
  • fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forrmng regions, turn and turn-forrnmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta ampmpathic regions, flexible regions, surface-forming regions, substrate bmdmg region, and high antigemc index regions
  • fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide comprising an amino acid sequence 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.
  • Fragments of the polypeptides of the mvention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these variants may be employed as intermediates for producmg the full-length polypeptides ofthe mvention Polynucleotides It is an object ofthe mvention to provide polynucleotides that encode yloV polypeptides, particularly polynucleotides that encode a polypeptide herem designated yloV
  • the polynucleotide compnses a region encodmg yloV polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a variant thereof
  • This mvention provides that this full length gene is essential to the growth and/or survival of an organism that possesses it, such as Streptococcus pneumoniae
  • isolated nucleic acid molecules encoding and/or expressing yloV polypeptides and polynucleotides, particularly Streptococcus pneumoniae yloV polypeptides and polynucleotides, mcluding, for example, unprocessed RNAs, nbozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • mcluding for example, unprocessed RNAs, nbozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vanants thereof, and compositions compnsmg the same
  • Another aspect ofthe mvention relates to isolated polynucleotides, including at least one full length gene, that encodes a yloV polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof
  • yloV polypeptide from Streptococcus pneumoniae compnsmg or consisting of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof Using the information provided herem, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 2], or a variant thereof Using the information provided herem, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 2], or a variant thereof Using the information provided herem, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 2], or a variant thereof Using the information provided herem, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 2], or a variant thereof Using the information provided herem, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 2], or a variant thereof Using the information provided herem, such as a polynucleo
  • a polynucleotide ofthe mvention encodmg yloV polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bactena us g Streptococcus pneumoniae 0100993 cells as starting matenal, followed by obtaining a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1]
  • a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 m E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, derived from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions
  • the present mvention provides for an isolated polynucleotide compnsmg or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the 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 mvention, including homologs and orthologs from species other than Streptococcus pneumoniae may be obtamed by a process that compnses the steps of screening an appropnate library under stringent hybndization conditions with a labeled
  • a coding sequence for a mature polypeptide or a fragment thereof by itself as well as a coding sequence for a mature polypeptide or a fragment m reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence
  • the polynucleotide ofthe mvention may also compnse at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, teirnination signals (such as rho-dependent and rho-independent teirnination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals.
  • the polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids.
  • a marker sequence that facilitates purification of a fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al, Cell 37: 767 (1984), both of that may be useful in purifying polypeptide sequence fused to them.
  • Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
  • a preferred embodiment ofthe invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 1666 set forth in SEQ ID NO:l of Table 1, both of that encode a yloV polypeptide.
  • 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 ofthe 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 j and its 3' end nucleic acid residue is at the right, bound to R3.
  • Any stretch of nucleic acid residues denoted by either Ri 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 ofthe above formula is a closed, circular polynucleotide, that 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 preferred embodiments ofthe 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 ofthe invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms ofthe same taxonomic genus.
  • a polynucleotide ofthe invention may also be obtained, for example, from organisms ofthe 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 Streptococcus pneumoniae yloV having an amino acid sequence set out in Table 1 [SEQ ID NO:2].
  • 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 comprise coding and or non-coding sequences.
  • the invention further relates to variants ofthe polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2]. Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
  • yloV variants that have the amino acid sequence of yloV polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5,
  • Prefened isolated polynucleotide embodiments also include polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l , or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO: l.
  • polynucleotide fragments such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l , or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 con
  • prefened embodiments of the invention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding yloV polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such polynucleotides.
  • Most highly prefened are polynucleotides that comprise a region that is at least 95% are especially prefened.
  • those with at least 97% are highly prefened among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly prefened, with at least 99% being the more prefened.
  • Prefened embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ED NO : 1 ] .
  • polynucleotides that hybridize, particularly under stringent conditions, to yloV polynucleotide sequences, such as those polynucleotides in Table 1.
  • the invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein.
  • the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein.
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.
  • the invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NO: l under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:l or a fragment thereof; and isolating said polynucleotide sequence.
  • Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein.
  • the polynucleotides ofthe invention may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding yloV and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a yloV gene.
  • Such probes generally will comprise 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 prefened probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs.
  • a coding region of a yloV gene may be isolated by screening using a DNA sequence provided in
  • Table 1 [SEQ ID NO:l] to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene ofthe invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members ofthe library the probe hybridizes to.
  • polynucleotides ofthe invention that are ohgonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used m the processes herein as described, but preferably for PCR, to determme whether or not the polynucleotides identified herem m whole or m part are transc ⁇ bed m bacte ⁇ a in infected tissue It is recognized that such sequences will also have utility m diagnosis ofthe stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional am o or carboxyl-termmal ammo acids, or amino acids mte ⁇ or to a mature polypeptide (when a mature form has more than one polypeptide chain, for mstance) Such sequences may play a role m processing of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from a mature protem by cellular enzymes For each and every polynucleotide ofthe mvention there is provided a polynucleotide complementary to it It is prefened that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary
  • a precursor protem, havmg a mature form ofthe polypeptide fused to one or more prosequences may be an mactive form ofthe polypeptide When prosequences are removed such inactive precursors generally are activated Some or all ofthe prosequences may be removed before activation Generally, such precursors are called proproterns
  • any contiguous fragment of SEQ ID NO 2 which retains at least 20%, preferably at least 50%, of an activity ofthe polypeptide encoded by the gene for SEQ ID NO 2 is within the invention, as are conespondmg fragment which are 70%, 80%, 90%, 95%,97%, 98% or 99% identical to such contiguous fragments
  • the contiguous fragment compnses at least 70% ofthe ammo acid residues of SEQ ID NO 2, preferably at least 80%, 90% or 95% ofthe residues
  • a polynucleotide ofthe mvention may encode a mature protem, a mature protem plus a leader sequence (that may be refened to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotem, that is a precursor to a proprotein, havmg a leader sequence and one or more prosequences, that generally are removed du ⁇ ng processing steps that produce active and mature forms ofthe polypeptide
  • the mvention also relates to vectors that comp ⁇ se a polynucleotide or polynucleotides of the mvention, host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides ofthe mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs de ⁇ ved from the DNA constructs ofthe mvention
  • Recombinant polypeptides ofthe present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compnsmg expression systems Accordingly, in a further aspect, the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides ofthe present mvention, to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides ofthe mvention by recombinant techniques
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention
  • Introduction of a polynucleotide mto the host cell can be effected by methods desc ⁇ bed in many standard laboratory manuals, such as Davis, et al , BASIC METHODS LN 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, micromjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection
  • appropnate hosts include bactenal cells, such as cells of streptococci, staphylococci, enterococci E coh, streptomyces, cyanobactena, Bacillus subtihs, and Streptococcus pneumoniae, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus, insect cells such as cells of Drosophila S2 and Spodoptera Sf9, ammal 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
  • bactenal cells such as cells of streptococci, staphylococci, enterococci E coh, streptomyces, cyanobactena, Bacillus subtihs, and Streptococcus pneumoniae
  • vectors include, among others, chromosomal-, episomal- and virus-de ⁇ ved vectors, for example, vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retrovrruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids
  • the expression system constructs may compnse control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and
  • This invention is also related to the use of yloV polynucleotides and polypeptides ofthe invention for use as diagnostic reagents.
  • Detection of yloV polynucleotides and/or polypeptides in 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 comprising the yloV gene or protein may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein.
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials.
  • Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis.
  • RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways.
  • amplification characterization ofthe species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide ofthe organism.
  • Deletions and insertions can be detected by a change in size ofthe amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species ofthe same genus or a different strain ofthe same species.
  • Point mutations can be identified by hybridizing amplified DNA to labeled yloV 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).
  • an array of ohgonucleotides probes compnsmg yloV 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 va ⁇ ety of questions m molecular genetics including gene expression, genetic linkage, and genetic va ⁇ ability (see, for example, Chee et al , Science, 274 610 (1996))
  • the present mvention relates to a diagnostic kit that compnses (a) a polynucleotide ofthe present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present mvention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibody to
  • This mvention also relates to the use of polynucleotides ofthe present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide ofthe mvention, preferable, SEQ ED NO 1, that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression ofthe polynucleotide
  • Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a va ⁇ ety of techniques, such as those desc ⁇ bed elsewhere herem
  • the mvention further provides a process for diagnosing, disease, preferably bactenal mfections, more preferably infections caused by Streptococcus pneumoniae, compnsmg determining from a sample denved from an individual, such as a bodily material, an mcreased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a yloV polynucleotide can be measured using any on ofthe methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods
  • a diagnostic assay in accordance with the mvention for detecting over-expression of yloV polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techniques that can be used to determme levels of a yloV polypeptide, m a sample denved from a host, such as a bodily mate ⁇ al, are well-known to those of skill m the art Such assay methods clude radiormmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays
  • Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libranes, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Co gan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
  • Polypeptides and polynucleotides ofthe present mvention are responsible for many biological functions, including many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize
  • the present mvention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide ofthe mvention, as well as related polypeptides and polynucleotides
  • agonists or antagonists e g , inhibitors
  • Compounds may be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical libranes, and natural product mixtures
  • Such agonists and antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of yloV polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Cohgan et
  • polypeptides, polypeptides and antibodies that bind to and or interact with a polypeptide ofthe present invention may also be used to configure screemng methods for detectmg the effect of added compounds on the production of mRNA and/or polypeptide in cells
  • an ELISA assay may be constructed for measunng secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screening compounds to identify those that enhance (agonist) or block (antagonist) the action of yloV polypeptides or polynucleotides, particularly those compounds that are bacte ⁇ static and/or bactencidal
  • the method of screening may mvolve high-throughput techmques
  • a synthetic reaction mix 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, compnsmg yloV polypeptide and a labeled substrate or hgand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a yloV agomst or antagonist
  • the ability of the candidate molecule to agonize or antagonize the yloV polypeptide is reflected m decreased binding ofthe labeled hgand or decreased production of product from such substrate Molecules that b
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of yloV polypeptide dimers, t ⁇ mers, tetramers or higher order structures, or structures formed by yloV polypeptide bound to another polypeptide yloV polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing ofthe two labeled species and excitation ofthe donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence ofthe 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 yloV polypeptide self-association as well as an association of yloV polypeptide and another polypeptide or small molecule.
  • YloV polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric.
  • Solution protein can then passed over the yloV polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index.
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for yloV polypeptide self-association as well as an association of yloV polypeptide and another polypeptide or small molecule.
  • a scintillation proximity assay may be used to characterize the interaction between an association of yloV polypeptide with another yloV polypeptide or a different polypeptide .
  • YloV polypeptide can be coupled to a scintillation-filled bead. Addition of radio-labeled yloV polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid. Thus, signal is emitted upon yloV polypeptide binding and compounds that prevent yloV polypeptide self-association or an association of yloV polypeptide and another polypeptide or small molecule will diminish signal.
  • methods for identifying compounds that bind to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the invention comprising: contacting a polypeptide and/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction ofthe 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 binding or interaction of the compound with the polypeptide and/or polynucleotide.
  • an assay for yloV agonists is a competitive assay that combines yloV and a potential agonist with yloV-binding molecules, recombinant yloV binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay.
  • YloV can be labeled, such as by radioactivity or a colorimetric compound, such that the number of yloV molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness ofthe potential antagonist.
  • a polypeptide and/or polynucleotide of the present mvention may also be used in a method for the structure-based design of an agonist or antagonist ofthe polypeptide and/or polynucleotide, by (a) determining m the first instance the three- dimensional structure ofthe 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 or antagonist, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced bindmg s ⁇ te(s), reactive s ⁇ te(s), and/or motif(s), and
  • the present mvention provides methods of treatmg abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of yloV polypeptide and/or polynucleotide
  • the expression and/or activity ofthe polypeptide and/or polynucleotide is m excess, several approaches are available.
  • One approach compnses admmistenng to an mdividual m need thereof an inhibitor compound (antagonist) as herem descnbed, optionally m combmation with a pharmaceutically acceptable earner, m an amount effective to inhibit the function and/or expression ofthe polypeptide and/or polynucleotide, such as, for example, by blocking the bmdmg of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition
  • soluble forms ofthe 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 ofthe yloV polypeptide and/or polypeptide
  • expression ofthe gene encoding endogenous yloV polypeptide can be inhibited using expression blocking techniques
  • This blocking may be targeted against any step m gene expression, but is preferably targeted agamst transcription and/or translation
  • An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988))
  • ohgonucleotides that form t ⁇ ple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360)
  • These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
  • the invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection.
  • the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial yloV proteins that mediate tissue damage and or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.
  • yloV agonists and antagonists preferably bacteristatic or bactericidal agonists and antagonists.
  • antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.
  • Antagonists ofthe invention include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression.
  • Antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing yloV-induced activities, thereby preventing the action or expression of yloV polypeptides and/or polynucleotides by excluding yloV polypeptides and/or polynucleotides from binding.
  • Antagonists ofthe invention also include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding 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 antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules).
  • Prefened antagonists include compounds related to and variants of yloV.
  • polypeptide antagomsts examples include antibodies or, m some cases, oligonucleotides or proteins that are closely related to the ligands. substrates, receptors, enzymes, etc . as the case may be, ofthe polypeptide, e g , a fragment ofthe ligands, substrates, receptors, enzymes, etc , or small molecules that bmd to the polypeptide ofthe present mvention but do not elicit a response, so that the activity ofthe polypeptide is prevented
  • Small molecules ofthe mvention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons It is preferred that these small molecules are organic molecules
  • Hehcobacter pylori herem "H pylori" bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, 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 gastnc adenocarcmoma, classifying the bacterium as a Group I (definite) carcinogen
  • Preferred antimicrobial compounds of the mvention agonists and antagomsts of yloV polypeptides and/or polynucleotides found using screens provided by the invention, or known in the art, particularly nanow-spectrum antibiotics, should be useful in the treatment of H pylori infection
  • Bodily mate ⁇ al(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, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy mate ⁇ als
  • D ⁇ sease(s) means any disease caused by or related to infection by a bacte ⁇ a.
  • mcludmg for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospmal fluid
  • “Host cell(s)” is a cell that has been introduced (e g , transformed or transfected) or is capable of introduction (e g , transformation or transfection) by an exogenous polynucleotide sequence
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, including but not limited to those desc ⁇ bed m
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO:l
  • y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the amino- or carboxy-termmal positions ofthe 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 withm the reference sequence, and wherem said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtractmg that product from said total number of amino acids m
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids m SEQ ID NO 2
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest integer prior to subtractmg it from x a
  • Ind ⁇ v ⁇ dual(s) means a multicellular eukaryote, mcludmg, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human
  • Isolated means altered “by the hand of man” from its natural state, / e , if it occurs m nature, it has been changed or removed from its o ⁇ ginal 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 introduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said orgamsm, which orgamsm may be living or non-living
  • Organ ⁇ sm(s) means a (I) prokaryote, mcludmg but not limited to, a member of the genus
  • Streptococcus Staphylococcus, Bordetella, Corynebacterium, Mycobactenum, Neissena, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Eschench a, Salmonella, Kle ⁇ siella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further mcludmg, but not limited
  • Polynucleotide(s) generally refers to any polynbonucleotide or polydeoxy ⁇ bonucleotide, that 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 t ⁇ ple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hyb ⁇ d molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions
  • polynucleotide as used herem refers to t ⁇ ple-stranded regions compn
  • Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from posttranslational natural processes and may be made by entirely synthetic methods, as well
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides ofthe mvention mtroduced or transformed mto a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides ofthe mvention
  • Va ⁇ ant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant 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 amino acid residue may or may not be one encoded by the genetic code.
  • the present invention also includes include variants of each of the polypeptides of the invention, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics.
  • substitutions are among Ala, Val, Leu and He; 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 prefened are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
  • a variant of a polynucleotide or polypeptide may be a naturally occu ing such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • Example 1 Strain selection, Library Production and Sequencing
  • the polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: l] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E. coli.
  • the sequencing data from two or more clones comprising overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO: l.
  • Libraries may be prepared by routine methods, for example: Methods 1 and 2 below.
  • Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures.
  • DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated mto 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 appropnate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E cob infected with the packaged library The library is amplified by standard procedures Example 2 yloV Characterization
  • RNAase free, DNAase free, DNA and protem free preparations of RNA obtained are suitable for Reverse Transcnption PCR (RT-PCR) usmg umque pnmer pairs designed from the sequence of each gene of Streptococcus pneumoniae 0100993 Using this procedure it was possible to demonstrate t ⁇ &tyloV s transcibed during infection.
  • Streptococcus pneumoniae 0100993 is grown either on TSA/5%horse blood plates or in AGCH medium overnight, 37°C, 5%C0 2 Bacte ⁇ a are then collected and resuspended m phosphate- buffered salme to an A ⁇ oo of approximately 0 4 Mice are anaesthetized with lsofluorane and 50ml of bactenal suspension (approximately 2 x 10 5 bacteria) is administered mtranasally usmg a pipetman Mice are allowed to recover and have food and water ad libitum After 48 hours, the mice are euthanized by carbon dioxide overdose, and lungs are aseptically removed and snap-frozen m liquid nitrogen b) Isolation of Streptococcus pneumoniae 0100993 RNA from infected tissue samples
  • RNA preparation is extracted with chloroform/isoamyl alcohol, and precipitated with DEPC- treated/Isopropanol Precipitation Solution (BIO101) RNA
  • RNA isolation is assessed by running samples on 1% agarose gels 1 x TBE gels stained with ethidium bromide are used to visualise total RNA yields
  • 2 2M formaldehyde gels are run and vacuum blotted to Hybond-N (Amersham)
  • the blot is then hybridised with a 32 P-labelled ohgonucletide probe, of sequence 5 ' AACTGAGACTGGCTTTAAGAGATTA 3 ' [SEQ ID NO 3], specific to 16S rRNA of Streptococcus pneumoniae
  • the size ofthe hybridising band is compared to that of control RNA isolated from in vitro grown Streptococcus pneumoniae 0100993 in the Northern blot Conect sized bactenal 16S rRNA bands can be detected in total RNA samples which show degradation ofthe mammalian RNA when visualised on TBE gels
  • DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with 20 units of RNAase-free DNAasel (GenHunter) m the buffer supplied m a final volume of 57 microhters
  • DNAase was inactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL, Life Technologies) according to the manufacturers protocol DNAase treated RNA was resuspended in 100 microhtres of DEPC treated water with the addition of Rnasin as described before
  • PCR reactions are set up on ice m 0 2ml tubes by adding the following components 43 microhtres PCR Master Mix (Advanced Biotechnologies Ltd ), 1 rmcrohtre PCR pnmers designed for the yloV gene (optimally 18-25 basepairs m length and designed to possess similar annealing temperatures), each primer at lOmM initial concentration, and 5 microhtres cDNA
  • PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 as follows 2 minutes at 94 °C, then 50 cycles of 30 seconds each at 94 °C, 50 °C and 72 °C followed by 7 minutes at 72 °C and then a hold temperature of 20 °C (the number of cycles is optimally 30-50 to determine the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of the starting quantity of cDNA from the RT reaction is to be made), 10 rmcrohtre aliquots are then run out on 1% 1 x TBE gels stained with ethidmm bromide, with PCR product, if present, sizes estimated by compa ⁇ son to a 100 bp DNA Ladder (Gibco BRL, Life Technologies) Alternatively if the PCR products are conveniently labelled by the use of a labelled PCR primer (e g labelled at the 5 'end with a dye) a suitable aliquot of the PCR product is run out on
  • RT/PCR controls may include +/- reverse transc ⁇ ptase reactions, 16S rRNA p ⁇ mers or DNA specific primer pairs designed to produce PCR products from non-transcribed Streptococcus pneumoniae 0100993 genomic sequences To test the efficiency of the primer pairs they are used in DNA PCR with Streptococcus pneumoniae 0100993 total DNA PCR reactions are set up and run as described above using approx 1 microgram of DNA in place ofthe cDNA
  • allelic replacement cassette was generated using PCR technology
  • the cassette consisted of a pair of 500bp chromosomal DNA fragments flanking an erythromycm resistance gene
  • the chromosomal DNA sequences are the 500bp preceding and following the DNA sequence encoding the yloV gene contained m Seq ID NO 1
  • allelic replacement cassette was introduced mto S pneumomae R6 by transformation Competent cells were prepared according to published protocols DNA was mtroduced into the cells by incubation of ng quantities of allelic replacement cassette with 10" cells at 30°C for 30 minutes The cells were transferred to 37°C for 90 minutes to allow expression ofthe erythromycm resistance gene Cells were plated m agar contammg lug erythromycm per ml Following incubation at 37°C for 36 hours, colomes are picked and grown overnight m Todd-Hewitt broth supplemented with 0 5% yeast extract Typically 1000 transformants contammg the appropnate allelic replacement are obtamed If no transformants are obtained in three separate transformation experiments as was the case for this gene yloV, then the gene is considered as being essential in vitro

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pulmonology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

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

Description

yloV
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 ofthe unknown function family, as well as their vanants, herein referred to as "yloV," "yloV polynucleotιde(s)," and "yloV polypeptιde(s)" as the case may be
BACKGROUND OF THE INVENTION The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia. meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one ofthe more intensively studied microbes For example, much of our early understanding that DNA is, in fact, the genetic matenal was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe Despite the vast amount of research with S pneumoniae, many questions concerning the virulence of this microbe remain It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics
The frequency of Streptococcus pneumoniae infections has πsen dramatically in the past few decades This has been attnbuted 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 Streptococcus pneumoniae strains that are resistant to some or all ofthe standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics," that is, high throughput genome- or gene-based biology This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics relies heavily on the vanous tools of biornformatics to identify gene sequences of potential interest from the many 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 yloV 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 m 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 yloV, in particular yloV polypeptides and yloV polynucleotides, recombinant matenals 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 yloV expression or activity
Vanous changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled in the art from reading the following descnptions and from reading the other parts ofthe present disclosure
DESCRIPTION OF THE INVENTION
The invention relates to yloV polypeptides and polynucleotides as descnbed in greater detail below In particular, the invention relates to polypeptides and polynucleotides of a yloV of Streptococcus pneumoniae, that is related by amino acid sequence homology to YloV protein [Bacillus subtihs]
>gι|2633956|emb|CAB13457| polypeptide The invention relates especially to yloV having a 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 ofthe invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including πbopolynucleotides
TABLE 1 yloV Polynucleotide and Polypeptide Sequences
(A) Streptococcus pneumoniae yloV polynucleotide sequence [SEQ ID NO 1]
5'-GTGTCAAAAATTACTACTAGCTTATTTCAAGAAATGGTGCAGGCTGCATCAACTCGCTTGAATAAGCAAG CTGAATATGTCAATTCATTAAACGTCTTTCCAGTTCCAGATGGAGATACTGGGACAAATATGGGAATGAC CATTGAAAATGGTGCTAAAGAAGTTGCAGACAAGCCAGCTTCTACAGTTGGAGAAGTAGCGAGCATTCTT GCCAAAGGGCTTTTGATGGGTGCGCGTGGGAACTCAGGAGTGATTACGTCTCAGCTTTTCCGTGGATTTT CCCAAGCTATCAAGGATAAAGACGAGTTAACAGGTCAAGACTTGGCTCTGGCCTTCCAATCAGGTGTGGA AGTTGCCTATAAGGCAGTGATGAAACCTGTTGAAGGAACGATTTTAACAGTTTCTCGTGGAGCTGCTATC GGTGCTAAGAAAAAAGCTGAGCAAACAGATGACGCTGTTGAAGTCATGCGCGCAGCCTTGGAAGGTGCTA AAACAGCTCTAGCTAAAACACCAGACATGCTTCCAGTATTGAAAGAAGTTGGCGTTGTGGACTCAGGTGG TCAAGGATTGGTCTTCATCTACGAAGGTTTCCTTTCAGCCCTTACTGGCGAATATATTGCATCTGAGGAC TTTGTAGCGACTCCTGCCAACATGAGTGAGATGATCAATGTAGAGCATCATAAGTCTGTAGCTGGTCACG TAGCGACTGAGGACATCACGTTTGGTTACTGTACTGAAATCATGGTAGCTCTTAAGCAAGGTCCAACCTA TGCTAAAGATTTTGACTACGATGAATTCCGTAACTACTTGGATGAGCTTGGGGATTCTCTCCTCGTTGTT AACGATGATGAAATTGTCAAAGTCCATGTTCATACAGAAGATCCAGGACTTGTTATGCAAGAAGGTCTCA AATATGGTAGCTTGGTCAAGGTAAAAGTTGACAATATGCGTAACCAACACGAAGCACAGGTTGAGAAAGA AGCTACTCAAGTTATCAAGTCGGCTGAAGAAAAAGAGTATGCTTTGATTGCTGTGGTGGCTGGTAAAGGT CTAGCAGATATCTTCTGTTCTCAAGGCGTGGATTATGTTATCGAAGGCGGTCAAACCATGAACCCTTCAA CAGAAGACTTTATCAAGGCTGTTGAACAGGTCAATGCCCGTAACATCATCTTCTTGCCAAACAACAAGAA CATCTTCATGGCAGCTCAATCTGCGGCAGAAGTTTTGGAGCAACCAGCAGTAGTGGTAGAGGCTCGCACT CTTCCTCAAGGTATGACAAGTCTTCTTGCCTTTGATCCAAGCAAGTCCATTGAAGAAAATCAAGAGCGTA TGACAGCTGCTCTTAGCGATGTCGTTAGCGGAAGCGTCACAACAGCCGTGCGTGATACAACGATCGATGG CTTAGAAATCCATGAAAACGATAATCTAGGTATGGTGGATGGAAAAATTCTTGTGTCAAACCCTGATATG CACCAAACATTGACTGAAACCTTGAAACATATGTTGGATGAAGACAGTGAAATCGTAACCTTCTATGTCG GTGAAGACGGAAGCGAAGAACTTGCCAATGAAATCGCTCAAGAAATCGTAGAAGAATTCGAAGACGTTGA AGTCGAGATTCACCAAGGTCAACAACCTGTTTACCCATACCTATTTAGTGTGGAATAA-3 '
(B) Streptococcus pneumoniae yloV polypeptide sequence deduced from a polynucleotide sequence m this table [SEQ rD NO:2].
NH2-vsκiττsLFQEMVQAASTRLNKQAEYVNSLNVFPVPDGDTGTNMGMTIENGAKEVADKPASTVGEVASlL
AKGLLMGARGNSGVITSQLFRGFSQAIKDKDELTGQDLALAFQSGVEVAY? AVMKPVEGTILTVSRGAAI GAKKKAEQTDDAVEVMRAALEGAKTALAKTPDMLPVLKEVGλA^DSGGQGLVFIYEGFLSALTGEYIASED FVATPANMSEMINVEHHKSVAGHVATEDITFGYCTEIMVALKQGPTYAKDFDYDEFRNYLDELGDSLLW NDDEIVKλ HVHTEDPGLVMQEGLKYGSLVKVKVDNMRNQHEAQVEKEATQVIKSAEEKEYALIAWAGKG LADI FCSQGVDYVIEGGQTMNPSTEDFIKAVEQVNARNIIFLPNNKNIFMAAQSAAEVLEQPAWVEART LPQGMTSLLAFDPSKSIEENQERMTAALSDWSGSVTTAVRDTTIDGLEIHENDNLGMVDGKILVSNPDM HQTLTETLKHMLDEDSEIVTFYVGEDGSEELANEIAQEIVEEFEDVEVEIHQGQQPVYPYLFS Ε-COOH
Deposited materials
A deposit comprising a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 IRY, Scotland on 11 April 1996 and assigned deposit number 40794. The deposit was described as Streptococcus pneumoniae 0100993 on deposit.
On 17 April 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly deposited with the NCIMB and assigned deposit number 40800. The Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain" or as "the DNA ofthe deposited strain." The deposited strain compnses a full length yloV gene The sequence of the polynucleotides comprised in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descnption 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 restnction or condition released to the public upon the issuance of a patent The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C §112 A license may be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted
In one aspect ofthe invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain, which polypeptide is compnsed in the deposited strain Further provided by the invention are yloV polynucleotide sequences in the deposited strain, such as DNA and RNA. and amino acid sequences encoded thereby Also provided by the invention are yloV polypeptide and polynucleotide sequences isolated from the deposited strain
Polypeptides
YloV polypeptide of the invention is substantially phylogenetically related to other proteins of the unknown function family
In one aspect ofthe invention there are provided polypeptides of Streptococcus pneumoniae referred to herein as "yloV" and "yloV polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same
Among the particularly preferred embodiments of the invention are variants of yloV polypeptide encoded by naturally occurring alleles of a yloV gene
The present invention further provides for an isolated polypeptide that (a) compnses or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
The polypeptides ofthe invention include a polypeptide of Table 1 [SEQ ID NO 2] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of yloV, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also include portions of such polypeptides with such portion of the polypeptide generally comprising at least 30 am o acids and more preferably at least 50 amino acids
The mvention also mcludes a polypeptide consisting of or compnsmg a polypeptide ofthe formula X-(Rι)m-(R2)-(R3)n-Y wherein, at the ammo terminus, X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, R\ and R3 are any ammo 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 R2 is an amino acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R2 is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy terminal ammo acid residue is at the nght, covalently bound to R3 Any stretch of ammo acid residues denoted by either Ri or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Other preferred embodiments of the mvention are provided where m is an mteger between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500
It is most preferred that a polypeptide of the mvention is denved from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms ofthe same taxonomic genus A polypeptide of the mvention may also be obtained, for example, from organisms ofthe same taxonomic family or order
A fragment is a variant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention As with yloV polypeptides, fragments may be "free-standing," or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle contmuous region m a smgle larger polypeptide
Preferred fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a contmuous seπes of residues that mcludes an ammo- and/or carboxyl-termmal am o acid sequence Degradation forms ofthe polypeptides of the mvention produced by or in a host cell, particularly a Streptococcus pneumoniae, are also preferred Further preferred are fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forrmng regions, turn and turn-forrnmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta ampmpathic regions, flexible regions, surface-forming regions, substrate bmdmg region, and high antigemc index regions
Further preferred 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.
Fragments of the polypeptides of the mvention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these variants may be employed as intermediates for producmg the full-length polypeptides ofthe mvention Polynucleotides It is an object ofthe mvention to provide polynucleotides that encode yloV polypeptides, particularly polynucleotides that encode a polypeptide herem designated yloV
In a particularly preferred embodiment of the mvention the polynucleotide compnses a region encodmg yloV polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a variant thereof This mvention provides that this full length gene is essential to the growth and/or survival of an organism that possesses it, such as Streptococcus pneumoniae
As a further aspect ofthe mvention there are provided isolated nucleic acid molecules encoding and/or expressing yloV polypeptides and polynucleotides, particularly Streptococcus pneumoniae yloV polypeptides and polynucleotides, mcluding, for example, unprocessed RNAs, nbozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vanants thereof, and compositions compnsmg the same
Another aspect ofthe mvention relates to isolated polynucleotides, including at least one full length gene, that encodes a yloV polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof
In another particularly preferred embodiment of the invention there is a yloV polypeptide from Streptococcus pneumoniae compnsmg or consisting of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof Using the information provided herem, such as a polynucleotide sequence set out m Table 1 [SEQ ID
NO 1], a polynucleotide ofthe mvention encodmg yloV polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bactena us g Streptococcus pneumoniae 0100993 cells as starting matenal, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1], typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 m E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, derived from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the original 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 sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descnbed by Maniatis, T , Fntsch, 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 m particular Screemng By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencing 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 Streptococcus pneumoniae 0100993 Moreover, each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open reading frame encodmg a protein havmg about the number of amino acid residues set forth m Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled m the art The polynucleotide of SEQ ID NO 1 , between nucleotide number 1 and the stop codon that begins at nucleotide number 1666 of SEQ ID NO 1. encodes the polypeptide of SEQ ID NO 2
The initial ammo acid encoded by the codon "GTG" is indicated in SEQ ID NO 2 as valrne However, m one embodiment of the mvention, this initial ammo acid of a polypeptide of the mvention is methionine
In a further aspect, the present mvention provides for an isolated polynucleotide compnsmg or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the 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 mvention, including homologs and orthologs from species other than Streptococcus pneumoniae, may be obtamed by a process that compnses the steps of screening an appropnate library under stringent hybndization conditions with a labeled or detectable probe consisting of or compnsmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones compnsmg said polynucleotide sequence The mvention provides a polynucleotide sequence identical over its entire length to a coding sequence
(open reading frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment m reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence The polynucleotide ofthe mvention may also compnse at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, teirnination signals (such as rho-dependent and rho-independent teirnination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of a fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al, Cell 37: 767 (1984), both of that may be useful in purifying polypeptide sequence fused to them. Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
A preferred embodiment ofthe invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 1666 set forth in SEQ ID NO:l of Table 1, both of that encode a yloV 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 ofthe molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end ofthe 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 ofthe 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 Rj and its 3' end nucleic acid residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either Ri 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 prefened embodiment, X and Y together define a covalent bond, the polynucleotide ofthe above formula is a closed, circular polynucleotide, that 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 preferred embodiments ofthe 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 prefened that a polynucleotide ofthe invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms ofthe same taxonomic genus. A polynucleotide ofthe invention may also be obtained, for example, from organisms ofthe 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 Streptococcus pneumoniae yloV 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 comprise coding and or non-coding sequences.
The invention further relates to variants ofthe polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2]. Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
Further particularly prefened embodiments are polynucleotides encoding yloV variants, that have the amino acid sequence of yloV polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5,
1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination.
Especially prefened among these are silent substitutions, additions and deletions, that do not alter the properties and activities of yloV polypeptide.
Prefened isolated polynucleotide embodiments also include polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l , or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO: l.
Further prefened embodiments of the invention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding yloV polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such polynucleotides. Most highly prefened are polynucleotides that comprise a region that is at least 95% are especially prefened. Furthermore, those with at least 97% are highly prefened among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly prefened, with at least 99% being the more prefened. Prefened embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ED NO : 1 ] .
In accordance with certain prefened embodiments of this invention there are provided polynucleotides that hybridize, particularly under stringent conditions, to yloV polynucleotide sequences, such as those polynucleotides in Table 1.
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. A specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O. lx SSC at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NO: l under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:l or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the invention, for instance, the polynucleotides ofthe invention, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding yloV and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a yloV gene. Such probes generally will comprise at least 15 nucleotide residues or base pairs. Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs. Particularly prefened probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs. A coding region of a yloV gene may be isolated by screening using a DNA sequence provided in
Table 1 [SEQ ID NO:l] to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene ofthe invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members ofthe library the probe hybridizes to. There are several methods available and well known to those skilled m the art to obtam full- length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al , PNAS USA 85 8998-9002, 1988) Recent modifications ofthe technique, exemplified by the Marathon™ technology (Clontech Laboratories Ine ) for example, have significantly sunphfied 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 ofthe DNA using a combination of gene specific and adaptor specific oligonucleotide primers The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific pnmer that anneals further 3' in 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 sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full- length PCR using the new sequence information for the design ofthe 5' primer The polynucleotides and polypeptides of 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 ofthe invention that are ohgonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used m the processes herein as described, but preferably for PCR, to determme whether or not the polynucleotides identified herem m whole or m part are transcπbed m bacteπa in infected tissue It is recognized that such sequences will also have utility m diagnosis ofthe stage of infection and type of infection the pathogen has attained
The mvention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional am o or carboxyl-termmal ammo acids, or amino acids mteπor to a mature polypeptide (when a mature form has more than one polypeptide chain, for mstance) Such sequences may play a role m processing of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from a mature protem by cellular enzymes For each and every polynucleotide ofthe mvention there is provided a polynucleotide complementary to it It is prefened that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary
A precursor protem, havmg a mature form ofthe polypeptide fused to one or more prosequences may be an mactive form ofthe polypeptide When prosequences are removed such inactive precursors generally are activated Some or all ofthe prosequences may be removed before activation Generally, such precursors are called proproterns
As will be recognized, the entire polypeptide encoded by an open reading frame is often not required for activity Accordingly, it has become routine m molecular biology to map the boundaπes of the primary structure required for activity with N-terminal and C-terminal deletion experiments These experiments utilize exonuclease digestion or convement restnction sites to cleave coding nucleic acid sequence For example, Promega (Madison, WI) sell an Erase-a-base™ system that uses Exonuclease HI designed to facilitate analysis ofthe deletion products (protocol available at www promega com) The digested endpornts can be repaired (e g , by ligatoon to synthetic linkers) to the extent necessary to preserve an open reading frame In this way, the nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity Nucleic acid sequences encodmg such fragments of SEQ ID NO 2 and vanants thereof as descnbed herem are within the mvention, as are polypeptides so encoded
As is known m the art, portions ofthe N-terminal and/or C-terminal sequence of a protein can generally be removed without serious consequence to the function ofthe protein The amount of sequence that can be removed is often quite substantial The nucleic acid cutting and deletion methods used for creating such deletion variants are now quite routine Accordingly, any contiguous fragment of SEQ ID NO 2 which retains at least 20%, preferably at least 50%, of an activity ofthe polypeptide encoded by the gene for SEQ ID NO 2 is within the invention, as are conespondmg fragment which are 70%, 80%, 90%, 95%,97%, 98% or 99% identical to such contiguous fragments In one embodiment, the contiguous fragment compnses at least 70% ofthe ammo acid residues of SEQ ID NO 2, preferably at least 80%, 90% or 95% ofthe residues
In sum, a polynucleotide ofthe mvention may encode a mature protem, a mature protem plus a leader sequence (that may be refened to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotem, that is a precursor to a proprotein, havmg a leader sequence and one or more prosequences, that generally are removed duπng processing steps that produce active and mature forms ofthe polypeptide
Vectors, Host Cells, Expression Systems
The mvention also relates to vectors that compπse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides ofthe mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs deπved from the DNA constructs ofthe mvention
Recombinant polypeptides ofthe present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compnsmg expression systems Accordingly, in a further aspect, the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides ofthe present mvention, to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides ofthe mvention by recombinant techniques
For recombinant production of the polypeptides of the mvention, host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto the host cell can be effected by methods descπbed in many standard laboratory manuals, such as Davis, et al , BASIC METHODS LN 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, micromjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection
Representative examples of appropnate hosts include bactenal cells, such as cells of streptococci, staphylococci, enterococci E coh, streptomyces, cyanobactena, Bacillus subtihs, and Streptococcus pneumoniae, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus, insect cells such as cells of Drosophila S2 and Spodoptera Sf9, ammal 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 vaπety of expression systems can be used to produce the polypeptides ofthe mvention Such vectors mclude, among others, chromosomal-, episomal- and virus-deπved vectors, for example, vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retrovrruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may compnse control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression in this regard The appropπate DNA sequence may be inserted mto the expression system by any of a vaπety of well-known and routme techniques, such as. for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra) In recombinant expression systems m eukaryotes, for secretion of a translated protem mto the lumen of the endoplasmic reticulum, mto the peπplasmic space or mto the extracellular environment, appropπate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals Polypeptides ofthe invention 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 protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of yloV polynucleotides and polypeptides ofthe invention for use as diagnostic reagents. Detection of yloV polynucleotides and/or polypeptides in 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 comprising the yloV gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein.
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials. Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways. Using amplification, characterization ofthe species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide ofthe organism. Deletions and insertions can be detected by a change in size ofthe amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species ofthe same genus or a different strain ofthe same species. Point mutations can be identified by hybridizing amplified DNA to labeled yloV 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 array of ohgonucleotides probes compnsmg yloV 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 vaπety of questions m molecular genetics including gene expression, genetic linkage, and genetic vaπability (see, for example, Chee et al , Science, 274 610 (1996)) Thus in another aspect, the present mvention relates to a diagnostic kit that compnses (a) a polynucleotide ofthe present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present mvention, 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 compπse a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others
This mvention also relates to the use of polynucleotides ofthe present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide ofthe mvention, preferable, SEQ ED NO 1, that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression ofthe polynucleotide Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a vaπety of techniques, such as those descπbed elsewhere herem The differences m a polynucleotide and/or polypeptide sequence between orgamsms possessing a first phenotype and orgamsms possessmg 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 (alle c vaπations) m a polynucleotide and/or polypeptide ofthe mvention may also be detected at the polynucleotide or polypeptide level by a vaπety of techniques, to allow for serotyping, for example For example, RT-PCR can be used to detect mutations in the RNA It is particularly prefened to use RT-PCR m 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 pπmers complementary to a polynucleotide encoding yloV polypeptide can be used to identify and analyze mutations The mvention further provides these pπmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These pπmers may be used for, among other things, amplifying yloV DNA and/or RNA isolated from a sample denved from an mdividual, such as a bodily matenal The pπmers 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 and/or prognose the infection or its stage or course, or to serotype and or classify the infectious agent
The mvention further provides a process for diagnosing, disease, preferably bactenal mfections, more preferably infections caused by Streptococcus pneumoniae, compnsmg determining from a sample denved from an individual, such as a bodily material, an mcreased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a yloV polynucleotide can be measured using any on ofthe 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 in accordance with the mvention for detecting over-expression of yloV polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techniques that can be used to determme levels of a yloV polypeptide, m a sample denved from a host, such as a bodily mateπal, are well-known to those of skill m the art Such assay methods clude radiormmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays
Antagonists and Agonists - Assays and Molecules
Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libranes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Co gan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
Polypeptides and polynucleotides ofthe present mvention are responsible for many biological functions, including many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize
(e g ,ιnhιbιt) the function ofthe polypeptide or polynucleotide Accordingly, m a further aspect, the present mvention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide ofthe mvention, as well as related polypeptides and polynucleotides In general, agonists or antagonists (e g , inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds may be identified from a vaπety of sources, for example, cells, cell-free preparations, chemical libranes, and natural product mixtures Such agonists and antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of yloV polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)) The screenmg methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protem ofthe polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may mvolve competition with a labeled competitor Further, these screenmg methods may test whether the candidate compound results a signal generated by activation or rnhibition ofthe polypeptide or polynucleotide, usmg detection systems appropπate to the cells compnsmg 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 ofthe candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists, m the absence of an agonist or antagonist, by testing whether the candidate compound results in inhibition of activation ofthe polypeptide or polynucleotide, as the case may be Further, the screenmg methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide ofthe present invention, to form a mixture, measuring yloV polypeptide and or polynucleotide activity m the mixture, and comparing the yloV polypeptide and/or polynucleotide activity ofthe mixture to a standard Fusion proteins, such as those made from Fc portion and yloV polypeptide, as herein descπbed, can also be used for high-throughput screening assays to identify antagonists ofthe polypeptide ofthe present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al , 1 Mol Recognition, 8 52-58 (1995), and K Johanson et al , 1 Biol Chem, 270(16) 9459-9471 (1995))
The polynucleotides, polypeptides and antibodies that bind to and or interact with a polypeptide ofthe present invention may also be used to configure screemng methods for detectmg the effect of added compounds on the production of mRNA and/or polypeptide in cells For example, an ELISA assay may be constructed for measunng secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
The mvention also provides a method of screening compounds to identify those that enhance (agonist) or block (antagonist) the action of yloV polypeptides or polynucleotides, particularly those compounds that are bacteπstatic and/or bactencidal The method of screening may mvolve high-throughput techmques 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, compnsmg yloV polypeptide and a labeled substrate or hgand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a yloV agomst or antagonist The ability of the candidate molecule to agonize or antagonize the yloV polypeptide is reflected m decreased binding ofthe labeled hgand or decreased production of product from such substrate Molecules that b d gratuitously, i e , without mducmg the effects of yloV polypeptide are most likely to be good antagonists Molecules that bmd well and, as the case may be, mcrease the rate of product production from substrate, mcrease signal transduction, or mcrease chemical channel activity are agomsts Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by usmg a reporter system Reporter systems that may be useful in this regard mclude but are not limited to coloπmetnc, labeled substrate converted mto product, a reporter gene that is responsive to changes m yloV polynucleotide or polypeptide activity, and binding assays known m the art Polypeptides ofthe invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known m the art These techmques include, but are not limited to, hgand binding and crosshnkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^ \), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or purification, and mcubated with a source ofthe putative receptor (e g , cells, cell membranes, cell supematants, tissue extracts, bodily materials) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists ofthe polypeptide that compete with the binding ofthe polypeptide to its receptor(s), if any Standard methods for conductmg such assays are well understood m the art The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational conelation time or tumbling rate Protem complexes, such as formed by yloV polypeptide associating with another yloV polypeptide or other polypeptide, labeled to compnse a fluorescently- labeled molecule will have higher polaπzation values than a fluorescently labeled monomeπc protem It is preferred that this method be used to characterize small molecules that disrupt polypeptide complexes
Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of yloV polypeptide dimers, tπmers, tetramers or higher order structures, or structures formed by yloV polypeptide bound to another polypeptide yloV polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing ofthe two labeled species and excitation ofthe donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence ofthe acceptor Compounds that block dimeπzation will inhibit fluorescence energy transfer
Surface plasmon resonance can be used to monitor the effect of small molecules on yloV polypeptide self-association as well as an association of yloV polypeptide and another polypeptide or small molecule. YloV polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric. Solution protein can then passed over the yloV polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index. This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for yloV polypeptide self-association as well as an association of yloV polypeptide and another polypeptide or small molecule.
A scintillation proximity assay may be used to characterize the interaction between an association of yloV polypeptide with another yloV polypeptide or a different polypeptide . YloV polypeptide can be coupled to a scintillation-filled bead. Addition of radio-labeled yloV polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid. Thus, signal is emitted upon yloV polypeptide binding and compounds that prevent yloV polypeptide self-association or an association of yloV polypeptide and another polypeptide or small molecule will diminish signal.
In other embodiments of the invention there are provided methods for identifying compounds that bind to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the invention comprising: contacting a polypeptide and/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction ofthe 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 binding or interaction of the compound with the polypeptide and/or polynucleotide.
Another example of an assay for yloV agonists is a competitive assay that combines yloV and a potential agonist with yloV-binding molecules, recombinant yloV binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. YloV can be labeled, such as by radioactivity or a colorimetric compound, such that the number of yloV molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness ofthe potential antagonist. It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present mvention may also be used in a method for the structure-based design of an agonist or antagonist ofthe polypeptide and/or polynucleotide, by (a) determining m the first instance the three- dimensional structure ofthe 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 or antagonist, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced bindmg sιte(s), reactive sιte(s), and/or motif(s), and
(d) testing whether the candidate compounds are indeed agonists or antagonists
It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps
In a further aspect, the present mvention provides methods of treatmg abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of yloV polypeptide and/or polynucleotide
If the expression and/or activity ofthe polypeptide and/or polynucleotide is m excess, several approaches are available One approach compnses admmistenng to an mdividual m need thereof an inhibitor compound (antagonist) as herem descnbed, optionally m combmation with a pharmaceutically acceptable earner, m an amount effective to inhibit the function and/or expression ofthe polypeptide and/or polynucleotide, such as, for example, by blocking 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 ofthe 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 ofthe yloV polypeptide and/or polypeptide
In still another approach, expression ofthe gene encoding endogenous yloV polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step m gene expression, but is preferably targeted agamst transcription and/or translation An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, ohgonucleotides that form tπple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo 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 antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection. In particular, the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial yloV proteins that mediate tissue damage and or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques. In accordance with yet another aspect of the invention, there are provided yloV agonists and antagonists, preferably bacteristatic or bactericidal agonists and antagonists.
The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases. Antagonists ofthe invention include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression. Antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing yloV-induced activities, thereby preventing the action or expression of yloV polypeptides and/or polynucleotides by excluding yloV polypeptides and/or polynucleotides from binding.
Antagonists ofthe invention also include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules). Prefened antagonists include compounds related to and variants of yloV. Other examples of polypeptide antagomsts mclude antibodies or, m some cases, oligonucleotides or proteins that are closely related to the ligands. substrates, receptors, enzymes, etc . as the case may be, ofthe polypeptide, e g , a fragment ofthe ligands, substrates, receptors, enzymes, etc , or small molecules that bmd to the polypeptide ofthe present mvention but do not elicit a response, so that the activity ofthe polypeptide is prevented
Small molecules ofthe mvention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons It is preferred that these small molecules are organic molecules
Hehcobacter pylori (herem "H pylori") bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, 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 gastnc adenocarcmoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the mvention (agonists and antagomsts of yloV polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art, particularly nanow-spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H pylori -induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastnc ulcers and gastntis
All publications and references, mcludmg but not limited to patents and patent applications, cited m this specification are herein incorporated by reference m their entirety as if each mdividual publication or reference were specifically and individually indicated to be incorporated by reference herem as being fully set forth Any patent application to which this application claims priority 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
"Bodily mateπal(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, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy mateπals "Dιsease(s)" means any disease caused by or related to infection by a bacteπa. mcludmg , for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospmal fluid
"Host cell(s)" is a cell that has been introduced (e g , transformed or transfected) or is capable of introduction (e g , transformation or transfection) by an exogenous polynucleotide sequence
"Identity," as known in 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, including but not limited to those descπbed m
(Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Bwcomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Gnftm, 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, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Caπllo, 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 m publicly available computer programs Computer program methods to determme 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 companson 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 comparison 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 in (1) and (2) below. (1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO:l, or:
nn ≤ xn " (xn • Y)>
wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO:l, y is 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 prior to subtracting it from xn. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the amino- or carboxy-termmal positions ofthe 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 withm the reference sequence, and wherem said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtractmg that product from said total number of amino acids m 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 m SEQ ID NO 2, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherem any non-mteger product of xa and y is rounded down to the nearest integer prior to subtractmg it from xa
"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 oπginal 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 introduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present m said orgamsm, which orgamsm may be living or non-living "Organιsm(s)" means a (I) prokaryote, mcludmg but not limited to, a member of the genus
Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobactenum, Neissena, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Eschench a, Salmonella, Kleώsiella, 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 pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecahs, Streptococcus faecium, Streptococcus durans, Neissena gonorrheae, Ne ssena menmgit ds, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dpthenae, Gardnerella vaginahs, Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actinomyctes israelu, Listena monocytogenes, Bordetella pertus s, Bordatella parapertusis, Bordetella bronchiseptica, Eschench a cob, Shigella dysentenae, Haemophύus inβuenzae, Haemophύus aegyptius, Haemophύus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typh , Citrobacter freundn, Proteus mirabihs, Proteus wlgans, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysentem, Shigella flexnen, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perfhngens, Clostndium tetani, Clostndium botuhnum, Treponema palhdum, Rickettsia nckettsii and Chlamydia trachomitis, (n) an archaeon, mcludmg but not limited to Archaebacter, and (m) 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 lactis, or Candida albicans
"Polynucleotide(s)" generally refers to any polynbonucleotide or polydeoxyπbonucleotide, that 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 tπple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybπd 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 tπple-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 ofthe molecules, but more typically mvolve only a region of some ofthe molecules One ofthe molecules of a tnple-helical region often is an oligonucleotide As used herem, the term "polynucleotide(s)" also mcludes DNAs or RNAs as descnbed above that compπse one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herem Moreover, DNAs or RNAs compnsmg unusual bases, such as rnosine, or modified bases, such as tπtylated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vaπety 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 characteπstic of viruses and cells, mcludmg, for example, simple and complex cells "Polynucleotιde(s)" also embraces short polynucleotides often refened to as olιgonucleotide(s) "Polypeptide(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptide(s)" refers to both short chains, commonly refened to as peptides, ohgopeptides and ohgomers and to longer chains generally refened to as proteins Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids "Polypeptide(s)" mclude those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed in basic texts and m 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 compπse 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 teπn i Modifications mclude, for example, acetylation, acylation, ADP-πbosylation. amidation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a pid or hpid denvative, covalent attachment of phosphotidyhnositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, lodrnation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racem zation, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-πbosylation, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins, such as argmylation, and ubiquitmation 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 m 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, km. N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from posttranslational natural processes and may be made by entirely synthetic methods, as well
"Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides ofthe mvention mtroduced or transformed mto a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides ofthe mvention
"Vaπant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant 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 amino acid residue may or may not be one encoded by the genetic code. The present invention also includes include variants of each of the polypeptides of the invention, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and He; 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 prefened are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination. A variant of a polynucleotide or polypeptide may be a naturally occu ing such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans. EXAMPLES
The examples below are carried out using standard techniques, that are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention. Example 1 Strain selection, Library Production and Sequencing
The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: l] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E. coli. The sequencing data from two or more clones comprising overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO: l. Libraries may be prepared by routine methods, for example: Methods 1 and 2 below.
Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 according to standard procedures and size-fractionated by either of two methods. Method 1
Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures. DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated mto 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 appropnate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E cob infected with the packaged library The library is amplified by standard procedures Example 2 yloV Characterization
The determination of expression during infection of a gene from Streptococcus pneumoniae Excised lungs from a 48 hour respiratory tract infection of Streptococcus pneumoniae
0100993 m the mouse is efficiently disrupted and processed in the presence of chaotropic agents and RNAase inhibitor to provide a mixture of ammal and bactenal RNA The optimal conditions for disruption and processmg to give stable preparations and high yields of bactenal RNA are followed by the use of hybridisation to a radiolabelled oligonucleotide specific to Streptococcus pneumoniae 16S RNA on Northern blots The RNAase free, DNAase free, DNA and protem free preparations of RNA obtained are suitable for Reverse Transcnption PCR (RT-PCR) usmg umque pnmer pairs designed from the sequence of each gene of Streptococcus pneumoniae 0100993 Using this procedure it was possible to demonstrate t\ι&tyloV s transcibed during infection.
a) Isolation of tissue infected with Streptococcus pneumoniae 0100993 from a mouse animal model of infection (lungs)
Streptococcus pneumoniae 0100993 is grown either on TSA/5%horse blood plates or in AGCH medium overnight, 37°C, 5%C02 Bacteπa are then collected and resuspended m phosphate- buffered salme to an Aβoo of approximately 0 4 Mice are anaesthetized with lsofluorane and 50ml of bactenal suspension (approximately 2 x 105 bacteria) is administered mtranasally usmg a pipetman Mice are allowed to recover and have food and water ad libitum After 48 hours, the mice are euthanized by carbon dioxide overdose, and lungs are aseptically removed and snap-frozen m liquid nitrogen b) Isolation of Streptococcus pneumoniae 0100993 RNA from infected tissue samples
Infected tissue samples, m 2-ml cryo-strorage tubes, are removed from -80°C storage mto a dry ice ethanol bath In a microbiological safety cabmet the samples are disrupted up to eight at a time while the remaining samples are kept frozen m the dry ice ethanol bath To disrupt the bacteria within the tissue sample, 50-100 mg of the tissue is transfered to a FastRNA tube contaimng a silica/ceramic matnx (BIO 101) Immediately, 1 ml of extraction reagents (FastRNA reagents, BIO 101) are added to give a sample to reagent volume ratio of approximately 1 to 20 The tubes are shaken in a reciprocatmg shaker (FastPrep FP120, BIO 101) at 6000 rpm for 20-120 sec The crude RNA preparation is extracted with chloroform/isoamyl alcohol, and precipitated with DEPC- treated/Isopropanol Precipitation Solution (BIO101) RNA preparations are stored in this lsopropanol solution at -80°C if necessary The RNA is pelleted (12,000g for 10 mm ), washed with 75% ethanol (v/v in DEPC-treated water), air-dned for 5-10 mm, and resuspended m 0 1 ml of DEPC-treated water, followed by 5-10 minutes at 55 °C Finally, after at least 1 mmute on ice, 200 units of Rnasin (Promega) is added RNA preparations are stored at -80 °C for up to one month For longer term storage the RNA precipitate can be stored at the wash stage ofthe protocol m 75% ethanol for at least one year at -20 °C
Quality of the RNA isolated is assessed by running samples on 1% agarose gels 1 x TBE gels stained with ethidium bromide are used to visualise total RNA yields To demonstrate the isolation of bactenal RNA from the mfected tissue 1 x MOPS, 2 2M formaldehyde gels are run and vacuum blotted to Hybond-N (Amersham) The blot is then hybridised with a 32P-labelled ohgonucletide probe, of sequence 5 ' AACTGAGACTGGCTTTAAGAGATTA 3 ' [SEQ ID NO 3], specific to 16S rRNA of Streptococcus pneumoniae The size ofthe hybridising band is compared to that of control RNA isolated from in vitro grown Streptococcus pneumoniae 0100993 in the Northern blot Conect sized bactenal 16S rRNA bands can be detected in total RNA samples which show degradation ofthe mammalian RNA when visualised on TBE gels
c) The removal of DNA from
Figure imgf000031_0001
RNA
DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with 20 units of RNAase-free DNAasel (GenHunter) m the buffer supplied m a final volume of 57 microhters
The DNAase was inactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL, Life Technologies) according to the manufacturers protocol DNAase treated RNA was resuspended in 100 microhtres of DEPC treated water with the addition of Rnasin as described before
d) The preparation of cDNA from RNA samples derived from infected tissue
3 microgram samples of DNAase treated RNA are reverse transcnbed using a SuperScπpt Preamphfication System for First Strand cDNA Synthesis kit (Gibco BRL, Life Technologies) according to the manufacturers instructions 150 nanogram of random hexamers is used to pπme each reaction Controls without the addition of SuperScπptll reverse transcπptase are also run Both +/- RT samples are treated with RNaseH before proceeding to the PCR reaction
e) The use of PCR to determine the presence of a bacterial cDNA species
PCR reactions are set up on ice m 0 2ml tubes by adding the following components 43 microhtres PCR Master Mix (Advanced Biotechnologies Ltd ), 1 rmcrohtre PCR pnmers designed for the yloV gene (optimally 18-25 basepairs m length and designed to possess similar annealing temperatures), each primer at lOmM initial concentration, and 5 microhtres cDNA
PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 as follows 2 minutes at 94 °C, then 50 cycles of 30 seconds each at 94 °C, 50 °C and 72 °C followed by 7 minutes at 72 °C and then a hold temperature of 20 °C (the number of cycles is optimally 30-50 to determine the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of the starting quantity of cDNA from the RT reaction is to be made), 10 rmcrohtre aliquots are then run out on 1% 1 x TBE gels stained with ethidmm bromide, with PCR product, if present, sizes estimated by compaπson to a 100 bp DNA Ladder (Gibco BRL, Life Technologies) Alternatively if the PCR products are conveniently labelled by the use of a labelled PCR primer (e g labelled at the 5 'end with a dye) a suitable aliquot of the PCR product is run out on a polyacrylamide sequencing gel and its presence and quantity detected using a suitable gel scanning system (e g ABI Prism™ 377 Sequencer using GeneScan™ software as supplied by Perkin Elmer)
RT/PCR controls may include +/- reverse transcπptase reactions, 16S rRNA pπmers or DNA specific primer pairs designed to produce PCR products from non-transcribed Streptococcus pneumoniae 0100993 genomic sequences To test the efficiency of the primer pairs they are used in DNA PCR with Streptococcus pneumoniae 0100993 total DNA PCR reactions are set up and run as described above using approx 1 microgram of DNA in place ofthe cDNA
Pnmer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are unmformative Of those which give the correct size product with DNA PCR two classes are distinguished m RT/PCR 1 Genes which are not transcnbed in vivo reproducibly fail to give a product m RT/PCR, and 2 Genes which are transcnbed in vivo reproducibly give the conect size product m RT PCR and show a stronger signal m the +RT samples than the signal (if at all present) m -RT controls Using this procedure it was possible to show that yloV was transcnbed m S pneumomae m the mouse animal model of infection at 48 hours of infection Example 3
Demonstration of gene essentiality to bacterial viability
An allelic replacement cassette was generated using PCR technology The cassette consisted of a pair of 500bp chromosomal DNA fragments flanking an erythromycm resistance gene The chromosomal DNA sequences are the 500bp preceding and following the DNA sequence encoding the yloV gene contained m Seq ID NO 1
The allelic replacement cassette was introduced mto S pneumomae R6 by transformation Competent cells were prepared according to published protocols DNA was mtroduced into the cells by incubation of ng quantities of allelic replacement cassette with 10" cells at 30°C for 30 minutes The cells were transferred to 37°C for 90 minutes to allow expression ofthe erythromycm resistance gene Cells were plated m agar contammg lug erythromycm per ml Following incubation at 37°C for 36 hours, colomes are picked and grown overnight m Todd-Hewitt broth supplemented with 0 5% yeast extract Typically 1000 transformants contammg the appropnate allelic replacement are obtamed If no transformants are obtained in three separate transformation experiments as was the case for this gene yloV, then the gene is considered as being essential in vitro

Claims

What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
(i) an isolated polypeptide comprising an amino acid having at least 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2, (iii) an isolated polypeptide that is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polyncleotide sequence of SEQ ID NO:l.
2. An isolated polynucleotide selected from the group consisting of:
(i) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide that has at least 95% identity to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2;
(ii) an isolated polynucleotide comprising a polynucleotide sequence that has at least 95% identity over its entire length to a polynucleotide sequence encoding the polypeptide of SEQ ED
NO:2;
(iii) an isolated polynucleotide comprising a nucleotide sequence that has at least 95% identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1 ;
(iv) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ED
NO:2;
(v) an isolated polynucleotide that is the polynucleotide of SEQ ID NO: 1;
(vi) an isolated polynucleotide of at least 30 nucleotides in length obtainable by screening an appropriate library under stringent hybridization conditions with a probe having the sequence of SEQ
ID NO: 1 or a fragment thereof of of at least 30 nucleotides in length;
(vii) an isolated polynucleotide encoding a mature polypeptide expressed by the yloV gene comprised in the Streptococcus pneumoniae; and
(viii) a polynucleotide sequence complementary to said isolated polynucleotide of (i), (ii), (iii), (iv), (v), (vi) or (vii).
3. A method for the treatment of an individual:
(i) in need of enhanced activity or expression of or immunological response to the polypeptide of claim 1 comprising the step of: administering to the individual a therapeutically effective amount of an antagonist to said polypeptide; or (ii) having need to inhibit activity or expression ofthe polypeptide of claim 1 comprising:
(a) administering to the individual a therapeutically effective amount of an antagonist to said polypeptide; or
(b) adniinistering to the individual a nucleic acid molecule that inhibits the expression of a polynucleotide sequence encoding said polypeptide;
(c) administering to the individual a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate, or receptor; or
(d) administering to the individual an amount of a polypeptide that induces an immunological response to said polypeptide in said individual.
4. A process for diagnosing or prognosing a disease or a susceptibility to a disease in an individual related to expression or activity ofthe polypeptide of claim 1 in an individual comprising the step of:
(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in an organism in said individual; or
(b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said individual.
5. A process for producing a polypeptide selected from the group consisting of:
(i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2;
(ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2;
(iii) an isolated polypeptide that is the amino acid sequence of SEQ ID NO:2, and
(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l, comprising the step of culturing a host cell under conditions sufficient for the production ofthe polypeptide.
6. A process for producing a host cell comprising an expression system or a membrane thereof expressing a polypeptide selected from the group consisting of: (i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2;
(ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2;
(iii) an isolated polypeptide that is the amino acid sequence of SEQ ID NO:2, and
(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l, said process comprising the step of transforming or transfecting a cell with an expression system comprising a polynucleotide capable of producing said polypeptide of (i), (ii), (iii) 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 (i), (ii), (iii) or (iv).
7. A host cell or a membrane expressing a polypeptide selected from the group consisting of: (i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID
NO:2;
(ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptide that is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l.
8. An antibody immunospecific for the polypeptide of claim 1.
9. A method for screening to identify compounds that agonize or that inhibit the function ofthe polypeptide of claim 1 that comprises a method selected from the group consisting of:
(a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound;
(b) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor;
(c) testing whether the candidate compound results in a signal generated by activation or inhibition ofthe polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide; (d) mixing a candidate compound with a solution comprising a polypeptide of claim 1, to form a mixture, measuring activity ofthe polypeptide in the mixture, and comparing the activity ofthe mixture to a standard; or
(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.
10. An agonist or antagonist to the polypeptide of claim 1.
PCT/US2000/021869 1999-08-12 2000-08-10 yloV WO2001011970A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US37289199A 1999-08-12 1999-08-12
US09/372,891 1999-08-12

Publications (1)

Publication Number Publication Date
WO2001011970A1 true WO2001011970A1 (en) 2001-02-22

Family

ID=23470045

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/021869 WO2001011970A1 (en) 1999-08-12 2000-08-10 yloV

Country Status (1)

Country Link
WO (1) WO2001011970A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034998A1 (en) * 1996-03-20 1997-09-25 Human Genome Sciences, Inc. Human cytokine polypeptide

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034998A1 (en) * 1996-03-20 1997-09-25 Human Genome Sciences, Inc. Human cytokine polypeptide

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENESEQ ON STN May 1998 (1998-05-01), BARASH ET AL.: "n-Geneseq35", XP002932248 *
OHGI ET AL.: "Expression of RNase Rh from rhizopus niveus in yeast and characterization of the secreted proteins", JOURNAL OF BIOCHEMISTRY, vol. 109, pages 776 - 785, XP002932249 *

Similar Documents

Publication Publication Date Title
US6268177B1 (en) Isolated nucleic acid encoding nucleotide pyrophosphorylase
WO2000027427A1 (en) trmD
US6251631B1 (en) nadE from Streptococcus pneumoniae
US6270762B1 (en) tdk
WO2000026377A1 (en) POLYNUCLEOTIDES AND POLYPEPTIDES ENCODING metK ISOLATED FROM $i(STREPTOCCOCCUS PNEUMONIAE)
US6326172B1 (en) ytgP
US20060115831A1 (en) Map
WO2001007463A1 (en) Trer
WO2000065026A2 (en) YycG
WO2001011970A1 (en) yloV
WO2000018797A1 (en) Map
EP1130969A1 (en) Murf2 polypeptides and polynucleotides
WO2000043491A2 (en) yhxB
WO2001007458A1 (en) lacR
WO2000024754A1 (en) infB POLYPEPTIDE AND POLYNUCLEOTIDES OF STREPTOCOCCUS PNEUMONIAE
WO2001024635A1 (en) AsuE
WO2000068365A1 (en) tRNA METHYLTRANSFERASES FROM STREPTOCOCCUS PNEUMONIAE
WO2000071555A2 (en) 509rr
WO2001008486A1 (en) CoaA POLYPEPTIDES AND POLYNUCLEOTIDES AND METHODS THEREOF
WO2000070075A1 (en) STREPTOCOCCUS PNEUMONIAE yerS
WO2000068140A1 (en) Signal transduction response regulator family of staphylococcus
WO2001016351A1 (en) DnaE
WO2001021764A1 (en) A pcra helicase of staphylococcus aureus
WO2000049033A1 (en) yybQ
WO2000068427A1 (en) yphC

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载