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WO2001011970A1 - POLYPEPTIDES ET POLYNUCLEOTIDES yloV - Google Patents

POLYPEPTIDES ET POLYNUCLEOTIDES yloV Download PDF

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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
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WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
sequence
ofthe
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PCT/US2000/021869
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English (en)
Inventor
Martin K. R. Burnham
Magdalena Zalacain
Sanjoy Biswas
Patrick V. Warren
John D. Holmes
Alison F. Chalker
Stephanie Van Horn
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Smithkline Beecham Corporation
Smithkline Beecham Plc
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Publication of WO2001011970A1 publication Critical patent/WO2001011970A1/fr

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    • 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

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Abstract

L'invention concerne des polypeptides et des polynucléotides yloV codant pour lesdits polypeptides yloV, et des procédés de production de ces polypeptides à l'aide de techniques de recombinaison. L'invention concerne également des techniques d'utilisation des polypeptides yloV pour cribler des composés antibactériens.
PCT/US2000/021869 1999-08-12 2000-08-10 POLYPEPTIDES ET POLYNUCLEOTIDES yloV WO2001011970A1 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1997034998A1 (fr) * 1996-03-20 1997-09-25 Human Genome Sciences, Inc. Polypeptides cytokines humains

Patent Citations (1)

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

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 *

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