EP1032582A1 - recJ POLYPEPTIDE - Google Patents

recJ POLYPEPTIDE

Info

Publication number
EP1032582A1
EP1032582A1 EP98959529A EP98959529A EP1032582A1 EP 1032582 A1 EP1032582 A1 EP 1032582A1 EP 98959529 A EP98959529 A EP 98959529A EP 98959529 A EP98959529 A EP 98959529A EP 1032582 A1 EP1032582 A1 EP 1032582A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
polynucleotide
compnsmg
recj
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98959529A
Other languages
English (en)
French (fr)
Inventor
Magdalena Zalacain
James R. Brown
Sanjoy Biswas
Richard L. Warren
Lisa K. Shilling
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Publication of EP1032582A1 publication Critical patent/EP1032582A1/de
Withdrawn legal-status Critical Current

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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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses
  • the invention relates to novel polynucleotides and polypeptides of the recJ (single-stranded-DNA specific exonuclease) family, hereinafter referred to as "recJ"
  • 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 of the 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
  • RecJ is a smgle-stranded DNA exonuclease that functions methylation-directed mismatch repair recJ mutants decrease recombination which is mdependent of RecBCD but dependent upon RecE or RecF Inhibitors of the proteins mvolved m this type of DNA repair could prevent the bacte ⁇ um from establishing or maintaining infection of the host and thereby have utility in antibacterial therapy
  • Streptococcus pneumoniae infections has nsen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an mcreasmg population of people with weakened immune systems It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics This phenomenon has created a demand for both new anU-microbial agents, vaccmes, and diagnostic tests for this organism
  • polypeptides of the invention possess amino acid sequence homology to a known H influenzae recJ protem See Swiss-prot, Accession number P45112 and
  • the polynucleotide compnses a region encodmg recJ polypeptides compnsmg a sequence set out m Table 1 [SEQ ID NO 1] which mcludes a full length gene, or a variant thereof
  • isolated nucleic acid molecules encodmg recJ particularly Streptococcus pneumoniae recJ
  • mcludmg mRNAs particularly Streptococcus pneumoniae recJ
  • cDNAs cDNAs
  • genomic DNAs genomic DNAs
  • Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsmg the same
  • a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization
  • particularly preferred embodiments of the mvention are naturally occurring allelic vanants of recJ and polypeptides encoded thereby
  • a polypeptide or polynucleotide of the mvention compnsmg contactmg a polypeptide or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide or polynucleotide with the compound, and determinmg whether the compound bmds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detectmg the presence or absence of a
  • recJ agonists and antagonists preferably bactenostatic or bactenocidal agonists and antagonists
  • compositions compnsmg a recJ polynucleotide or a recJ polypeptide for administration to a cell or to a multicellular organism
  • the mvention relates to novel recJ polypeptides and polynucleotides as descnbed m greater detail below
  • the mvention relates to polypeptides and polynucleotides of a novel recJ of Streptococcus pneumoniae, which is related by amino acid sequence homology to H influenzae recJ polypeptide
  • the mvention relates especially to recJ havmg the nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively, and to the recJ nucleotide sequences of the DNA m the deposited stram and ammo acid sequences encoded thereby TABLE 1 recJ Polynucleotide and Polypeptide Sequences
  • a deposit containing a Streptococcus pneumoniae 0100993 stram has been deposited with the National Collections of Industnal and Marine Bactena Ltd (herem “NCIMB"), 23 St Machar Dnve, Aberdeen AB2 1RY, Scotland on 11 Apnl 1996 and assigned deposit number 40794
  • the deposit was descnbed as Streptococcus peumnoiae 0100993 on deposit On 17 Apnl 1996 a Streptococcus peumnoiae 0100993 DNA library in E coli was similarly depositedwith the NCIMB and assigned deposit number 40800
  • the Streptococcus pneumoniae stram deposit is refened to herem as "the deposited stram" or as "the DNA of the deposited stram "
  • the deposited stram contams the full length recJ gene
  • a license may be required to make, use or sell the deposited stram, and compounds denved therefrom, and no such license is hereby granted Polypeptides
  • polypeptides of the mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (m particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of recJ, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO l]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2and more preferably at least 90% similanty (more preferably at least 90% identity) to a polypeptide of Table 1 [SEQ ID NO 2] and still more preferably at least 95% similanty (still more preferably at least 95% identity) to a polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids
  • the mvention also mcludes polypeptides of the formula
  • R 1 X-(R 1 ) m -(R 2 )-(R 3 ) n -Y
  • X is hydrogen, and at the carboxyl terminus
  • Y is hydrogen or a metal
  • Rj and R3 are any ammo acid residue
  • m is an mteger between 1 and 1000 or zero
  • n is an mteger between 1 and 1000 or zero
  • R 2 is an ammo acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 In the formula above R 2 is onented so that its ammo terminal residue is at the left, bound to and its carboxy terminal residue is at the nght, bound to R3
  • Any stretch of ammo acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer
  • a fragment is a vanant polypeptide havmg an ammo acid sequence that entirely
  • Prefened fragments m include, for example, truncation polypeptides havmg a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a contmuous senes of residues that mcludes the ammo terminus, or a contmuous senes of residues that mcludes the carboxyl terminus
  • Degradation forms of the polypeptides of the mvention m a host cell, particularly a Streptococcus pneumoniae are also prefened
  • fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-formmg regions, turn and turn-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate bmdmg region, and high antigenic mdex regions
  • Vanants that are fragments of the polypeptides of the mvention may be employed for producmg the conespondmg full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the fiill-length polypeptides of the mvention
  • X or "Xaa” may also be used m descnbmg certain polypeptides of the invention "X” and “Xaa” mean that any of the twenty naturally occurmg amino acids may appear at such a designated position in the polypeptide sequence Polynucleotides
  • Another aspect of the mvention relates to isolated polynucleotides. mcludmg the full length gene, that encode the recJ polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof Usmg the information provided herem, such as a polynucleotide sequence set out m Table 1
  • a polynucleotide of the mvention encodmg recJ polypeptide may be obtamed usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bactena usmg Streptococcus pneumoniae 0100993 cells as starting matenal, followed by obtaining a full length clone
  • a polynucleotide sequence of the invention such as a sequence given m Table 1 [SEQ ID NO 1]
  • a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 E coli 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 usmg strmgent conditions By sequencmg the individual clo
  • the DNA sequence set out m Table 1 [SEQ ID NO 1] contams an open reading frame encodmg a protem havmg about the number of ammo acid residues set forth m Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known m the art
  • RecJ of the mvention is structurally related to other proteins of the recJ (single-stranded- DNA specific exonuclease) family, as shown by the results of sequencmg the DNA encodmg recJ of the deposited stram See Swiss-prot, Accession number P45112 and Fleischmann et al , Science. 269 (5223). 496-512 (1995) Also see Ukita and Ikeda, J Bactenol. 178(8). 2362- 2367 (1996), Garzon et al , J Mol Gen Genet. 250(5). 570-580 (1996), and Lovett and Sutera et al , Genetics. 140(1). 27-45 (1995)
  • the invention provides a polynucleotide sequence identical over its entire length to a codmg sequence m Table 1 [SEQ ID NO 1] Also provided by the mvention is the codmg sequence for the mature polypeptide or a fragment thereof, by itself as well as the codmg sequence for the mature polypeptide or a fragment m reading frame with other codmg sequence, such as those encodmg a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence
  • the polynucleotide may also contam non-coding sequences, mcludmg for example, but not limited to non-coding 5' and 3' sequences, such as the transcnbed, non-translated sequences, termmation signals, nbosome bmdmg sites, sequences that stabilize mRNA, mtrons, polyadenylation signals, and additional codmg sequence which encode additional ammo acids
  • a marker sequence that facilitates punfication of the fused polypeptide
  • a prefened embodiment of the mvention is a polynucleotide compnsmg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 2233 set forth SEQ ID NO 1 of Table 1 , both of which encode the rec J polypeptide
  • the mvention also mcludes polynucleotides of the formula
  • X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal
  • Ri and R3 is any nucleic acid residue
  • m is an mteger between 1 and 3000 or zero
  • n is an mteger between 1 and 3000 or zero
  • R is a nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from Table 1 In the polynucleotide formula above R 2 is onented so that its 5' end residue is at the left, bound to R ⁇ and its 3' end residue is at the nght, bound to R3 Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer In a prefened
  • polynucleotides of the inventions are denved from Streptococcus pneumoniae, however, they may preferably be obtamed from organisms of the same taxonomic genus They may also be obtamed.
  • polynucleotide encodmg a polypeptide encompasses polynucleotides that mclude a sequence encodmg a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae recJ havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2]
  • the term also encompasses polynucleotides that mclude a smgle contmuous region or discontinuous regions encodmg the polypeptide (for example, mterrupted by mtegrated phage or an insertion sequence or editmg) together with additional regions, that also may contam codmg and/or non-coding sequences
  • the mvention further relates to vanants of the polynucleotides descnbed herem that encode for vanants of the polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Vanants that are fragments of the polynucleotides of the mvention may be used to synthesize fiill- length polynucleotides of the mvention
  • prefened embodiments are polynucleotides encoding recJ vanants, that have the ammo acid sequence of recJ polypeptide of Table 1 [SEQ ID NO 2] m which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, deleted or added, m any combination Especially prefened among these are silent substitutions, additions and deletions, that do not alter the properties and activities of recJ
  • polynucleotides that are at least 70% identical over their entire length to a polynucleotide encodmg recJ polypeptide havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides
  • most highly prefened are polynucleotides that compnse a region that is at least 80% identical over its entire length to a polynucleotide encodmg recJ polypeptide of the deposited stram and polynucleotides complementary thereto hi this regard, polynucleotides at least 90% identical over their entire length to the same are particularly prefened, and among these particularly prefened polynucleotides, those with 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
  • the mvention further relates to polynucleotides that hybndize to the herem above-descnbed sequences
  • the mvention especially relates to polynucleotides that hybndize under stnngent conditions to the herem above-descnbed polynucleotides
  • strmgent conditions and “stnngent hybndization conditions” mean hybndization will occur only if there is at least 95% and preferably at least 97% identity between the sequences
  • strmgent hybndization conditions is overnight mcubation at 42°C m a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm
  • polynucleotide assays of the mvention may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encodmg recJ and to isolate cDNA and genomic clones of other genes that have a high sequence similanty to the recJ gene
  • probes generally will compnse at least 15 bases
  • probes will have at least 30 bases and may have at least 50 bases
  • Particularly prefened probes will have at least 30 bases and will have 50 bases or less
  • the codmg region of the recJ gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an ohgonucleotide probe A labeled ohgonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to
  • polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herem relatmg to polynucleotide assays
  • Polynucleotides of the mvention that are oligonucleotides derived from the sequences of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herein as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcnbed m bactena in infected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of mfection and type of mfection the pathogen has attained
  • the mvention also provides polynucleotides that may encode a polypeptide that is the mature protem plus additional ammo or carboxyl-termmal ammo acids, or ammo acids mtenor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance)
  • Such sequences may play a role m processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things
  • the additional ammo acids may be processed away from the mature protem by cellular enzymes
  • a precursor protem, havmg the mature form of the polypeptide fused to one or more prosequences may be an mactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • N may also be used m descnbmg certain polynucleotides of the invention "N" means that any of the four DNA or RNA bases may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a base that when taken m combination with adjacent nucleotide positions, when read m the correct reading frame, would have the effect of generatmg a premature termmation codon m such readmg frame
  • a polynucleotide of the mvention may encode a mature protein, a mature protem plus a leader sequence (which 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 preproprotein, which is a precursor to a proprotein, havmg a leader sequence and one or more prosequences, which generally are removed dunng processmg steps that produce active and mature forms of the polypeptide Vectors, host cells, expression
  • the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques
  • Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs 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 descnbed m many standard laboratory manuals, such as Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Sprmg Harbor Laboratory Press, Cold Sprmg Harbor,
  • appropnate hosts include bactenal cells, such as streptococci, staphylococci, enterococci E cob, streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells, and plant cells
  • bactenal cells such as streptococci, staphylococci, enterococci E cob, streptomyces and Bacillus subtihs cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
  • vectors include, among others, chromosomal, episomal and virus-denved vectors, e g , 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 and retroviruses, 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 contam 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
  • appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography
  • high performance liquid chromatography is employed for punfication
  • Well known techmques for refoldmg protem may be employed to regenerate active conformation when the polypeptide is denatured dunng isolation and or punfication Diagnostic Assays
  • This mvention is also related to the use of the recJ polynucleotides of the mvention for use as diagnostic reagents Detection of recJ m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease Eukaryotes (herem also "md ⁇ v ⁇ dual(s)"), particularly mammals, and especially humans, particularly those mfected or suspected to be infected with an organism compnsmg the recJ gene may be detected at the nucleic acid level by a vanety of techniques
  • Nucleic acids for diagnosis may be obtamed from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin
  • Genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification technique pnor to analysis RNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characterization of the species and stram of prokaryote present m an mdividual, may be made by an analysis of the genotype of the prokaryote gene
  • Deletions and insertions can be detected by a change in size of the amphfied product m companson to the genotype of a reference sequence
  • Pomt mutations can be identified by hybndizmg amplified DNA to labeled recJ polynucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m
  • RT-PCR can be used to detect mutations It is particularly prefened to used 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 or RT-PCR
  • PCR primers complementary to a nucleic acid encodmg recJ can be used to identify and analyze mutations Examples of representative primers are shown below m Table 2
  • R 1 X-(R 1 ) m -(R 2 )-(R 3 ) n -Y
  • X is hydrogen
  • Y is hydrogen or a metal
  • Rj and R3 is any nucleic acid residue
  • m is an mteger between 1 and 20 or zero
  • n is an mteger between 1 and 20 or zero
  • R is a primer sequence of the mvention, particularly a primer sequence selected from Table 2
  • R 2 is onented so that its 5' end residue is at the left, bound to R j and its 3' end residue is at the nght, bound to R3
  • Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer bemg complementary to a region of a polynucle
  • the mvention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end
  • These primers may be used for, among other things, amplifying recJ DNA isolated from a sample denved from an mdividual
  • the primers may be used to amplify the gene isolated from an infected mdividual such that the gene may then be subject to vanous techmques for elucidation of the DNA sequence In this way, mutations m the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections by Streptococcus pneumoniae, comprising determining from a sample derived from an individual a mcreased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of recJ polynucleotide can be measured usmg any on of the methods well known m the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods
  • a diagnostic assay m accordance with the mvention for detectmg over- expression of recJ protem compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a recJ protem, m a sample denved from a host are well-known to those of skill m the art
  • Assay techmques that can be used to determine levels of a recJ protem, m a sample denved from a host are well-known to those of skill m the art
  • Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Antibodies
  • the polypeptides of the mvention or vanants thereof, or cells expressmg them can be used as an immunogen to produce antibodies lmmunospecific for such polypeptides
  • Antibodies as used herem mcludes monoclonal and polyclonal antibodies, chimenc, smgle chain, simiamzed antibodies and humanized antibodies, as well as Fab fragments, mcludmg the products of an Fab lmmunolglobulm expression library
  • Antibodies generated against the polypeptides of the mvention can be obtamed by administering the polypeptides or epitope-bearmg fragments, analogues or cells to an ammal, preferably a nonhuman, usmg routme protocols
  • an ammal preferably a nonhuman, usmg routme protocols
  • any technique known m the art that provides antibodies produced by contmuous cell line cultures can be used Examples mclude vanous techmques, such as those m Kohler, G and Milstem, C , Nature 256 495-497 (1975), Kozbor et al , Immunology Today 4 72 (1983), Cole et al , pg 77-96 m MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Liss, Inc (1985)
  • phage display technology may be utilized to select antibody genes with bmdmg activities towards the polypeptide either from repertoires of PCR amphfied v-genes of lymphocytes from humans screened for possessing anti-recJ or from naive hbranes (McCafferty, J et al , (1990), Nature 348, 552-554, Marks, J et al , (1992) Biotechnology 10, 779-783)
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T et al , (1991) Nature 352, 624-628) If two antigen bmdmg domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptides to purify the polypeptides by affinity chromatography
  • antibodies against recJ- polypeptide may be employed to treat infections, particularly bactenal infections
  • polypeptide variants mclude antigenically, epitopically or lmmunologically equivalent vanants that form a particular aspect of this mvention
  • antigenically equivalent derivative encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protem or polypeptide accordmg to the mvention, mterfere with the immediate physical interaction between pathogen and mammalian host
  • immunologically equivalent derivative encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies m a vertebrate, the antibodies act to interfere with the immediate physical mteraction between pathogen and mammalian host
  • the polypeptide such as an antigenically or lmmunologically equivalent derivative or a fusion protem thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken
  • the fusion protem may provide stability to the polypeptide
  • the antigen may be associated, for example by conjugation, with an lmmunogenic carrier protein for example bovme serum albumin (BSA) or keyhole limpet haemocyanin (KLH)
  • BSA bovme serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide compnsmg multiple copies of the protein or polypeptide, or an antigenically or lmmunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogemcity so as to obviate the use of a carrier
  • the antibody or variant thereof is modified to make it less lmmunogenic in the mdividual
  • the antibody may most preferably be "humanized", where the compl mentarity determinmg reg ⁇ on(s) of the hybndoma-de ⁇ ved antibody has been transplanted mto a human monoclonal antibody , for example as described in Jones, P et al (1986), Nature 321, 522-525 or Tempest et al , (1991) Biotechnology 9, 266-273
  • a polynucleotide of the invention m genetic immunization will preferably employ a suitable deliver ⁇ ' method such as direct injection of plasmid DNA mto muscles (Wolff et al , Hum Mol Genet 1992, 1 363, Manthorpe et al , Hum Gene Ther 1963 4, 419), delivery of DNA complexed with specific protem carriers (Wu et al , J Biol Chem 1989 264.16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA, 1986 83,9551), encapsulation of DNA in various forms of hposomes (Kaneda et al , Science 1989 243,375), particle bombardment (Tang et al , Nature 1992, 356 152, Eisenbraun et al , DNA Cell Biol 1993, 12 791) and in vivo mfection usmg cloned retroviral vectors (Seeger et al , direct
  • Polypeptides of the mvention may also be used to assess the bmdmg of small molecule substrates and hgands m, for example, cells, cell-free preparations, chemical libranes, and natural product mixtures
  • substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
  • the mvention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagomst) the action of recJ polypeptides or polynucleotides, particularly those compounds that are bactenostatic and/or bactenocidal
  • the method of screening may mvolve high-throughput techmques For example, to screen for agonists or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof
  • an assay for recJ antagomsts is a competitive assay that combmes recJ and a potential antagomst with recJ-bmdmg molecules, recombinant recJ bmdmg molecules, natural substrates or hgands, or substrate or gand mimetics.
  • RecJ can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of recJ molecules bound to a bmdmg molecule or converted to product can be determmed accurately to assess the effectiveness of the potential antagomst
  • Potential antagomsts mclude small orgamc molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide or polypeptide of the mvention and thereby inhibit or extinguish its activity
  • Potential antagomsts also may be small orgamc molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule, without mducmg recJ-mduced activities, thereby preventmg the action of recJ by excluding recJ from bmdmg
  • Potential antagomsts m clude a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small orgamc molecules, peptides or peptide-like molecules
  • Other potential antagomsts m clude antisense molecules (see Okano, J Neurochem 56 560 (1991), OI1GODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a descnption of these molecules)
  • Prefened potential antagomsts m include compounds related to and vanants of red
  • Each of the DNA sequences provided herein may be used m the discovery and development of antibacterial compounds
  • the encoded protem, upon expression, can be used as a target for the screening
  • the mvention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to mterfere with the initial physical mteraction between a pathogen and mammalian host responsible for sequelae of infection
  • the molecules of the invention may be used m the prevention of adhesion of bacteria, in particular gram positive bacteria, to mammalian extracellular matrix proteins on m-dwelhng devices or to extracellular matrix protems in wounds, to block red protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosme kmases (Rosenshme et al , Infect lmmun 60 2211 (1992), to block bacterial adhesion between mammalian extracellular matrix proteins and bacterial recJ proteins that mediate tissue damage and, to block the normal progression of pathogenesis in infections initiated other than by the implantation of m-dwelhng devices or by other surgical techmques
  • the antagomsts and agonists of the mvention may be employed, for instance, to inhibit and treat diseases
  • Hehcobacter pylori herem H pylori
  • bactena infect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastritis
  • International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylon International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm
  • the mtemational Agency for Research on Cancer recently recognized a cause-and- effect relationship between H pylori and gastric adenocarcmoma, classifying the bactenum as a Group I (defimte) carcmogen
  • Preferred antimicrobial compounds of the mvention found usmg screens provided by the invention, particularly broad- spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H pylori -induced cancers, such
  • Another aspect of the invention relates to a method for mducmg an immunological response in an individual, particularly a mammal which compnses inoculating the mdividual with recJ, or a fragment or vanant thereof, adequate to produce antibody and/ or T cell immune response to protect said mdividual from mfection, particularly bactenal infection and most particularly Streptococcus pneumoniae mfection Also provided are methods whereby such immunological response slows bacterial replication
  • Yet another aspect of the mvention relates to a method of mducmg immunological response in an individual which comprises dehvermg to such mdividual a nucleic acid vector to direct expression of recJ, or a fragment or a variant thereof, for expressmg recJ, or a fragment or a vanant thereof in vivo in order to mduce an immunological response, such as, to produce antibody and/ or T cell immune response, mcludmg, for example, cytokine-producing T cells or cytotoxic
  • a further aspect of the mvention relates to an immunological composition which, when introduced mto an individual capable or having induced withm it an immunological response, mduces an immunological response m such mdividual to a recJ or protein coded therefrom wherein the composition comprises a recombinant recJ or protein coded therefrom compnsmg DNA which codes for and expresses an antigen of said recJ or protem coded therefrom
  • the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that ansmg from CTL or CD4+ T cells
  • a recJ polypeptide or a fragment thereof may be fused with co-protem which may not by itself produce antibodies, but is capable of stabilizing the first protem and producmg a fused protein which will have lmmunogenic and protective properties
  • fused recombinant protem preferably further comprises an antigenic co-protem, such as hpoprotem D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubihze the protein and facilitate production and purification thereof
  • the co-protem may act as an adjuvant m the sense of providing a generalized stimulation of the immune system
  • the co-protein may be attached to either the ammo or carboxy terminus of the first protem
  • compositions particularly vaccme compositions, and methods compnsmg the polypeptides or polynucleotides of the mvention and lmmunostimulatory DNA sequences, such as those described in Sato, Y et al Science 273 352 (1996)
  • this mvention provides methods usmg the descnbed polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacterial cell surface protems m DNA constructs used in such genetic immunization experiments m ammal models of infection with Streptococcus pneumoniae will be particularly useful for identifying protem epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of bactenal infection, particularly Streptococcus pneumoniae mfection, m mammals, particularly humans
  • the polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect agamst invasion of bacteria, for example by blocking adherence of bactena to damaged tissue
  • tissue damage include wounds m skin or connective tissue caused, e g , by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds m the mucous membranes, such as the mouth, mammary glands, urethra or vagma
  • the mvention also mcludes a vaccme formulation which compnses an lmmunogenic recombinant protem of the mvention together with a suitable earner Smce the protem may be broken down m the stomach, it is preferably administered parenterally, mcludmg, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal
  • Formulations suitable for parenteral administration mclude aqueous and non-aqueous stenle injection solutions which may contam anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation msotomc with the bodily fluid, preferably the blood, of the mdividual, and aqueous and non-aqueous stenle suspensions which may mclude suspending agents or thickemng agents
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dned condition
  • compositions, kits and administration The mvention also relates to compositions compnsmg the polynucleotide or the polypeptides discussed above or their agonists or antagomsts
  • the polypeptides of the mvention may be employed m combination with a non-sterile or stenle earner or earners for use with cells, tissues or orgamsms, such as a pharmaceutical earner suitable for administration to a subject
  • Such compositions compnse for instance, a media additive or a therapeutically effective amount of a polypeptide of the mvention and a pharmaceutically acceptable earner or excipient
  • Such earners may mclude, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof
  • the formulation should suit the mode of administration
  • the mvention further relates to diagnostic and pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions
  • compositions may be admimstered m any effective, convenient manner mcludmg, for instance, administration by topical, oral, anal, vaginal, intravenous, mtrapentoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others
  • the active agent may be administered to an individual as an mjectable composition, for example as a sterile aqueous dispersion, preferably isotonic
  • compositions may be formulated for topical application for example in the form of omtments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contam appropriate conventional additives, mcludmg, for example, preservatives, solvents to assist drug penetration, and emollients m omtments and creams
  • topical formulations may also contain compatible conventional carriers, for example cream or omtment bases, and ethanol or oleyl alcohol for lotions
  • Such earners may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation
  • the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg
  • the physician m any event will determine the actual dosage which will be most suitable for an mdividual and will vary with the age, weight and response of the particular mdividual
  • the above dosages are exemplary of the average case
  • In-dwellmg devices mclude surgical implants, prosthetic devices and catheters, I e , devices that are mtroduced to the body of an mdividual and remam m position for an extended time
  • Such devices mclude, for example, artificial jomts, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters
  • composition of the invention may be administered by injection to achieve a systemic effect agamst relevant bacteria shortly before insertion of an in-dwelhng device Treatment may be continued after surgery during the m-body time of the device
  • composition could also be used to broaden pe ⁇ operative cover for any surgical technique to prevent bacterial wound infections, especially Streptococcus pneumoniae wound infections
  • compositions of this mvention may be used generally as a wound treatment agent to prevent adhesion of bactena to matnx protems exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis
  • the composition of the mvention may be used to bathe an indwelling device immediately before insertion
  • the active agent will preferably be present at a concentration of 1 ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices
  • a vaccme composition is conveniently in mjectable form Conventional adjuvant
  • D ⁇ sease(s) means and disease caused by or related to infection by a bactena, mcludmg otitis media, conjunctivitis, pneumoma, bacteremia, menmgitis, smusitis, pleural empyema and endocarditis, and most particularly menmgitis, such as for example infection of cerebrospinal fluid
  • “Host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determmed by companng 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 strmgs of such sequences
  • Identity can be readily calculated by known methods, mcludmg but not limited to those descnbed m (Computational Molecular Biology, Lesk, A M , ed , Oxford Umversity Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M , and Griffin.
  • Polynucleotide embodiments further include an isolated polynucleotide compnsmg a polynucleotide sequence havmg at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1 , wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, mcludmg transition and transversion.
  • alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups withm the reference sequence, and wherein said number of nucleotide alterations is determmed by multiplying the total number of nucleotides m SEQ ID NO 1 by the mteger defining the percent identity divided by 100 and then subtractmg that product from said total number of nucleotides m SEQ ID NO 1, or
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides m SEQ ID NO 1
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator
  • any non-mteger product of x n and y is rounded down to the nearest mteger pnor to subtractmg it from x n
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, nussense or frameshift mutations m this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 1, that is it may be 100% identical, or it may include up to a certain mteger number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity
  • Such alterations are selected from the group consistmg of at least one nucleic acid deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleic acids m the reference sequence or m one or more contiguous groups withm the reference sequence
  • the number of nucleic acid alterations for a given percent identity is determmed by multiplying the total number of nucleic acids in SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtractmg that product from said total number of nucleic acids m SEQ ID NO 1,
  • Polypeptide embodiments further mclude an isolated polypeptide compnsmg a polypeptide havmg at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain mteger number of ammo acid alterations as compared to the reference sequence,
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids m SEQ ID NO 2
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest mteger pnor to subtractmg it from x a
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may mclude up to a certain mteger number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity
  • Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or m one or more contiguous groups withm the reference sequence
  • the number of ammo acid alterations for a given % identity is determmed 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 ammo acids in SEQ ID NO 2, or
  • n a is the number of ammo acid alterations
  • x a is the total number of amino acids m SEQ ID NO 2
  • y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest mteger pnor to subtractmg it from x a
  • Isolated means altered “by the hand of man” from its natural state, l e , if it occurs m nature, it has been changed or removed from its onginal environment, or both
  • a polynucleotide or a polypeptide naturally present m a living organism 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
  • Polynucleotide(s) generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotide(s)” mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions or single-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions
  • polynucleotide as used herem refers to tnple-stranded regions compnsmg RNA or
  • 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, oligopeptides and o gomers and to longer chains generally refened to as proteins
  • Polypeptides may contam ammo acids other than the 20 gene encoded ammo acids
  • Polypeptide(s)” mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed m 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 m a given polypeptide Also, a given polypeptide may contam many types of modifications Modifications
  • covalent attachment of flavin covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or hpid denvative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, pid attachment, sulfation, gamma- carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation.
  • Polypeptides may be branched or cyclic, with or without branching Cychc, branched and branched circular polypeptides may result from post-translational natural processes and
  • 'Nanant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical vanant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below
  • a typical vanant of a polypeptide differs m ammo 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 vanant and reference polypeptide may differ in ammo acid
  • the polynucleotide havmg a DNA sequence given m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E cob
  • the sequencmg data from two or more clones contaimng overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence m SEQ ID NO 1 Libraries may be prepared by routme methods, for example Methods 1 and 2 below
  • Total cellular DNA is mechanically sheared by passage through a needle m order to size-fractionate accordmg to standard procedures
  • DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI, the library 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 restnction enzymes appropriate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library The library is amphfied by standard procedures Example 2
  • RNAase free, DNAase free, DNA and protein free preparations of RNA obtained are suitable for Reverse Transcription PCR (RT-PCR) using unique primer pairs designed from the sequence of each gene of Streptococcus pneumoniae 0100993 a) Isolation of tissue infected with Streptococcus pneumoniae 0100993 from a mouse animal model of infection (lungs).
  • RT-PCR Reverse Transcription PCR
  • Streptococcus pneumoniae 0100993 is grown either on TSA/5%horse blood plates or m AGCH medium overnight, 37°C, 5%C0 2 Bactena are then collected and resuspended m phosphate-buffered saline to an A ⁇ oo of approximately 0 4 Mice are anaesthetized with isofluorane and 50ml of bacterial suspension (approximately 2 x 10 5 bactena) is admimstered lntranasally 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 in 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 preparation
  • RNA preparations are stored at -80 °C for up to one month
  • the RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one year at -20 °C
  • RNA isolation Quality of the RNA isolated is assessed by running samples on 1 % agarose gels 1 x TBE gels stained with ethtdium bromide are used to visualise total RNA yields To demonstrate the isolation of bactenal RNA from the infected tissue 1 x MOPS.
  • DNAase was inactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL, Life Technologies) accordmg to the manufacturers protocol DNAase treated RNA was resuspended m 100 microhtres of DEPC treated water with the addition of Rnasin as descnbed before d)
  • TRIzol LS Reagent Gibco BRL, Life Technologies
  • DNAase treated RNA was resuspended m 100 microhtres of DEPC treated water with the addition of Rnasin as descnbed before d
  • the preparation of cDNA from RNA samples derived from infected tissue. 3 microgram samples of DNAase treated RNA are reverse transcribed using a SuperScnpt Preamphfication System for First Strand cDNA Synthesis kit (Gibco BRL.
  • PCR Master Mix (Advanced Biotechnologies Ltd ), 1 microhtre PCR primers (optimally 18-25 basepairs in length and designed to possess similar annealing temperatures), each primer at 10 mM initial concentration, and 5 microhtres cDNA
  • PCR reactions are run on a Perkm 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 microhtre ahquots are then run out on 1% 1 x TBE gels stamed with ethidium bromide, with PCR product, if present, sizes estimated by comparison 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 seque
  • RT PCR controls may mclude +/- reverse transc ⁇ ptase reactions, 16S rRNA primers or DNA specific primer pairs designed to produce PCR products from non-transcribed Streptococcus pneumoniae 0100993 genomic sequences
  • RT/PCR are PCR failures and as such are umnformative 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 in RT/PCR, and (2) Genes which are transcribed in vivo reproducibly give the correct size product m RT/PCR and show a stronger signal in the +RT samples than the signal (if at all present) in -RT controls

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WO1999026959A1 (en) 1999-06-03

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