WO2001053458A2 - Methods and reagents for performing antimicrobial compound screening - Google Patents

Methods and reagents for performing antimicrobial compound screening Download PDF

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Publication number
WO2001053458A2
WO2001053458A2 PCT/US2001/001786 US0101786W WO0153458A2 WO 2001053458 A2 WO2001053458 A2 WO 2001053458A2 US 0101786 W US0101786 W US 0101786W WO 0153458 A2 WO0153458 A2 WO 0153458A2
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Prior art keywords
polypeptide
seq
era
polynucleotide
identity
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PCT/US2001/001786
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French (fr)
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WO2001053458A9 (en
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Andrei Nicolae Lupas
Kenneth H. Pearce, Jr.
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Smithkline Beecham Corporation
Smithkline Beecham P.L.C.
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Publication of WO2001053458A2 publication Critical patent/WO2001053458A2/en
Publication of WO2001053458A9 publication Critical patent/WO2001053458A9/en

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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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses.
  • the invention relates to polynucleotides and polypeptides of the Era family, as well as their variants, hereinafter referred to as "Era binding domain,” "ERA binding domain polynucleotide(s),” and “ERA binding domain polypeptide(s).”
  • Era binding domain Era binding domain
  • ERA binding domain polynucleotide(s) and "ERA binding domain polypeptide(s).”
  • the era gene in Escherichia coli codes for an essential protein.
  • the protein, Era is a GTPase and is able to autophosphorylate itself at a serine and/or threonine residue (March PE., 1992. Membrane-associated GTPases in bacteria. Molecular Microbiology 6: 1253-1257). It is found in both soluble and membrane compartments of E. coli cells and has been found to be localized to the membrane at points consistent with a role in septation or nucleoid segregation (Gollop, N. & March P.E., 1991. Localization of the membrane binding sites of Era in Escherichia coli. Research in Microbiology 142: 301-307).
  • Homologous proteins have been found in a variety of bacteria and show functional complementation (Pillutla, R.C., Sharer, J.D., Gulati, P.S., Wu, E., Yamashita, Y., Lerner, C.G., Inouye, M., & March, P.E., 1995.
  • Cross- species complementation of the indispensable Escherichia coli era gene highlights amino acid regions essential for activity. Journal of Bacteriology 177: 2194-2196).
  • polypeptides and polypeptides that have a present benefit of, among other things, being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease. Certain of the polypeptides of the invention possess significant amino acid sequence homology to a known Era from B.suhtilis protein.
  • the present invention relates to Era binding domains, in particular ERA binding domain polypeptides and ERA binding domain polynucleotides, recombinant materials and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of microbial diseases, amongst others.
  • the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting ERA binding domain expression or activity.
  • the invention also provides methods for antagonizing an interaction between ERA and the ERA binding site. Further provided by the invention are methods for antagonizing an interaction between ERA and the ERA binding site.
  • ERA and the ERA binding site where said binding site is a site on the genomic DNA or an
  • RNA and DNA are RNA and DNA, either or both of which comprise a ERA binding site.
  • Particularly preferred embodiments of the invention are methods of the invention used to identify antimicrobial compounds.
  • Preferred embodiments are also provided whereby polynucleotide and/or FtsZ binding is demonstrated using polarization.
  • ERA is involved in late localization or early septation, and have devised methods to screen for compounds that agonize or antagonize the polymerization reaction in late post chromosomal septation. Based on their studies, the applicants posit that ERA guides the FtsZ ring formation.
  • One method useful to show such interaction is immunoprecipitation of DNA with ERA and ERA dimers, trimer or tetramer.
  • a column with DNA or RNA to bind ERA or ERA dimer, trimer or tetramer followed by PCR may be used to show the same.
  • ERA comprises a GAP-like domain comprising arginine within a binding pocket where the GTPase domain is formed
  • this interaction among others, can be used for screening antimicrobial compounds as described herein.
  • Screening methods to identify compounds that modulate, agonize or antagonize GTP::Arg-GAP interactions, are provided herein.
  • Preferred embodiments of these screens include, for example, quenching, tumbling, RIA and scintillation proximity assays.
  • Preferred embodiments of the invention also include, for example, a method for screening compounds that alter an activity of ERA comprising the step of providing ERA and nucleotide and assaying nucleotide exchange activity, such as by using cycles of GTP.
  • the invention further provides proteins related to ERA, discovered by the applicants that can be used in the methods of the invention.
  • proteins include, but are not limited to, GTP-GTP-GAP (ORF which uses GTPase activity, for example, as a screen) and GAP(-euk)- GTP-conserved sequence (also referred to as THDF). Both of these proteins comprise a GTPase domain.
  • polypeptides and polynucleotides of the invention are believed to be essential for the survival of the related bacteria.
  • inhibitors of ERA and other polynucleotides and polypeptides of the invention are useful to treat bacterial infections.
  • the invention relates to ERA binding domain polypeptides and polynucleotides as described in greater detail below.
  • the invention relates to polypeptides and polynucleotides of Streptococcus pneumoniae, which is related by amino acid sequence homology to ERA binding domain from B.subtilis polypeptide.
  • the invention relates especially to those having the nucleotide and amino acid sequences comprising ERA binding domains set out in Table 1.
  • Table 1 sets forth, among other things, a multiple alignment of ERA binding domain sequences with their closest relatives (the 50k proteins, containing two GTPase domains, and ThdF) and with human Ras - the closest relative with known structure.
  • ERA binding domains for identifying compounds that agonize or antagonize the interaction between an ERA binding domain and its cognate binding molecule, such as a nucleotide or polynucleotide, especially an RNA polynucleotide.
  • the GTPase domain is separated from the KH domain by an intervening sequence of about 50 residues. Database searches with this sequence do not yield significant matches to other proteins.
  • the only entirely conserved residue in this sequence is an arginine, which is always preceeded by a large hydrophobic residue.
  • an arginine either present in the same polypeptide (Ga) or in a separate protein (RasGAP and RhoGAP) is required for efficient hydrolysis of the bound nucleotide (hence the name GAP for GTPase activating protein).
  • GAP GTPase activating protein
  • the domain separating the GTPase and KH domains of ERA is proposed to be the ERA-specific GAP. Domains containing an invariant arginine are observed in two closely related GTPases that are also present in all fully sequenced eubacterial genomes: a 50kD open reading frame and ThdF, believed to be involved in thiophene and furan oxidation. In the 50k protein the region bears distant similarity to eGAP:
  • ERA from Escherichia coli was truncated at Prol74 (eliminating the GTPase domain) and the C-terminal part of the sequence, corresponding to:
  • PEATHHFPEDYITDRSQRFMASEIIREKMRFLGAE PYSVTV ⁇ IERFVSNERGGYDINGLI VEREGQK KMVIGNKGAKIKTIGIEARKD QEMFEAPVH E VKVKSGADDERA RSLGYVDD fSEQ ID NO:1] was used to perform a PSI-Blast search at: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph- psi_blast on the non-redundant NCBI database.
  • the output of the program is shown below herein in Table 1, with the significance of the matches given in brackets. The threashhold for significance was set at 0.01.
  • sequences above the threashhold correspond to the already known ERA homologues from various organisms. Although no other sequences were identified above the threashhold, inspection of the sequences in the non-significant part of the search shows a surprising enrichment in proteins known to bind RNA (underlined) or single-stranded DNA (in italics).
  • the alignment of ERA with these sequences reveals a region of similarity centered around the motif: [alphatic]-[aliphatic]-I-G-x-x-G where x is frequently lysine.
  • a comparison of the identified RNA and ssDNA-binding proteins showed that these were also only similar to each other in this domain.
  • ERA_COXBU [SEQ ID NO: 6] mkptyCGYAAIIGRPNVGKST LNOLLEOKISITSRKPOTTRYOI G-VKTFK
  • YPHC_BACSU 1 [SEQ ID NO: 13] mgKPWAIVGRPNVGKSTIFNRIAGERISIVEDTPGVTRDRIYS-SAE NYDFN IDTGGIDIG- Y329_MYCGE 1[SEQ ID NO: 14]
  • YPHC_BACSU 2 [SEQ ID NO: 17] ...VIOFCLIGRPNVGKSSLVNAM GEERVIVSNVAGTTRDAVDT-SFTYN
  • GKLVF AAMKVDgekgesdasalwslg slrl974 1[SEQ ID NO.-39]
  • DSEFLPEIREQANLA AEAKAAIFWDGQQGPTASD ⁇ EIAQW RQQ
  • GELTD ⁇ PEEVLMAELIEEAALQG ⁇ /RDELPHSLAWIDEVspregrddllDVHAALYVERDSQKGIvT [SEQ ID NO: 79] orf_syn DPGPYYYPP- DLVTDQPERFIMAELIREQILLLTRQEVPHSVAIAIEKVeetper TNVFAAITVERGSQKGIII
  • h helix
  • s strand (assigned by similarity to proteins of known structure)
  • red residues involved in phosphate binding and hydrolysis
  • blue residues involved in guanine ring recognition magenta: putative GTPase activating arginine motif (by analogy to GAPs of known structure)
  • green KH domain signature motif
  • cyan lysine(s) presumed to bind the polynucleotide backbone
  • the output of the program is shown below, with the significance of the matches given in brackets.
  • the threshhold for significance was set at 0.01.
  • the sequences above the threshhold correspond to the already known ERA homologues from various organisms. Although no other sequences were identified above the threshhold, inspection of the sequences in the non-significant part of the search shows a surprising enrichment in proteins known to bind RNA (underlined) or single-stranded DNA (in italics).
  • the alignment of ERA with these sequences reveals a region of similarity centered around the motif: [alphatic]-[aliphatc]-I-G-x- x-G[SEQ ID NO: 100] where x is frequently lysine.
  • RNA and ssDNA-binding proteins showed that these were also only similar to each other in this domain.
  • Medline searches showed that the domain had been described in 1993 as the KH domain (Siomi et al. Nucl. Acids Res. 21:1193) and its structure was determined by NMR in 1996 (Musco et al. Cell 85:237).
  • An evaluation of the significance of the similarity between ERA sequences and a representative set of KH domains in MACAW yielded probabilities of chance occurrence less than le "20 .
  • the analysis used BLASTP 2.0.3 to search Non-redundant GenBank CDS
  • PROTEIN ERA >gi j 1228147 (U48415 ) Era [ Salmonella typhimurium] Length 301
  • HLELWVKVKSG ADDE LRSLGYVDDL Sbjct: 274 HLELWVKVKSGWADDESRLRSLGYVDDL 301 sp
  • PROTEIN ERA >gi
  • Escherichia coli >gi
  • HLELWVKVKSG ADDERALRSLGYVDDL[SEQ ID NO: 105] Sbjct: 274 HLELWVKVKSGWADDERALRSLGYVDDL 301 [SEQ ID NO: 106] gi I 2627217 >gi
  • PROTEIN ERA >gi
  • Haemophilus influenzae (strain Rd KW20) >gi
  • PROTEIN S3 (BS3) (BS2) >gi
  • GIVIGRN 300 [SEQ ID NO: 217]
  • RS3_HALMA >sp
  • KMVIGNKGAKIKTIG 105 [SEQ ID NO:257]
  • PROTEIN S3 >gi
  • Methanococcus jannaschii >gi
  • PROTEIN S3 >gi 11685377 (U78193) ribosomal protein S3 [Borrelia burgdorferi] Length 93
  • 2073527 (U80032) weak similarity to human NuMA protein (PIR:A42184) and S. cerevisiae glucoamylase S1/S2 precursor
  • PROTEIN S3 (HS4) >gi
  • Halobacterium halobium Length 302
  • Gap Penalties Existence: 11, Extension: 1 Number of Hits to DB: 20918269 Number of Sequences: 275942 Number of extensions: 782086 Number of successful extensions: 2522
  • the GTPase domain is separated from the KH domain by an intervening sequence of about 50 residues. Database searches with this sequence do not yield significant matches to other proteins.
  • the only entirely conserved residue in this sequence is an arginine, which is always preceeded by a large hydrophobic residue.
  • GTPases notably Ras, Rho, and heterotrimerc G-protein a subunit
  • an arginine either present in the same polypeptide (Ga) or in a separate protein (RasGAP and RhoGAP) is required for efficient hydrolysis of the bound nucleotide (hence the name GAP for GTPase activating protein).
  • GAP GTPase activating protein
  • RasGAP and RhoGAP the arginine is always preceeded by a large hydrophobic residue. The role of this arginine in efficient nucleotide hydrolysis is emerging as a common feature of all P-loop NTPases.
  • the domain separating the GTPase and KH domains of ERA is proposed to be the ERA-specific GAP. Domains containing an invariant arginine are observed in two closely related GTPases that are also present in all fully sequenced eubacterial genomes: a 50kD open reading frame and ThdF, believed to be involved in thiophene and furan oxidation. In the 50k protein the region bears distant similarity to eGAP:
  • Y329_MYCGE 2 LYNQPPL-FKGKRLQITYAVQTKSQIPHFVLFCNDpKYLHFSYARFLENK- IRENFGF NSVPISLYFKSKNarirtkpev[SEQ ID NO: 356] orf_mycle 2 AATPPPV-RGGKQPRILFATQATARPPTFVLFTt—GFLEACYRRFLERR-
  • RhoGAP RhoGAP
  • LQQEGLYRVSSTREKCKRLRRKLlrgkstphlgnkd [ S ⁇ Q ID NO : 367 ] CHIN_HUMAN . . . . ttlvkahttkRPMWDMCIREIESrg — LNSEG YRVSGFSDLIEDVKMAFdrdgekadisvnm f SEQ ID NO .- 368 ]
  • gtp-binding protein Mycobacterium tuberculosis [SEQ ID NO:382] MTQDGTWVDESDWQLDDS ⁇ IAESGAAPWAWGRPNVGKSTLVNRILGRREAWQDIPGVTRDRVCYDAL WTGRRFWQDTGGWEPNAKGLQRLVAEQASVAMRTADAVILWDAGVGATAADEAAARILLRSGKPVFLA ANKVDSEKGESDAAALWSLGLGEPHAISAMHGRGVADLLDGVLAALPEVGESASASGGPRRVALVGKPNV GKSSLLNKLAGDQRSWHEAAGTTVDPVDSLIELGGDVWRFVDTAGLRRKVGQASGHEFYASVRTHAAID SAEVAIVLIDASQPLTEQDLRVISMVIEAGRALVLAYNKWDLVDEDRRELLQREIDRELVQVRWAQRVNI SAKTGRAVHKLVPAMEDALASW
  • gtp-binding protein [Treponema pallidum] [SEQ ID NO: 383] MKGQDVILCDGGRHFSYKVLPRWIVGRPNVGKSTLFNRLLGRRRSITSNTSGVTRDSIEETVILRGFPL RLVDTSGFTVFSEKKASRQHIDTLVLEQTYKSIQCADKILLVLDGTCESA ⁇ DEEVIQYLRPYWGKLIAAV NKTEGGEEVHYNYARYGFSTLICVSAEHGRNIDALERAIIQNLFSVDERRELPKDDWRLAIVGKPNTGK STLMNYLMRRTVSLVCDRAGTTRDWTGHVEFKQYKFIIADTAGIRKRQKVYESIEYYSVIRAISILNAV DIVLYIVDARDGFSEQDKKIVSQISKRNLGVIFLLNKWDLLEGSTSLIAKKKRDVRTAFGKMNFVPWPV SAKTGHGISDALHCVCKIFAQLNTKVETSAL
  • gtp-binding protein [Borrelia burgdorferi] [SEQ ID NO: 385] MLSYKKVLIVGRPNVGKSALFNRILDTKRSITESTYGVTRDLVE ⁇ VCKVDSFKFKLIDTGGFTILKDEIS KIWQKVLSSLEKVDLILLVLDIN ⁇ ILLEDYQIIERLRKYSSKWLVLNKVDTKDKECLAHEFHNLGFKR YFLVSAAHCRGITKLRDFLKVEVGEVGIESGADIKVGIIGKPNSGKSTLINYLSGNEIAIVSDQPGTTRD FIKTKFTRNGKVFEWDTAGIRRRARVNEIVEYYSVNRALKVIDMVDIVFLLIDVQEKLTSQDKKIAHYV TKKGKGIVIVFSKWDLVDESKGYFEALKSHVKFFFPILNFAPIFRISVHKRIGLDSLFK ⁇ SFKLKDQLEL KTSTP
  • a deposit containing 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 1RY, Scotland on 11 April 1996 and assigned deposit number 40794. The deposit was described as Streptococcus pneumoniae 0100993 on deposit. On 17 April 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly deposited with the NCIMB and assigned deposit number 40800.
  • the Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain.”
  • the deposited strain contains the full length Era gene.
  • the sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein.
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure.
  • the strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent.
  • the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. ⁇ 112.
  • a license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain, which polypeptide is contained in the deposited strain.
  • ERA binding domain polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby.
  • ERA binding domain polypeptide and polynucleotide sequences isolated from the deposited strain are also provided.
  • Era polypeptide of the invention is structurally related to other proteins of the Era family.
  • polypeptides of various bacteria referred to herein as "Era” and "Era polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • Era polypeptides of various bacteria referred to herein as "Era” and "Era polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • variants of Era polypeptide encoded by naturally occurring alleles of the Era gene are particularly preferred embodiments of the invention.
  • the present invention further provides for an isolated polypeptide which:
  • (a) comprises or consists of an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to a polypeptide comprising an ERA binding domain sequence selected from Table 1 over the entire length of such sequence in Table l;
  • polypeptides of the invention include a polypeptide comprising an ERA binding domain sequence selected from Table 1 (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of Era, and also those which have at least 70% identity to a polypeptide comprising an ERA binding domain sequence selected from Table 1 or the relevant portion, preferably at least 80% identity to a polypeptide comprising an ERA binding domain sequence selected from Table 1 and more preferably at least 90% identity to a polypeptide comprising an ERA binding domain sequence selected from Table 1 and still more preferably at least 95% identity to a polypeptide comprising an ERA binding domain sequence selected from Table 1 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • the invention also includes a polypeptide consisting of or comprising a polypeptide of the formula:
  • R 2 is oriented so that its amino terminal amino acid residue is at the left, covalently bound to R j and its carboxy terminal amino acid residue is at the right, covalently bound to R3.
  • Any stretch of amino acid residues denoted by either R or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polypeptide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • a fragment is a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention.
  • fragments may be "free-standing,” or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence comprising an ERA binding domain sequence selected from Table 1, or of variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl- terminal amino acid sequence.
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Streptococcus pneumoniae, are also preferred.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • biologically active fragments are those fragments that mediate activities of Era, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Streptococcus pneumoniae or the ability to initiate, or maintain cause Disease in an individual, particularly a human.
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • X or "Xaa” may also be used in describing certain polypeptides of the invention.
  • X and “Xaa” mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the polypeptide sequence.
  • polynucleotides that encode ERA binding domain polypeptides particularly polynucleotides that encode the polypeptide herein designated Era.
  • the polynucleotide comprises a region encoding ERA binding domain polypeptides comprising a sequence comprising an ERA binding domain sequence selected from Table 1 which includes a full length gene, or a variant thereof. The Applicants believe that this full length gene is essential to the growth and/or survival of an organism which possesses it, such as Streptococcus pneumoniae.
  • isolated nucleic acid molecules encoding and/or expressing ERA binding domain polypeptides and polynucleotides, particularly Streptococcus pneumoniae ERA binding domain polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs.
  • Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.
  • Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a ERA binding domain polypeptide having a deduced amino acid sequence of Table 1 and polynucleotides closely related thereto and variants thereof.
  • a ERA binding domain polypeptide from Streptococcus pneumoniae comprising or consisting of an amino acid sequence of Table 1, or a variant thereof.
  • a polynucleotide sequence encoding a polypeptide sequence set out in Table 1 a polynucleotide of the invention encoding ERA binding domain polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Streptococcus pneumoniae 0100993 cells as starting material, followed by obtaining a full length clone.
  • a polynucleotide sequence of the invention such as a polynucleotide sequence encoding a polypeptide given in Table 1
  • a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide, 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.
  • sequencing is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence.
  • sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).
  • each DNA sequence encoding a polypeptide sequence set out in Table 1 contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art.
  • the present invention provides for an isolated polynucleotide comprising or consisting of: (a) a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to a sequence encoding a sequence comprising an ERA binding domain sequence selected from Table 1 over the entire length of such sequence selected from Table 1 ; (b) a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or 100% exact, to the amino acid sequence of a sequence comprising an ERA binding domain sequence selected from Table 1, over the entire length of such sequence selected from Table 1.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled or detectable probe consisting of or comprising a region encoding a sequence from Table 1 or a fragment thereof; and isolating a full-length gene and or genomic clones containing said polynucleotide sequence.
  • the invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) comprising an ERA binding domain sequence comprising an ERA binding domain sequence selected from Table 1. Also provided by the invention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence.
  • the polynucleotide of the invention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho- independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals.
  • the polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of the 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 which 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.
  • the invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:
  • R 1 X-(R 1 ) m -(R 2 )-(R 3 ) n -Y
  • X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond
  • Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond
  • each occurrence of R j and R3 is independently any nucleic acid residue or modified nucleic acid residue
  • m is an integer between 1 and 3000 or zero
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1 or a modified nucleic acid sequence thereof.
  • R is oriented so that its 5' end nucleic acid residue is at the left, bound to R ⁇ and its 3' end nucleic acid residue is at the right, bound to R3.
  • Any stretch of nucleic acid residues denoted by either R ⁇ and/or R 2 , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary.
  • m and/or n is an integer between 1 and 1000.
  • Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polynucleotide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae ERA binding domain having an amino acid sequence set out in Table 1.
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.
  • the invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence comprising an ERA binding domain sequence selected from Table 1. Fragments of a polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
  • ERA binding domain variants that have the amino acid sequence of ERA binding domain polypeptide comprising an ERA binding domain sequence selected from Table 1 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of ERA binding domain polypeptide.
  • polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding ERA binding domain polypeptide having an amino acid sequence set out in Table 1, and polynucleotides that are complementary to such polynucleotides.
  • polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding ERA binding domain polypeptide and polynucleotides complementary thereto.
  • polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred.
  • those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
  • 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.
  • stringent conditions and “stringent hybridization conditions” mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences.
  • 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 0. lx SSC at about 65°C.
  • Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
  • the invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library containing the complete gene for a polynucleotide sequence encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1 under stringent hybridization conditions with a probe having the sequence derived from a polynucleotide encoding a polypeptide sequence selected from Table 1 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 of the invention may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding ERA binding domain and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the ERA binding domain 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 preferred probes will have at least 20 nucleotide residues or base pairs and will have less than 30 nucleotide residues or base pairs.
  • a coding region of a ERA binding domain gene may be isolated by screening using a DNA sequence encoding a region of a polypeptide set forth in Table 1 as an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • RACE Rapid Amplification of cDNA ends
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
  • polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relating to polynucleotide assays.
  • polynucleotides of the invention that are oligonucleotides derived from a sequence of Table 1 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
  • the invention also provides polynucleotides that encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be processed away from the mature protein by cellular enzymes.
  • a precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
  • proproteins In addition to the standard A, G, C, T/U representations for nucleotides, the term "N" may also be used in describing certain polynucleotides of the invention.
  • N means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.
  • a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
  • a leader sequence which may be referred to as a preprotein
  • a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
  • the invention also relates to vectors that comprise a polynucleotide ' or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, etal, BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • bacterial cells such as cells of streptococci, staphylococci, enterococci E. coli, streptomyces, cyanobacteria, Bacillus subtilis, and 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
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells
  • plant cells such as cells of a gymnosperm or angiosperm.
  • vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picomavirases and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook etal, MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
  • Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification. Diagnostic, Prognostic, Serotyping and Mutation Assays
  • This invention is also related to the use of ERA binding domain polynucleotides and polypeptides of the invention for use as diagnostic reagents.
  • Detection of ERA binding domain 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 ERA binding domain 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 of the 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 of the organism.
  • Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species.
  • Point mutations can be identified by hybridizing amplified DNA to labeled ERA binding domain 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 oligonucleotides probes comprising ERA binding domain nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al, Science, 274: 610 (1996)).
  • the present invention relates to a diagnostic kit which comprises:
  • a polynucleotide of the present invention preferably the nucleotide sequence encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1, or a fragment thereof ;
  • polypeptide of the present invention preferably the polypeptide set forth in Table 1 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to a polypeptide comprising an ERA binding domain sequence selected from Table 1.
  • kits may comprise a substantial component.
  • a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others.
  • This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents.
  • Detection of a mutated form of a polynucleotide of the invention preferably, a sequence comprising an ERA binding domain sequence selected from Table 1, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide.
  • Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.
  • the nucleotide sequences of the present invention are also valuable for organism chromosome identification.
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an organism's chromosome, particularly to a Streptococcus pneumoniae chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention may be an important step in correlating those sequences with pathogenic potential and/or an ecological niche of an organism and/or drug resistance of an organism, as well as the essentiality of the gene to the organism.
  • the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data may be found on-line in a sequence database.
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (coinheritance of physically adjacent genes) or mating studies, such as by conjugation.
  • the differences in a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined. If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype.
  • Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example.
  • RT-PCR can be used to detect mutations in the RNA. It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan.
  • RNA, cDNA or genomic DNA may also be used for the same purpose, PCR.
  • PCR primers complementary to a polynucleotide encoding ERA binding domain polypeptide can be used to identify and analyze mutations.
  • primers may be used for, among other things, amplifying ERA binding domain DNA and/or RNA isolated from a sample derived from an individual, such as a bodily material.
  • the primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various techniques for elucidation of the polynucleotide sequence. In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent.
  • the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Streptococcus pneumoniae, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1. Increased or decreased expression of a ERA binding domain polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
  • a diagnostic assay in accordance with the invention for detecting over- expression of ERA binding domain 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 determine levels of a ERA binding domain polypeptide, in a sample derived from a host, such as a bodily material, are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.
  • Differential Expression The polynucleotides and polynucleotides of the invention may be used as reagents for differential screening methods.
  • differential screening and differential display methods There are many differential screening and differential display methods known in the art in which the polynucleotides and polypeptides of the invention may be used.
  • the differential display technique is described by Chuang et al., J. Bacteriol 775:2026-2036 (1993). This method identifies those genes which are expressed in an organism by identifying mRNA present using randomly-primed RT-PCR. By comparing pre-infection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF "unknowns.”
  • IVET In Vivo Expression Technology (IVET) is described by Camilli et al, Proc. Nat'l. Acad. Sci. USA. 97:2634-2638 (1994). IVET identifies genes up-regulated during infection when compared to laboratory cultivation, implying an important role in infection. ORFs identified by this technique are implied to have a significant role in infection establishment and/or maintenance. In this technique random chromosomal fragments of target organism are cloned upstream of a promoter-less recombinase gene in a plasmid vector. This construct is introduced into the target organism which carries an antibiotic resistance gene flanked by resolvase sites.
  • the resistant pool is introduced into a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance.
  • the chromosomal fragment carried by each antibiotic sensitive bacterium should carry a promoter or portion of a gene normally upregulated during infection. Sequencing upstream of the recombinase gene allows identification of the up regulated gene.
  • RT-PCR may also be used to analyze gene expression patterns.
  • messenger RNA is isolated from bacterial infected tissue, e.g., 48 hour murine lung infections, and the amount of each mRNA species assessed by reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs.
  • the determination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed in the infected tissue. Analysis of gene transcription can be carried out at different times of infection to gain a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacterials.
  • the bacterial mRNA preparation need not be free of mammalian RNA. This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species which are very short lived in the bacterium (in the order of 2 minute halflives).
  • the bacterial mRNA is prepared from infected murine lung tissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL) for very short periods of time, subsequent processing according to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA.
  • the process is optimized by finding those conditions which give a maximum amount of Streptococcus pneumoniae 16S ribosomal RNA as detected by probing Northerns with a suitably labeled sequence specific oligonucleotide probe.
  • a 5' dye labeled primer is used in each PCR primer pair in a PCR reaction which is terminated optimally between 8 and 25 cycles.
  • the PCR products are separated on 6% polyacrylamide gels with detection and quantification using GeneScanner (manufactured by ABI).
  • the polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high density arrays or grids. These high density arrays are particularly useful for diagnostic and prognostic purposes.
  • a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention may be used for probing, such as using hybridization or nucleic acid amplification, using a probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual.
  • Such a presence may indicate the presence of a pathogen, particularly Streptococcus pneumoniae, and may be useful in diagnosing and/or prognosing disease or a course of disease.
  • a grid comprising a number of variants of the polynucleotide sequences of the invention are preferred. Also preferred is a comprising a number of variants of a polynucleotide sequence encoding polypeptide sequences of Table 1.
  • polypeptides and polynucleotides of the invention or variants thereof, or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively.
  • antibodies against ERA binding domain polypeptides or polynucleotides there are provided antibodies against ERA binding domain polypeptides or polynucleotides .
  • Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and/or polynucleotides of the invention, or epitope- bearing fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols.
  • any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, pg.
  • phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-Era or from naive libraries (McCafferty, et al, (1990), Nature 348, 552-554; Marks, et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et al, (1991) Nature 352: 628).
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for example, affinity chromatography.
  • Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form a particular aspect of this invention.
  • a polypeptide or polynucleotide of the invention such as an antigenically or immunologically equivalent derivative or a fusion protein of the polypeptide is used as an antigen to immunize a mouse or other animal such as a rat or chicken.
  • the fusion protein may provide stability to the polypeptide.
  • the antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin, keyhole limpet haemocyanin or tetanus toxoid.
  • an immunogenic carrier protein for example bovine serum albumin, keyhole limpet haemocyanin or tetanus toxoid.
  • a multiple antigenic polypeptide comprising multiple copies of the polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
  • the antibody or variant thereof is modified to make it less immunogenic in the individual.
  • the antibody may most preferably be "humanized," where the complimentarity determining region or regions of the hybridoma- derived antibody has been transplanted into a human monoclonal antibody, for example as described in lones et al. (1986), Nature 321, 522-525 or Tempest et al, (1991) Biotechnology 9, 266-273.
  • a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
  • particularly preferred embodiments of the invention are naturally occurring allelic variants of ERA binding domain polynucleotides and polypeptides encoded thereby.
  • a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al, Hum Mol Genet (1992) 1: 363, Manthorpe et al, Hum. Gene Ther. (1983) 4: 419), delivery of DNA complexed with specific protein carriers (Wu et al, J Biol Chem.
  • Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al, Current Protocols in Immunology 1(2): Chapter 5 (1991).
  • Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned. It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides and polynucleotides. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned.
  • Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of ERA binding domain polypeptides and polynucleotides; or may be structural or functional mimetics thereof (see Coligan et al, Current Protocols in Immunology l(2):Chapter 5 (1991)).
  • the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve competition with a labeled competitor.
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be.
  • the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring ERA binding domain polypeptide and/or polynucleotide activity in the mixture, and comparing the ERA binding domain polypeptide and/or polynucleotide activity of the mixture to a standard.
  • Fusion proteins such as those made from Fc portion and ERA binding domain polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. lohanson et al, J Biol Chem, 270(16):9459-9471 (1995)).
  • Preferred screening methods include, for example, screening for an interaction between ERA binding domain and any agent can be measured with physical techniques such as fluorescence polarization (FP), flourescence energy transfer (FET), surface plasmon resonance (SPR), scintillation proximity assay (SPA), and radioimmune assay (RIA).
  • FP fluorescence polarization
  • FET flourescence energy transfer
  • SPR surface plasmon resonance
  • SPA scintillation proximity assay
  • RIA radioimmune assay
  • a scintillation proximity assay can be used to characterize the interaction between ERA binding domain and an agent. Either ERA binding domain or the agent is coupled to a scintillation-filled bead. Addition of radio-labelled ERA binding domain or agent to the coupled beads results in binding where the radioactive source molecule is in close proximity to the scintillation fluid. Thus, signal is emitted upon binding.
  • polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells.
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of ERA binding domain polypeptides or polynucleotides, particularly those compounds that are bacteriostatic and or bacteriocidal.
  • the method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising ERA binding domain polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a ERA binding domain agonist or antagonist.
  • the ability of the candidate molecule to agonize or antagonize the ERA binding domain polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate.
  • Molecules that bind gratuitously, i.e., without inducing the effects of ERA binding domain polypeptide are most likely to be good antagonists.
  • Molecules that bind well and, as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists. Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system.
  • Reporter systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in ERA binding domain polynucleotide or polypeptide activity, and binding assays known in the art.
  • Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, 1 ⁇ 1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supematants, tissue extracts, bodily materials). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.
  • methods for identifying compounds which 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 pennit 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 of the polypeptide and/or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and/or polynucleotide.
  • an assay for ERA binding domain agonists is a competitive assay that combines ERA binding domain and a potential agonist with Era-binding molecules, recombinant ERA binding domain binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay.
  • ERA binding domain can be labeled, such as by radioactivity or a colorimetric compound, such that the number of ERA binding domain molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
  • Potential antagonists include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression.
  • Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing Era-induced activities, thereby preventing the action or expression of ERA binding domain polypeptides and/or polynucleotides by excluding ERA binding domain polypeptides and/or polynucleotides from binding.
  • Potential antagonists 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 potential antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules).
  • Preferred potential antagonists include compounds related to and variants of Era.
  • polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • polypeptides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide. It is preferred that these cassettes comprise 5' and 3' restriction sites to allow for a convenient means to ligate the cassettes together when desired. It is further preferred that these cassettes comprise gene expression signals known in the art or described elsewhere herein.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for a polypeptide and/or polynucleotide of the present invention; or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises:
  • polypeptide and/or polynucleotide of the present invention which polypeptide preferably comprises an ERA binding domain sequence selected from Table 1, and which polynucleotide is preferably a polynucleotide encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1.
  • a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide and/or polynucleotide, by: (a) determining in the first instance the three-dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof; (b) deducing the three-dimensional structure for the likely reactive site(s), binding site(s) or motif(s) of an agonist, antagonist or inhibitor;
  • One approach comprises administering to an individual in need thereof an inhibitor compound (antagonist) as herein described, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous polypeptide and/or polynucleotide may be administered. Typical examples of such competitors include fragments of the ERA binding domain polypeptide and/or polypeptide.
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGi, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
  • expression of the gene encoding endogenous ERA binding domain polypeptide can be inhibited using expression blocking techniques.
  • This blocking may be targeted against any step in gene expression, but is preferably targeted against 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, 7 Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • oligonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al., Nucleic Acids Res (1979) 3:173; Cooney et al, Science (1988) 241:456; Dervan et al, Science (1991) 251:1360). These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
  • Each of the polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds.
  • the encoded protein, upon expression,- can be used as a target for the screening of antibacterial drugs.
  • 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 ERA binding domain protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al., Infect. Immun.
  • This invention provides a method of screening drugs to identify those which are antibacterial by measuring the ability of the drug to interfere with the biosynthesis of
  • E.coli UDP- ⁇ acetylenolpyruvylglucosamine reductase catalyses the reduction of UDP-N-acetylglucosamine enolpyruvate with the concommitanst oxidation of ⁇ ADPH.
  • UDP-N-acetylenolpyruvylglucosamine is incubated with ⁇ ADPH in the presence of the UDP-N-acetylenolpyruvylglucosamine reductase protein to generate ⁇ ADP which can be measured spectrophotometrically at 340nm (Benson, T.E., Marquardt, J.L., Marquardt, A.C., Etzkorn, FA. & Walsh, C.T., [1993], Biochemistry, 32,
  • ERA binding domain agonists and antagonists preferably bacteriostatic or bacteriocidal agonists and antagonists.
  • the antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.
  • H. pylori Helicobaeter 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 Helicobaeter Pylori (International Agency for Research on Cancer, Lyon, France, http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agency for Research on Cancer recently recognized a cause-and- effect relationship between H. pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen.
  • Preferred antimicrobial compounds of the invention should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H. pylori-mdaced cancers, such as gastrointestinal carcinoma. Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis.
  • Vaccines agonists and antagonists of ERA binding domain polypeptides and/or polynucleotides found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics.
  • Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of ERA binding domain polynucleotide and/or polypeptide, or a fragment or a variant thereof, for expressing ERA binding domain polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not.
  • an immunological response such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not.
  • a further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, induces an immunological response in such individual to a ERA binding domain polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant ERA binding domain polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said ERA binding domain polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention.
  • the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells.
  • a ERA binding domain polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or immunogenic properties, and preferably protective properties.
  • fused recombinant protein preferably further comprises an antigenic co-protein, such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co-protein which solubilizes the protein and facilitates production and purification thereof.
  • the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protein.
  • the co- protein may be attached to either the amino- or carboxy-terminus of the first protein.
  • compositions particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996). .
  • polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Streptococcus pneumoniae.
  • Such experiments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Streptococcus pneumoniae infection, in mammals, particularly humans.
  • a polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue.
  • tissue damage include wounds in skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina.
  • the invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier.
  • each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • compositions, kits and administration have been described with reference to certain ERA binding domain polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant polypeptides or polynucleotides.
  • compositions comprising a ERA binding domain polynucleotide and/or a ERA binding domain polypeptide for administration to a cell or to a multicellular organism.
  • the invention also relates to compositions comprising a polynucleotide and/or a polypeptides discussed herein or their agonists or antagonists.
  • the polypeptides and polynucleotides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual.
  • compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient.
  • a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides, polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
  • the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
  • the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams.
  • Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
  • Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
  • the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides, polynucleotides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • composition will be adapted to the route of administration, for instance by a systemic or an oral route.
  • Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
  • penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like.
  • the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg.
  • the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual.
  • the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time.
  • Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.
  • CAPD continuous ambulatory peritoneal dialysis
  • composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device.
  • composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especially Streptococcus pneumoniae wound infections.
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins 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 invention 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 vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response.
  • a suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
  • Antibody(ies) as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
  • Antigenically equivalent derivative(s) as used herein encompasses a polypeptide, polynucleotide, or the equivalent of either which will be specifically recognized by certain antibodies which, when raised to the protein, polypeptide or polynucleotide according to the invention, interferes with the immediate physical interaction between pathogen and mammalian host.
  • Bispecific antibody(ies) means an antibody comprising at least two antigen binding domains, each domain directed against a different epitope.
  • Bodily material(s) means any material derived from an individual or from an organism infecting, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy materials.
  • Disease(s) means any disease caused by or related to infection by a bacteria, 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.
  • Fusion protein(s) refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232262].
  • “Host cell(s)” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
  • Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990).
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • Parameters for polypeptide sequence comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to a reference sequence of Table 1, wherein said polynucleotide sequence may be identical to a reference sequence of Table 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 of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in a given sequence by the integer defining the percent identity divided by 100 and
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in a given sequence
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding a polypeptide of Table 1 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • a polynucleotide sequence of the present invention may be identical to a reference sequence of Table 1, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of amino acids in a polypeptide of Table 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in said polypeptide, or:
  • n n is the number of amino acid alterations
  • x n is the total number of amino acids in a polypeptide in Table 1
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of a polypeptide of Table 1, wherein said polypeptide sequence may be identical to a reference sequence of Table 1 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in a polypeptide of Table 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in said poly
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in a polypeptide of Table 1
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • a polypeptide sequence of the present invention may be identical to the reference sequence of a polypeptide of Table 1, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in a polypeptide of Table 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in said sequence, or:
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in a polypeptide of Table 1
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • immunologically equivalent derivative(s) as used herein encompasses a polypeptide, polynucleotide, or the equivalent of either which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
  • Immunospecific means that characteristic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the invention or the polynucleotides of the invention than its affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the prior art.
  • “Individual(s)” means a multicellular eukaryote, including, 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, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living.
  • Organism(s) means a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas
  • Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotide(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • the term "polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • the term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)” also embraces short polynucleotides often referred to as oligonucleotide(s).
  • Polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
  • Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxy lation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation,
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • “Subtraction set” is one or more, but preferably less than 100, polynucleotides comprising at least one polynucleotide of the invention
  • Variant(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 in 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 mclude 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.
  • variants 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 preferred 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 occurring 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.
  • 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 into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coli infected with the packaged library.
  • the library is amplified by standard procedures.
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures.
  • EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library.
  • the library is amplified by standard procedures.
  • An allelic replacement cassette is generated using PCR technology.
  • the cassette typically consists of a pair of 500bp chromosomal DNA fragments flanking an erythromycin resistance gene.
  • the chromosomal DNA sequences are usually the 500bp preceding and following the gene of interest.
  • Attempts are made to introduce the allelic replacement cassette into S. pneumoniae R6 or S. pneumoniae 100993 by transformation. Competent cells are prepared according to published protocols. DNA is introduced into the cells by incubation of 500ng of allelic replacement cassette with 10" cells at 30°C for 30 minutes. The cells are transferred to 37 ⁇ C for 90 minutes to allow expression of the erythromycin resistance gene.

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Abstract

The invention provides ERA binding domain polypeptides and polynucleotides encoding ERA binding domain polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ERA binding domain polypeptides to screen for antibacterial compound.

Description

METHODS AND REAGENTS FOR PERFORMING ANTIMICROBIAL COMPOUND SCREENING
FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, the invention relates to polynucleotides and polypeptides of the Era family, as well as their variants, hereinafter referred to as "Era binding domain," "ERA binding domain polynucleotide(s)," and "ERA binding domain polypeptide(s)." Methods using such polynucleotides and polypeptides in antimicrobial compound screening are also provided. BACKGROUND OF THE INVENTION
The era gene in Escherichia coli codes for an essential protein. The protein, Era is a GTPase and is able to autophosphorylate itself at a serine and/or threonine residue (March PE., 1992. Membrane-associated GTPases in bacteria. Molecular Microbiology 6: 1253-1257). It is found in both soluble and membrane compartments of E. coli cells and has been found to be localized to the membrane at points consistent with a role in septation or nucleoid segregation (Gollop, N. & March P.E., 1991. Localization of the membrane binding sites of Era in Escherichia coli. Research in Microbiology 142: 301-307). Homologous proteins have been found in a variety of bacteria and show functional complementation (Pillutla, R.C., Sharer, J.D., Gulati, P.S., Wu, E., Yamashita, Y., Lerner, C.G., Inouye, M., & March, P.E., 1995. Cross- species complementation of the indispensable Escherichia coli era gene highlights amino acid regions essential for activity. Journal of Bacteriology 177: 2194-2196).
Clearly, there exists a need for polynucleotides and polypeptides, such as the ERA binding domain embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease. Certain of the polypeptides of the invention possess significant amino acid sequence homology to a known Era from B.suhtilis protein.
SUMMARY OF THE INVENTION
The present invention relates to Era binding domains, in particular ERA binding domain polypeptides and ERA binding domain polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of microbial diseases, amongst others. In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting ERA binding domain expression or activity.
The invention also provides methods for antagonizing an interaction between ERA and the ERA binding site. Further provided by the invention are methods for antagonizing an interaction between
ERA and the ERA binding site, where said binding site is a site on the genomic DNA or an
RNA or both simultaneously.
Still further provided by the invention are methods for antagonizing an interaction between the RNA and DNA, either or both of which comprise a ERA binding site. Particularly preferred embodiments of the invention are methods of the invention used to identify antimicrobial compounds.
Preferred embodiments are also provided whereby polynucleotide and/or FtsZ binding is demonstrated using polarization.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The applicants have made the important discovery that ERA is involved in late localization or early septation, and have devised methods to screen for compounds that agonize or antagonize the polymerization reaction in late post chromosomal septation. Based on their studies, the applicants posit that ERA guides the FtsZ ring formation. One method useful to show such interaction is immunoprecipitation of DNA with ERA and ERA dimers, trimer or tetramer. Moreover, a column with DNA or RNA to bind ERA or ERA dimer, trimer or tetramer followed by PCR may be used to show the same. The recognition that ERA comprises a GAP-like domain comprising arginine within a binding pocket where the GTPase domain is formed, lead to the present invention, whereby this interaction, among others, can be used for screening antimicrobial compounds as described herein. Screening methods to identify compounds that modulate, agonize or antagonize GTP::Arg-GAP interactions, are provided herein. Preferred embodiments of these screens include, for example, quenching, tumbling, RIA and scintillation proximity assays.
Preferred embodiments of the invention also include, for example, a method for screening compounds that alter an activity of ERA comprising the step of providing ERA and nucleotide and assaying nucleotide exchange activity, such as by using cycles of GTP.
The invention further provides proteins related to ERA, discovered by the applicants that can be used in the methods of the invention. These proteins include, but are not limited to, GTP-GTP-GAP (ORF which uses GTPase activity, for example, as a screen) and GAP(-euk)- GTP-conserved sequence (also referred to as THDF). Both of these proteins comprise a GTPase domain.
The polypeptides and polynucleotides of the invention are believed to be essential for the survival of the related bacteria. Thus, inhibitors of ERA and other polynucleotides and polypeptides of the invention are useful to treat bacterial infections.
The invention relates to ERA binding domain polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of Streptococcus pneumoniae, which is related by amino acid sequence homology to ERA binding domain from B.subtilis polypeptide. The invention relates especially to those having the nucleotide and amino acid sequences comprising ERA binding domains set out in Table 1. Table 1 sets forth, among other things, a multiple alignment of ERA binding domain sequences with their closest relatives (the 50k proteins, containing two GTPase domains, and ThdF) and with human Ras - the closest relative with known structure. Herein described is also a method for identifying this domain in ERA, and methods using polypeptides comprising such ERA binding domains for identifying compounds that agonize or antagonize the interaction between an ERA binding domain and its cognate binding molecule, such as a nucleotide or polynucleotide, especially an RNA polynucleotide.
In ERA, the GTPase domain is separated from the KH domain by an intervening sequence of about 50 residues. Database searches with this sequence do not yield significant matches to other proteins. The only entirely conserved residue in this sequence is an arginine, which is always preceeded by a large hydrophobic residue.
In other GTPases, notably Ras, Rho, and heterotrimeric G-protein a subunit, which have been extensively investigated by X-ray crystallography, an arginine, either present in the same polypeptide (Ga) or in a separate protein (RasGAP and RhoGAP) is required for efficient hydrolysis of the bound nucleotide (hence the name GAP for GTPase activating protein). In RasGAP and RhoGAP, the arginine is always preceeded by a large hydrophobic residue. The role of this arginine in efficient nucleotide hydrolysis is emerging as a common feature of all
P-loop NTPases.
In view of this, the domain separating the GTPase and KH domains of ERA is proposed to be the ERA-specific GAP. Domains containing an invariant arginine are observed in two closely related GTPases that are also present in all fully sequenced eubacterial genomes: a 50kD open reading frame and ThdF, believed to be involved in thiophene and furan oxidation. In the 50k protein the region bears distant similarity to eGAP:
TABLE 1 Era Polynucleotide and Polypeptide Sequence Comprising ERA Binding Domains
Identification of the KH domain in ERA
In order to identify potential similarities between the ERA family and other sequences present in public databases, ERA from Escherichia coli was truncated at Prol74 (eliminating the GTPase domain) and the C-terminal part of the sequence, corresponding to:
PEATHHFPEDYITDRSQRFMASEIIREKMRFLGAE PYSVTVΞIERFVSNERGGYDINGLI VEREGQK KMVIGNKGAKIKTIGIEARKD QEMFEAPVH E VKVKSGADDERA RSLGYVDD fSEQ ID NO:1] was used to perform a PSI-Blast search at: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph- psi_blast on the non-redundant NCBI database. The output of the program is shown below herein in Table 1, with the significance of the matches given in brackets. The threashhold for significance was set at 0.01. The sequences above the threashhold correspond to the already known ERA homologues from various organisms. Although no other sequences were identified above the threashhold, inspection of the sequences in the non-significant part of the search shows a surprising enrichment in proteins known to bind RNA (underlined) or single-stranded DNA (in italics). The alignment of ERA with these sequences reveals a region of similarity centered around the motif: [alphatic]-[aliphatic]-I-G-x-x-G where x is frequently lysine. A comparison of the identified RNA and ssDNA-binding proteins showed that these were also only similar to each other in this domain. Medline searches showed that the domain had been described in 1993 as the KH domain (Siomi et al. Nucl. Acids Res. 21:1193) and its structure was determined by NMR in 1996 (Musco et al. Cell 85:237). An evaluation of the significance of the similarity between ERA sequences and a representative set of KH domains in MACAW yielded probabilities of chance occurrence less than le'20. BLASTP 2.0.3 was used in this analysis to search the Non- redundant GenBank CDS (translations+PDB+SwissProt+SPupdate+PIR) and resulted in 275,942 sequences or 82,475,789 total letters. Results of PSI-Blast iteration 3 of Sequences above threshhold are shown in Table 1. Putative mitochondrial targeting seq. »> GTPase domain ssssssss hhhhhhhhhh ssssssssss sssssssss
ERA_ECO I[SEQ ID NO: 2] msidksyCGFIAIVGRP VGKSTLLMKL GOKISITSRKAOTIRHRIVG--IHTEG
AYQAIYVDTPGHMEe-
ERA_HAEIN[SEQ ID NO : 3 ] mteqfdktyCGFIAIVGRPNVGKSTL NKILGOKISITSRKAOTTRHRIVG- KTEG
AYQEIYVDTPGHIEe-- BEX_BACSU[SEQ ID NO: 4] mtnesfkSGFVSIIGRPNVGKSTFLNRVIGOKIAIMSDKPOTTRNKVOG-VLTTG
TSQTIFIDTPGIHKP
ERA_STRMU[SEQ ID NO: 5] msfkSGFVAILGRPNVGKSTF NHVMGOKIAIMSDKAOTTRNKI G-1YTTD KEQIVFIDTPGIHKP
ERA_COXBU [SEQ ID NO: 6] mkptyCGYAAIIGRPNVGKST LNOLLEOKISITSRKPOTTRYOI G-VKTFK
DIQVIYVDTPGHAGt—
ERA_HELPY[SEQ ID NO: 7] mmktkAGFVALIGKPNAGKST NTL NAHLAVSHKANATRKMKC-
IVPFKdkewyESQIIF DTPGLHHQ orf_myctu[SEQ ID NO: 8] mtefhSGFVCLVGRPNTGIST TNALVGAKVAITSTRPQTRHAIRG-IVHSD
DFQII VDTPGLHRP orf_syn[SEQ ID NO: 9] mdipnttatiatipqapagfrSGFVAIVGRPNVGKSTLMNQLVGQKIAITSPVAQTRNRLQG-IITTP--
—SSQII DΓPGIHKP— ERA_MYCGΞ[SEQ ID NO: 10]
MKVLKVGV GPTNAG^TLINFLHNDDS MVSSMNNTTLLSISTEVINQA NKNIVFIDVPGFTEK YQN2_CAEEL[SEQ ID NO: 11] miikrlnf serif staststsyektpicrpatkCLOLAVIGAPNVGKSL TNS IRCPLSAVSSKMDTTTRN
ISA-SICSD STQLVFVpSPGAVSTshv est_ uman[SEQ ID NO: 12] rmrdeqdvllvhhpdmpensrv RVVLLGAPNAGKSTI-SNQ LGRKVFPVSRKVHTTRCQALG-VITEK-- ETQVI LDTPGIISPgkq
YPHC_BACSU 1[SEQ ID NO: 13] mgKPWAIVGRPNVGKSTIFNRIAGERISIVEDTPGVTRDRIYS-SAE NYDFN IDTGGIDIG- Y329_MYCGE 1[SEQ ID NO: 14]
MFWAIIGRTWGKSTLFNRLIQKPI^IVSDTPNTTRDRIFG-IGEW KRKIAFID GGLIAK--
50k_mycle 1[SEQ ID NO: 15] ms qdg tws des dwe 1 dds dlaefGPWAWGRPNVGKST VNRI GRREAWODVPGVTRDRVSY- DAMWT GRRFWQOTGGWEPD slrl974 1[SEQ ID NO: 16] ms PIVAIIGRPNVGRSTFVNRLAGNOOAIVHDOPGIIRDRTYR- PAFWR
DRDFQWDTGG VFNd —
YPHC_BACSU 2 [SEQ ID NO: 17] ...VIOFCLIGRPNVGKSSLVNAM GEERVIVSNVAGTTRDAVDT-SFTYN
QQEFVIVDTAGMRKKgkv
Y329_MYCGE 2 [SEQ ID NO: 18]
...KIRFCVIGKPNVGKSSLINO VKONRVLVSNESGTTRDAIDV-P KVN
GEKFL IDTAGIKRKgki orf_mycle 2 [SEQ ID NO:19]
...LRRVA VGKPNVGi^S LNK AGDQRSWHEAAGTTVDPVDS- IEMG
GRVWRFVDTAG RRKvgq slrl974 2 [SEQ ID NO :20]
...EIKVAIVGRPNVGiSS LNA TGΞQRAIVSPISGT RDAIDM-WERN GQKYRLIDTAGIRRKknv
THDF_ECO I[SEQ ID NO: 21]
...GMKWIAGRPNAGKSS LNA AGREAAIVTDIAGTTRDVLRX-XIHID
GMPLHI IDTAGLREA
THDF_MYCGE[SEQ ID NO: 22] ...PFKIAIIGETNVGKSSLLNA LNODKAIVSNIKGSTRDWEG-DFNLN
GYLIKILDTAGIRKH
THDF_SYNY3 [ SEQ ID NO : 23 ]
...G KVAIVGOPNVGKSS NAWSRTDRAIVTDLPGTTRDWES-O VVE
GIPIQVLDΓAGIRE — SSI_YEAST ...GIKLV GAPNVGKSSLVNS TNDDISIVSDIPGTTRDSIDA-MINVN
GYKVIICDTAGIREKS—
RASH_HUMA [SEQ ID NO:24] mtEYKVWGAGGVGKSALtiqliqnhfv
DEYDPTIEDSYRkQWIDge TCL DILDTAGQEEY
hhhhhhhhhhhhh ssssssss hhhhhhhhhhhhhhhh ssssss hhhhhhhhhhh ERA_ECO I [ SEQ ID NO:25] KRAINRLMNKAASSSIGDVELVIFWEg-
TRWTPDDEMVLNK REG KAPVILAVISI.WENVQEKADLLPHLQFLASQMN
ERA_HAEIN[SEQ ID NO:26] KRAINRLMNRAASSAIGDVDLIIFWDg- HWNADDEMVLNKLRNA KAPλA/ AINKVIKI NKDDL PFITD SSKFN BΞX_BACSU[SEQ ID NO: 27]
KH LGDFMMKVAQNT REVDLILFMINAEEGYGKGDEFIIEKLQTM STPVFLIVNKIDKIHPD-
Q L IDEYRKRYP
ERA_STRMU[SEQ ID NO: 28]
KTA GDFMVESAYSTLREVDTV FMVPADEKRGKGDN IIΞRLKAA KVPVILVINKIDKVHPD- QL ΞQIDDFRNQMD
ERA_COXBU [ SEQ ID No: 29] ERTINRYMNRTARGA RDVDAIVFVIΞ —
PHWESQDAWVLDNLKEI ETPVFLVINKVDKIKNRAELLP IEKVSSLYA
ERA_HELPY[SEQ ID NO: 30]
EKLLNQCM SQALKAMGDAE CVFLASvhddlkgyee f Isle QKPHILALSKΣøTATHK- QVLQKLQEYQQYDSq orf_myctu[SEQ ID NO:31]
RT GKR NDLVRETYAAVDVIG CIPADEAIGPGDR IVEQLRSTgpaNTT WIV KIDKVPKE-
KWAQLVAVSE VTn orf_syn[SEQ ID NO: 32] HHELGRVLVKNAIQAIHSVD WF VDSSAT GRGDRFWD QKT
DGPVWGLNKQDQQPPDqreelnasyetltenh
ERA_MYCGE [ SEQ ID NO:33] HSNYELITKEIRKALSGIDVLL WRsdqnnkieflktqlqqlkryq-
NLTRIF INKFHQKSLSevnkaiileefkpqktieinllk YQN2_CAEEL[SEQ ID
NO : 34] rqtmkktSATSGDRVLQDPERA QRAQHVLWQDstapgayihhrvlhmlhrys —
HVPSI VMNKIDLVMRRsdllplveiltngqlsdnqqistkpaqigr est_human[SEQ ID NO: 35] k
RHHLΞ S EDPWKSMΞSAD Vl/LVDvsdkwtrnqlspqllrcltkysQIPSVLVMMKVIlCLKQ svll eltaaltegwngkklkmrqafhshp
YPHC_BACSU 1[SΞQ ID NO: 36]
DEPF AQIRQQAEIAMDEADVIIFMVNGREGVTAADEEVAKILYRT
KKPλΛ/LAλ^KLDntemraniydfyslg
Y329_MYCGE 1[SEQ ID NO:37] QTPLQQLIALQVQAALSQAKAIIF VSLQEQ NSDDFYVAKV KKNk--
D PVI WNKAEnfnpktaeetlkdyyslg 50k_mycle 1[SEQ ID NO: 38]
AKGLKRLVAEQASVA RTADAVILWDVGVGATDADEAAARILLRS
GKLVF AAMKVDgekgesdasalwslg slrl974 1[SEQ ID NO.-39] DSEFLPEIREQANLA AEAKAAIFWDGQQGPTASDΞEIAQW RQQ
SVPVILATHKCEspdqgaiqaaefwhlg
YPHC_BACSU 2 [SEQ ID NO: 40]
YETTEKYSVLRA KAIDRSEWAWLDGEEGIIEQDKRIAGYAHΞA
GKAλ/VIVVI>IKWDAVDKDestmkefeenirdhfqfld Y329_MYCGE 2 NMGIETASYIKTKLAIARSNVI MVDGSKPISEQDEVIGGLAQAA
LIPVII λ/NKWDLVLKNnnttnaykkmlklhfkhld orf_mycle 2 [SEQ ID NO: 41]
ASGHEFYASVRTHGAIDSAEWIM IDASEPLTGQDQRVLSMVIDA
GRAIiVLAFNKWDLVDEDrcdllereidrelvqvr — slrl974 2 [SEQ ID NO:42]
DYGAEFFGINRAFKAIRRADWLFVLDVLDGVTEQDLK AGRIIED
GRAVV VINKWDAVΞKDsy t iyehreqlmar ly f md
THDF_ECOLI [ SEQ ID NO: 3]
SDEVERIGIERA QEIEQADRVIFMYDgtttdavdpaeiwpefiarlpaK PITWRNKADitgetlgms evn
THDF_MYCGE [ SEQ ID NO: 44]
KSGLΞKAGIKKSFESIKQAN VIY LDathpkkdlelisffkkn KKDFFVFYNKKDlit
THDF_SYNY3 [SEQ ID NO.-45] ADQVEQIGVERSRKAAQQAD VLLTVDahqgwteadqliyeqvk DRPLI VINKIDlgradlvs-
MSS1_YEAST[SEQ ID NO: 46]
SDKIEMLGIDRAKKKSVQSD CLFIVDptdlskllpedilahlssktfgNKRIIIWlKSDlvsddemtk vlnklqtrlgs RASH_HUMAN [SEQ ID NO:47] samrdqymRTGEGFLCVFAINntksfedihqyreqikrvkdsdDVPMVLVGNBCDlaartvesrqaqdla rsy
GTPase domain »><« eGAP domainfSEQ ID NO.-48] sssss hhhhhhhhhhhh ERA_ECOLI[SEQ ID NO: 49] F DIVPISAETG NVDTIAAIVRKHL ERA_HAEIN
FAHIVPISAQRGN NVHE EKIVRQSL [SEQ ID NO:50]
BEX_BACSU
FKEIVPISft ΞGN NIETLLAQIEAYL [SEQ ID NO: 51] ERA_STRMU
FQEIVPISΛ QGN NVSH VDLLVDHL [SEQ ID NO: 52]
ERA_COXBU
FQKITPLSAKTGD QVGT EQAVHQLM [SEQ ID NO: 53]
ERA_HELPY FLA VP SAKKSQ N NALLECISQHL [SEQ ID NO:54] orf_myctu
AAEIVPVSA TGD RVD LIDV AAA [SEQ ID NO: 55] orf_syn
GWPCFKFSft TGE GLSNFQSALEARL [SEQ ID NO: 56] ERA_MYCGE
FDKN F SIFKQV elrynifrkdi [SEQ ID NO: 57]
YQN2_CAEEL lgkslstniqssssfkpsdekwqsqfreliqkptwkcsysetrslfrticgwsgFERVFFVSSLNGE-- GIDE RDHL SIS [SEQ ID NO: 58] est_human gthcpspavkdpntqsvgnpqrigwph
FKEIFMLSALSQE DVKT KQY LTQA [SEQ ID NO:59]
YPHC_BACSU 1
FGEPYPISGTHGL G GDL DAVAEHFknipetkyneev- [SEQ ID NO: 60]
Y329_MYCGE 1 FGRPWISAAHGI GIGDLMD LVKQNqllpnennddlak [SEQ ID NO: 61]
50k_mycle 1
LGEPHAISRMHGR GVADLLDKVLAALpnvaestsldggl [SEQ ID NO: 62] slrl974 1
LGEPYPMSAIHGS GTGDLLDALLEYLpapqeepeede— [SEQ ID NO: 63] YPHC_BACSU 2
YAPILFMSALTKK RIHT MPAIIKASENHS RVQTNVLNDVIMDAV[SEQ ID NO: 64]
Y329_MYCGE 2
FAPVLFISVKNQ RLNTIFEQ KIIQSQLET VATPLLNDVIQQAQ [SEQ ID NO : 65] orf_mycle 2 WAQRVNISAKTGR AVQKLVPAMENSLAS DTRIATGP NIWIKAW [SEQ ID NO: 66] slrl974 2
WAEMIFVSAQTGL RVQKI DCVDIAAQEHRRRVTTAVINEVLEEAV [ SEQ ID NO: 67] THDF__ECOLI
GHALIRLSARTg EGVDVLRNHLKQS ... [SEQ ID NO: 68]
THDF_MYCGE
NKFENSISAKQkdik-ELVDLLTKYINEF ... [SEQ ID NO: 69] THDF_SYNY3
YPPEITN VLTaaaanLGIEALENAIIΞQ . . . [ SEQ ID NO : 70 ]
MSS1__YEAST
KYPILSVSCKTk EGIESLISTLTSN . . . [ SEQ ID NO : 71 ]
RASH_HU AN GIPYIETSAKTRQ GVEDAFYTLVREIrqh . . . [ SEQ ID O : 72 ] eGAP domain >» «< sssssss hhhhhh
ERA_ECOLI PEATHHFPΞ-
DYITDRSQRFMASEIIREKLMRFLGAELPYSVTVEIERFvsnergg-YDINGLILVEREGQKKMVI [SEQ ID NO: 73]
ERA_HAEIN REGVHHFPE-
DYVTDRSQRFMASEIIREKLMRFTGEELPYSVTVEIEQFkvnergt-YEINGLILVEREGQKK Vl [SEQ ID NO: 74]
BEX_BACSU PEGPQFYPS- DQVTDHPERFIISELIREKVLHLTREEIPHSIAVAIESIkgqdngs-VHVAATIVVΞRDSQKGIVI [SEQ ID NO:75]
ERA_STRMU EEGFQYFPA-
DQITDHPERFLVSEMIREKVLLLTREEIPHSVAWIDSMardeethk-IHIRATIMVERDSQKGIII [SEQ ID NO:76] ERA_COXBU PESPFYFPP-
EQVTDRSDQFMASEIIRE LMRLLGQΞIPYSLAVTLIEFrkeeki IRISAVI VEKKSQKGIVI
[SEQ ID NO: 77]
ERA_HELPY SPSAWLFEK-
DLMSDEKMRDIYKEIIRESLFDFLSDEIPYESDVMIDKFieeeri DKλ/YARIIVEKESQKKIVT [SEQ ID NO:78] orf_myctu PAGPAYYPD-
GELTDΞPEEVLMAELIEEAALQGλ/RDELPHSLAWIDEVspregrddllDVHAALYVERDSQKGIvT [SEQ ID NO: 79] orf_syn DPGPYYYPP- DLVTDQPERFIMAELIREQILLLTRQEVPHSVAIAIEKVeetper TNVFAAITVERGSQKGIII
[SEQ ID NO: 80] ΞRA_MYCGE
NFIDANNDDFKILEGIiREQIIFYCKNEIPHIARIEIIEKsfnkeknl-L IHLVISVPKLSQKKIII
[SEQ ID NO: 81]
YQN2_CAEEL PQGEWKMQD- GMPTGESAQQLCIDSIRAAVLDTTPSDVAYTVQIRISEWeeqgev LQIVGEIRCQKPRDGSLII
[SEQ ID NO: 82] est_human QPGPWEYHS-
AVLTSQTPEEICANIIREKLLEHLPQEVPYNVqqktavweegpgge-LVIQQKLLVPKESYVKI-C-r
[SEQ ID NO: 83] YPHC_BACSU 2 AMNPTPT-HNGSRLKIYYATQVSVKPPSFWFVNDpELMHFSYERFLENR-
IRDAFGF EGTPIKIFARARK
[SEQ ID NO: 84]
Y329_MYCGE 2 LYNQPPL-FKGKRLQITYAVQTKSQIPHFVLFCNDpKYLHFSYARFLENK-
IRENFGF NSVPISLYFKSKNarirtkpev [SEQ ID NO: 85] orf_mycle 2 AATPPPV-RGGKQPRILFATQATARPPTFVLFT —GFLEACYRRFLERR-
UffiTFGF EGSPIRINVRVREkrglkrr
[SEQ ID NO: 86] s lrl974 2 SWHSPPTtRQGKQGKIYYGTQVSTQPPAIALFVNDpNRFNDNYRRYIEKQ- ERKQLGF FGSPIRLFWRGKKvremegsrnratkv
[ SEQ ID NO : 87 ]
KH domain >» hhhhhhhhh ssssss ssssss hhhhhhhhhhh
ERA_ECOLI Ga TKGiAKIKTIGIEARKDMQEMFΞAPVHLEL VKVKSG ADDERALRSLGYvddl [ SEQ ID NO : 88 ]
ERA_HAΞIN
GSAGGQKIKTIGMEARADMERLFDNKVHLEL VKVKSGWADDERALRSLGYmde [ SEQ ID NO : 89 ]
BEX_BACSU
GKKGSLLKEVGKRARADIEALLGSRVYLEL VKVQKD RNKMSQLRDFGFkedey[SEQ ID NO: 90] ERA_STRMU
G-SI«S-^LKKIGQMARRDIELMLGDKVYLETVWKVKKN RDKKLDLADFGYnkkey[SEQ ID NO: 91]
ERA_COXBU
GKGGERLKRVGTNARLDMEK FGKRVFLQL VKVKSGWADNERLLRELGFee [ SEQ ID NO : 92 ]
ERA_HΞLPY G3SNGVNIKRIGTNARLKMQEVGEKKVFLNLQVIAQKSWSKEEKSLQKLGYihrrnrd [ SEQ ID NO : 93 ] or f _myc tu GKGG^RLREVGTAARSQIENLLGTKVYLDLRVKVAKN QRDPKQLGRLGF [ SEQ
ID NO : 94 ] orf_syn
GQ iQSMLQAIGTAARQQIQKLISGDVYLKLFVKVΞPKWRQSRQQLLEFGYrvee [ SEQ ID NO : 95 ]
ERA_MYCGE GaXNAEMIKAIGIATRKKLLNHFDCDIFIDIFVKTekqklpvysf lsk [ SEQ ID NO : 96 ] YQN2_CAΞEL GK3GKRISEIGRRVNEHLHSLFQRQLYARLIVTHngklitqsk [SEQ ID NO : 97 ] est_human GPKftTVISQIAQEAGHDLMDIFLCDVDIRLSVKLlk [ SEQ ID NO : 98 ]
Legend: h = helix; s = strand (assigned by similarity to proteins of known structure) red: residues involved in phosphate binding and hydrolysis; blue: residues involved in guanine ring recognition magenta: putative GTPase activating arginine motif (by analogy to GAPs of known structure) green: KH domain signature motif; cyan: lysine(s) presumed to bind the polynucleotide backbone
Identification of the KH domain in ERA
In order to identify potential similarities between the ERA family and other sequences present in public databases, ERA from Escherichia coli was truncated at Pro 174 (eliminating the GTPase domain) and the C-terminal part of the sequence, corresponding to:
PEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYSVTVEIERFVSNERGGYDINGLILVEREGQK KMVIGNKGAKIKTIGIEARKDMQEMFEAPVHLΞL VKVKSG ADDΞRALRSLGYVDD [ SEQ ID NO : 99 ] was used to perform a PSI-Blast search at: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph- psi_blast on the non-redundant NCBI database.
The output of the program is shown below, with the significance of the matches given in brackets. The threshhold for significance was set at 0.01. The sequences above the threshhold correspond to the already known ERA homologues from various organisms. Although no other sequences were identified above the threshhold, inspection of the sequences in the non-significant part of the search shows a surprising enrichment in proteins known to bind RNA (underlined) or single-stranded DNA (in italics). The alignment of ERA with these sequences reveals a region of similarity centered around the motif: [alphatic]-[aliphatc]-I-G-x- x-G[SEQ ID NO: 100] where x is frequently lysine. A comparison of the identified RNA and ssDNA-binding proteins showed that these were also only similar to each other in this domain. Medline searches showed that the domain had been described in 1993 as the KH domain (Siomi et al. Nucl. Acids Res. 21:1193) and its structure was determined by NMR in 1996 (Musco et al. Cell 85:237). An evaluation of the significance of the similarity between ERA sequences and a representative set of KH domains in MACAW yielded probabilities of chance occurrence less than le"20. The analysis used BLASTP 2.0.3 to search Non-redundant GenBank CDS
(translations+PDB+SwissProt+SPupdate+PIR) yielding 275,942 sequences or 82,475,789 total letters. Results of PSI-Blast iteration 3 showing Sequences above threshhold are shown below herein in Table 1.
sp|Q56057|ERA_SALTY GTP-BINDING PROTEIN ERA [Salmonella typhimurium]
(2e-58) sp|P06616|ERA_ΞCOLI GTP-BINDING PROTEIN ERA [Escherichia coli]
(3e-58) gi 12627217 (AB003804) GTP-binding protein [Streptococcus gordonii]
(5e-58) sp|P37214|ERA_STRMU GTP-BINDING PROTEIN ERA HOMOLOG [Streptococcus mutans] (7e-58) sp|P43728|ERA_HAEIN GTP-BINDING PROTEIN ERA [Haemophilus influenzae]
(6e-57) sp|P42182|BEX_BACSU BEX PROTEIN [Bacillus subtilis]
(2e-56) sp|P51836|ERA_COXBU GTP-BINDING PROTEIN ERA HOMOLOG [Coxiella burnetii] (9e-54) gnl I PID| e315161 (Z95208) unknown [Mycobacterium tuberculosis]
(le-52) pir| |RGECGT transforming protein homolog ras - Escherichia coli (le-52) pir| |S72606 B1937_F3_102 protein - Mycobacterium leprae
(2e-50) gnl|PID|dl010672 (D63999) GTP-binding protein Era [Synechocystis sp . ]
(2e-49) sp|P56059 |ΞRA_HELPY GTP-BINDING PROTEIN ERA HOMOLOG [Helicobaeter pylori] (7e-49) gi 11800154 (AC000108) ORF3 [Helicobaeter pylori]
(le-48) gi 12240044 (AF005773) putative GTP-binding protein [Streptococcus thermophilus] (le-39) sp|Q09523 |YQN2_CAEEL HYPOTHETICAL 43.9 KD GTP-BINDING PROTEIN E02H1.2 IN CHROMOSOME II (2e-36) spjP47627|ERA_MYCGΞ GTP-BINDING PROTEIN ERA HOMOLOG [Mycoplasma genitalium] (4e-31) sp|P75210|ERA_MYCPN GTP-BINDING PROTEIN ERA HOMOLOG [Mycoplasma pneumoniae] (5e-29) gi 11066959 (Z36752) E02H1.2 [Caenorhabditis eleganns]
(le-20) gnl|PID|ell87522 (Z47075) E02H1.2 [Caenorhabditis elegans]
(8e-19)
Sequences below threshhold (shown below) sp|P21465|RS3_BACSU 3 OS RIBOSOMAL PROTEIN S3 (BS3) (BS2) [Bacillus subtilis] (0.015) sp|P23309|RS3_BACST 30S RIBOSOMAL PROTEIN S3 (BS2) [Bacillus stearothermophilus] (0.026) gi I 470377 (U00047) similar to yeast protein HX and E. coli PNP [Caenorhabditis elegans] (0.099) sp]P29157 |NUSA_THECE NUSA PROTEIN HOMOLOG [Thermococcus celer]
(0.38) pir) |S41652 hnRNP X protein - mouse (fragment) (0.38) gi 11575609 (U69127) FUSE binding protein 3 [Homo sapiens]
(0.50) gnl |PID|e236367 (Z71175) penylacetyl-CoA ligase [Pseudomonas putida]
(0.66) gnl|PID|e248342 (Z73969) C12D8.1a [Caenorhabditis elegans] (1.1) gnl|PID|e248405 (Z73969) C12D8.1b [Caenorhabditis elegans]
(1-1) sp|P2028l|RS3_HALMA 30S RIBOSOMAL PROTEIN S3 (HMAS3) (HSl) [Haloareula marismortui] (1.5) pir||B36933 Era homolog - Streptococcus mutans (fragment)
(1.9) sp|Q10093 |YAOE_SCHPO HYPOTHETICAL 138.8 KD PROTEIN C11D3.14C [Schizosaccharomyces pombe] (2.5) gi 11575607 (U69126) FUSE binding protein 2 [Homo sapiens] (2.5) gnl|PID|e309998 (Z82053) T26E3.2 [Caenorhabditis elegans]
(2.5) spjP46772|RS3_THEMA 3 OS RIBOSOMAL PROTEIN S3 [Thermotoga maritima]
(3.3) pirl lA53184 myc far upstream element-binding protein - human
(3.3) gi 12648654 (AE000972) transcription termination factor NusA [Archaeoglobus fulgidus] (3.3) gi|4790 (X69584) YKL253 [Saccharomyces cerevisiae]
(4.3) sp|P32860|YKE0_YEAST HYPOTHETICAL 29.2 KD PROTEIN IN SPC42-PTM1 REGION [S. cerevisiae] (4.3) sp|Q58235|FKB2_METJA FKBP-TYPE PEPTIDYL-PROLYL CIS-TRANS ISOMERASE [M. jannaschii] (4.3) sp|P94273|RS3_BORBU 30S RIBOSOMAL PROTEIN S3 [Borrelia burgdorferi]
(4.3) gnl|PID|dl020206 (AB001721) ORF3 [Leptospira interrogans]
(4.3) gi 12055427 (U94832) KSRP [Homo sapiens]
(4.3) gi 12073527 (U80032) weak similarity to human NuMA protein [Caenorhabditis elegans] (4.3) gi 12286200 (AF010578) polynucleotide phosphorylase [Pisum sativum]
(4.3) pir||s23464 vigilin - chicken [Gallus gallus]
(4.3) gnl I PID| ell85191 (Z99112) similar to hypothetical proteins [Bacillus subtilis] (4.3) sp|P15009|RS3_HALHA 30S RIBOSOMAL PROTEIN S3 (HS4) Halobacterium halobium (5.7) bbs 1161148 (S75665) PSI=P element somatic inhibitor [Drosophila sp. ] (5. 7) sp|P17697|CLUS_BOVIN CLUSTΞRIN PRECURSOR (GLYCOPROTΞIN III)
(7.4) sp I P451581 EX5A_HAEIN EXODEOXYRIBONUCLEASE V ALPHA CHAIN [Haemophilus influenzae] ( 7. 4) gnl | PlD| dl009000 (D49426) magnesium chelatase subunit [Anabaena variabilis] (7.4) gi|2621322 (AΞ000813) conserved protein [Methanobacterium thermoautotrophicum] (9.8) Alignments (shown below) sp I Q56057 I ERA_SALTY >s | Q56057 | ERA_SALTY GTP-BINDING
PROTEIN ERA >gi j 1228147 (U48415 ) Era [ Salmonella typhimurium] Length = 301
Score = 224 bits ( 565 ) , Expect = 2e-58
Identities = 141/ 148 ( 95% ) , Positives = 143 / 148 ( 96%)
Query : 1
ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASΞIIREKLMRFLGAELPYS 60 ISAETG+NVDTIA IVRKHLPEA HHFPEDYITDRSQRFMASEIIREKLMRFL
AELPYS
Sbjct: 154 ISAETGMNVDTIAGIVRKHLPEAIHHFPΞDYITDRSQRFMASEIIREKLMRFLRAELPYS 213
Query: 61
VTVEIΞRFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV 120 [SEQ ID NO:101]
VTVEIERFV+NERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV [ SEQ ID NO: 102] Sbjct: 214
VTVEIΞRFVTNERGGYDINGLILVΞREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV 273 [SEQ ID NO: 103]
Query: 121 HLEL VKVKSGWADDΞRALRSLGYVDDL 148
HLELWVKVKSG ADDE LRSLGYVDDL Sbjct: 274 HLELWVKVKSGWADDESRLRSLGYVDDL 301 sp|P06616|ERA_ECOLI >sp | P06616 | ERA_ECOLI GTP-BINDING
PROTEIN ERA >gi | 62870 | pir | | S44713 GTP-binding protein era
Escherichia coli >gi | 998396 |bbs | 166732 GTPase, Era [Escherichia coli, Peptide Mutant, 301 aa] >gi| 416295 (M14658) era
[Escherichia coli] >gi|98764l|gnl|PID|dl011565 (D64044) GTP binding protein [Escherichia coli] >gi 11033155 (U36841) GTP-binding protein [Escherichia coli] >gi] 1788919 (AE000343) GTP-binding protein [Escherichia coli]
Length = 301
Score = 223 bits (563), Expect = 3e-58 Identities = 148/148 (100%) , Positives = 148/148 (100%)
Query: 1 ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60
ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS Sbjct: 154 ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 213
Query: 61 VTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV 120
VTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIΞARKDMQEMFEAPV Sbjct: 214 VTVEIERFVSNERGGYDINGLILVERΞGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV 273
Query: 121 HLEL VKVKSG ADDERALRSLGYVDDL 148 [SEQ ID NO: 104]
HLELWVKVKSG ADDERALRSLGYVDDL[SEQ ID NO: 105] Sbjct: 274 HLELWVKVKSGWADDERALRSLGYVDDL 301 [SEQ ID NO: 106] gi I 2627217 >gi| 2627217 (AB003804) GTP-binding protein [Streptococcus gordonii]
Length = 299
Score = 222 bits (561), Expect = 5e-58
Identities = 61/145 (42%), Positives = 85/145 (58%), Gaps = 1/145 (0%)
Query: 1 ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60 ISA G NV + 1+ ++L E +FPED ITD +RF+ SE+IREK++ E+P+S
Sbjct: 150 ISALQGNNVSRLVDILSENLEEGFQYFPEDQITDHPERFLVSEMIREKVLMLTREΞIPHS 209
Query: 61 VTVEIERFVSNERGG- YDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAP 119
V V 1+ +E I I+VER+ QK ++IG G+ +K IG AR+D++ M Sbjct: 210 VAWIDSMKRDΞETDKVHIRATIMVERDSQKGIIIGKGGSMLKKIGSMARRDIELMLGDK 269
Query: 120 VHLELWVKVKSGWADDERALRSLGY 144 [SEQ ID NO: 107]
V LE WVKVK W D + L GY[SEQ ID NO: 108] Sbjct: 270 VFLETWVKVKKNWRDKKLDLADFGY 294 [SEQ ID NO: 109]
sp|P37214|ERA_STRMU >sp | P37214 | ERA_STRMU GTP-BINDING PROTEIN ERA HOMOLOG Length = 299 Score = 222 bits (560), Expect = 7e-58
Identities = 62/145 (42%), Positives = 85/145 (57%), Gaps = 1/145 (0%)
Query: 1
ISAΞTGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60 ISA G NV + ++ HL Ξ +FP D ITD +RF+ SE+IREK++
E+P+S
Sbj ct : 150 ISALQGNNVSHLVDLLVDHLEΞGFQYFPADQITDHPERFLVSEMIREKVLLLTREEIPHS 209
Query : 61 VTVEIERFVSNERGG-
YDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAP 119
V V 1+ +E I I+VER+ QK ++IG KGA +K IG AR+D++ M Sbj ct : 210 VAWIDSMARDEETHKIHIRATIMVERDSQKGIIIGKKGAMLKKIGQMARRDIELMLGDK 269
Query: 120 VHLELWVKVKSGWADDERALRSLGY 144 [SEQ ID NO: 110] V+LΞ WVKVK W D + L GY[SEQ ID NO: 111] Sbjct: 270 VYLETWVKVKKNWRDKKLDLADFGY 29 [SEQ ID NO: 112]
sp|P43728|ERA_HAEIN >sp | P43728 | ERA_HAEIN GTP-BINDING
PROTEIN ERA >gi | 1074173 |pir | |F64042 GTP-binding protein (era) homolog -
Haemophilus influenzae (strain Rd KW20) >gi| 1572957 (U32687)
GTP-binding protein (era) [Haemophilus influenzae] Length = 302
Score = 219 bits (552) , Expect = 6e-57 Identities = 113/147 (76%), Positives = 125/147 (84%) Query: 1
ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60 ISA+ G NV + IVR+ L E HHFPEDY+TDRSQRFMASEIIREKLMRF G
ΞLPYS
Sbjct: 156 ISAQRGNNVHΞLEKIVRQSLREGVHHFPEDYVTDRSQRFMASEIIREKLMRFTGEELPYS 215
Query: 61
VTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV 120
VTVEIE+F NΞRG Y+INGLILVERΞGQKKMVIG G KIKTIG+EAR DM+ +F+
V
Sbjct: 216 VTVEIEQFKVNERGTYEINGLILVEREGQKKMVIGAGGQKIKTIGMEARADMERLFDNKV 275
Query: 121 HLELWVKVKSGWADDERALRSLGYVDD 147 [SEQ ID NO: 113]
HLELWVKVKSGWADDERALRSLGY+D+ [SEQ ID NO: 114] Sbjct: 276 HLELWVKVKSGWADDERALRSLGYMDE 302 [SEQ ID NO: 115]
sp I P42182 I BΞX_BACSU >sp | P42182 | BEX_BACSU BEX PROTEIN
>gi I 606745 (TJ18532) Bex [Bacillus subtilis] >gi| 1303826 (D84432) YqfH
[Bacillus subtilis] >gi | 2634961 | gnl | PID| ell85795 (Z99116) GTP-binding protein [Bacillus subtilis] >gi | 2634975 | gnl | PID| ell83759 (Z99117) GTP-binding protein [Bacillus subtilis] Length = 301
Score = 217 bits (548 ) , Expect = 2e-56 Identities = 58/147 (39%) , Positives = 90/147 ( 60%)
Query : 1
ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASΞIIREKLMRFLGAELPYS 60 ISA G N++T+ A + +LPE +P D +TD +RF+ SE+IREK++ E+P+S Sbjct: 153
ISALEGNNIETLLAQIEAYLPEGPQFYPSDQVTDHPERFIISELIREKVLHLTREEIPHS 212
Query: 61
VTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV 120 + V IE + G + I+VER+ QK +VIG KG+ +K +G AR D++ + +
V
Sbjct: 213 IAVAIΞSIKGQDNGSVHVAATIWERDSQKGIVIGKKGSLLKEVGKRARADIΞALLGSRV 272
Query: 121 HLELWVKVKSGWADDERALRSLGYVDD 147 [SEQ ID NO: 116] +LELWVKV+ W + LR G+ +D[SEQ ID NO: 117] Sbjct: 273 YLELWVKVQKDWRNKMSQLRDFGFKED 299 [SEQ ID NO: 118]
sp|P51836|ERA_COXBU >sp | P51836 | ΞRA_COXBU GTP-BINDING PROTEIN ERA HOMOLOG >gi ) 2126359 |pir | | S60768 GTP-binding protein era -
Coxiella burnetii >gi| 439872 (L27436) GTP-binding protein [Coxiella burnetii] Length = 295
Score = 208 bits (525), Expect = 9e-54
Identities = 73/146 (50%), Positives = 101/146 (69%), Gaps = 1/146
(0%)
Query: 1 ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60 +SA+TG V T+ V + +PE+ +FP + +TDRS +FMASEIIREKLMR LG E+PYS
Sbj ct : 151
LSAKTGDQVGTLEQAVHQLMPESPFYFPPEQVTDRSDQFMASEIIREKLMRLLGQEIPYS 210 Query: 61
VTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQΞMFEAPV 120
+ V + F Ξ+ 1+ +1 VE++ QK +VIG G ++K +G AR DM++ F V
Sbjct: 211 LAVTLIEFRKEEKI- IRISAVIWVEKKSQKGIVIGKGGERLKRVGTNARLDMEKWFGKRV 269
Query: 121 HLELWVKVKSGWADDERALRSLGYVD 146 [SEQ ID NO: 119]
L+LWVKVKSGWAD+ER LR LG+ +[SEQ ID NO: 120] Sbjct: 270 FLQLWVKVKSGWADNERLLRELGFEE 295 [SEQ ID NO: 121]
gnl|PID|e315161 >gnl | PID | e315161 (Z95208) unknown [Mycobacterium tuberculosis] Length = 300
Score = 204 bits (515), Expect = le-52
Identities = 47/146 (32%), Positives = 81/146 (55%), Gaps = 2/146 (1%)
Query: 1 ISAETGLNVDTIAAIVRKHLPEATHHFPΞDYITDRSQRFMASEIIREKLMRFLGAELPYS 60 +SA TG VD + ++ LP ++P+ +TD + + +E+IRE ++ + ELP+S
Sbjct: 155 VSAMTGDRVDLLIDVLAAALPAGPAYYPDGΞLTDEPEEVLMAELIREAALQGVRDELPHS 214
Query: 61 VTVEIERFVSNERGG — YDINGLILVEREGQKKMVIGNKGAKIKTIGIΞARKDMQEMFEA 118
+ V 1+ E D++ + VER+ QK +VIG GA+++ +G AR ++ +
Sbj ct : 215 LAWIDΞVSPREGRDDLIDVHAALYVERDSQKGIVIGKGGARLREVGTAARSQIENLLGT 274 Query : 119 PVHLELWVKVKSGWADDERALRSLGY 144 [ SEQ ID NO : 122 ] V+L+L VKV W D + L LG+ [ SEQ ID NO : 123 ] Sbjct: 275 KVYLDLRVKVAKNWQRDPKQLGRLGF 300 [SEQ ID NO:124]
pir I |RGΞCGT >pir| | RGECGT transforming protein homolog ras - Escherichia coli Length = 316
Score = 204 bits (515), Expect = le-52
Identities = 136/136 (100%), Positives = 136/136 (100%)
Query: 1
ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60
ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS Sbjct: 154 ISAΞTGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 213
Query: 61 VTVΞIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV 120
VTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV Sbjct: 214 VTVEIΞRFVSNΞRGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV 273
Query: 121 HLELWVKVKSGWADDE 136 [SEQ ID NO: 125] HLELWVKVKSGWADDE [SEQ ID NO: 126]
Sbjct: 274 HLELWVKVKSGWADDE 289 [SEQ ID NO:127] pir||S72606 >pir||S72606 B1937_F3_102 protein -
Mycobacterium leprae >gi| 466988 (U00016) B1937_F3_102 [Mycobacterium leprae]
Length = 302
Score = 197 bits (497), Expect = 2e-50
Identities = 44/146 (30%), Positives = 81/146 (55%), Gaps = 2/146 (1%)
Query: 1 ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIRΞKLMRFLGAELPYS 60 +SA TG VD + ++ LP ++ +TD + + +E+IRE ++ + ELP+S
Sbjct: 157 VSAVTGEQVDVLIDVLAAALPPGPAYYSAGELTDEPEELLMAELIREAVLEGVHDELPHS 216
Query: 61 VTVEIERFVSNERGG— YDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEA 118
+ V 1+ G D++ ++ VER QK +VIG GA+++ +GI AR+ ++++ Sbjct: 217 LAWIDEVSPRAGRGDLIDVHAVLYVERPSQKGIVIGKSGARLREVGIAARRQIEKLLGT 276
Query: 119 PVHLELWVKVKSGWADDERALRSLGY 144 [SEQ ID NO: 128]
++L+L V V W + + L LG+[SEQ ID NO: 129] Sbjct: 277 NIYLDLHVNVAKNWQRNPKQLGRLGF 302 [SEQ ID NO: 130]
gnl|PID|dl010672 >gnl | PID| dl010672 (D63999)
GTP-binding protein Era [Synechocystis sp.] Length = 315
Score = 194 bits (488), Expect = 2e-49
Identities = 46/146 (31%), Positives = 82/146 (55%), Gaps = 1/146 (0%)
Query: 2 SAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYSV 61 SA TG + + + L +++P D +TD+ +RF+ +E+IRE+++ E+P+SV Sbjct: 170 SALTGEGLSNFQSALEARLDPGPYYYPPDLVTDQPERFIMAELIREQILLLTRQEVPHSV 229
Query: 62 TVEIERFVSNERGGYDINGLILVERΞGQKKMVIGNKGAKIKTIGIΞARKDMQEMFEAPVH 121
+ IE+ ++ I VER QK ++IG KG+ ++ IG AR+ +Q++
V+ Sbjct: 230 AIAIEKV-
EETPERTNVFAAITVERGSQKGIIIGQKGSMLQAIGTAARQQIQKLISGDVY 288 Query : 122 LΞLWVKVKSGWADDERALRSLGYVDD 147 [ SEQ ID NO : 131 ]
L+L+VKV+ W + L GY + [ SEQ ID NO : 132 ]
Sbj ct : 289 LKLFVKVEPKWRQSRQQLLEFGYRVE 314 [ SEQ ID NO : 133 ]
sp|P56059|ERA_HELPY >sp | P56059 | ERA_HΞLPY GTP-BINDING
PROTEIN ERA HOMOLOG >gi | 2313632 (AE000566) GTP-binding protein (era)
[Helicobaeter pylori] Length = 302
Score = 192 bits (483), Expect = 7e-49
Identities = 56/145 (38%), Positives = 84/145 (57%), Gaps = 1/145 (0%) Query: 1 ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60
+SA+ N++ + + +HL + F +D ++D R + ElIRE L FL E+PY Sbjct: 153 LSAKKSQNLNALLECISQHLSPSAWLFEKDLMSDEKMRDIYKEIIRESLFDFLSDEIPYE 212
Query: 61
VTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQΞMFEAPV 120
V I++F+ ER + I+VE+E QKK+VIG G IK IG AR MQE+ E
V
Sbjct: 213 SDVMIDKFIEEERID- KVYARIIVEKESQKKIVIGKNGVNIKRIGTNARLKMQEVGEKKV 271
Query: 121 HLELWVKVKSGWADDERALRSLGYV 145 [SEQ ID NO: 134]
L L V + W+ +E++L+ LGY+[SΞQ ID NO: 135] Sbjct: 272 FLNLQVIAQKSWSKEΞKSLQKLGYI 296 [SEQ ID NO:136]
gi|l800154 >gi|l800154 (AC000108) ORF3 [Helicobaeter pylori] Length = 301
Score = 191 bits (481), Expect = le-48
Identities = 57/145 (39%), Positives = 85/145 (58%), Gaps = 1/145 (0%) Query : 1
ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60 [SEQ ID
NO: 137]
+SA+ N++T+ + K+L + F +D ++D R + EIIRE L FL E+PY[SEQ ID NO:138] Sbjct: 152
LSAKKSQNLNTLLECISKYLSPSAWLFEKDLMSDEKMRDIYKΞIIRESLFDFLSDEIPYE 211 [SEQ ID NO: 139]
Query: 61
VTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPV 120 [SEQ
ID NO: 140]
V I++F+ ER + I+VE+E QKK+VIG G IK IG AR MQE+ E V{SEQ ID
NO: 141] Sbjct: 212 SDVMIDKFIEEERID-
KVYARIIVEKESQKKIVIGKNGVNIKRIGTSARLKMQEVGEKKV 270 [SEQ ID NO: 142]
Query: 121 HLELWVKVKSGWADDERALRSLGYV 145 [SEQ ID NO: 143] L L V + W+ +E++L+ LGY+[SEQ ID NO: 144] Sbjct: 271 FLNLQVIAQKSWSKEEKSLQKLGYI 295 [SEQ ID NO: 145]
gi I 2240044 >gi| 2240044 (AF005773) putative GTP-binding protein [Streptococcus thermophilus] Length = 108
Score = 162 bits (405) , Expect = le-39
Identities = 48/102 (47%), Positives = 64/102 (62%), Gaps = 1/102 (0%)
Query: 30 DYITDRSQRFMASΞIIREKLMRFLGAELPYSVTVEIERFVSNΞRGG- YDINGLILVEREG 88 [SEQ ID NO:146]
D ITD +RF+ SE+IREK+++ E+P+SV V 1+ +E I I+VER+ [SEQ ID NO: 147] Sbjct: 1 DQITDHPERFLVSEMIREKVLQLTREEIPHSVAWIDSMKRDEETDTVHIRATIMVERDS 60 [SEQ ID NO:148] Query: 89 QKKMVIGNKGAKIKTIGIEARKDMQEMFEAPVHLELWVKVKS 130 [SEQ ID NO: 149 ]
QK +VIG KGA +K IG ARKD++ M V LE WVKVK [SEQ ID nO:150] Sbjct: 61 QKGIVIGKKGAMLKKIGTLARKDIΞLMLGDKVFLETWVKVKK 102 [SEQ ID NO:151] sp|Q09523 |YQN2_CAEEL >sp|Q09523 |YQN2_CAEEL
HYPOTHETICAL 43.9 KD GTP-BINDING PROTEIN E02H1.2 IN CHROMOSOME II
Length = 394
Score = 151 bits (377) , Expect = 2e-36
Identities = 23/126 (18%), Positives = 52/126 (41%), Gaps = 1/126 (0%)
Query: 1 ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60 [SEQ ID
NO: 152]
+S+ G +D + + P+ + T S + + + IR ++ +++
Y+[SEQ ID nO:153]
Sbjct: 259 VSSLNGEGIDΞLRDHLMSISPQGEWKMQDGMPTGΞSAQQLCIDSIRAAVLDTTPSDVAYT 318 [SEQ
ID NO: 154]
Query: 61
VTVEIERFVSNERGGYDINGLILVΞREGQKKMVIGNKGAKIKTIGIEARKDMQEMFΞAPV 120 [SEQ ID NO: 155]
V + I + I G I ++ ++IG G +1 IG + + +F+
+[SΞQ ID NO:156] Sbjct: 319 VQIRISEWEΞQGEV- LQIVGEIRCQKPRDGSLIIGKGGKRISEIGRRVNEHLHSLFQRQL 377 [SEQ ID NO: 157]
Query: 121 HLELWV 126 [SEQ ID NO: 158]
+ L V[SEQ ID NO-.159] Sbjct: 378 YARLIV 383 [SEQ ID NO:160]
sp|P47627|ΞRA_MYCGE >sp I P47627 | ERA_MYCGE GTP-BINDING PROTEIN ERA HOMOLOG >gi | 1361527 | pir | | H64242 GTP-binding protein era homolog (spg) ho olog - Mycoplasma genitalium (SGC3)
Figure imgf000028_0001
(U39724) GTP-binding protein era homolog [Mycoplasma genitalium] Length = 290
Score = 133 bits (332), Expect = 4e-31 Identities = 29/102 (28%), Positives = 56/102 (54%), Gaps = 1/102 (0%)
Query: 30 DYITDRSQRFMASEIIREKLMRFLGAELPYSVTVEI- ERFVSNERGGYDINGLILVEREG 88 [SEQ ID NO: 161]
++I + F E +RΞ+++ + E+P+ +EI E+ + E+ 1+ +1 V +[SEQ ID NO: 162] Sbjct: 178
NFIDANNDDFKILEGLREQIIFYCKNEIPHIARIEIIEKSFNKEKNLLKIHLVISVPKLS 237 [SEQ ID NO: 163]
Query: 89 QKKMVIGNKGAKIKTIGIEARKDMQEMFEAPVHLELWVKVKS 130 [SEQ ID NO:164]
QKK++IG IK IGI RK + F+ + ++++VK +[SEQ ID NO: 165] Sbjct: 238 QKKIIIGKNAΞMIKAIGIATRKKLLNHFDCDIFIDIFVKTEK 279 [SEQ ID NO:166]
sp|P75210|ERA_MYCPN >sp | P75210 | ERA_MYCPN GTP-BINDING
PROTEIN ERA HOMOLOG >gi ) 2146514 |pir ] | S73600 probable GTP- binding protein spg - Mycoplasma pneumoniae (SGC3) (ATCC 29342)
Figure imgf000028_0002
(AE000027) Mycoplasma pneumoniae, GTP-binding protein era homolog; similar to Swiss-Prot Accession Number P37214, from S. mutans [Mycoplasma pneumoniae]
Length = 291 Score = 126 bits (314), Expect = 5e-29
Identities = 25/100 (25%) , Positives = 56/100 (56%) , Gaps = 1/100 (1%) Query: 30 DYITDRSQRFMASEIIREKLMRFLGAELPYSVTVEI- ERFVSNERGGYDINGLILVEREG 88 [SEQ ID NO: 167] +++ + F E +RE+++++ Ξ+P+ V +EI ++ + ++ I V
+[SEQ ID NO: 168] Sbjct: 178
EFLDADTDNFKILEALREQIIKYCSEEIPHWRLEIVDKSFDQAKNLLKLHLSISVPKLS 237 [SEQ ID NO: 169]
Query: 89 QKKMVIGNKGAKIKTIGIEARKDMQEMFEAPVHLΞLWVKV 128 [SEQ ID NO: 170]
QKK++IG +K IG++ R+ + E ++ V +EL+V+[SEQ ID NO: 171] Sbjct: 238 QKKIIIGRNAQMVKKIGMQMRQKLLEYYDCNVFVELFVRT 277 [SEQ ID NO:172]
gi 11066959 >gi| 1066959 (Z36752) E02H1.2 [Caenorhabditis elegans] Length = 92
Score = 98.7 bits (242), Expect = le-20
Identities = 17/82 (20%), Positives = 36/82 (43%), Gaps = 1/82 (1%)
Query: 45
IREKLMRFLGAELPYSVTVEIΞRFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTI 104 [SEQ ID NO: 173]
IR ++ +++ Y+V + I + I G I ++ ++IG G +1 I [SEQ ID NO: 174]
Sbjct: 1 IRAAVLDTTPSDVAYTVQIRISEWEEQGΞV- LQIVGEIRCQKPRDGSLIIGKGGKRISEI 59 [SEQ ID NO:175]
Query: 105 GIΞARKDMQEMFEAPVHLELWV 126 [SEQ ID NO: 176] G + + +F+ ++ L V[SEQ ID NO: 177]
Sbjct: 60 GRRVNEHLHSLFQRQLYARLIV 81 [SEQ ID NO: 178] gnl|PID|ell87522 >gnl | PID| ell87522 (Z47075) E02H1.2 [Caenorhabditis elegans] Length = 333
Score = 92.8 bits (227), Expect = 8e-19 Identities = 11/74 (14%) , Positives = 29/74 (38%) Query: 1
ISAETGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYS 60 [SEQ ID NO: 179] +S+ G +D + + P+ + T S + + + IR ++ +++
Y+[SEQ ID NO: 180] Sbjct: 259
VSSLNGEGIDELRDHLMSISPQGEWKMQDGMPTGESAQQLCIDSIRAAVLDTTPSDVAYT 318 [SEQ ID NO:181]
Query: 61 VTVEIERFVSNERG 74 [SEQ ID NO: 182]
V + I +[SEQ ID NO: 183] Sbjct: 319 VQIRISΞWEΞQGEV 332 [SEQ ID NO:184]
sp|P21465|RS3_BACSU >sp | P21465 | RS3_BACSU 3 OS RIBOSOMAL
PROTEIN S3 (BS3) (BS2) >gi | 786159 | gnl | PID | dl009470 (D50302) Ribosomal
Protein S3 [Bacillus subtilis] >gi | 2632389 | gnl | PID| ell82055 (Z99104) ribosomal protein S3 (BS3) [Bacillus subtilis] Length = 218
Score = 38.7 bits (88), Expect = 0.015 Identities = 20/103 (19%), Positives = 36/103 (34%), Gaps = 19/103
(18%)
Query: 22
EATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYSVTVEIERFVSNERGGYDINGL 81 [SEQ ID
NO: 185] Ξ+ + +DY + E l ++L VΞIΞR +[SEQ ID
NO:186]
Sbj ct : 19 ESKWYAGKDYADFLHΞDLKIREYISKRLSD ASVSKVEIERAANRVNITIHTA-
- 70 [SEQ ID NO:187]
Query: 82 ILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPVHLEL 124 [SEQ ID NO:188]
+ MVIG G++++ RK + + VH+ +[SEQ ID NO: 189] Sbj ct : 71 KPGMVIGKGGSEVE ALRKALNSLTGKRVHINI 102 [SEQ ID
NO : 190 ]
gi 11165309 >gi| 1165309 (U43929) S3 [Bacillus subtilis] Length = 218
Score = 38.7 bits (88), Expect = 0.015
Identities = 20/103 (19%), Positives = 36/103 (34%), Gaps = 19/103
(18%)
Query: 22
EATHHFPEDYITDRSQRFMASΞIIREKLMRFLGAELPYSVTVEIERFVSNERGGYDINGL 81 [SEQ ID
NO:191]
E+ + +DY + E l ++L VEIER +[SEQ ID NO:192]
Sbj ct : 19 ESKWYAGKDYADFLHEDLKIREYISKRLSD ASVSKVΞIERAANRVNITIHTA-
- 70 [SEQ ID NO: 193]
Query: 82 ILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPVHLEL 124 [SEQ ID NO: 194]
+ MVIG G++++ RK + + VH+ +[SEQ ID NO: 195]
Sbjct: 71 KPGMVIGKGGSEVE ALRKALNSLTGKRVHINI 102 [SEQ ID
NO: 196] sp|P23309|RS3_BACST >sp | P23309 | RS3_BACST 30S RIBOSOMAL PROTEIN S3 (BS2) >gi | 80223 | pir | | S10613 ribosomal protein S3
- Bacillus stearothermophilus >gi| 580921 (X54994) ribosomal protein S3 [Bacillus stearothermophilus] Length = 218
Score = 37.9 bits (86), Expect = 0.026
Identities = 21/109 (19%), Positives = 40/109 (36%), Gaps = 20/109
(18%) Query : 22
EATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYSVTVEIERFVSNERGGYDINGL 81 [SEQ ID
NO: 197]
E+ + +DY + E l ++L VEIER +[SEQ ID NO: 198]
Sbjct: 19 ESRWYAEKDYADLVHEDLKIREYINKRLQD AAVSRVEIERAANRVNVTIHTA-
- 70 [SEQ ID NO: 199]
Query: 82 ILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPVHLELWVKVKS 130 [SEQ ID NO:200]
+ MVIG G++++ RK + ++ H+ + V++K[SEQ ID NO: 201]
Sbjct: 71 KPGMVIGKGGSEVE ALRKALTQLTGKRΞHINI-VEIKK 107 [SEQ
ID NO: 202]
gi 1470377 >gi| 470377 (U00047) similar to yeast protein HX and E. coli PNP [Caenorhabditis elegans] Length = 768
Score = 35.9 bits (81), Expect = 0.099
Identities = 11/34 (32%), Positives = 16/34 (46%), Gaps = 1/34 (2%) Query: 80 GLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQ 113 [SEQ ID NO: 203] G ++V R M+IG G IK + E +Q[SEQ ID NO: 204] Sbjct: 453 GEVIVPRLS-AGMIIGKGGEMIKRLAAETGTKIQ 485 [SEQ ID NO.-205]
sp|P29157|NUSA_THECE >sp | P29157 | NUSA_THECE NUSA PROTEIN
HOMOLOG >gi|9924l|pir| |S18712 hypothetical protein X (rpl30-rpsl2 intergenic region) - Thermococcus celer >gi| 48144 (X67313) ORF-X gene product [Thermococcus celer] >gi| 58411 (X60305) orf-x [Thermococcus celer]
Length = 145 Score = 34.0 bits (76), Expect = 0.38
Identities = 20/98 (20%), Positives = 40/98 (40%), Gaps = 18/98 (18%)
Query: 62 TVEIERFVSNERGGYDINGLILVERΞGQKKMVIGNKGAKIKTIGIEARKDMQ - 113 [SEQ ID NO:206]
++ + + R I V ++G+ + +G KGA +K + K+++[SEQ ID NO: 207]
Sbjct: 22 ATVLDCLIDDRRNRL
IFVIKKGEMGLALGKKGANVKRVQNMIGKEIEVIEHSENP 76 [SEQ ID NO: 208]
Query: 114 EMFEAPVHLELWVKVKSGWADDΞRA LRSLGYVD 146 [SEQ ID NO: 209]
E F ++ L VKVK ++R L +G +[SEQ ID NO: 210]
Sbjct: 77 EEFLRNIYKSLGVKVKRVHITEKRDGKRVALLDIGPRE 114 [SEQ ID NO:211] pir| |S41652 >pir | |S41652 hnRNP X protein - mouse (fragment)
Length = 108
Score = 34.0 bits (76), Expect = 0.38
Identities = 13/49 (26%) , Positives = 20/49 (40%)
Query: 65 IERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQ 113 [SEQ ID NO: 212]
+++ +1+ L LV Q +IG G KIK I +Q[SEQ ID NO: 213]
Sbjct: 21 VKKMREESGARINISELRLWPASQCGSLIGKGGCKIKEIRESTGAQVQ 69 [SEQ ID NO: 214]
gi 11575609 >gi| 1575609 (U69127) FUSE binding protein 3 [Homo sapiens] Length = 600
Score = 33.6 bits (75), Expect = 0.50 Identities = 21/76 (27%), Positives = 28/76 (36%), Gaps = 12/76 (15%) Query : 38
RFMASEIIREKLMRFLGAELPYSVTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNK 97 [SEQ ID NO:215]
R M EIIREK + V + G ++ V R +VIG[SEQ ID NO:216]
Sbjct: 253 REMVLEIIREK DQADFRGVRGDFNSRMGGGSIEV SVPRFAV-
GIVIGRN 300 [SEQ ID NO: 217]
Query: 98 GAKIKTIGIEARKDMQ 113 [SEQ ID NO:218] G IK I +A +Q[SEQ ID NO:219]
Sbjct: 301 GEMIKKIQNDAGVRIQ 316 [SEQ ID NO: 220]
Score = 30.1 bits ( 66 ) , Expect = 5.7
Identities = 7/23 (30%), Positives = 13/23 (56%)
Query: 91 KMVIGNKGAKIKTIGIEARKDMQ 113 [SEQ ID NO: 221]
+VIG G IK+I ++ ++[SEQ ID NO:222] Sbjct: 395 GLVIGKGGENIKSINQQSGAHVE 417 [SEQ ID NO:223]
gnl|PID|e236367 >gnl | PID | e236367 (Z71175) penylacetyl-CoA ligase [Pseudomonas putida] Length = 439
Score = 33.2 bits (74), Expect = 0.66
Identities = 21/92 (22%), Positives = 37/92 (39%), Gaps = 8/92 (8%)
Query: 30 DYITDRSQRFMASEI
IREKLMRFLGAΞLPYSVTVEIERFVSNERGGYDINGLI 82 [SEQ ID NO: 224] IT RS + I E++++ Y + + + +
++[SEQ ID NO: 225] Sbjct: 326
GKITGRSDDMLIIRGVNVFPTQIEEQVLKIKQLSEMYEIHLYRNGNLDSVEVHVELRAEC 385 [SEQ ID NO:226]
Query: 83 LVEREGQKKMVIGNKGAKIKT-IGIEARKDMQ 113 [SEQ ID NO: 227] EGQ+K+VIG +IKT IGI + +Q[SEQ ID NO: 228] Sbjct: 386 QHLDEGQRKLVIGELSKQIKTYIGISTQVHLQ 417 [SEQ ID NO: 229]
gnl|PID|e248342 >gnl | PID| e248342 (Z73969) C12D8.1a [Caenorhabditis elegans]
Length = 589
Score = 32.4 bits (72), Expect = 1.1
Identities = 12/42 (28%), Positives = 20/42 (47%), Gaps = 1/42 (2%)
Query: 72 ERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQ 113 [SEQ ID NO: 230]
GG G ++V R ++IG +G IK + +E +Q[SEQ ID NO: 231] Sbjct: 221 GGGGASARGEVWPRSSV-GIIIGKQGDTIKRLAMETGTKIQ 261 [SEQ ID NO:232]
Score = 29.3 bits (64), Expect = 9.8
Identities = 19/99 (19%), Positives = 35/99 (35%), Gaps = 14/99 (14%)
Query: 46 REKLMRFLGAΞLPYSVTVEIERFVS NERGGYDINGLILVΞREG
QKKM 92 [SEQ ID' NO: 233]
R ++ ++ Y T I V + GG ++ G ++ +
+[SEQ ID NO: 234] Sbjct: 273 RCAVIMGTRDQI-
YRATERITELVKKSTMQQGGGGNVAGAMVSNEASTFYMSVPAAKCGL 331 [SEQ ID NO: 235] Query: 93 VIGNKGAKIKTIGIEARKDMQEMFEAPVHLELWVKVKSG 131 [SEQ ID NO:236]
VIG G IK I Ξ+ + + + + V V G[SEQ ID NO:237] Sbjct: 332 VIGKGGETIKQINSESGAHCELSRDPTGNADEKVFVIKG 370 [SEQ ID NO: 238]
gnl|PID|e248405 >gnl | PID| e248405 (Z73969) C12D8.1b [Caenorhabditis elegans] Length = 611
Score = 32.4 bits (72), Expect = 1.1
Identities = 12/42 (28%) , Positives = 20/42 (47%) , Gaps = 1/42 (2%) Query: 72 ERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQ 113 [SEQ ID NO: 239]
GG G ++V R ++IG +G IK + +E +Q[SEQ ID NO: 240] Sbjct: 243 GGGGASARGE WPRSSV-GIIIGKQGDTIKRLAMΞTGTKIQ 283 [SEQ ID NO:241]
Score = 29.3 bits (64), Expect = 9.8
Identities = 19/99 (19%) , Positives = 35/99 (35%) , Gaps = 14/99 (14%)
Query: 46 REKLMRFLGAELPYSVTVEIERFVS NERGGYDINGLILVEREG
QKKM 92 [SEQ ID NO: 242] R ++ ++ Y T I V + GG ++ G ++ +
+[SEQ ID NO: 243]
Sbjct: 295 RCAVIMGTRDQI-
YRATERITΞLVKKSTMQQGGGGNVAGAMVSNEASTFYMSVPAAKCGL 353 [SEQ ID NO.-244]
Query: 93 VIGNKGAKIKTIGIEARKDMQEMFEAPVHLELWVKVKSG 131 [SEQ ID NO:245] VIG G I I E+ + + + + V V G[SEQ ID NO: 246]
Sbjct: 354 VIGKGGETIKQINSΞSGAHCELSRDPTGNADEKVFVIKG 392 [SEQ ID NO:247]
sp|P2028l|RS3_HALMA >sp | P202811 RS3_HALMA 3 OS RIBOSOMAL PROTEIN S3 (HMAS3) (HSl) >gi | 70852 | ir | | R3HS3S ribosomal protein S3 -
Haloareula marismortui >gi| 148807 (J05222) ribosomal protein S3
[Haloareula marismortui] Length = 304
Score = 32.0 bits (71), Expect = 1.5
Identities = 16/55 (29%), Positives = 24/55 (43%), Gaps = 9/55 (16%)
Query: 62 TVEIERFVSNERG GYDIN GLILVΞRΞGQKKMVIGNKGAKIKTIGIE
107 [SEQ ID NO-.248] +1+ F + E G G D+ G +V + + MVIG G 1+ I E [ SEQ
ID NO : 249 ]
Sbjct: 14 RTQIDEFFAEELGRAGYGGMDVAKTPMGTQIVLKAEKPGMVIGKGGKNIRKITTE 68 [SEQ ID NO:250]
pir]]B36933 >pir|]B36933 Era homolog - Streptococcus mutans ( fragment) Length = 169
Score = 31.7 bits (70), Expect = 1.9
Identities = 8/20 (40%) , Positives = 11/20 (55%)
Query: 1 ISAETGLNVDTIAAIVRKHL 20 [SEQ ID NO:246] ISA G NV + ++ HL[SEQ ID NO: 247] Sbjct: 150 ISALQGNNVSHLVDLLVDHL 169 [SEQ ID NO:248]
sp I Q10093 I YAOE_SCHPO >sp | Q10093 | YAOE_SCHPO
HYPOTHETICAL 138.8 KD PROTEIN C11D3.14C IN CHROMOSOME I
Figure imgf000037_0001
(Z68166) unknown [Schizosaccharomyces pombe] Length = 1260
Score = 31.3 bits (69), Expect = 2.5
Identities = 23/71 (32%), Positives = 35/71 (48%), Gaps = 2/71 (2%)
Query: 43 ΞIIREKLMRFLGAΞLPYSVTVEIERFVSNERGGYDINGLILVEREGQK- KMVIGNKGAKI 101 [SEQ ID nO:251]
E IR L G E+P V +++ R S R G + L+ER+G+K +1 +[SEQ ID NO:252]
Sbjct: 48 EGIRRVLCYASGEEIPRKVPLDLTRVSS- IRMGTTVATNALLERKGEKTAFIITEGFRNL 106 [SEQ ID NO: 253]
Query: 102 KTIGIEARKDM 112 [SEQ ID NO:254] IG +AR D+[SEQ ID NO: 255] Sbjct: 107 VEIGNQARPDL 117 [SEQ ID NO:256]
gi 11575607 >gi| 1575607 (U69126) FUSE binding protein 2 [Homo sapiens] Length = 652
Score = 31.3 bits (69), Expect = 2.5
Identities = 19/68 (27%), Positives = 23/68 (32%), Gaps = 8/68 (11%)
Query: 47 EKLMRFLGAΞLPYSVTVEIERFVSNERGGYDINGLILVEREGQK-
KMVIGNKGAKIKTIG 105 [SEQ ID NO:257]
E +M L V F G I G I V +VIG G IK
I [ SEQ ID NO : 258 ]
Sbjct: 241 EMVMDILRN VTKAGFGDRNEYGSRIGGGIDVPVPRHSVGWIGRSGEMIKKIQ 293 [SEQ ID NO: 259]
Query: 106 IEARKDMQ 113 [SEQ ID NO:260]
+A +Q[SEQ ID NO: 261] Sbjct: 294 NDAGVRIQ 301[SEQ ID NO:262]
gnl|PID|e309998 >gnl | PID| e309998 (Z82053) T26E3.2 [Caenorhabditis elegans] Length = 365
Score = 31.3 bits (69), Expect = 2.5
Identities = 13/60 (21%), Positives = 20/60 (32%), Gaps = 7/60 (11%)
Query: 5 TGLNVDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEIIRΞKLMRFLGAELPYSVTVE 64 [SEQ ID
NO: 263]
G + + + K + P R A E I E ++R + E YS V[SEQ ID
NO:264]
Sbjct: 84 QGDDTΞVLLIQEAKKSCRGKWYMPAG RVEAGETIΞEAWREVKEETGYSCDW 136 [SEQ ID NO: 265] sp|P46772|RS3_THEMA >sp | P46772 |RS3_THEMA 3OS RIBOSOMAL
PROTEIN S3 >gi|625510|pir| |S40194 ribosomal protein S3 - Thermotoga maritima >gi| 581804 (Z21677) ribosomal protein S3 [Thermotoga maritima]
Length = 209
Score = 30.9 bits (68), Expect = 3.3
Identities = 12/60 (20%), Positives = 23/60 (38%), Gaps = 7/60 (11%)
Query: 65
IERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPVHLEL 124 [SEQ ID NO:266]
I IN + R ++IG KG++I R++++ F V + +[SEQ ID NO:267]
Sbjct: 51 ISEIYVERPDAERINITVKTARP GIIIGRKGSEI
TSLREELERKFNRRWINI 103 [SEQ ID NO: 268]
pir||A53184 >pir||A53184 myc far upstream element-binding protein - human >gi| 60152 (U05040) FUSE binding protein [Homo sapiens] Length = 644
Score = 30.9 bits (68), Expect = 3.3 Identities = 9/23 (39%), Positives = 12/23 (52%) Query: 91 KMVIGNKGAKIKTIGIEARKDMQ 113 [SEQ ID NO:269] +VIG G IK I +A +Q[SEQ ID NO:270] Sbjct: 288 GIVIGRNGEMIKKIQNDAGVRIQ 310 [SEQ ID NO:271]
gi I 2648654 >gi| 2648654 (AE000972) transcription termination-antitermination factor NusA, putative [Archaeoglobus fulgidus] Length = 139 Score = 30.9 bits (68), Expect = 3.3
Identities = 11/40 (27%), Positives = 17/40 (42%), Gaps = 2/40 (5%)
Query: 72 ERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKD 111 [SEQ ID NO: 272] E+ G I ++ V K +VIG G I A++[SEQ ID NO: 273] Sbjct: 93 EKEGSKI-AWSVS-PDLKGIVIGKGGKNINKAKELAKRH 130 [SEQ ID NO:274]
gi|4790 >gi|4790 (X69584) YKL253 [Saccharomyces cerevisiae] Length = 230
Score = 30.5 bits (67), Expect = 4.3
Identities = 14/55 (25%), Positives = 24/55 (43%), Gaps = 4/55 (7%) Query: 29 ΞDYITDRSQRFMASEIIREKLMRFLGAΞLPYSVTVEIERF VSNERGGYDIN
79 [SEQ ID NO:275]
+D++T R + 1+ +++ L +L Y V + F GGY IN [SEQ
ID NO.-276]
Sbjct: 86 DDFLTINKDRMVHWNSIKPEIIDLLTKQLAYGEDVISKEFHAVQEEEGEGGYKIN 140 [SEQ ID NO; 277]
sp|P32860|YKE0_YEAST >sp | P32860 | YKE0_YEAST HYPOTHETICAL 29.2 KD PROTEIN IN SPC42-PTM1 INTERGENIC REGION >gi|539300|pir| |S37861 nitrogen fixation protein nifU homolog YKL040c - yeast ( Saccharomyces cerevisiae) >gi| 486050 (Z28040) ORF YKL040c [Saccharomyces cerevisiae] Length = 256
Score = 30.5 bits (67), Expect = 4.3
Identities = 14/55 (25%), Positives = 24/55 (43%), Gaps = 4/55 (7%)
Query: 29 EDYITDRSQRFMASEIIREKLMRFLGAELPYSVTVEIERF VSNERGGYDIN
79 [SEQ ID NO:278] +D++T R + 1+ +++ L +L Y V + F GGY IN [ SEQ
ID NO : 2793
Sbj ct : 86 DDFLTINKDRMVHWNSIKPEIIDLLTKQLAYGEDVISKEFHAVQEEEGEGGYKIN 140 [ SEQ ID NO : 280 ]
sp|Q58235|FKB2_METJA >sp ] Q58235 | FKB2_METJA PUTATIVE
FKBP-TYPE PEPTIDYL-PROLYL CIS-TRANS ISOMERASE MJ0825 (PPIASE) (ROTAMASE) >gi|2129178|pir| |A64403 peptidylprolyl isomerase
(EC
5.2.1.8) - Methanococcus jannaschii >gi| 1591512 (U67526) rotamase, peptidyl-prolyl cis-trans isomerase [Methanococcus jannaschii]
Length = 240
Score = 30.5 bits (67), Expect = 4.3
Identities = 13/50 (26%), Positives = 21/50 (42%), Gaps = 2/50 (4%)
Query: 52 FLGAELPYSVTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKI 101 [SEQ ID NO:281]
G E+ Y ++IE V +++ + V R K+ I N KI[SEQ ID NO:282]
Sbjct: 143 LAGKEVKY--RIKIEEWDDKKNIVKEIVKMYVPRLSDVKVTIRNGTVKI 190 [SEQ ID NO:283]
sp|P94273|RS3_BORBU >sp | P94273 | S3_BORBU 3 OS RIBOSOMAL
PROTEIN S3 >gi 11685377 (U78193) ribosomal protein S3 [Borrelia burgdorferi] Length = 93
Score = 30.5 bits (67), Expect = 4.3
Identities = 18/62 (29%), Positives = 29/62 (46%), Gaps = 8/62 (12%) Query: 44
IIREKLMRFLGAELPYSVTVEIERFVSNERGGYDINGLILVEREGQKKMVIGNKGAKIKT 103 [SEQ ID NO:284] +IR ++M+FL +EI R + +1+ R G VIG KG+
++[SEQ ID NO:285]
Sbjct: 38 LIRREIMKFLKGIKFDISDIΞIIRNNPQ KVTWIVTPRPGS
VIGLKGSNLEK 89 [SEQ ID NO: 286]
Query: 104 IG 105 IG Sbjct: 90 IG 91
gnl|PID|dl020206 >gnl | PID| dl020206 (AB001721) ORF3 [Leptospira interrogans]
Length = 236
Score = 30.5 bits (67), Expect = 4.3
Identities = 20/109 (18%), Positives = 44/109 (40%), Gaps = 14/109 (12%)
Query: 44 IIREKLM—RFLGAELPYSVTVEIERFVSNERGGY DINGLILVEREGQK-
KMVIGNK 97 [SEQ ID NO: 287]
I+R ++ ++ + V + + + + E D++G I VE ++ +VIG +[SEQ ID NO: 288] Sbjct: 53
IVRAYVISRDLPAEKIIHGWLTVLKKMGIEAEWGMGDVDGKIYVEVASKESGLVIGKR 112 [SEQ ID NO: 289]
Query: 98 GAKIKTIGIEARKDMQEMF—EAPVHLELWVKVKSGWADD-ERALRSLG 143 [SEQ
ID NO:290]
G+ + I + + L++ + D E +L LG[SEQ ID
NO: 291]
Sbjct: 113 GSTLDAIQFILNLMVDSKIRHGRKIVLDI ESYRDKRΞLSLVRLG 156 [SEQ ID NO: 292] gi 12055427 >gi| 2055427 (U94832) KSRP [Homo sapiens] Length = 711
Score = 30.5 bits (67), Expect = 4.3 Identities = 15/47 (31%), Positives = 18/47 (37%), Gaps = 1/47 (2%)
Query: 68 FVSNERGGYDINGLILVEREGQK-KMVIGNKGAKIKTIGIEARKDMQ 113 [SEQ ID NO: 293]
F G I G I V +VIG G IK I +A +Q[SEQ ID NO:294] Sbjct: 311 FGDRNEYGSRIGGGIDVPVPRHSVGWIGRSGEMIKKIQNDAGVRIQ 357 [SEQ ID NO:295]
gi I 2073527 >gi | 2073527 (U80032) weak similarity to human NuMA protein (PIR:A42184) and S. cerevisiae glucoamylase S1/S2 precursor
(SP:P08640) [Caenorhabditis elegans] Length = 1624
Score = 30.5 bits (67), Expect = 4.3
Identities = 15/70 (21%), Positives = 26/70 (36%), Gaps = 2/70 (2%)
Query: 21
PEATHHFPEDYITDRSQRFMASEIIREKLMRFLGAELPYSVTVEIERFVSNERGGYDING 80 [SEQ ID NO:296]
E+ ++ E+ I D S + E E++ E Y +E + E
D+[SEQ ID NO: 297]
Sbjct: 858
DESEYYDDEEEIEDESDEYEDΞEYWDEEIEYEDEEEYEYEDEEVLEEYGDEΞEEDIDMKS 917 [SEQ ID N0:298]
Query: 81 LIL—VEREG 88 [SEQ ID NO:299] + VE Ξ[SEQ ID NO: 300]
Sbjct: 918 VESSDVEΞES 927 [SEQ ID NO: 301]
gi I 2286200 >gi| 2286200 (AF010578) polynucleotide phosphorylase [Pisum sativum]
Length = 897 Score = 30.5 bits (67), Expect = 4.3 Identities = 14/67 (20%), Positives = 29/67 (42%), Gaps = 3/67 (4%)
Query: 40 MASΞIIREKLMRFLGAELPYSVTVEIERFVSNERGGYDINGLILVEREGQKKM— VIGNK 97 [SEQ ID NO:302]
+ I+RE L++ + + ++ + LI V + K+ +IG+[SEQ ID NO: 303] Sbjct: 650 ITLPIMREALLQ-
ARDGRKHILGEMMKSLPPPAKRLSKYAPLIHVMKVRPDKINLIIGSG 708 [SEQ ID NO:304]
Query: 98 GAKIKTI 104 [SEQ ID NO: 305] G K+K+I[SEQ ID NO.-306] Sbjct: 709 GKKVKSI 715 [SEQ ID NO:307]
pir||S23464 >pir||S23464 vigilin - chicken
>gi|2281033 | gnl | PID | e331110 (X65292) vigilin [Gallus gallus]
Length = 1270
Score = 30.5 bits (67), Expect = 4.3
Identities = 14/59 (23%), Positives = 26/59 (43%), Gaps = 5/59 (8%)
Query: 47 EKLMRFLGAELPYSVTVEIΞRFVSNERGGYDINGLILVE-REGQKKMVIGNKGAKIKTI 104[SEQ ID NO:308]
E + +L +V +++ ++ I VE ++ Q K VIG KG ++ I [SEQ ID NO: 309] Sbjct: 266 EIVFTGEKEQLAQAVA-RVKKIYΞEKK KKTTTIAVEVKKSQHKYVIGRKGNSLQΞI
320 [SEQ ID NO:310]
gnl|PID|ell85191 >gnl | PID| ell85191 (Z99112) similar to hypothetical proteins [Bacillus subtilis] Length = 81
Score = 30.5 bits (67), Expect = 4.3 Identities = 9/38 (23%), Positives = 12/38 (30%), Gaps = 1/38 (2%)
Query: 71 NERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEA 108 [SEQ ID NO: 311] E I + V + VIG +G K I [SEQ ID NO: 312] Sbjct: 28 REETDQKIALRLSVHK-SDTGKVIGKQGRTAKAIRTAV 64[SEQ ID NO:313]
sp|P15009|RS3_HALHA >sp | P15009 | RS3_HALHA 30S RIBOSOMAL
PROTEIN S3 (HS4) >gi | 81068 |pir | | S11598 ribosomal protein S3 -
Halobacterium halobium Length = 302
Score = 30.1 bits (66), Expect = 5.7 Identities = 10/35 (28%), Positives = 17/35 (48%), Gaps = 4/35 (11%)
Query: 83 LVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFE 117 [SEQ ID NO: 314] +V + + MVIG G 1+ I ++E F+[SEQ ID NO: 315]
Sbjct: 44 IVLKAEKPGMVIGKGGKNIRKITT QLEERFD 74 [SEQ ID NO:316] prf I |1404256A >prf | | 1404256A ribosomal protein HS4 [Halobacterium halobium] Length = 83
Score = 30.1 bits (66), Expect = 5.7 Identities = 10/35 (28%), Positives = 17/35 (48%), Gaps = 4/35 (11%)
Query: 83 LVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFE 117 [SEQ ID NO: 317]
+V + + MVIG G 1+ I ++Ξ F+[SΞQ ID NO: 318] Sbjct: 44 IVLKAEKPGMVIGKGGKNIRKITT QLEERFD 74 [SEQ ID NO: 319]
bbs 1161148 >bbs| 161148 (S75665) PSI=P element somatic inhibitor [Drosophila, Peptide, 796 aa] [Drosophila sp.] Length = 796
Score = 30.1 bits ( 66) , Expect = 5.7
Identities = 8/29 (27%) , Positives = 12/29 (40%) Query: 91 KMVIGNKGAKIKTIGIEARKDMQEMFΞAP 119 [SEQ ID NO:320]
+VIG G 1+ I E +Q +[SEQ ID NO:321] Sbjct: 328 GWIGKGGDMIRKIQTECGCKLQFIQGKN 356 [SEQ ID NO:322] dbj I |AB006961_9 >dbj | |AB006961_9 (AB006961) ribosomal protein S3 [Halobacterium halobium] Length = 302
Score = 30.1 bits (66), Expect = 5.7 Identities = 10/35 (28%), Positives = 17/35 (48%), Gaps = 4/35 (11%)
Query: 83 LVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFE 117 [SEQ ID NO: 323]
+V + + MVIG G 1+ I ++E F+[SEQ ID NO: 324] Sbjct: 44 IVLKAEKPGMVIGKGGKNIRKITT QLEERFD 74 [SEQ ID NO:325]
sp|P17697|CLUS_BOVIN >sp | P17697 | CLUS_BOVIN CLUSTERIN
PRECURSOR (GLYCOPROTEIN III) (GPIII) >gi | 108702 | pir | |A35744 clusterin precursor - bovine >gi| 163115 (J05391) glycoprotein III precursor [Bos taurus] Length = 439
Score = 29.7 bits (65), Expect = 7.4
Identities = 9/36 (25%) , Positives = 15/36 (41%)
Query: 9 VDTIAAIVRKHLPEATHHFPEDYITDRSQRFMASEI 44 [SEQ ID NO: 326] + + HL +HFP T+ + R + El [SEQ ID NO:327] Sbjct: 245 IHQAQQAMDAHLQRTPYHFPTMEFTΞNNDRTVCKEI 280 [SEQ ID NO: 328] S I P45158 I EX5A_HAΞIN >sp | P45158 | EX5A_HAΞIN
EXODEOXYRIBONUCLEASE V ALPHA CHAIN >gi | 1074067 |pir | |E64116 exodeoxyribonuclease V (recD) homolog - Haemophilus influenzae (strain Rd KW20) >gi 11574782 (U32811) exodeoxyribonuclease V (recD)
[Haemophilus influenzae] Length = 640 Score = 29.7 bits (65), Expect = 7.4
Identities = 19/139 (13%), Positives = 51/139 (36%), Gaps = 5/139 (3%)
Query: 13 AAIVRKHLPEATHHFPΞDYITDRSQRFMASEIIREKLMRFLGAELPY- SVTVΞIERFVS- 70 [SEQ ID NO: 329]
V + P A + + + I+ L ++ Y + ++Ξ++[SEQ ID NO -.330] Sbjct: 110 QDPVNQIAPMAFQFGALYFYRAWQDEYRIVQYIKNTLKKYRTLAFSYDEIHQKLEKYFPE 169 [SEQ ID NO: 331]
Query: 71
NERGGYDINGLILVEREGQKKMVIGNKGAKIKTIGIEARKDMQEMFEAPVHLELWVKVK 129 [SEQ ID NO:332]
Ξ+ + + + ++ G G T +QE+F+ +H++L[SEQ ID NO:333]
Sbjct: 170 KQEKTDWQKVAVATAIK- SPFSIITGGPGTGKTTTVTRLLLVLQELFDCKLHIKLVAPTG 228 [SEQ ID NO:334]
Query: 130 SGWADDERALRS-LGYVDD 147 [SEQ ID NO:335]
+ E ++++ LG++ +[SEQ ID NO: 336] Sbjct: 229 KAASRLEESIKNALGFMQE 247 [SEQ ID NO: 337] gnl|PID|dl009000 >gnl | PID | dl009000 (D49426) magnesium chelatase subunit [Anabaena variabilis]
Length = 338
Score = 29.7 bits (65), Expect = 7.4
Identities = 14/53 (26%), Positives = 23/53 (42%), Gaps = 2/53 (3%)
Query: 6 GLNVDTIAAIVRKHLPEATHHFPΞDYITDRSQRFMASEIIRΞKLMRFLGAELP 58 [SEQ ID NO: 338]
G T + LPE + + +D S + S+ +R+K GAE+P[SEQ ID NO:339] Sbjct: 14 GTGKSTTIRALADLLPEIPWANDPFNSDPSDPDLMSDEVRQK—SGTGAEIP 6 [SEQ ID NO:340] gi | 2621322 >gi | 2621322 (AE000813 ) conserved protein [Methanobacterium thermoautotrophicum] Length = 335
Score = 29.3 bits (64), Expect = 9.8
Identities = 10/50 (20%), Positives = 21/50 (42%), Gaps = 4/50 (8%)
Query: 50 MRFLGAELPY-SVTVΞIERFVSNERGGYDINGLILVEREGQKKMVIGNKG 98 [SEQ ID NO:341]
+R + ++ + + 1 V +E+ + LI+ K +IG G[SEQ ID
NO: 342]
Sbjct: 12 IRDIRKDIGHGDADIRIVDWLDΞKRD SLLI APDRSDKSAIIGKGG 58 [SEQ ID
NO: 343] CPU time: 21.71 user sees. 0.30 sys . sees 22.01 total sees .
Database : Non-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR Posted date: Dec 1, 1997 7:51 AM
Number of letters in database: 82,475,789 Number of sequences in database: 275,942
Lambda K H 0.318 0.150 0.406
Gapped
Lambda K H 0.270 0.0525 0.230
Matrix: BLOSUM62
Gap Penalties: Existence: 11, Extension: 1 Number of Hits to DB: 20918269 Number of Sequences: 275942 Number of extensions: 782086 Number of successful extensions: 2522
Number of sequences better than 10 : 55
Number of HSP's better than 10.0 without gapping: 30
Number of HSP's successfully gapped in prelim test: 25 Number of HSP's that attempted gapping in prelim test: 2449
Number of HSP's gapped (non-prelim): 85 length of query: 148 length of database: 82475789 effective HSP length: 52 effective length of query: 96 effective length of database: 68126805 effective search space: 6540173280
T: 11
A: 40 XI: 16 ( 7.3 bits)
X2: 38 (14.8 bits)
X3: 64 (24.9 bits)
SI: 41 (21.5 bits)
S2: 65 (29.6 bits)
Role of the domain separating the ERA GTPase and KH domains as an eGAP (ERA- specific GTPase activating protein domain).
In ERA, the GTPase domain is separated from the KH domain by an intervening sequence of about 50 residues. Database searches with this sequence do not yield significant matches to other proteins. The only entirely conserved residue in this sequence is an arginine, which is always preceeded by a large hydrophobic residue.
In other GTPases, notably Ras, Rho, and heterotrimerc G-protein a subunit, which have been extensively investigated by X-ray crystallography, an arginine, either present in the same polypeptide (Ga) or in a separate protein (RasGAP and RhoGAP) is required for efficient hydrolysis of the bound nucleotide (hence the name GAP for GTPase activating protein). In RasGAP and RhoGAP, the arginine is always preceeded by a large hydrophobic residue. The role of this arginine in efficient nucleotide hydrolysis is emerging as a common feature of all P-loop NTPases. In view of this, the domain separating the GTPase and KH domains of ERA is proposed to be the ERA-specific GAP. Domains containing an invariant arginine are observed in two closely related GTPases that are also present in all fully sequenced eubacterial genomes: a 50kD open reading frame and ThdF, believed to be involved in thiophene and furan oxidation. In the 50k protein the region bears distant similarity to eGAP:
ERA_ECOLI PEATHHFPE- DYITDRSQRFMASEIIREKLMRFLGAELPYSVTVEIERFvsnergg— [SEQ ID NO: 344]
ERA_HAΞIN REGVHHFPE-
DYVTDRSQRFMASEIIREKLMRFTGEELPYSVTVEIEQFkvnergt— [SEQ ID NO: 345]
BEX_BACSU PEGPQFYPS-
DQVTDHPERFIISELIREKVLHLTREΞIPHSIAVAIESIkgqdngs— [SEQ ID NO:346] ERA_STRMU EEGFQYFPA-
DQITDHPERFLVSEMIREKVLLLTREEIPHSVAWIDSMardeethk-[SEQ ID NO:347]
ERA_COXBU PESPFYFPP-
EQVTDRSDQFMASEIIREKLMRLLGQEIPYSLAVTLIEFrkeeki [SEQ ID NO: 348]
ERA_HELPY SPSAWLFEK- DLMSDEKMRDIYKΞIIRESLFDFLSDEIPYESDVMIDKFieeeri [SEQ ID NO:349] orf_myctu PAGPAYYPD-
GELTDEPEEVLMAELEREAALQGVRDELPHSLAWIDEVspregrddl [SEQ ID NO : 350 ] orf_syn DPGPYYYPP-
DLVTDQPΞRFIMAELIREQILLLTRQEVPHSVAIAIEKVeetper [SEQ ID NO: 351] ERA_MYCGE
NFIDANNDDFKILEGI-EQIIFYCKNEIPHIARIΞIIEKsfnkeknl-[SEQ ID NO:352]
YQN2_CAEEL PQGEWKMQD-
GMPTGΞSAQQLCIDSIRAAVLDTTPSDVAYTVQIRISEWeeqgev [SEQ ID NO:353] est_human QPGPWEYHS- AVLTSQTPEEICANIIBEKLLEHLPQΞVPYNVqqktavweegpgge--[SEQ ID NO:354]
YPHC_BACSU 2 AMNPTPT-HNGSRLKIYYATQVSVKPPSFWFVNDpELMHFSYERFLENR-
IRDAFGF EGTPIKIFARARK[SEQ ID NO: 355]
Y329_MYCGE 2 LYNQPPL-FKGKRLQITYAVQTKSQIPHFVLFCNDpKYLHFSYARFLENK- IRENFGF NSVPISLYFKSKNarirtkpev[SEQ ID NO: 356] orf_mycle 2 AATPPPV-RGGKQPRILFATQATARPPTFVLFTt—GFLEACYRRFLERR-
LRETFGF EGSPIRINVRVREkrglkrr[SEQ ID NO: 357] slrl974 2 SWHSPPTtRQGKQGKIYYGTQVSTQPPAIALFVNDpNRFNDNYRRYIΞKQ- RKQLGF FGSPIRLFWRGKKvremegsrnr[SEQ ID NO:358]
whereas in ThdF, it appears distantly related to RhoGAP:
THDF_SYNY3 mqlEDTIAAIATAivpq
QGSICSVV LSGPQSLTIAKTLFdapgnqtweshriltSEQ ID NO:359] THDF_MYCGE mksEINIFALATApf
NSALHIIRFSGPDVYEILNKITnkkitrkgmqiqrt [ SEQ ID NO : 360 ]
THDF_ECOLI msdND IVAQATPpg
RGGVGILRISGFKAREVAETVLgklpkpryadylp f [ SEQ ID NO : 361 ] MSS1_YΞAST mnsas flqsrlisrs flvrrslkrysglakpytfqQPTIYALSTPanq
TSAIAHRISGTHAKYIYNRLVdsstvppirkailr t SEQ ID NO : 362 ]
THDF_HAEIN mr Idf ke f f mKETIVAQATApg
RGGIGH-RVSGPLATKVAQAILgkcpkprmadylpf [ SEQ ID NO : 363 ]
THDF_PSEPU mntvRETIAAIATAqg RGGVG-Λ RLSGPLAAKAGLLITgrtltprhahygpf [ SEQ ID NO : 3643
THDF_BACSU MDTIAAISTPmg
EGAIAIVRLSGPEAIQIADKIYkgpkgktlssvesh rSEQ ID NO : 365 ] hhhhhhhhhhhhhhhh hhhhhhhhhhh 3 BPl_MOUSE . . . . rthlqdlgrdlALPIEACVLLLLSegm-
QEEEΘ-F LAAGASVLKRLKQTMasdphsleef csg [ SEQ ID NO : 366 ]
RN_DROME . . . . sdyaprvapmVPALIVHCVTEIEArg —
LQQEGLYRVSSTREKCKRLRRKLlrgkstphlgnkd [ SΞQ ID NO : 367 ] CHIN_HUMAN . . . . ttlvkahttkRPMWDMCIREIESrg — LNSEG YRVSGFSDLIEDVKMAFdrdgekadisvnm f SEQ ID NO .- 368 ]
RGCIJHUMAN . . . . ekf iqssgqpVPLWESCIRFINLng —
LQHEGIER\7SGAQLRVSEIRDAFergedplvegctap [ SEQ ID NO : 369 ]
RGA1_YEAST
. . . . varcnyennelPMILSVCIDFIESdeenMRSEGIYRKSGSQLVIEEIEKQFsawkvqqntetpn [ SEQ ID NO : 370 ]
BCR_HUMAN ....avvtkrerskVPYIVRQCVEEIERrg--
MEEVGIYRVSGVATDIQALKAAFdvnnkdvsvmmse[SEQ ID NO:371] >gill730915lsplP50743IYPHC_BACSU HYPOTHETICAL 48.8 KD GTP-BINDING PROTEIN IN CMK-GPSA INTERGENIC REGION [Bacillus subtilis][SEQ ID NO:372] MGKPWAIVGRPNVGKSTIFNRIAGERISIVEDTPGVTRDRIYSSAΞWLNYDFNLIDTGGIDIGDEPFLA QIRQQAEIAMDEADVIIFMVNGREGVTAADEEVAKILYRTKKPWLAVNKLDNTEMRANIYDFYSLGFGE PYPISGTHGLGLGDLLDAVAEHFKNIPETKYNEEVIQFCLIGRPNVGKSSLVNAMLGEERVIVSNVAGTT RDAVDTSFTYNQQEFVIVDTAGMRKKGKVYETTEKYSVLRALKAIDRSEWAWLDGEEGIIEQDKRIAG YAHEAGKAWIWNKWDAVDKDESTMKEFEENIRDHFQFLDYAPILFMSALTKKRIHTLMPAIIKASENH SLRVQTNVLNDVIMDAVAMNPTPTHNGSRLKIYYATQVSVKPPSFWFVNDPELMHFSYERFLENRIRDA FGFEGTPIKIFARARK >gi|2313967 (AE000594) gtp-binding protein homologue (yphc) [Helicobaeter pylori] [SEQ ID NO: 373]
MNTSHKTLKTIAILGQPNVGKSSLFNRLARΞRIAITSDFAGTTRDINKRKIALNGHEVELLDTGGMAKDA LLSKEIKALNLKAAQMSDLILYWDGKSIPSDEDLKLFREVFKINPNCFLVINKIDNDKEKERAYAFSSF GMPKSFNISVSHNRGISALIDAVLSALDLNQIIEQDLDADILESLETPNNALEΞΞIIQVGIIGRVNVGKS SLLNALTKKERSLVSSVAGTTIDPIDETILIGDQKICFVDTAGIRHRGKILGIΞKYALERTQKALEKSHI ALLVLDVSAPFVELDΞKISSLADKHSLGIILVLNKWDIRYAPYEEIIATLKRKFRFLEYAPVITTSCLKA RHIDΞIKHKIIEVYECFSKRIPTSLLNSVINQATQKHPLPSDGGKLVKVYYATQFATKPPQISLIMNRPK ALHFSYKRYLINTLRKΞFNFLGTPLILNAKDKKSAQQN
>gi|l549379 (U62737) putative protein [Synechococcus PCC7942][SEQ ID NO:374]
EFFGINRSFKAIRRADVCLLVIDVLDGVTDQDQKLAGRIEEDGRACVIWNKWDAHEKDSSTIYEVERQL RDRLYFLDWAPMIFVSALTGQRVEKILDQVNTWEQHRRRVGTSVINEVLGDAIAWRTPPTTRQGRQGRI YYGTQVTTQPPSFTLFVNDPKLFGESYRRYIERQFRESLGFSGTPIRLFWRGKKSRΞLERGANRATRV
>gi|l65329l|gnl|PID|dl018939 (D90912) hypothetical protein [Synechocystis sp.] - slrl974[SEQ ID NO: 375] MSLPIVAIIGRPNVGKSTFVNRLAGNQQAIVHDQPGITRDRTYRPAFWRDRDFQWDTGGLVFNDDSEFL PEIREQANLALAEAKAAIFWDGQQGPTASDEEIAQWLRQQSVPVILAVNKCESPDQGAIQAAEFWHLGL GEPYPMSAIHGSGTGDLLDALLEYLPAPQEEPEEDEIKVAIVGRPNVGKSSLLNALTGEQRAIVSPISGT TRDAIDMWERNGQKYRLIDTAGIRRKKNVDYGAEFFGINRAFKAIRRADWLFVLDVLDGVTEQDLKLA GRIIΞDGRAWLVINKWDAVΞKDSYTIYEHREQLMARLYFMDWAEMIFVSAQTGLRVQKILDCVDIAAQΞ HRRRVTTAVINEVLEEAVSWHSPPTTRQGKQGKIYYGTQVSTQPPAIALFVNDPNRFNDNYRRYIΞKQFR KQLGFFGSPIRLFWRGKKVREMEGSRNRATKV
>gi|249512l|sp|P75309|Y329_MYCPN HYPOTHETICAL 32.7 KD GTP-BINDING PROTEIN MG329 HOMOLOG [Mycoplasma pneumoniae] - First GTPase domain is deleted. [SEQ ID NO: 376] MDRLVKDNQLPPYHGSSETNPEVRFCVIGKPNVGKSSLINQLVQQNRVLVSDESGTTRDAIDIPLRVNGQ NYLLIDTAGIRRKGKIAPGIEAASYGKTQLAIARSNIILLMVDGSKPLSEQDEIIGGLAQAALIPVIILV NKWDLVQKDSNTMAKFKKQLQSQFQHLSFAPIVFISVKNNKRLHTIFEQLQIIQEQLTKKISTSLLNDVI QQAQLFNQAPLFKGGRLQVTYAVQTHSQTPHFVLFCNDPKFVHFSYARFLENKIRESFGFSAVPITLYFK SKNARIRGVAKT
>gi|l723150|sp|P4757l|Y329_MYCGE HYPOTHETICAL 50.2 KD GTP-BINDING PROTEIN MG329 [Mycoplasma genitalium] [SEQ ID NO: 377]
MFTVAIIGRTNVGKSTLFNRLIQKPMAIVSDTPNTTRDRIFGIGEWLKRKIAFIDTGGLIAKQTPLQQLI ALQVQAALSQAKAIIFLVSLQEQLNSDDFYVAKVLKKNKDKPVILWNKAENFNPKTAEETLKDYYSLGF GRPWISAAHGIGIGDLMDLLVKQNQLLPNENNDDLAKIRFCVIGKPNVGKSSLINQLVKQNRVLVSNES GTTRDAIDVPLKVNGEKFLLIDTAGIKRKGKINMGIETASYIKTKLAIARSNVILLMVDGSKPISEQDEV IGGLAQAALIPVIILVNKWDLVLKNNNTTNAYKKMLKLHFKHLDFAPVLFISVLKNQRLNTIFEQLKIIQ SQLETKVATPLLNDVIQQAQLYNQPPLFKGKRLQITYAVQTKSQIPHFVLFCNDPKYLHFSYARFLENKI RENFGFNSVPISLYFKSKNARIRTKPEV
>gi 11788858 (AE000337) f503; this 503 aa orf is 72 pet identical (12 gaps) to 488 residues of an approx. 512 aa protein yl36_haein sw: p44536 [Escherichia coli] [SEQ ID NO:378]
MRCLMIYKNEALNMVPWALVGRPNVGKSTLFNRLTRTRDALVADFPGLTRDRKYGRAEIEGREFICIDT GGIDGTEDGVΞTRMAEQSLLAIEEADWLFMVDARAGLMPADEAIAKHLRSREKPTFLVANKTDGLDPDQ AWDFYSLGLGEIYPIAASHGRGVLSLLEHVLLPWMEDLAPQEEVDEDAEYWAQFEAEENGEEEEEDDFD PQSLPIKLAIVGRPNVGKSTLTNRILGEΞRVWYDMPGTTRDSIYIPMERDGREYVLIDTAGVRKRGKIT DAVEKFSVIKTLQAIEDANWMLVIDAREGISDQDLSLLGFILNSGRSLVIWNKWDGLSQEVKEQVKET LDFRLGFIDFARVHFISALHGSGVGNLFESVREAYDSSTRRVGTSMLTRIMTMAVEDHQPPLVRGRRVKL KYAHAGGYNPPIWIHGNQVKDLPDSYKRYLMNYFRKSLDVMGSPIRIQFKΞGENPYANKRNTLTPTQMR KRKRLMKHIKKNK
>gi|ll75159 | sp| P44536 | 136_HAEIN HYPOTHETICAL GTP-BINDING PROTEIN HI0136 [Haemophilus influenzae] [SEQ ID NO:379]
MATPWALVGRPNVGKSTLFNRLTRTRDALVADFPGLTRDRKYGHAHIAGYDFIVIDTGGIDGTEEGVEE KMAEQSLLAIDEADIVLFLVDARAGLTAADIGIANYLRQRQNKITVWANKTDGIDADSHCAEFYQLGLG EIEQIAASQGRGVTQLMEQVLAPFAEKMENADENDRTSEEEQDEWEQEFDFDSEEDTALIDDALDEELEE EQDKNIKIAIVGRPNVGKSTLTNRILGEDRVWFDMPGTTRDSIYIPMERDGQQYTLIDTAGVRKRGKVH LAVEKFSVIKTLQAIQDANWLLTIDARENISDQDLSLLGFILNAGRSLVIWNKWDGLDQDVKDRVKSE LDRRLDFIDFARVHFISALHGSGVGNLFDSIKEAYACATQKMTTSLLTRILQMATDEHQPPMIGGRRIKL KYAHPGGYNPPIIWHGNQMDKLPDSYKRYLSNYYRKSLKIIGSPIRLLFQEGSNPFAGRKNKLTPNQLR KRKRLMKFIKKAKR
>gi 12827027 (AF008210) orf453 hypothetical protein [Buchnera aphidicola] [SEQ ID NO:380]
MLPIIVLIGRTNVGKSTLFNILSKTRNALVADYPGLTRDRNYGYCYLKENKKITIVDTAGINFKSQKIEK QSHEQTLKAIKECDGILFLVNARDGVMPEΞYEISRKIRKYΞKKTILVINKIDGIKEISKINEFYSLGFKE NIKISASHNQGINNLISKYLTPWINSKFKEKKLEKISQEHSKKΞKNSVKIACIGKPNVGKSTLINSLLMK KRMITSNKAGTTLDTVLVPIKYNYKNYIFIDTAGMSKKKSKTNKIEKFCKIKTLQTIEKSHLTLLIIDAK DQISKQDLLLSSFIEKSGKPLIIVINKCDLLSLKΞKKNLENLIKKQLKCNFFSKIHFISALNNEGTVELF KSIDTSYHTSQKKIKTSQVMKIMHKAVKKHQPPIINGRRIKLKYAHLGNSNPIEIMIHGNQVKNLSLCYK KYLKNFFYKTLKMNGTPIKIQFKETMNPYISKK
>gi I 2145946 | pir I I S72953 probable GTP-binding protein - Mycobacterium leprae [ SEQ ID NO : 381 ]
MSQDGTWSDESDWELDDSDLAEFGPWAWGRPNVGKSTLVNRILGRREAWQDVPGVTRDRVSYDAMWT GRRFWQDTGGWEPDAKGLKRLVAEQASVAMRTADAVILWDVGVGATDADEAAARILLRSGKLVFLAAN KVDGEKGESDASALWSLGLGEPHAISAMHGRGVADLLDKVLAALPNVAΞSTSLDGGLRRVALVGKPNVGK SSLLNKLAGDQRSWHEAAGTTVDPVDSLIEMGGRVWRFVDTAGLRRKVGQASGHEFYASVRTHGAIDSA EWIMLIDASEPLTGQDQRVLSMVIDAGRALVLAFNKWDLVDEDRCDLLEREIDRELVQVRWAQRVNISA KTGRAVQKLVPAMENSLASWDTRIATGPLNIWIKAWAATPPPVRGGKQPRILFATQATARPPTFVLFTT GFLEACYRRFLERRLRETFGFΞGSPIRINVRVREKRGLKRR
>gi|2326756|gnl|PID|e332860 (Z98268) gtp-binding protein [Mycobacterium tuberculosis] [SEQ ID NO:382] MTQDGTWVDESDWQLDDSΞIAESGAAPWAWGRPNVGKSTLVNRILGRREAWQDIPGVTRDRVCYDAL WTGRRFWQDTGGWEPNAKGLQRLVAEQASVAMRTADAVILWDAGVGATAADEAAARILLRSGKPVFLA ANKVDSEKGESDAAALWSLGLGEPHAISAMHGRGVADLLDGVLAALPEVGESASASGGPRRVALVGKPNV GKSSLLNKLAGDQRSWHEAAGTTVDPVDSLIELGGDVWRFVDTAGLRRKVGQASGHEFYASVRTHAAID SAEVAIVLIDASQPLTEQDLRVISMVIEAGRALVLAYNKWDLVDEDRRELLQREIDRELVQVRWAQRVNI SAKTGRAVHKLVPAMEDALASWDTRIATGPLNTWLTEVTAATPPPVRGGKQPRILFATQATARPPTFVLF TTGFLEAGYRRFLERRLRETFGFDGSPIRVNVRVREKRAGKRR
>gi| 1732241 (U70661) gtp-binding protein [Treponema pallidum] [SEQ ID NO: 383] MKGQDVILCDGGRHFSYKVLPRWIVGRPNVGKSTLFNRLLGRRRSITSNTSGVTRDSIEETVILRGFPL RLVDTSGFTVFSEKKASRQHIDTLVLEQTYKSIQCADKILLVLDGTCESAΞDEEVIQYLRPYWGKLIAAV NKTEGGEEVHYNYARYGFSTLICVSAEHGRNIDALERAIIQNLFSVDERRELPKDDWRLAIVGKPNTGK STLMNYLMRRTVSLVCDRAGTTRDWTGHVEFKQYKFIIADTAGIRKRQKVYESIEYYSVIRAISILNAV DIVLYIVDARDGFSEQDKKIVSQISKRNLGVIFLLNKWDLLEGSTSLIAKKKRDVRTAFGKMNFVPWPV SAKTGHGISDALHCVCKIFAQLNTKVETSALNTALKDWVTSYPPPRKYGHVSLKYLVQVSVRPIEFLLFA NRPDRIPENYVRFLQNRIREDLGLDSIPVKLTIRKNCRKR
>gi|2415535|gnl|PID|e349830 (AJ000745) era protein [Campylobacter jejuni] - incorrect annotation [SEQ ID NO:384] TLIENATRAHPLPHDYGKSVKIYYAVQYDLAPPKKAKIMDRPKALHFSYKRYLQDQIRKEFNFEGVPLPI
>gi 12688422 (AE001153) gtp-binding protein [Borrelia burgdorferi] [SEQ ID NO: 385] MLSYKKVLIVGRPNVGKSALFNRILDTKRSITESTYGVTRDLVEΞVCKVDSFKFKLIDTGGFTILKDEIS KIWQKVLSSLEKVDLILLVLDINΞILLEDYQIIERLRKYSSKWLVLNKVDTKDKECLAHEFHNLGFKR YFLVSAAHCRGITKLRDFLKVEVGEVGIESGADIKVGIIGKPNSGKSTLINYLSGNEIAIVSDQPGTTRD FIKTKFTRNGKVFEWDTAGIRRRARVNEIVEYYSVNRALKVIDMVDIVFLLIDVQEKLTSQDKKIAHYV TKKGKGIVIVFSKWDLVDESKGYFEALKSHVKFFFPILNFAPIFRISVHKRIGLDSLFKΞSFKLKDQLEL KTSTPDLNKMLNLWIKDYHLNISHKIKYITQVSTNPVKFILFANKIKNFPNSYYNYLVNNLRKIGYKNIP ILVΞLKEKIRDLK>gi|2851487|sp|P25522|THDF_ECOLI THIOPHENE AND FURAN OXIDATION PROTEIN THDF [Escherichia coli] [SEQ ID NO: 386]
MSDNDTIVAQATPPGRGGVGILRISGFKAREVAETVLGKLPKPRYADYLPFKDADGSVLDQGIALWFPGP NSFTGEDVLELQGHGGPVILDDLLKRILTIPGLRIARPGEFSERAFLNDKLDLAQAEAIADLIDASSEQA ARSALNSLQGAFSARVNHLVEALTHLRIYVEAAIDFPDΞEIDFLSDGKIEAQLNDVIADLDAVRAEARQG SLLREGMKWIAGRPNAGKSSLLNALAGREAAIVTDIAGTTRDVLREHIHIDGMPLHIIDTAGLREASDE VERIGIERAWQΞIEQADRVLFMVDGTTTDAVDPAEIWPEFIARLPAKLPITWRNKADITGETLGMSEVN GHALIRLSARTGΞGVDVLRNHLKQSMGFDTNMEGGFLARRRHLQALEQAAEHLQQGKAQLLGAWAGELLA EELRLAQQNLSEITGEFTSDDLLGRIFSSFCIGK >gijll74669 |sp|P43730 |THDF_HAEIN POSSIBLE THIOPHENE AND FURAN OXIDATION PROTEIN THDF [Haemophilus influenzae] [SEQ ID NO:387] MRLDFKEFFMKETIVAQATAPGRGGIGILRVSGPLATKVAQAILGKCPKPRMADYLPFKDADGTILDQGI ALYFKSPNSFTGEDVLELQGHGGQWLDLLLKRILQIDGIRLARPGEFSEQAFLNDKLDLAQAEAIADLI DATSEQAVRSALKSLQGEFSKKVNELVDSVIYLRTYVEASIDFPDEEIDFLADGKIEANLRGIINQLEDV RSEAKQGSILREGMKWIAGRPNAGKSSLLNALAGREAAIVTDIAGTTRDVLREHIHIDGMPLHIIDTAG LRDAIDEVERIGISRAWTEIEQADRIILMLDSSDPESADLSKVRSEFLAKLPSTLPVTIVRNKIDLNGEQ ASESEQGGYQMISLSAQTHDGVQLLREHLKQAMGFQTGMEGGFLARRRHLDALDKAAEHLQIGLVQLTEF HAGELLAEΞLRLVQSYLSΞITGQFTSDDLLGNIFSSFCIGK
>gi|l780759|gnl|PID|e290700 (Y10436) orf452; translated orf similarity to swiss-prot: thdf_psepu thiophene [SEQ ID NO: 388] and furan oxidation protein of pseudomonas putida [Coxiella burnetii] MTYVFPETIAAQATPSGRGGIGWRVSGEKTKAIAQKILGCVPKPRYATFVKFRDSGSVIDΞGIALYFPK PNSFTGEDVLELHGHGGPWMDRLLNTVLKAGARQARPGEFSERAFLNNKIDLAQAEAVADLINASSEQA ARSAMRSLQGEFSKRIHQLVDALIQLRMYIEASIDFPEEEIDFLADERIKETLENLTHQVQEIEKTAKQG ALLREGITWIAGEPNVGKSSLLNLLSGQETAIVTDIAGTTRDIIRESIHIDGLPIHWDTAGLRLTEDV VΞKEGVRRTQKAVQQADLLLLMIDASKPTEDFKKIIAQWFSENDNKIPTLIVENKIDLIGΞAPRKENKEY PHIKLSVKTRAGVELLKNHLKNTAGFEATHENNFIARRRHCDAIARASAFLKNANNHLLNQKAGELVAED LKLAQNALSEITGEFTSDDLLGKIFSEFCIGK
>gi|l35727|sp|P25755|THDF_PSEPU POSSIBLE THIOPHENE AND FURAN OXIDATION PROTEIN THDF [Pseudomonas putida] [SEQ ID NO:389] MNTVRETIAAIATAQGRGGVGIVRLSGPLAAKAGLLITGRTLTPRHAHYGPFRDDEGLVLDEGIALFFPG PNSFTGEDVLELQGHGGPWLDMLLQRCVQVGCRLARPGEFSERAFLNDKLDLAQAEAIADLIEASSSQA ARNALRSLQGEFSRRVHSLTEALIALRIYVEAAIDFPEEEIDFLADGHVLSMLDAVRSELSTVQREAGQG ALLRDGMTWIAGRPNAGKSSLLNQLAGREAAIVTDIAGTTRDILREHIHIDGMPLHWDTAGLRDTDDH VEKIGVERALKAIGEADRVLLWDSTAPEASDPFALWPEFLAQRPDPAKVTLIRNKADLSGERVALΞQCD DGHVTITLSAKGDDTGLQLLRDHLKGCMGYEQTAESGFSARRRHLDALRQASEHLEHGRAQLTLAGAGEL LAEDLRQAQHALGEITGAFSSDDLLGRIFSSFCIGK
>gi)2495118|sp|Q44633 ]THDF_BUCAP POSSIBLE THIOPHENE AND FURAN OXIDATION PROTEIN THDF [Buchnera aphidicola] [SEQ ID NO: 390] MIRNDTIIAQVTCPGKSAVGILRVSGIHANQVAFAVLGKIPKPRFATYSKFFDESKKVLDEGISLWFPAP FSLTGEDVLELQGHGNPFIMDLLIKRILCLKNIKIRIAQPGEFCQRAFLNGKIDLIQAEAIDDLINSETE SWRASLNSLHGNFSFYIQKIIKKLIEFRTNIEASIDFSEENIDFDFNIFIMSNFEKLNDKFLKIKNIVS EGSLIREAKRIVIVGPPNAGKSSLLNVLSCRDRAIVTDLPGTTRDVLYENINIHGISCEIIDTAGLRETE DKIEKIGIQRSWEMIKNSDHVLYVMDKTISLEDQKKTSIQFMKQISSYNIQEVTFVLNKNDLVEDFCGIT KIENLLFISISALTGQGIDILKKHLSNRQKDKSQEGLFIARRRHIHQIDLSYCELLKAQKNWLKYKNIEL LAΞSLNIINKLLGEITGEFTSSDLLKRIFSTFCIGK >gi|2754806 (AF003957) thdf [Myzus persicae primary endosymbiont] [SEQ ID NO:391]
MIHIETIVAPVTSPGKSAVSILRISGSQTKIVAKKVLGSIPKARFATYSKFLGKNSWLDQGISLWFPAP FSFTGEDVLELQGHGSPLIMDLLIKRITSIENVRMAKPGEFSΞRAFLNGKIDLIQAEAIDDLINSETESS IRASLHSLQGDFSSHIKQLISTVIEFRTNIESSIDFSEEEIDIDLKSLIYIKLKΞLEEKFIKTKKVISEG SLLKEGKKIVIAGPPNAGKSSLLNALSHSNRAIVTKIPGTTRDLLYENISINGISCQLIDTAGLRDTKNE IERIGIFRAWEVIKKADHVLFVIDKTTKQSEQKKICNEFIQNISVNNIQITFILNKNDLVQDKFNTEKIE SLLFINISARTGQGIDKLRKHIVKIAKNENKEGVFIARRRHINQINLAYNΞFLMAKKKWIISKNIELLAE SLRLINRFLGΞITGRFTSNDLLKRIFSSFCIGK
>gi J2495120]spjP73839|THDF_SYNY3 POSSIBLE THIOPHENE AND FURAN OXIDATION PROTEIN THDF [Synechocystis sp . ] [SEQ ID NO:392] MQLEDTIAAIATAIVPQQGSIGWRLSGPQSLTIAKTLFDAPGNQTWESHRILYGHVRHPQTKAAIDEAL LLLMLAPRSYTKEDWΞFQCHGGIMPVQQVLQLCLQQGARLAQPGEFSFRAFLNGRLDLTQAESISELVG AQSPQAAAIALAGLQGKLAQPIRDLRNTCLDILAEVEARIDFEDDLPPLDEDSIRQQLQNLYQQLEDILN TAQRGELLRTGLKVAIVGQPNVGKSSLLNAWSRTDRAIVTDLPGTTRDWΞSQLWEGIPIQVLDTAGIR ETADQVEQIGVERSRKAAQQADLVLLTVDAHQGWTEADQLIYEQVKDRPLILVINKIDLGRADLVSYPPE ITNTVLTAAAANLGIEALENAIIEQVNQTNLSPQNLDFAINQRQΞAVLTEAQLALKQLQQTMAEQLPLDF WTIDLRLAINALGQVTGETVTESVLDRIFSRFCIGK
>gi|l351237|sp|P47254|THDF_MYCGE POSSIBLE THIOPHENE AND FURAN OXIDATION PROTEIN THDF [Mycoplasma genitalium] [SEQ ID NO: 393] MKSEINIFALATAPFNSALHIIRFSGPDVYEILNKITNKKITRKGMQIQRTWIVDENNKRIDDVLLFKFV SPNSYTGEDLIEISCHGNMLIVNEICALLLKKGGVYAKPGEFTQRSFLNGKMSLQQASAVNKLILSPNLL VKDIVLNNLAGEMDQQLEQIAQQVNQLVMQMEVNIDYPEYLDEQVELSTLNNKVKLIIEKLKRIIENSKQ LKKLHDPFKIAIIGETNVGKSSLLNALLNQDKAIVSNIKGSTRDWEGDFNLNGYLIKILDTAGIRKHKS GLEKAGIKKSFESIKQANLVIYLLDATHPKKDLELISFFKKNKKDFFVFYNKKDLITNKFENSISAKQKD IKELVDLLTKYINEFYKKIDQKIYLIENWQQILIEKIKEQLEQFLKQQKKYLFFDVLVTHLREAQQDILK LLGKDVGFDLVNΞIFNNFCLGK
>gi I 2495119 | sp | P75104 | HDF_MYCPN POSSIBLE THIOPHENE AND FURAN OXIDATION PROTEIN THDF [Mycoplasma pneumoniae] [SEQ ID NO.-394]
MDTKQTMFALATAPFNSAIHIIRLSGPDVYRIINQITNKEVKPLGMRIQRVWLIDHNQKKVDDVLLFKFV APNSYTGEDLIEISCHGSMVIVNEIIGLLLKHGAVQAQPGEFTQRGYLNGKMSLNQAASVNNLVLSPNTT LKDVALNALAGQVDARLΞPLVEKLGQLVMQMEVNLDYPEYTDΞQRELVTMNQAWQITQILNQIWGQDQ LQRLKDPFKIAIIGNTNVGKSSLLNALLDQDKAIVSAIKGSTRDIVEGDFALNGHFVKILDTAGIRQHQS ALΞKAGIQKTFGAIKTANLVIYLLDARQPEPDPKIIARLKKLKKDFFLVHNKADLVQQSFQVSISAKQKQ IQPLVDLLTQYLHQFYSVEQNQLYLISDWQTILLQKAIAELEHFLIKQQNCLFFDILWΉLRAAHEYILQ VLGKNTNYDLINEIFKHFCLGK >gi|l35725|sp|P2581l|THDF_BACSU POSSIBLE THIOPHENE AND FURAN OXIDATION PROTEIN THDF [Bacillus subtilis] [SEQ ID NO: 395]
MDTIAAISTPMGEGAIAIVRLSGPΞAIQIADKIYKGPKGKTLSSVESHTIHYGHIVDRPSDRWEEVMVS VLKAPRTFTREDVIEINCHGGIVTVNQVLQLALREGARLAEPGEFTKRAFLNGRIDLSQAEAVMDLIRAK TDRAMNVAMNQMEGRLSALVRRLRSEILETLAHVEVNIDYPEYDDVEEMTHQILVEKATAVKKEIETLLR TSEQGKILREGLSTVIIGRPNVGKSSLLNSLVHEAKAIVTDIPGTTRDVIΞEYVNVRGVPLRLVDTAGIR ETEDIVERIGVERSRQVLKEADLILLVLNYSEELSEEDVKLFEAVEGMDVIVILNKTDLEPKIDTERVRE LANGRPWTTSLLKEEGINDLEEAIQSLFYTGAIESGDLTYVSNTRHITILQQAKRAIEDALSGIEQDVP IDMVQIDLTRCWELLGEIIGDSVHESLIDQLFSQFCLGK
>gi|l524359|gnl|PID|e245928 (X98090) gtpase [Synechocystis PCC6803] [SEQ ID NO.-396] MQLEDTIARIATAIVPQQGSIGWRLSGPQSLTIAKTLFDAPGNQTWESHRILYGHVRHPQTKAAIDEAL LLLMLAPRSYTKEDWEFQCHGGIMPVQQVLQLCLQQGARLAQPGEFSFRAFLNGRLDLTQAΞSHSELVG AQSPQAGAIALAGLQGKLAQPIRDLRNTCLDILAEVEARIDFEDDLPPLDEDSIRQQLQNLYQQLEDILN TAQAGELLRTGLKVAIVGQPNVGKSSLLNAWSRTDRAIVTDLPGTTRDWESQLWEGIPIQVLDTAGIR ΞTADQVEQIGVERSRKAAQQADLVLLTVDAHQGWTEADQLIYEQVKDRPLILVINKIDLGRADLVSYPPE ITNTVLTAAAANLGIEALENAIIEQVNQTNLSPQNLDFAINQRQEAVLTEAQLALKQLQQTMAEQLPLDF WTIDLRLAMNALGEVTGETVTESVLDRIFSRFCIGK
>gi 12314627 (AE000645) thiophene and furan oxidizer (tdhf) [Helicobaeter pylori] [SEQ ID NO: 397 ] MKNTSSSTTLTMNDTIAAIATPLGKGAISIIKISGHNALNILKQLTQKQDFTPRYAYVHDIFSNGVLLDK ALVIYFKAPYSFTGΞDVCEIQCHGSPLLAQNILQACLNLGARLAKAGEFSKKAFLNHKMDLSEIEASVQL ILCEDESVLNALARQLKGELKIFIEEARGNLLKLLASSEVLIDYSEEDIPSDFLDGVSLNLEKQIASFKD LLDFSNAQKQRNKGHALSIVGKPNAGKSSLLNAMLLΞERALVSDIKGTTRDTIEEVIΞLKGHKVRLIDTA GIRESADKIERLGIEKSLKSLENCDIILGVFDLSKPLEKEDFNLIDTLNRAKKPCIWLNKNDLAPKLEL EILKSYLKIPYTLLETNTLNSKACLKDLSQKISAFFPKLDTQNKLLLTSLAQKIALENAITELQNAKNHL ΞTLELFSYHILSAIENLNLLTRPYETSQMLDSMFSEFCLGK
>gi|l729932|sp|P53364|THDF_BORBU THIOPHENE AND FURAN OXIDATION PROTEIN THDF [Borrelia burgdorferi] [SEQ ID NO:398]
MSKFFERDDDIVALATPFLSSALCVIRSSGASSISKFSKIFSNHSALNSASGNTIHYGYILDSENGCKVD ΞWVCLYAPKSFTGQDAIEVMAHGSVIGIKKIIDLFLKSGFRMAEPGΞFTLRAFLAKKIDLTKAEAIHEI IFAKTNKTYSLAVNKLSGALFVKIDAIKKSILNFLSAVSVYLDYEVDDHEISIPFDLILSSKAELKKLIN SYKVYEKIDNGVALVLAGSVNAGKSSLFNLFLKKDRSIVSSYPGTTRDYIEASFΞLDGILFNLFDTAGLR DADNFVERLGIEKSNSLIKEASLVIYVIDVSSNLTKDDFLFIDSNKSNSKILFVLNKIDLKINKSTEEFV RSKVLNSSNLIMISTKNLEGIDILYDKIRALISYERVEIGLDDIIISSNRQMQLLEKAYALILDLLSKID RQVSYDMLAFDAYEIINCLGEITGEVSSEDVLDNMFKNFCLGK >gi|2780770|gnl|PID|dl025285 (AB010203) orf4; putative [Leptospira interrogans] [SEQ ID NO:399]
MSGPEALTISSSFLFSKNKFLSPSEILPRTAIQCVFQIGDRKIDQILFFYFKSPNSYTGEDLCEFHFHGN PILLREALDAIFRAGARPAKQGEFSRRAFLNΞKLDLTEVEAIGRLISARSRFELELAQKNVFGEVTRFTS NLRSQLISLKAECEAEIDFSTEDLTYESLEERKTRIENVKSLCQTLISKSSSAEKLIQQFRIVLYGEPNT GKSSLMNVLLGKERSIISEIPGTTRDYISΞEIFLEGIPVRLVDTAGVRETTDHIΞKLGIERSEKΞFQSAD VRLFLVDVSKKENWKEFINKSRERLEGSILIANKIDILNSSWDRNLFSDVKDLIVLΞISCKTKEGISNLL DAIKERTGKLGHSEDYVLLEERQRYHFETIVRCLDKTLHLLKEGAPAEIYIQEINYALAEIGEVNGKVDT EEVLGRIFSKFCVGK
>gi |2126491 |pir I |PC6012 thdF protein - Rhodobacter sphaeroides
(fragment)
[SEQ ID NO: 400] MDTIYALASARGKAGVAVLRLSGPRSHEAVQAFGVPLPSLRHAALRRLTWNGEVLDEALVLLFGAGASFT GETSAELHLHGSPAAVSSVLRVLSGLPG
>gi|l709142 | sp | P32559 |MSS1_YEAST MITOCHONDRIAL GTPASE MSS1 PRECURSOR [SEQ ID NO:401] MNSASFLQSRLISRSFLVRRSLKRYSGLAKPYTFQQPTIYALSTPANQTSAIAIIRISGTHAKYIYNRLV DSSTVPPIRKAILRNIYSPSSCSVKPHDQKESKILLDTSLLLYFQAPYSFTGEDVLELHVHGGKAWNSI LKAIGSLHDRSSGKDIRFALPGDFSRRAFQNGKFDLTQLEGIKDLIDSETESQRRSALSSFNGDNKILFE NWRΞTIIENMAQLTAIIDFADDNSQEIQNTDΞIFHNVEKNIICLRDQIVTFMQKVEKSTILQNGIKLVLL GAPNVGKSSLVNSLTNDDISIVSDIPGTTRDSIDAMINVNGYKVIICDTAGIREKSSDKIEMLGIDRAKK KSVQSDLCLFIVDPTDLSKLLPEDILAHLSSKTFGNKRIIIWNKSDLVSDDEMTKVLNKLQTRLGSKYP ILSVSCKTKEGIESLISTLTSNFESLSQSSADASPVIVSKRVSEILKNDVLYGLEEFFKSKDFHNDIVLA TENLRYASDGIAKITGQAIGIEEILDSVFSKFCIGK
Deposited materials A deposit containing 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 1RY, Scotland on 11 April 1996 and assigned deposit number 40794. The deposit was described as Streptococcus pneumoniae 0100993 on deposit. On 17 April 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly deposited with the NCIMB and assigned deposit number 40800. The Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain." The deposited strain contains the full length Era gene. The sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein. The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. § 112.
A license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain, which polypeptide is contained in the deposited strain. Further provided by the invention are ERA binding domain polynucleotide sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby. Also provided by the invention are ERA binding domain polypeptide and polynucleotide sequences isolated from the deposited strain. Polypeptides
Era polypeptide of the invention is structurally related to other proteins of the Era family. In one aspect of the invention there are provided polypeptides of various bacteria referred to herein as "Era" and "Era polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same. Among the particularly preferred embodiments of the invention are variants of Era polypeptide encoded by naturally occurring alleles of the Era gene.
The present invention further provides for an isolated polypeptide which:
(a) comprises or consists of an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to a polypeptide comprising an ERA binding domain sequence selected from Table 1 over the entire length of such sequence in Table l;
(b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to a sequence selected from a polypeptide comprising an ERA binding domain sequence selected from Table 1 over the entire length of said sequence selected from Table 1; (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of an ERA binding domain sequence selected from Table 1 sequence, over the entire length of such sequence selected from Table 1.
The polypeptides of the invention include a polypeptide comprising an ERA binding domain sequence selected from Table 1 (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of Era, and also those which have at least 70% identity to a polypeptide comprising an ERA binding domain sequence selected from Table 1 or the relevant portion, preferably at least 80% identity to a polypeptide comprising an ERA binding domain sequence selected from Table 1 and more preferably at least 90% identity to a polypeptide comprising an ERA binding domain sequence selected from Table 1 and still more preferably at least 95% identity to a polypeptide comprising an ERA binding domain sequence selected from Table 1 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids. The invention also includes a polypeptide consisting of or comprising a polypeptide of the formula:
X-(R1)m-(R2)-(R3)n-Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety described herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety described herein for modified polypeptides, R^ and R3 are any amino acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R2 is an amino acid sequence of the invention, particularly an amino acid sequence comprising an ERA binding domain sequence selected from selected from Table 1 or modified forms thereof. In the formula above, R2 is oriented so that its amino terminal amino acid residue is at the left, covalently bound to Rj and its carboxy terminal amino acid residue is at the right, covalently bound to R3. Any stretch of amino acid residues denoted by either R or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
It is most preferred that a polypeptide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
A fragment is a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention. As with ERA binding domain polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence comprising an ERA binding domain sequence selected from Table 1, or of variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl- terminal amino acid sequence. Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Streptococcus pneumoniae, are also preferred. Further preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
Also preferred are biologically active fragments, which are those fragments that mediate activities of Era, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Streptococcus pneumoniae or the ability to initiate, or maintain cause Disease in an individual, particularly a human.
Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
In addition to the standard single and triple letter representations for amino acids, the term "X" or "Xaa" may also be used in describing certain polypeptides of the invention. "X" and "Xaa" mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the polypeptide sequence.
Polynucleotides
It is an object of the invention to provide polynucleotides that encode ERA binding domain polypeptides, particularly polynucleotides that encode the polypeptide herein designated Era. In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding ERA binding domain polypeptides comprising a sequence comprising an ERA binding domain sequence selected from Table 1 which includes a full length gene, or a variant thereof. The Applicants believe that this full length gene is essential to the growth and/or survival of an organism which possesses it, such as Streptococcus pneumoniae.
As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressing ERA binding domain polypeptides and polynucleotides, particularly Streptococcus pneumoniae ERA binding domain polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.
Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a ERA binding domain polypeptide having a deduced amino acid sequence of Table 1 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a ERA binding domain polypeptide from Streptococcus pneumoniae comprising or consisting of an amino acid sequence of Table 1, or a variant thereof. Using the information provided herein, such as a polynucleotide sequence encoding a polypeptide sequence set out in Table 1, a polynucleotide of the invention encoding ERA binding domain polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Streptococcus pneumoniae 0100993 cells as starting material, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence encoding a polypeptide given in Table 1, typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions. By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence. Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence. Moreover, each DNA sequence encoding a polypeptide sequence set out in Table 1 contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art.
In a further aspect, the present invention provides for an isolated polynucleotide comprising or consisting of: (a) a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to a sequence encoding a sequence comprising an ERA binding domain sequence selected from Table 1 over the entire length of such sequence selected from Table 1 ; (b) a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or 100% exact, to the amino acid sequence of a sequence comprising an ERA binding domain sequence selected from Table 1, over the entire length of such sequence selected from Table 1. A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than Streptococcus pneumoniae, may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled or detectable probe consisting of or comprising a region encoding a sequence from Table 1 or a fragment thereof; and isolating a full-length gene and or genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) comprising an ERA binding domain sequence comprising an ERA binding domain sequence selected from Table 1. Also provided by the invention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence. The polynucleotide of the invention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho- independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al, Cell 37: 767 (1984), both of which 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.
The invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:
X-(R1)m-(R2)-(R3)n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of Rj and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1 or a modified nucleic acid sequence thereof. In the polynucleotide formula above, R is oriented so that its 5' end nucleic acid residue is at the left, bound to R\ and its 3' end nucleic acid residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either R\ and/or R2, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where, in a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary. In another preferred embodiment m and/or n is an integer between 1 and 1000. Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
It is most preferred that a polynucleotide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae ERA binding domain having an amino acid sequence set out in Table 1. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence comprising an ERA binding domain sequence selected from Table 1. Fragments of a polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding ERA binding domain variants, that have the amino acid sequence of ERA binding domain polypeptide comprising an ERA binding domain sequence selected from Table 1 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of ERA binding domain polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding ERA binding domain polypeptide having an amino acid sequence set out in Table 1, and polynucleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding ERA binding domain polypeptide and polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences. 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 0. lx SSC at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library containing the complete gene for a polynucleotide sequence encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1 under stringent hybridization conditions with a probe having the sequence derived from a polynucleotide encoding a polypeptide sequence selected from Table 1 or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein. As discussed elsewhere herein regarding polynucleotide assays of the invention, for instance, the polynucleotides of the invention, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding ERA binding domain and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the ERA binding domain gene. Such probes generally will comprise at least 15 nucleotide residues or base pairs. Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs. Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have less than 30 nucleotide residues or base pairs.
A coding region of a ERA binding domain gene may be isolated by screening using a DNA sequence encoding a region of a polypeptide set forth in Table 1 as an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to. There are several methods available and well known to those skilled in the art to obtain full-length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al, PNAS USA 85, 8998- 9002, 1988). Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product
(typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relating to polynucleotide assays.
The polynucleotides of the invention that are oligonucleotides derived from a sequence of Table 1 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
The invention also provides polynucleotides that encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.
For each and every polynucleotide of the invention there is provided a polynucleotide complementary to it. It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary. A precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins. In addition to the standard A, G, C, T/U representations for nucleotides, the term "N" may also be used in describing certain polynucleotides of the invention. "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.
In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
Vectors, Host Cells, Expression Systems
The invention also relates to vectors that comprise a polynucleotide 'or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques.
For recombinant production of the polypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, etal, BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as cells of streptococci, staphylococci, enterococci E. coli, streptomyces, cyanobacteria, Bacillus subtilis, and 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; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells, such as cells of a gymnosperm or angiosperm. A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picomavirases and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook etal, MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
In recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification. Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of ERA binding domain polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of ERA binding domain 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 ERA binding domain gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein. Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials. Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways. Using amplification, characterization of the 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 of the organism. Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled ERA binding domain polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics. Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried out with or without denaturing agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al, Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as Rnase, VI and SI protection assay or a chemical cleavage method. See, for example, Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising ERA binding domain nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al, Science, 274: 610 (1996)).
Thus in another aspect, the present invention relates to a diagnostic kit which comprises:
(a) a polynucleotide of the present invention, preferably the nucleotide sequence encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1, or a fragment thereof ;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide set forth in Table 1 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to a polypeptide comprising an ERA binding domain sequence selected from Table 1.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others. This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide of the invention, preferably, a sequence comprising an ERA binding domain sequence selected from Table 1, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.
The nucleotide sequences of the present invention are also valuable for organism chromosome identification. The sequence is specifically targeted to, and can hybridize with, a particular location on an organism's chromosome, particularly to a Streptococcus pneumoniae chromosome. The mapping of relevant sequences to chromosomes according to the present invention may be an important step in correlating those sequences with pathogenic potential and/or an ecological niche of an organism and/or drug resistance of an organism, as well as the essentiality of the gene to the organism. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data may be found on-line in a sequence database. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (coinheritance of physically adjacent genes) or mating studies, such as by conjugation.
The differences in a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined. If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype.
Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations in the RNA. It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose, PCR. As an example, PCR primers complementary to a polynucleotide encoding ERA binding domain polypeptide can be used to identify and analyze mutations.
These primers may be used for, among other things, amplifying ERA binding domain DNA and/or RNA isolated from a sample derived from an individual, such as a bodily material. The primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various techniques for elucidation of the polynucleotide sequence. In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent.
The invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Streptococcus pneumoniae, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1. Increased or decreased expression of a ERA binding domain polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
In addition, a diagnostic assay in accordance with the invention for detecting over- expression of ERA binding domain 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 determine levels of a ERA binding domain polypeptide, in a sample derived from a host, such as a bodily material, are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays. Differential Expression The polynucleotides and polynucleotides of the invention may be used as reagents for differential screening methods. There are many differential screening and differential display methods known in the art in which the polynucleotides and polypeptides of the invention may be used. For example, the differential display technique is described by Chuang et al., J. Bacteriol 775:2026-2036 (1993). This method identifies those genes which are expressed in an organism by identifying mRNA present using randomly-primed RT-PCR. By comparing pre-infection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF "unknowns."
In Vivo Expression Technology (IVET) is described by Camilli et al, Proc. Nat'l. Acad. Sci. USA. 97:2634-2638 (1994). IVET identifies genes up-regulated during infection when compared to laboratory cultivation, implying an important role in infection. ORFs identified by this technique are implied to have a significant role in infection establishment and/or maintenance. In this technique random chromosomal fragments of target organism are cloned upstream of a promoter-less recombinase gene in a plasmid vector. This construct is introduced into the target organism which carries an antibiotic resistance gene flanked by resolvase sites. Growth in the presence of the antibiotic removes from the population those fragments cloned into the plasmid vector capable of supporting transcription of the recombinase gene and therefore have caused loss of antibiotic resistance. The resistant pool is introduced into a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance. The chromosomal fragment carried by each antibiotic sensitive bacterium should carry a promoter or portion of a gene normally upregulated during infection. Sequencing upstream of the recombinase gene allows identification of the up regulated gene.
RT-PCR may also be used to analyze gene expression patterns. For RT PCR using the polynucleotides of the invention, messenger RNA is isolated from bacterial infected tissue, e.g., 48 hour murine lung infections, and the amount of each mRNA species assessed by reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs. The determination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed in the infected tissue. Analysis of gene transcription can be carried out at different times of infection to gain a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacterials. Because of the gene specific nature of the PCR primers employed it should be understood that the bacterial mRNA preparation need not be free of mammalian RNA. This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species which are very short lived in the bacterium (in the order of 2 minute halflives). Optimally the bacterial mRNA is prepared from infected murine lung tissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL) for very short periods of time, subsequent processing according to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA. Preferably the process is optimized by finding those conditions which give a maximum amount of Streptococcus pneumoniae 16S ribosomal RNA as detected by probing Northerns with a suitably labeled sequence specific oligonucleotide probe. Typically a 5' dye labeled primer is used in each PCR primer pair in a PCR reaction which is terminated optimally between 8 and 25 cycles. The PCR products are separated on 6% polyacrylamide gels with detection and quantification using GeneScanner (manufactured by ABI).
Gridding and Polynucleotide Subtraction
Methods have been described for obtaining information about gene expression and identity using so called "high density DNA arrays" or grids. See, e.g., M. Chee et al., Science. 274:610-614 (1996) and other references cited therein. Such gridding assays have been employed to identify certain novel gene sequences, referred to as Expressed Sequence Tags (EST) (Adams et a., Science, 252:1651-1656 (1991)). A variety of techniques have also been described for identifying particular gene sequences on the basis of their gene products. For example, see International Patent Application No. WO91/07087, published May 30, 1991. In addition, methods have been described for the amplification of desired sequences. For example, see International Patent Application No. WO91/17271, published November 14, 1991.
The polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high density arrays or grids. These high density arrays are particularly useful for diagnostic and prognostic purposes. For example, a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention, may be used for probing, such as using hybridization or nucleic acid amplification, using a probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual. Such a presence may indicate the presence of a pathogen, particularly Streptococcus pneumoniae, and may be useful in diagnosing and/or prognosing disease or a course of disease. A grid comprising a number of variants of the polynucleotide sequences of the invention are preferred. Also preferred is a comprising a number of variants of a polynucleotide sequence encoding polypeptide sequences of Table 1. Antibodies
The polypeptides and polynucleotides of the invention or variants thereof, or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively.
In certain preferred embodiments of the invention there are provided antibodies against ERA binding domain polypeptides or polynucleotides .
Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and/or polynucleotides of the invention, or epitope- bearing fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985). Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the invention.
Alternatively, phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-Era or from naive libraries (McCafferty, et al, (1990), Nature 348, 552-554; Marks, et al, (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et al, (1991) Nature 352: 628). The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for example, affinity chromatography.
Thus, among others, antibodies against Era-polypeptide or Era-polynucleotide may be employed to treat infections, particularly bacterial infections. Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form a particular aspect of this invention.
A polypeptide or polynucleotide of the invention, such as an antigenically or immunologically equivalent derivative or a fusion protein of the polypeptide is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide. The antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin, keyhole limpet haemocyanin or tetanus toxoid. Alternatively, a multiple antigenic polypeptide comprising multiple copies of the polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be "humanized," where the complimentarity determining region or regions of the hybridoma- derived antibody has been transplanted into a human monoclonal antibody, for example as described in lones et al. (1986), Nature 321, 522-525 or Tempest et al, (1991) Biotechnology 9, 266-273.
In accordance with an aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of ERA binding domain polynucleotides and polypeptides encoded thereby.
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al, Hum Mol Genet (1992) 1: 363, Manthorpe et al, Hum. Gene Ther. (1983) 4: 419), delivery of DNA complexed with specific protein 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 liposomes (Kaneda et al, Science (1989) 243: 375), particle bombardment (Tang et al, Nature (1992) 356:152, Eisenbraun et al, DNA Cell Biol (1993) 12: 791) and in vivo infection using cloned retroviral vectors (Seeger et al., PNAS USA (1984) 81: 5849).
Antagonists and Agonists - Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al, Current Protocols in Immunology 1(2): Chapter 5 (1991).
Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned. It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides and polynucleotides. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of ERA binding domain polypeptides and polynucleotides; or may be structural or functional mimetics thereof (see Coligan et al, Current Protocols in Immunology l(2):Chapter 5 (1991)).
The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring ERA binding domain polypeptide and/or polynucleotide activity in the mixture, and comparing the ERA binding domain polypeptide and/or polynucleotide activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and ERA binding domain polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. lohanson et al, J Biol Chem, 270(16):9459-9471 (1995)). Preferred screening methods include, for example, screening for an interaction between ERA binding domain and any agent can be measured with physical techniques such as fluorescence polarization (FP), flourescence energy transfer (FET), surface plasmon resonance (SPR), scintillation proximity assay (SPA), and radioimmune assay (RIA). The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate. Upon binding of an agent to the labelled molecule, the tumbling rate will slow and thus the increase in fluorescence polarization can be directly measured.
A scintillation proximity assay can be used to characterize the interaction between ERA binding domain and an agent. Either ERA binding domain or the agent is coupled to a scintillation-filled bead. Addition of radio-labelled ERA binding domain or agent to the coupled beads results in binding where the radioactive source molecule is in close proximity to the scintillation fluid. Thus, signal is emitted upon binding.
The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of ERA binding domain polypeptides or polynucleotides, particularly those compounds that are bacteriostatic and or bacteriocidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising ERA binding domain polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a ERA binding domain agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the ERA binding domain polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i.e., without inducing the effects of ERA binding domain polypeptide are most likely to be good antagonists. Molecules that bind well and, as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists. Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in ERA binding domain polynucleotide or polypeptide activity, and binding assays known in the art.
Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, 1^1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supematants, tissue extracts, bodily materials). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.
In other embodiments of the invention there are provided methods for identifying compounds which 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 pennit 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 of the polypeptide and/or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and/or polynucleotide. Another example of an assay for ERA binding domain agonists is a competitive assay that combines ERA binding domain and a potential agonist with Era-binding molecules, recombinant ERA binding domain binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. ERA binding domain can be labeled, such as by radioactivity or a colorimetric compound, such that the number of ERA binding domain molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
Potential antagonists include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing Era-induced activities, thereby preventing the action or expression of ERA binding domain polypeptides and/or polynucleotides by excluding ERA binding domain polypeptides and/or polynucleotides from binding.
Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules). Preferred potential antagonists include compounds related to and variants of Era. Other examples of potential polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
Certain of the polypeptides of the invention are biomimetics, functional mimetics of the natural ERA binding domain polypeptide. These functional mimetics may be used for, among other things, antagonizing the activity of ERA binding domain polypeptide or as a antigen or immunogen in a manner described elsewhere herein. Functional mimetics of the polypeptides of the invention include but are not limited to truncated polypeptides. For example, preferred functional mimetics include, a polypeptide comprising an ERA binding domain sequence selected from Table 1 lacking 20, 30, 40, 50, 60, 70 or 80 amino- or carboxy-terminal amino acid residues, including fusion proteins comprising one or more of these truncated sequences. Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide. It is preferred that these cassettes comprise 5' and 3' restriction sites to allow for a convenient means to ligate the cassettes together when desired. It is further preferred that these cassettes comprise gene expression signals known in the art or described elsewhere herein. Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for a polypeptide and/or polynucleotide of the present invention; or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises:
(a) a polypeptide and/or a polynucleotide of the present invention;
(b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention;
(c) a cell membrane expressing a polypeptide and/or polynucleotide of the present invention; or
(d) antibody to a polypeptide and/or polynucleotide of the present invention; which polypeptide preferably comprises an ERA binding domain sequence selected from Table 1, and which polynucleotide is preferably a polynucleotide encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide and/or polynucleotide, by: (a) determining in the first instance the three-dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof; (b) deducing the three-dimensional structure for the likely reactive site(s), binding site(s) or motif(s) of an agonist, antagonist or inhibitor;
(c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding site(s), reactive site(s), and/or motif(s); and
(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors. It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps.
In a further aspect, the present invention provides methods of treating abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of ERA binding domain polypeptide and/or polynucleotide. If the expression and/or activity of the polypeptide and/or polynucleotide is in excess, several approaches are available. One approach comprises administering to an individual in need thereof an inhibitor compound (antagonist) as herein described, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous polypeptide and/or polynucleotide may be administered. Typical examples of such competitors include fragments of the ERA binding domain polypeptide and/or polypeptide.
In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGi, where fusion takes place at the hinge region. In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa. Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914.
In still another approach, expression of the gene encoding endogenous ERA binding domain polypeptide can be inhibited using expression blocking techniques. This blocking may be targeted against any step in gene expression, but is preferably targeted against 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, 7 Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Alternatively, oligonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney et al, Science (1988) 241:456; Dervan et al, Science (1991) 251:1360). These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
Each of the polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein, upon expression,- can be used as a target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection. In particular, the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block ERA binding domain protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al., Infect. Immun. 60:2211 (1992); to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial ERA binding domain 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.
This invention provides a method of screening drugs to identify those which are antibacterial by measuring the ability of the drug to interfere with the biosynthesis of
UDP-N-acetylmuramateby the enzyme.
It has been shown that E.coli UDP-Ν acetylenolpyruvylglucosamine reductase catalyses the reduction of UDP-N-acetylglucosamine enolpyruvate with the concommitanst oxidation of ΝADPH.
In a preferred embodiment, UDP-N-acetylenolpyruvylglucosamine is incubated with ΝADPH in the presence of the UDP-N-acetylenolpyruvylglucosamine reductase protein to generate ΝADP which can be measured spectrophotometrically at 340nm (Benson, T.E., Marquardt, J.L., Marquardt, A.C., Etzkorn, FA. & Walsh, C.T., [1993], Biochemistry, 32,
2024-2030) to provide a measurement of UDP-Ν-acetylenolpyruvylglucosamine reductase enzymatic activity. The decrease of enzymatic activity in this reaction would indicate the presence of an inhibitor.
In accordance with yet another aspect of the invention, there are provided ERA binding domain agonists and antagonists, preferably bacteriostatic or bacteriocidal agonists and antagonists.
The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.
Helicobaeter pylori (herein "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 Helicobaeter Pylori (International Agency for Research on Cancer, Lyon, France, http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agency for Research on Cancer recently recognized a cause-and- effect relationship between H. pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen. Preferred antimicrobial compounds of the invention (agonists and antagonists of ERA binding domain polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H. pylori-mdaced cancers, such as gastrointestinal carcinoma. Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis. Vaccines
There are provided by the invention, products, compositions and methods for assessing ERA binding domain expression, treating disease, assaying genetic variation, and administering a ERA binding domain polypeptide and/or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Streptococcus pneumoniae bacteria. Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with ERA binding domain polynucleotide and/or polypeptide, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Streptococcus pneumoniae infection. Also provided are methods whereby such immunological response slows bacterial replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of ERA binding domain polynucleotide and/or polypeptide, or a fragment or a variant thereof, for expressing ERA binding domain polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not. One example of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA- protein complex.
A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, induces an immunological response in such individual to a ERA binding domain polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant ERA binding domain polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said ERA binding domain polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells.
A ERA binding domain polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or immunogenic properties, and preferably protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co-protein which solubilizes the protein and facilitates production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protein. The co- protein may be attached to either the amino- or carboxy-terminus of the first protein.
Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996). .
Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof, which have been shown to encode non-variable regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Streptococcus pneumoniae. Such experiments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Streptococcus pneumoniae infection, in mammals, particularly humans.
A polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage include wounds in skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina. The invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
While the invention has been described with reference to certain ERA binding domain polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant polypeptides or polynucleotides. Compositions, kits and administration
In a further aspect of the invention there are provided compositions comprising a ERA binding domain polynucleotide and/or a ERA binding domain polypeptide for administration to a cell or to a multicellular organism. The invention also relates to compositions comprising a polynucleotide and/or a polypeptides discussed herein or their agonists or antagonists. The polypeptides and polynucleotides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides, polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation. In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Polypeptides, polynucleotides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The composition will be adapted to the route of administration, for instance by a systemic or an oral route. Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like.
For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.
The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device. In addition, the composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especially Streptococcus pneumoniae wound infections.
Many orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia. Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality. It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation.
In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis.
Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion. The active agent will preferably be present at a concentration of 1 μg/ml to lOmg/ml for bathing of wounds or indwelling devices. A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.
GLOSSARY The following definitions are provided to facilitate understanding of certain terms used frequently herein. "Antibody(ies)" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library. "Antigenically equivalent derivative(s)" as used herein encompasses a polypeptide, polynucleotide, or the equivalent of either which will be specifically recognized by certain antibodies which, when raised to the protein, polypeptide or polynucleotide according to the invention, interferes with the immediate physical interaction between pathogen and mammalian host.
"Bispecific antibody(ies)" means an antibody comprising at least two antigen binding domains, each domain directed against a different epitope.
"Bodily material(s) means any material derived from an individual or from an organism infecting, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy materials.. "Disease(s)" means any disease caused by or related to infection by a bacteria, 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. "Fusion protein(s)" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232262]. On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified.
"Host cell(s)" is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity. Parameters for polypeptide sequence comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4
A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
Parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3
Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons. A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below. (1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to a reference sequence of Table 1, wherein said polynucleotide sequence may be identical to a reference sequence of Table 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 of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in a given sequence by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in said given sequence, or:
nn ≤ n - (xn • y),
wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in a given sequence, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn. Alterations of a polynucleotide sequence encoding a polypeptide of Table 1 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations. By way of example, a polynucleotide sequence of the present invention may be identical to a reference sequence of Table 1, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of amino acids in a polypeptide of Table 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in said polypeptide, or:
nn < xπ - (xn • y),
wherein nn is the number of amino acid alterations, xn is the total number of amino acids in a polypeptide in Table 1, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn.
(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of a polypeptide of Table 1, wherein said polypeptide sequence may be identical to a reference sequence of Table 1 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in a polypeptide of Table 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in said polypeptide, or:
na < xa - (xa • y),
wherein na is the number of amino acid alterations, xa is the total number of amino acids in a polypeptide of Table 1, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of a polypeptide of Table 1, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in a polypeptide of Table 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in said sequence, or:
na < xa - (xa • y),
wherein na is the number of amino acid alterations, xa is the total number of amino acids in a polypeptide of Table 1, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa. "Immunologically equivalent derivative(s)" as used herein encompasses a polypeptide, polynucleotide, or the equivalent of either which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host. "Immunospecific" means that characteristic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the invention or the polynucleotides of the invention than its affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the prior art. "Individual(s)" means a multicellular eukaryote, including, 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, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said organism, which organism may be living or non-living.
"Organism(s)" means a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Cotynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Achaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans. "Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)" also embraces short polynucleotides often referred to as oligonucleotide(s).
"Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxy lation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol 182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
"Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the invention introduced or transformed into a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the invention.
"Subtraction set" is one or more, but preferably less than 100, polynucleotides comprising at least one polynucleotide of the invention
"Variant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in 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 mclude variants of each of the polypeptides of the invention, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred 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 occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans. EXAMPLES
The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention.
Example 1 Strain selection, Library Production and Sequencing
The polynucleotide having a DNA sequence given below in this Example 1 was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E. coli. The sequencing data from two or more clones containing overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in Example 1. Libraries may be prepared by routine methods, for example: Methods 1 and 2 below. Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 according to standard procedures and size-fractionated by either of two methods. Method 1
Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures. DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coli infected with the packaged library. The library is amplified by standard procedures. Method 2
Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures. EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library. The library is amplified by standard procedures.
(A) Sequences from Streptococcus pneumoniae era polynucleotide sequence
5 ' - ATGACTTTTAAATCAGGCTTTGTAGCCATTTTAGGACGTCCCAATGTTGGGAAGTCAACC TTTTTAAATCACGTTATGGGGCAAAAGATTGCCATCATGAGTGACAAGGCGCAGACAACG CGCAATAAAATCATGGGAATTTACACGACTGATAAGGAGCAAATTGTCTTTATCGACACA CCAGGGATTCACAAACCTAAAACAGCTCTCGGAGATTTCATGGTTGAGTCTGCCTACAGT ACCCTTCGCGAAGTGGACACTGTTCTTTTCATGGTGCCTGCTGATGAAGCGCGTGGTAAG GGGGACGATATGATTATCGAGCGTCTCAAGGCTGCCAAGGTTCCTGTGATTTTGGTGGTG AATAAAATCGATAAGGTCCATCCAGACCAGCTCTTGTCTCAGATTGATGACTTCCGTAAT CAAATGGACTTTAAGGAAATTGTTCCAATCTCAGCCCTTCAGGGAAATAACGTGTCTCGT CTAGTGGATATTTTGAGTGAAAATCTGGATGAAGGTTTCCAATATTTCCCGTCTGATCAA ATCACAGACCATCCAGAACGTTTCTTAGTTTCAGAAATGGTTCGCGAGAAAGTCTTGCAC CTAACTCGTGAAGAGATTCCGCATTCTGTAGCAGTAGTTGTTGACTCTATGAAACGAGAC GAAGAGACAGACAAGGTTCACATCCGTGCAACCATCATGGTCGAGCGCGATAGCCAAAAA GGGATTATCATCGGTAAAGGTGGCGCTATGCTTAAGAAAATCGGTAGCATGGCCCGTCGT GATATCGAACTCATGCTAGGAGACAAGGTCTTCCTAGAAACCTGGGTCAAGGTCAAGAAA AACTGGCGCGATAAAAAGCTAGATTTGGCTGACTTGGGCTATAATGAAAGAGAATACTAA- 3 '
(B) era polypeptide sequence deduced from the polynucleotide sequence in Example 1(A). NH2-MTFKSGFVAILGRPNVGKSTFLNHVMGQKIAIMSDKAQTTRNKIMGIYTTDKEQIVFIDT
PGIHKPKTALGDFMVESAYSTLREVDTVLFMVPADEARGKGDDMIIERLKAAKVPVILWNKIDKVHPDQ
LLSQIDDFRNQMDFKEIVPISALQGNNVSRLVDILSENLDEGFQYFPSDQ
ITDHPERFLVSEMVREKVLHLTREEIPHSVAVWDSMKRDEETDKVHIRATIMVERDSQK GIIIGKGGAMLKKIGSMARRDIELMLGDKVFLETWVKVKKNWRDKKLDLADLGYNEREY -COOH
Example 2 Essentiality of ERA binding domain from S.pneumoniae. An allelic replacement cassette is generated using PCR technology. The cassette typically consists of a pair of 500bp chromosomal DNA fragments flanking an erythromycin resistance gene. The chromosomal DNA sequences are usually the 500bp preceding and following the gene of interest. Attempts are made to introduce the allelic replacement cassette into S. pneumoniae R6 or S. pneumoniae 100993 by transformation. Competent cells are prepared according to published protocols. DNA is introduced into the cells by incubation of 500ng of allelic replacement cassette with 10" cells at 30°C for 30 minutes. The cells are transferred to 37^C for 90 minutes to allow expression of the erythromycin resistance gene. Cells are plated in agar containing lug erythromycin per ml. Following incubation at 37°C for 36 hours, any observed colonies are picked and grown overnight in Todd-Hewitt broth supplemented with 0.5% yeast extract. Typically, in positive control experiments carried out in parallel which target a non-essential gene, 10^-10^ transformants containing the appropriate allelic replacement are obtained. If erythromycin resistant colonies are only observed in transformation experiments using S. pneumoniae R6, DNA from these cells are used to transform S. pneumoniae 100993. The transformation procedure is identical to that for S. pneumoniae R6 except that a competence stimulating heptadecapeptide (Havarstein et al., (1995) P.N.A.S. 92, 11140-11144) is added at a concentration of lug/ml in the initial transformation mix. Mutants are selected by their ability to grow in agar containing lug erythromycin per ml.
If no transformants are obtained in three separate transformation experiments, then the target gene is considered as being essential in vitro. Based on these analyses Era from S.pneumoniae was shown to be essential in vitro.
Example 3 The determination of expression during infection of a gene from Streptococcus pneumoniae
Excised lungs from a 48 hour respiratory tract infection of Streptococcus pneumoniae
0100993 in the mouse is efficiently disrupted and processed in the presence of chaotropic agents and RNAase inhibitor to provide a mixture of animal and bacterial RNA. The optimal conditions for disruption and processing to give stable preparations and high yields of bacterial RNA are followed by the use of hybridisation to a radiolabelled oligonucleotide specific to Streptococcus pneumoniae 16S RNA on Northern blots. The RNAase free, DNAase free, DNA and 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)
Streptococcus pneumoniae 0100993 is grown either on TSA/5%horse blood plates or in AGCH medium overnight, 37°C, 5%C02. Bacteria are then collected and resuspended in phosphate-buffered saline to an A600 of approximately 0.4. Mice are anaesthetized with isofluorane and 50ml of bacterial suspension (approximately 2 x 105 bacteria) is administered intranasally using 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 Infected tissue samples, in 2-ml cryo-strorage tubes, are removed from -80°C storage into a dry ice ethanol bath. In a microbiological safety cabinet the samples are disrupted up to eight at a time while the remaining samples are kept frozen in the dry ice ethanol bath. To disrupt the bacteria within the tissue sample, 50-100 mg of the tissue is transfered to a FastRNA tube containing a silica/ceramic matrix (BIO101). Immediately, 1 ml of extraction reagents (FastRNA reagents, BIO 101) are added to give a sample to reagent volume ratio of approximately 1 to 20. The tubes are shaken in a reciprocating shaker (FastPrep FP120, BIO101) at 6000 rpm for 20-120 sec. The crude RNA preparation is extracted with chloroform isoamyl alcohol, and precipitated with DEPC-treated/Isopropanol Precipitation Solution (BIO101). RNA preparations are stored in this isopropanol solution at -80°C if necessary. The RNA is pelleted (12,000g for 10 min.), washed with 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 min, and resuspended in 0.1 ml of DEPC-treated water, followed by 5-10 minutes at 55 °C. Finally, after at least 1 minute on ice, 200 units of Rnasin (Promega) is added. RNA preparations are stored at -80 °C for up to one month. For longer term storage the RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one year at -20 °C.
Quality of the RNA isolated is assessed by running samples on 1% agarose gels. 1 x
TBE gels stained with ethidium bromide are used to visualise total RNA yields. To demonstrate the isolation of bacterial RNA from the infected tissue 1 x MOPS, 2.2M formaldehyde gels are run and vacuum blotted to Hybond-N (Amersham). The blot is then hybridised with a 32P-labelled oligonucletide probe, of sequence 5' AACTGAGACTGGCTTTAAGAGATTA 3', specific to 16S rRNA of Streptococcus pneumoniae. The size of the hybridising band is compared to that of control RNA isolated from in vitro grown Streptococcus pneumoniae 0100993 in the Northern blot. Correct sized bacterial 16S rRNA bands can be detected in total RNA samples which show degradation of the mammalian RNA when visualised on TBE gels, c) The removal of DNA from Streptococcus pneumoniae -derived RNA
DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with 20 units of RNAase-free DNAasel (GenHunter) in the buffer supplied in a final volume of 57 microliters .
The DNAase was inactivated and removed by treatment with TRIzol LS Reagent
(Gibco BRL, Life Technologies) according to the manufacturers protocol.
DNAase treated RNA was resuspended in 100 microlitres of DEPC treated water with the addition of Rnasin as described before. d) The preparation of cDNA from RNA samples derived from infected tissue
3 microgram samples of DNAase treated RNA are reverse transcribed using.a
Superscript Preamplification System for First Strand cDNA Synthesis kit (Gibco BRL, Life
Technologies) according to the manufacturers instructions. 150 nanogram of random hexamers is used to prime each reaction. Controls without the addition of SuperScriptll reverse transcriptase are also run. Both +/-RT samples are treated with RNaseH before proceeding to the PCR reaction e) The use of PCR to determine the presence of a bacterial cDNA species
PCR reactions are set up on ice in 0.2ml tubes by adding the following components: 43 microlitres PCR Master Mix (Advanced Biotechnologies Ltd.); 1 microlitre PCR primers (optimally 18-25 basepairs in length and designed to possess similar annealing temperatures), each primer at lOmM initial concentration; and 5 microlitres 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 microlitre aliquots are then run out on 1% 1 x TBE gels stained 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 sequencing gel and its presence and quantity detected using a suitable gel scanning system (e.g. ABI Prism™ 377 Sequencer using GeneScan™ software as supplied by Perkin Elmer). RT/PCR controls may include +/- reverse transcriptase reactions, 16S rRNA primers 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 of the cDNA.
Primer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are uninformative. Of those which give the correct size product with DNA PCR two classes are distinguished in RT PCR: 1. Genes which are not transcribed 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 in RT/PCR and show a stronger signal in the +RT samples than the signal (if at all present) in -RT controls
Based on these analyses it was discovered that the Streptococcus pneumoniae era gene was transcribed in vivo.

Claims

What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
(i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least:
(a) 70% identity;
(b) 80% identity;
(c) 90% identity; or
(d) 95% identity to an amino acid sequence comprising an ERA binding domain sequence selected from Table 1 over the entire length of such seqeucne of Table 1; (ii) an isolated polypeptide comprising an amino acid sequence comprising an ERA binding domain sequence selected from Table 1 or (iii) an isolated polypeptide which is an amino acid sequence comprising an ERA binding domain sequence selected from Table 1.
2. An isolated polynucleotide selected from the group consisting of:
(i) an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide that has at least (a) 70% identity;
(b) 80% identity;
(c) 90% identity; or
(d) 95% identity; to an amino acid sequence comprising an ERA binding domain sequence selected from Table 1, over the entire length of a sequence of Table 1 ;
(ii) an isolated polynucleotide comprising a nucleotide sequence that has at least:
(a) 70% identity
(b) 80% identity;
(c) 90% identity; or (d) 95% identity; over its entire length to a nucleotide sequence encoding a polypeptide of ;
(iii) an isolated polynucleotide comprising a nucleotide sequence which has at least:
(a) 70% identity;
(b) 80% identity; (c) 90% identity; or
(d) 95% identity; to that of a polynucleotide encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1 over the entire length of such polypeptide in Table 1; (iv) an isolated polynucleotide which is a polynucleotide encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1; or
(v) an isolated polynucleotide obtainable by screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of a polynucleotide encoding a polypeptide comprising an ERA binding domain sequence selected from Table 1 or a fragment thereof.
3. An antibody immunospecific for the polypeptide of claim 1.
4. A method for the treatment of an individual: (i) in need of enhanced activity or expression of the polypeptide of claim 1 comprising:
(a) administering to the individual a therapeutically effective amount of an agonist to said polypeptide; and/or
(b) providing to the individual an isolated polynucleotide comprising a nucleotide sequence encoding said polypeptide in a form so as to effect production of said polypeptide activity in vivo.; or
(ii) having need to inhibit activity or expression of the polypeptide of claim 1 comprising:
(a) administering to the individual a therapeutically effective amount of an antagonist to said polypeptide; and/or (b) administering to the individual a nucleic acid molecule that inhibits the expression of a nucleotide sequence encoding said polypeptide; and/or
(c) administering to the individual a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate , or receptor.
5. A process for diagnosing a disease or a susceptibility to a disease in an individual related to expression or activity of the polypeptide of claim 1 in an individual comprising: (a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in the genome of said individual; and/or
(b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said individual.
6. A method for screening to identify compounds that activate and/or that inhibit the function of the polypeptide of claim 1 which comprises a method selected from the group consisting of:
(a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing or comrpising the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound;
(b) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing or comprising the polypeptide) or a fusion protein thereof in the presence of a labeled competitor;
(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide;
(d) mixing a candidate compound with a solution containing a polypeptide of claim 1, to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard; (e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay, or (f) (1) contacting a composition comprising the polypeptide with the compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound; and
(2) determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the interaction of the compound with the polypeptide.
7. An agonist or an antagonist of the activity or expression polypeptide of claim 1.
8. An expression system comprising a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell.
9. A host cell comprising the expression system of claim 8 or a membrane thereof expressing the polypeptide of claim 1.
10. A process for producing a polypeptide of claim 1 comprising culturing a host cell of claim 9 under conditions sufficient for the production of said polypeptide.
11. A process for producing a host cell as defined in claim 9 comprising transforming or transfecting a cell with the expression system of claim 8 such the host cell, under appropriate culture conditions, produces a polypeptide of claim 1.
12. A host cell produced by the process of claim 11 or a membrane thereof expressing a polypeptide of claim 1.
PCT/US2001/001786 2000-01-18 2001-01-17 Methods and reagents for performing antimicrobial compound screening WO2001053458A2 (en)

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US60/176,870 2000-01-18

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Cited By (2)

* Cited by examiner, † Cited by third party
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US20110189693A1 (en) * 2010-02-03 2011-08-04 Industry-Academic Cooperation Foundation, Yonsei University Methods for Cancer Diagnosis, Anti-Cancer Drug Screening, and Test of Drug Effectiveness on the Basis of Phoshorylation of Ras at Thr-144 and Thr-148
WO2012162628A3 (en) * 2011-05-25 2014-06-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Lipopeptide inhibitors of ras oncoproteins

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110189693A1 (en) * 2010-02-03 2011-08-04 Industry-Academic Cooperation Foundation, Yonsei University Methods for Cancer Diagnosis, Anti-Cancer Drug Screening, and Test of Drug Effectiveness on the Basis of Phoshorylation of Ras at Thr-144 and Thr-148
KR101533469B1 (en) * 2010-02-03 2015-07-02 연세대학교 산학협력단 Methods for cancer diagnosis, anti-cancer drug screening, and test of drug effectiveness on the basis of phoshorylation of Ras at Thr-144 and Thr-148
WO2012162628A3 (en) * 2011-05-25 2014-06-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Lipopeptide inhibitors of ras oncoproteins
US9328142B2 (en) 2011-05-25 2016-05-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Lipopeptide inhibitors of RAS oncoproteins

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