This application claims benefit of GB application number 96190723, filed September 12, 1996 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, in these and in other regards, the invention relates to novel polynucleotides and polypeptides of the tryptophanyl tRNA syπthetase family, hereinafter referred to as "trpS BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, onus media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid Since its isolation more than 100 years ago, Streptococcus pneumomae has been one of the more intensively studied microbes For example, much of our early understanding that DNA is, in fact, the genetic mateπal was predicated on the work of Gπffith and of Avery, Macleod and McCarty using this microbe Despite the vast amount of research with 5 pneumomae. many questions concerning the virulence of this microbe remain It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics

The frequency of Streptococcus pneumomae infections has πsen dramatically in the past 20 years This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Streptococcus pneumomae strains which are resistant to some or all of the standard antibiotics This has created a demand for both new anti-microbial agents and diagnostic tests for this organism t-RNA synthetases have a pn ary role in protein synthesis according to the following scheme

Enzyme +ATP + AA ^**■ > Enzyme AA-AMP + PPi

Enzv e AA-AMP + t-RNA <=> Enzyme + AMP + AA-t-RNA

in which AA is an ammo acid

Inhibition of this process leads to a reduction in the levels of charged t-RNA and this tπggers a cascade of responses known as the stπngent response, the result ol which is the induction of a state of dormancy in the organism As such selective inhibitors of bacteπal t- RNA synthetase have potential as antibacteπal agents One example of such is mupiroαn which is a selective inhibitor of isoleucyl t-RNA synthetase Other t-RNA synthetases are now being examined as possible anti-bacteπal targets, this process being greatly assisted by the isolation of the synthetase

Clearly, there is a need for factors, such as the novel compounds of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disea.se There is also a need for identification and characteπzation of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.

The polypeptides of the invention have ammo acid sequence homoiogy to a known Clostπdium longisporum tryptophanyl tRNA synthetase protein SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have been identified as novel trpS polypeptides by homoiogy between the amino acid sequence set out in Table 1 [SEQ ID NO 2] and a known ammo acid sequence or sequences of other proteins such as Clostπdium longisporum tryptophanyl tRNA synthetase protein.

It is a further object of the invention to provide polynucleotides that encode trpS polypeptides, particularly polynucleotides that encode the poiypeptide herein designated trpS

In a particularly preferred embodiment of the invention the polynucleotide compπses a region encoding trpS polypeptides compπsing the sequence set out in Table 1 [SEQ ID NO 1] which includes a frill length gene, or a vaπant thereof.

In another particularly preferred embodiment of the invention there is a novel trpS protein from Streptococcus pneumomae compπsing the ammo acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof

In accordance with another aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature poiypeptide expressible by the Streptococcus pneumomae 0100993 strain contained in the deposited strain

A further aspect of the invention there are provided isolated nucleic acid molecules encoding trpS, particularly Streptococcus pneumomae trpS, including rnRNAs, cDNAs, genomic DNAs Further embodiments of the invention include biologically, diagnostically,

prophylactically, clinically or therapeutical ly useful vaπants thereof, and compositions compπsing the same

In accordance with another 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 occurπng allelic vaπants of tηpS and polypeptides encoded thereby other aspect of the invention there are provided novel polypeptides of Streptococcus pneumomae referred to herein as trpS as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vaπants thereof, and compositions compπsing the same Among the particularly preferred embodiments of the invention are vaπants of trpS poiypeptide encoded by naturally occurπng alleles of the trpS gene

In a preferred embodiment of the invention there are provided methods for producing the aforementioned trpS polypeptides

In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies

In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing trpS expression, treating disease, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleura! empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospmal fluid, assaying genetic vaπation, and administeπng a trpS poiypeptide or polynucleotide to an organism to raise an immunological response against a bacteπa, especially a Streptococcus pneumomae bacteπa

In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybπdize to tipS polynucleotide sequences, particularly under stπngent conditions

In certain preferred embodiments of the invention there are provided antibodies against trpS polypeptides

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 of a poiypeptide or polynucleotide of the invention compπsing contacting a poiypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the poiypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the

binding or interaction of the poiypeptide or polynucleotide with the compound, and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activitv of the poiypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the poiypeptide or polynucleotide In accordance with yet another aspect of the invention, there are provided trpS agonists and antagonists, preferably bacteπostatic or bacteπocidal agonists and antagonists

In a further aspect of the invention there are provided compositions compπsing a trpS polynucleotide or a trpS poiypeptide for administration to a cell or to a multicellular organism

Vaπous changes and modifications within the spiπt and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descπptions and from reading the other parts of the present disclosure GLOSSARY

The following definitions are provided to facilitate understanding of certain terms used frequently herein "Host cell" 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 poiypeptide sequences or two or more polynucleotide sequences, as determined by compaπng the sequences In the art, ' identity ' also means the degree of sequence relatedness between poiypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" and "similarity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Bιolog\, 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 Analyst*; in Molecular Biology, von Heinje, G , Academic Press, 1987, and Sequence Analysis Primer, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991 , and Canllo, H , and Lipman, D , SIAM J Applied Math . 48 1073 (1988) Preferred methods to determine identity are designed to give the largest match between the sequences tested Methods to determine identity and similarity are codified in publicly available computer programs Preferred computer program methods to determine identity and similarity 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) As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO 1 it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO 1 In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence Analogously , by a poiypeptide having an amino acid sequence having at least, for example, 95% identity to a reference ammo acid sequence of SEQ ID NO 2 is intended that the ammo acid sequence of the poiypeptide is identical to the reference sequence except that the poiypeptide sequence may include up to five amino acid alterations per each 100 am o acids of the reference am o acid of SEQ ID NO 2 In other words, to obtain a poiypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the am o acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of ammo acids up to 5% of the total am o acid residues in the reference sequence may be inserted into the reference sequence These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence

"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 oπginal environment, or both For example, a polynucleotide or a poiypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or poiypeptide separated from the coexisting mateπals of its natural state is "isolated", as the term is employed herein

"Polynucleotιde(s)" generally refers to any polyπbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" include, without limitation, single- and double-stranded DNA. DNA that is a mixture of single- and double-stranded regions or single-, double- and tπple-stranded regions, single- and double-.stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybπd molecules compπsing DNA and RNA that may be single-stranded or, more typically, double-stranded, or tπple-stranded regions, or a mixture of single- and double-stranded regions In addition, 'polynucleotide" as used herein refers to tπple-stranded regions compπsing 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 tπple-he cal region often is an oligonucleotide As used herein, the term "poiynucieotιde(s)" also includes DNAs or RNAs as descπbed above that contain one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is intended herein Moreover, DNAs or RNAs compπsing unusual bases, such as inosine, or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herein It will be appreciated that a great vaπety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art The term "polynucleotιde(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 characteπstic of viruses and cells, including, for example, simple and complex cells "Polynucleotιde(s)" also embraces short polynucleotides often referred to as olιgonucleotιde(s)

"Polypeptιde(s)" refers to any peptide or protein compπsing two or more amino acids joined to each other by peptide bonds or modified peptide bonds "Polypeptιde(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 ammo acids "Polypeptιde(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 descπbed 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 poiypeptide Also, a given poiypeptide may contain many types of modifications Modifications can occur anywhere in a poiypeptide,

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, cyciization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methyiation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma- car oxylation 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.

"Variant(s)" as the term is used herein, is a polynucleotide or poiypeptide that differs from a reference polynucleotide or poiypeptide 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 poiypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the poiypeptide encoded by the reference sequence, as discussed below. A typical variant of a poiypeptide differs in amino acid sequence from another, reference poiypeptide. Generally, differences are limited so that the sequences of the reference poiypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference poiypeptide 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. A variant of a polynucleotide or poiypeptide 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 vaπants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled aπisans DESCRIPTION OF THE INVENTION The invention relates to novel trpS polypeptides and polynucleotides as descπbed in greater detail below In particular, the invention relates to polypeptides and polynucleotides of a novel trpS of Streptococcus pneumomae, which is related by amino acid sequence homoiogy to Ciostπdium longisporum tryptophanyl tRNA synthetase poiypeptide The invention relates especially to trpS having the nucleotide and am o acid sequences set out in Table 1 [SEQ ID NO. 1 ] and Table 1 [SEQ ID NO. 2] respectively, and to the trpS nucleotide sequences of the DNA in the deposited strain and ammo acid sequences encoded thereby

TABLE 1 trpS Polynucleotide and Poiypeptide Sequences

(A) Sequences from Streptococcus pneumomae trpS polynucleotide sequence [SEQ ID NO 1 ]

5 ' -1 ATGACTAAAC CCATTATTTT AACAGGAGAC CGTCCAACAG GAAAATTGCA

51 TATTGGACAT TATGTTGGAA GTCTCAAAAA TCGAGTATTA TTACAGGAAG

101 AGGATAAGTA TGATATGTTT GTGTTCTTGG CTGACCAACA AGCCTTGACA

151 GATCATGCCA AAGATCCTCA AACCATTGTA GAGTCTATCG GAAATGTGGC

201 TTTGGATTAT CTTGCAGTTG GATTGGATCC AAATAAGTCA ACTATTTTTA

251 TTCAAAGCCA GATTCCAGAG TTGGCTGAGT TGTCTATGTA TTATATGAAT

301 CTAGTTTCGT TAGCACGTTT GGAGCGAAAT CCAACAGTCA AGACAGAGAT

351 TTCTCAGAAA GGATTTGGAG AAAGCATTCC GACAGGATTC TTGGTCTATC

401 CAATCGCTCA AGCAGCTGAT ATCACAGCTT TCAAGGCT.AA TTATGTTCCT

451 GTTGGGACAG ATCAGAAACC AATGATTGAG CAAACTCGTG AAATTGTTCG

501 TTCTTTTAAC AATGCATATA ACTGTGATGT CTTGGTAGAG CCGGAAGGTA

551 TTTATCCAGA AAATGAGAGA GCAGGGCGTT TGCCTGGTTT AGATGGAAAT

601 GCTAAAATGT CTAAATCACT AAATAATGGT ATTTATTTAG CTGATGATGC

651 GGATACTTTG CGTAAAAAAG TAATGAGTAT GTATACAGAT CCAGATCATA

701 TCCGCGTTGA GGATCCAGGT AAGATTGAGG GAAATATGGT TTTCCATTAT

751 CTAGATGTTT TTGGTCGTCC AGAAGATGCT CAAGAAATTG CTGATATGAA

801 AGAACGTTAT CAACGAGGTG GTCTTGGTGA TGTGAAGACC AAGCGTTATC

851 TACTTGAAAT ATTAGAACGT GAACTGGGTC CTATTCGTGA GCGCCGTATT

901 GAATTTGCTA AGGATATGGG AGAAGTTTAT AATATGATTC AAAAAGGTAG

951 TGAAAGAGCG CGTGAAGTTG CGGGTCAAAC CCTATCTGAG GTAAAAGGGG

1001 CAATGGGACT TCATTACTTT AACTAA-3 '

(B) tφS poiypeptide sequence deduced from the polynucleotide sequence in this table

[SEQ ID NO:2J.

NH.-l MTKPIILTGD RPTGKLHIGH YVGSLKNRVL LQEEDKYDMF VFLADQQALT

51 DHAKDPQTIV ESIGNVALDY LAVGLDPNKS TIFIQSQIPE LAELSMYYMN

101 LVSLARLER PTVKTEISQK GFGESIPTGF LVYPIAQAAD ITAFKANYVP

151 VGTDQKPMIE QTREIVRSFN NAYNCDVLVE PEGIYPENER AGRLPGLDGN

201 AKMSKSLNNG IYLADDADTL RKKVMSMYTD PDHIRVEDPG KIEGNMVFHY

251 LDVFGRPEDA QEIADMKERY QRGGLGDVKT KRYLLEILER ELGPIRERRI

301 EFAKDMGEVY NMIQKGSERA REVAGQTLSE VKGAMGLHYF N-COOH

(C) Polynucleotide sequence embodiments [SEQ ID Nθ:l]. X-.Rl-rr ¹ ATGACTAAAC CCATTATTTT AACAGGAGAC CGTCCAACAG GAAAATTGCA

51 TATTGGACAT TATGTTGGAA GTCTCAAAAA TCGAGTATTA TTACAGGAAG

101 AGGATAAGTA TGATATGTTT GTGTTCTTGG CTGACCAACA AGCCTTGACA

151 GATCATGCCA AAGATCCTCA AACCATTGTA GAGTCTATCG GAAATGTGGC

201 TTTGGATTAT CTTGCAGTTG GATTGGATCC AAATAAGTCA ACTATTTTTA

251 TTCAAAGCCA GATTCCAGAG TTGGCTGAGT TGTCTATGTA TTATATGAAT

301 CTAGTTTCGT TAGCACGTTT GGAGCGAAAT CCAACAGTCA AGACAGAGAT

351 TTCTCAGAAA GGATTTGGAG AAAGCATTCC GACAGGATTC TTGGTCTATC

401 CAATCGCTCA AGCAGCTGAT ATCACAGCTT TCAAGGCTAA TTATGTTCCT

451 GTTGGGACAG ATCAGAAACC AATGATTGAG CAAACTCGTG AAATTGTTCG

501 TTCTTTTAAC AATGCATATA ACTGTGATGT CTTGGTAGAG CCGGAAGGTA

551 TTTATCCAGA AAATGAGAGA GCAGGGCGTT TGCCTGGTTT AGATGGAAAT

601 GCTAAAATGT CTAAATCACT AAATAATGGT ATTTATTTAG CTGATGATGC

651 GGATACTTTG CGTAAAAAAG TAATGAGTAT GTATACAGAT CCAGATCATA

701 TCCGCGTTGA GGATCCAGGT AAGATTGAGG GAAATATGGT TTTCCATTAT

751 CTAGATGTTT TTGGTCGTCC AGAAGATGCT CAAGAAATTG CTGATATGAA

801 AGAACGTTAT CAACGAGGTG GTCTTGGTGA TGTGAAGACC AAGCGTTATC

851 TACTTGAAAT ATTAGAACGT GAACTGGGTC CTATTCGTGA GCGCCGTATT

901 GAATTTGCTA AGGATATGGG AGAAGTTTAT AATATGATTC AAAAAGGTAG

951 TGAAAGAGCG CGTGAAGTTG CGGGTCAAAC CCTATCTGAG GTAAAAGGGG

1001 CAATGGGACT TCATTACTTT AACTAA- (R ₂ ) _n -Y

(D) Poiypeptide sequence embodiments [SEQ ID NO:2|.

X-(R; _j _) _n -l MTKPIILTGD RPTGKLHIGH YVGSLKNRVL LQEEDKYDMF VFLADQQALT

51 DHAKDPQTIV ESIGNVALDY LAVGLDPNKS TIFIQSQIPE LAELSMYYMN

101 LVSLARLERN PTVKTEISQK GFGESIPTGF LVYPIAQAAD ITAFKANYVP

151 VGTDQKPMIE QTREIVRSFN NAYNCDVLVE PEGIYPENER AGRLPGLDGN

201 AKMSKSLNNG I LADDADTL RKKVMSMYTD PDHIRVEDPG KIEGNMVFHY

251 LDVFGRPEDA QEIADMKERY QRGGLGDVKT KRYLLEILER ELGPIRERRI

301 EFAKDMGEVY NMIQKGSERA REVAGQTLSE VKGAMGLHYF N-(R ₂ ) _n -Y

(E) Sequences from Streptococcus pneumoniae trpS polynucleotide ORF sequence [SEQ ID NO:3] .

5 ' -1 AAGGCTAATT ATGTTCCTGT TGGGACAGAT CAGAAACCAA TGATTGAGCA

51 AACTCGTGAA ATTGTTCGTT CTTTTAACAA TGCATATAAC TGTGATGTCT

101 TGGTAGAGCC GGAAGGTATT TATCCAGAAA ATGAGAGAGC AGGGCGTTTG

151 CCTGGTTTAG ATGGAAATGC TAAAATGTCT AAATCACTAA ATAATGGTAT

201 TTATTTAGCT GATGATGCGG ATACTTTGCG TAAAAAAGTA ATGAGTATGT

251 ATACAGATCC AGATCATATC CGCGTTGAGG ATCCAGGTAA GATTGAGGGA

301 AATATGGTTT TCCATTATCT AGATGTTTTT GGTCGTCCAG AAGATGCTCA

351 AGAAATTGCT GATATGAAAG AACGTTATCA ACGAGGTGGT CTTGGTGATG

401 TGAAGACCAA GCGTTATCTA CTTGAAATAT TAGAACGTGA ACTGGGTCCT

451 ATTCGTGAGC GCCGTATTGA ATTTGCTAAG GATATGGGAG AAGTTTATAA

501 TATGATTC.AA. AAAGGTAGTG AAAGAGCGCG TGAAGTTGCG GGTCAAACCC

551 TATCTGAGGT AAAAGGGGCA ATGGGACTTC ATTACTTTAA CTAA-3 '

(F) tφS poiypeptide sequence deduced from the polynucleotide ORF sequence in this table [SEQ ID NO:4).

NH. - l KANYVPVGTD QKPMIEQTRE IVRSFNNAYN CDVLVEPEGI YPENERAGRL

51 PGLDGNAKMS KSLNNGIYLA DDADTLRKKV MSMYTDPDHI RVEDPGKIEG

101 NMVFHYLDVF GRPEDAQEIA DMKERYQRGG LGDVKTKRYL LEILERELGP

151 IRERRIEFAK DMGEVYNMIQ KGSERAREVA GQTLΞEVKGA MGLHYFN-COOH

Deposited materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industπal and Maπne Bacteπa Ltd (herein "NCIMB"), 23 St. Machar Dnve, Aberdeen AB2 1RY, Scotland on 1 1 Apπl 1996 and assigned deposit number 40794 The deposit was descπbed as Streptococcus peumnoiae 0100993 on deposit

On 17 Apπl 1996 a Streptococcus peumnoiae 0100993 DNA library in E colt was similarly depositedwith the NCIMB and assigned deposit number 40800. The Streptococcus pneumomae 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 tφS gene The sequence of the polynucleotides contained in the deposited strain, as weli as the amino acid sequence of the poiypeptide encoded thereby, are controlling in the event of any conflict with any descπption 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 Puφoses of Patent Procedure The strain will be irrevocably and without restπction 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 §1 12. A license may be required to make, use or sell the deposited strain, and compounds deπved therefrom, and no such hcen.se is hereby granted. Polypeptides

The polypeptides of the invention include the poiypeptide of Table 1 [SEQ ID NO 2] (in particular the mature poiypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of tφS, and also those which have at least 70% identity to a poiypeptide of Table 1 [SEQ ID NOS.2 and 4] or the relevant portion, preferably at least 80% identity to a poiypeptide of Table 1 [SEQ ID NOS.2 and 4], and more preferably at least 90% similaπty (more preferably at least 90% identity) to a poiypeptide of Table 1 [SEQ ID NOS.2 and 4] and still more preferably at least 95% similanty (still more preferably at least 95% identity) to a poiypeptide of Table 1 [SEQ ID NOS:2 and 4] and also include portions of such polypeptides with such portion of the poiypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth in Table 1 (D) [SEQ ID NO 2] wherein, at the ammo terminus, X is hydrogen, and at the carboxyl terminus, Y is

hydrogen or a metal, R _| and R2 is any amino acid residue, and n is an integer between 1 and 1000 Any stretch of amino acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer

A fragment is a vaπant poiypeptide having an am o acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides As with tφS polypeptides fragments may be 'free-standing," or compπsed within a larger poiypeptide of which they form a part or region, most preferably as a single continuous region, a single larger poiypeptide

Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NOS 2 and 4], or of vaπants thereof, such as a continuous seπes of residues that includes the amino terminus, or a continuous seπes ot residues that includes the carboxyl terminus Degradation forms of the polypeptides of the invention in a host celi, particularly a Streptococcus pneumo ae, are also preferred Further preferred are fragments characteπzed by structural or functional attπbutes such as fragments that compπse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-form g 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 tφS, including those with a similar activity or an improved activity, or with a decreed undesirable activity Also included are those fragments that are antigenic or lmmunogenic in an animal, especially in a human Particularly preferred are fragments compπsing 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

Vaπants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length poiypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the invention Polynucleotides

Another aspect of the invention relates to isolated polynucleotides, including the full length gene, that encode the tφS poiypeptide having a deduced ammo acid sequence of Table I [SEQ ID NOS 2 and 4] and polynucleotides closely related thereto and vaπants thereof

Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NOS 1 and 3], a polynucleotide of the invention encoding tφS poiypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteπa using Streptococcus pneumo ae 0100993 cells as starting mateπal, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a sequence given in Table I [SEQ ID NOS 1 and 3], 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 ohgonucleotide, preferably a 17-mer or longer, derived from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions By sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence Conveniently, such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Maniatis, T , Fπtsch, E F and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spπng Harbor Laboratory Press, Cold Spπng Harbor, New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13 70) Illustrative of the invention the polynucleotide set out in Table 1 [SEQ ID NO 11 was discovered in a DNA hbrarv deπved from Streptococcus pneumoniae 0100993

The DNA sequence set out in Table 1 [SEQ ID NOS 1 ] contains an open reading frame encoding a protein having about the number of am o acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated using ammo acid residue molecular weight values well known in the art The polynucleotide of SEQ ID NO 1. between nucleotide number 1 through number 1023 encodes the poiypeptide of SEQ ID NO 2 The stop codon begins at nucleotide number 1024 of SEQ ID NO 1 tφS of the invention is structurally related to other proteins of the tryptophanyl tRNA synthetase family, as shown by the results of sequencing the DNA encoding tφS of the deposited strain The protein exhibits greatest homoiogy to Clostπdium longisporum tryptophanyl tRNA synthetase protein among known proteins tφS of Table I [SEQ ID NO 2] has about 66% identity over its entire length and about 81 % similaπty over its entire length with the ammo acid sequence of Clostπdium longisporum tryptophanyl tRNA synthetase poiypeptide

The invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Table I [SEQ ID NO 1 ] Also provided by the invention is the coding

sequence for the mature poiypeptide or a fragment thereof, by itself as well as the coding sequence for the mature poiypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence The polynucleotide may also contain non-coding sequences, including for example, but not limited to non-coding 5' and 3' sequences, such as the transcπbed, non-translated sequences, termination signals, πbosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional ammo acids For example, a marker sequence that facilitates puπfication of the fused poiypeptide 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 descπbed in Gentz et al , Proc Natl Acad Sci , USA 86 821-824 (1989), or an HA tag (Wilson et al , Cell 37 767 (1984) Polynucleotides of the invention also include, but are not limited to, polynucleotides compπsing a structural gene and its naturally associated sequences that control gene expression

A preferred embodiment of the invention is a polynucleotide of compπsing nucleotide 1 to 1023 or 1024 set forth in SEQ ID NO 1 of Table 1 which encode the trpS poiypeptide

The invention also includes polynucleotides of the formula set forth in Table 1 (C)[SEQ

ED NO 1 ] wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' end of the molecule,

Y is hydrogen or a metal, R _j and R2 is any nucleic acid residue, and n is an integer between I and 1000 Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1 , may be either a heteropolymer or a homopolymer, preferably a heteropolymer

The term "polynucleotide encoding a poiypeptide" as used herein encompasses polynucleotides that include a sequence encoding a poiypeptide of the invention, particularly a bacteπal poiypeptide and more particularly a poiypeptide of the Streptococcus pneumoniae tφS having the amino acid sequence set out in Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the poiypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences

The invention further relates to vanants of the polynucleotides descπbed herein that encode for vaπants of the poiypeptide having the deduced ammo acid sequence of Table 1 [SEQ ED NO 2] Vaπants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention

Further particularly preferred embodiments are polynucleotides encoding tφS vaπants, that have the ammo acid sequence of tφS poiypeptide of Table 1 [SEQ ID NO.2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, deleted or

added, in any combination Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of tφS

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding tφS poiypeptide having an amino acid sequence set out in Table 1 [SEQ ID NOS.2 and 4], and polynucleotides that are complementary to such polynucleotides Alternatively, most highly preferred are polynucleotides that compπse a region that is at least 80% identical over its entire length to a polynucleotide encoding tφS poiypeptide of the deposited strain 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 Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature poiypeptide encoded by the DNA of Table 1 [SEQ ID NO ^* l]

The invention further relates to polynucleotides that hybπdize to the herein above- descπbed sequences In this regard, the invention especially relates to polynucleotides that hybπdize under stπngent conditions to the herein above-descπbed polynucleotides As herein used, the terms "stπngent conditions" and "stπngeπt hybπdization conditions" mean hybπdization will occur only if there is at least 95% and preferably at least 97% identity between the sequences An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC ( 150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybπdization 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 Spπng Harbor, N Y , ( 1989), particularly Chapter 1 1 therein The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 or SEQ ID NO 3 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said

DNA sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers descπbed elsewhere herein

As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybπdization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding tφS and to isolate cDNA and genomic clones of other genes that have a high sequence similaπty to the tφS gene Such probes generally will compπse at least 15 bases Preferably, such probes will have at least 30 bases and may have at least 50 bases Particularly preferred probes will have at least 30 bases and will have 50 bases or less. For example, the coding region of the tφS gene may be isolated by screening using the

DNA sequence provided in SEQ ID NO 1 to synthesize an ohgonucleotide 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 hybπdizes to The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and mateπals for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays

Polynucleotides of the invention that are oligoπucleottdes derived from the sequences of SEQ ID NOS.1 and/or 2 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein whole or m part are transcribed in bacteπa 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 may encode a poiypeptide that is the mature protein plus additional amino or carboxyl-terminal am o acids, or ammo acids inteπor to the mature poiypeptide (when the mature form has more than one poiypeptide 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 ammo acids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the poiypeptide fused to one or more prosequences may be an inactive form of the poiypeptide. 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 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 ts a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed duπng processing steps that produce active and mature forms of the poiypeptide

Vectors, host cells, expression The invention also relates to vectors that compπse 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 deπved from the DNA constructs of the invention For recombinant production, host cells can be genetically engineered to incoφorate expression systems or portions thereof or polynucleotides of the invention Introduction of a polynucleotide into the host cell can be effected by methods descnbed in many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambroo et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spπng Harbor Laboratory Press, Cold Spπng Harbor, N Y (1989), such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic lipid-mediated transfection, eiectroporation, transduction, scrape loading, ballistic introduction and infection

Representative examples of appropπate hosts include bacteπal cells, such as streptococci, staphylococci, enterococci E coll, streptomyces and Bacillus subtilis cells, fungal cells, such as yeast cells and Aspergdlus cells, insect cells such as Drosophda S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells

A great vaπety of expression systems can be used to produce the polypeptides of the invention Such vectors include, among others, chromosomal, episomal and virus-deπved vectors, e g , vectors deπved from bacteπal plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deπved from combinations thereof,

such as those deπved from plasmid and bactenophage 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 poiypeptide in a host may be used for expression in this regard The appropπate DNA sequence may be inserted into the expression system by any of a vaπety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the peπplasmic space or into the extracellular environment, appropπate secretion signals may be coφorated into the expressed poiypeptide These signals may be endogenous to the poiypeptide or they may be heterologous signals

Polypeptides of the invention can be recovered and puπfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanoi 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 punfication Well known techniques for refolding protein may be employed to regenerate active conformation when the poiypeptide is denatured duπng isolation and or punfication Diagnostic Assays

This invention is also related to the use of the tφS polynucleotides of the invention for use as diagnostic reagents Detection of tφS tn a eukaryote, particularly a mammal, and especially a human will provide a diagnostic method for diagnosis of a disease Eukarvotes (herein aiso "ιndιvιdual(s)"), particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compπsing the tφS gene may be detected at the nucleic acid level by a vaπety of techniques

Nucleic acids for diagnosis may be obtained from an infected individual s cells and tissues, such as bone, blood, muscle, cartilage, and skin Genomic DNA mav be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique pπor to analysis RNA or cDNA may also be used in the same ways Using amplification, characteπzation of the species and strain of prokaryote present in an individual, may be made by an analvsis of the genotype ot the prokaryote gene Deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence Point mutations can be identified by hybπdizmg amplified DNA to labeled tφS polynucleotide

sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denatuπng agents, or by direct DNA sequencing See, e.g , Myers et al , Science, 230 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S 1 protection or a chemical cleavage method. See, e g , Cotton et al , Proc. Natl. Acad Set.. USA, 85 4397-4401 (1985).

Cells carrying mutations or polymoφhisms in the gene of the invention may also be detected at the DNA level by a vaπety of techniques, to allow for serotyping, for example For example, RT-PCR can be used to detect mutations It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan RNA or cDNA may also be used for the same purpose, PCR or RT-PCR As an example, PCR pπmers complementary to a nucleic acid encoding tφS can be used to identify and analyze mutations Examples of representative pπmers are shown below in Table 2

Table 2

Primers for amplification of trpS polynucleotides SEQ ID NO PRIMER SEQUENCE

5 5 ' -AAGGCTAATTATGTTCCTGTTGGG-3 '

6 5 ' -TTAGTTAAAGTAATGAAGTCCCAT-3 '

The invention further provides these pπmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end. These pπmers may be used for, among other things, amplifying tφS DNA isolated from a sample deπved from an individual. The pπmers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to vaπous techniques for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent

T e invention further provides a process for diagnosing, disease, preferably bacteπal infections, more preferably infections by Streptococcus pneumoniae, and most preferably otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as tor example infection ot cerebrospinal

fluid, compπsing determining from a sample derived from an individual a increased level of expression of polynucleotide having the sequence of Table 1 [SEQ ID NO 1 ] Increased or decreased expression of tφS polynucleotide can be measured using any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods

In addition, a diagnostic assay in accordance with the invention for detecting over- expression of tφS protein 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 tφS protein, in a sample deπved from a host are well-known to those of skill in the art Such assay methods include radioimmunoassays, competitive-binding assays. Western Blot analysis and ELISA assays

Antibodies

The polypeptides of the invention or variants thereof, or cells expressing them can be used as an immunogen to produce antibodies immunospecific for such polypeptides

"Antibodies" as used herein includes monoclonal and polyclonal antibodies, chimeπc, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunolglobulin expression library

Antibodies generated against the polypeptides of the invention can be obtained by administeπng the polypeptides or epitope-beaπng fragments, analogues or cells 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 vaπous 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, Ine (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 of this invention Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies Alternatively phage display technology may be utilized to select antibody genes with binding activities towards the poiypeptide either from repertoires of PCR amplified v- genes of lymphocytes from humans screened for possessing anti-tφS or from naive libraries (McCafferty, J et al , (1990), Nature 348, 552-554, Marks, J et al , ( 1992) Biotechnology

10, 779-783) The affinity of these antibodies can also be improved bv chain shuffling (Clackson, T et al , (1991 ) Nature 352, 624-628)

If two antigen binding domains are present each domain mav be directed against a different epitope - termed 'bispecific' antibodies The above-descπbed antibodies may be employed to isolate or to identih clones expressing the polypeptides to puπfy the polypeptides by affinity chromatography

Thus, among others, antibodies against tφS- poiypeptide may be employed to treat infections, particularly bacteπal infections and especially otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid

Poiypeptide vaπants include antigenically, epitopically or immunoiogically equivalent vaπants that form a particular aspect of this invention The term ' antigenically equivalent deπvative as used herein encompasses a poiypeptide or its equivalent w hich will be specifically recognized by certain antibodies which, when raised to the protein or poiypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host The term ' immunoiogically equivalent deπvative" as used herein encompasses a peptide or its equivalent 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 The poiypeptide, such as an antigenically or immunoiogically equivalent deπvative or a fusion protein thereof is used as an antigen to immunize a mouse or other anima! such as a rat or chicken The fusion protein may provide stability to the poiypeptide The antigen may be associated, for example by conjugation, with an immunogenic arner protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH) Alternatively a multiple antigenic peptide compπsing multiple copies of the protein or poiypeptide, or an antigenically or immunoiogically equivalent poiypeptide 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 comp mentaπty determining regιon(s) of the hybπdoma-deπved antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P et al (1986), Nature 321 , 522-525 or Tempest et al , ( 1991 ) Biotechnology 9, 266-273

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, Manthoφe et al., Hum Gene Ther 1963:4, 419), delivery of DNA complexed with specific protein earners (Wu et al., J Bwl 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., DM4 Cell Biol 1993, 12.791) and in vivo infection using cloned retrovirai vectors (Seeger et al., PNAS USA 1984 81,5849). Antagonists and agonists - assays and molecules

Polypeptides of the invention may also be used to assess the binding of small molecule substrates and gands in. for example, cells, cell-free preparations, chemical libraries, and natural product mixtures These substrates and ligands may be natural substrates and gands or may be structural or functional mimetics. See, e.g., Coligan et al.. Current Protocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of tφS polypeptides or polynucleotides, particularly those compounds that are bacteπostatic and or bacteπocidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising tφS poiypeptide and a labeled substrate or ligand of such poiypeptide is incubated in the absence or the presence of a candidate molecule that may be a tφS agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the tφS poiypeptide 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 tφS poiypeptide are most likely to be good antagonists. Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to coloπmetπc labeled substrate converted into product, a reporter gene that is responsive to changes in tφS polynucleotide or poiypeptide activity, and binding assays known in the art.

Another example of an assay for tφS antagonists is a competitive assay that combines tφS and a potential antagonist with tφS-bindmg molecules, recombinant tφS binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropπate

conditions for a competitive inhibition assay tφS can be labeled, such as by radioactivity or a coioπmetπc compound, such that the number of tφS 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 small organic molecules, peptides. polypeptides and antibodies that bind to a polynucleotide or poiypeptide of the invention and thereby inhibit or extinguish its activity Potential antagonists also may be small organic molecules, a peptide, a poiypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing tφS-induced activities, thereby preventing the action of tφS by excluding tφS from binding

Potential antagonists include a small molecule that binds to and occupies the binding site of the poiypeptide 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 descπption of these molecules) Preferred potential antagonists include compounds related to and vaπants of tφS

Each of the DNA sequences provided herein may be used in the discovery and development of antibacteπal compounds. The encoded protein, upon expression, can be used as a target for the screening of antibacteπal drugs Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shme-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 poiypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and 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 bacteria, to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block tφS protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al.. Infect. Immun. 60:221 1 ( 1992), to block bacterial adhesion between mammalian extracellular matrix proteins and bacterial tφS proteins that mediate tissue

damage and. to block the normal progression of pathogenesis in infections initiated other than by the implantation of m-dwelling devices or by other surgical techniques

The antagonists and agonists of the invention may be employed, for instance, to inhibit and treat otitis media, conjunctivitis, pneumonia, bacteremia. meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid

Hehcobacter 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 Hehcobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the international Agency for Research on Cancer recently recognized a cause-and-effect relationship between H p\lorι and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of tφS) found using screens provided by the invention, particularly broad-spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H pylori-inάuced cancers, such as gastrointestinal carcinoma Such treatment should also cure gastric ulcers and gastritis Vaccines 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 tφS, or a fragment or variant thereof, adequate to produce antibodv and/ or T cell immune response to protect said individual from infection, particularly bacteπal infection and most particularly Streptococcus pneumoniae infection Also provided are methods whereby such immunological response slows bacteπal 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 to direct expression of tφS, or a fragment or a variant thereof, for expressing tφS, 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, cytokme-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not One way 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 modified nucleic acid, or a DNA/RNA hybrid

A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunological response in such individual to a tφS or protein coded therefrom, wherein the composition comprises a recombinant tφS or protein coded therefrom comprising DNA which codes for and expresses an antigen of said tφS or protein coded therefrom The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells.

A tφS poiypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and 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, relatively large co-proteins which solubilize the protein and facilitate production and punfication thereof Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system 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 compπsing the polypeptides 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 descπbed polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacteπal cell surface proteins in DNA constructs used in such genetic immunization experiments in animal models of infection with Streptococcus pneumoniae 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 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

The poiypeptide 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 bacteπa to damaged tissue Examples of tissue damage include wounds in skin or connective tissue caused, e g , by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina

The invention also includes a vaccine formulation which comprises an immunogenic recombinant protein of the invention together with a suitable carrier Since the protein may be broken down the stomach, it 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, bacteπostats and solutes which render the formulation insotonic 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 ampules and vials and mav be stored in a freeze-dπed condition requiring only the addition of the sterile liquid earner immediately pπor 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 expeπmentation

While the invention has been described with reference to certain tφS protetn, it is to be understood that this covers fragments of the naturally occurπng protein and similar proteins with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant protein Compositions, kits and administration

The invention also relates to compositions compπsing the polynucleotide or the polypeptides discussed above or their agonists or antagonists The polypeptides of the invention may be employed in combination with a non-steπle or steπle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to a subject Such compositions compπse, for instance, a media additive or a therapeutically effective amount of a poiypeptide of the invention and a pharmaceutically acceptable earner or excipient Such earners may include, but are not limited to, saline, buffered saline, dextrose, water, glyceroi, ethanol and combinations thereof The formulation should suit the mode of administration The

invention further relates to diagnostic and pharmaceutical packs and kits compπsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention

Polypeptides 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 _, intrapeπtoneal, 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 steπle 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

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, l 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 bacteπa shortly before insertion of an tn-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 peπoperative cover for anv surgical technique to prevent bacteπal wound infections, especially Streptococcus pneumo ae wound infections Many orthopaedic 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 descπbed above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteπa to matπx 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 lμg/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

Each reference disclosed herein is incoφorated by reference herein in its entirety Any patent application to which this application claims priority is also incoφorated by reference herein in its entirety EXAMPLES

The examples below are earned out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise descπbed in detail The examples are illustrative, but do not limit the invention

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO I was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in £ coli The sequencing data from two or more clones containing overlapping Streptococcus pneumomae

DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared by routine methods, for example- Methods 1 and 2 below

Total cellular DNA is isolated from 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 I 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 restnction 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

Example 2 trpS Characterization

The enzyme mediated incorporation of radiolabelled ammo acid into tRNA may be measured by the uminoacylation method which measures amino acid-tRNA as tπchloroacetic acid-precipitable radioactivity from radiolabelled amino acid in the presence of tRNA and ATP (Hughes J, Mellows G and Soughton S, 1980, FEBS Letters, 122.322- 324) Thus inhibitors of tryptophanyl tRNA synthetase can be detected by a reduction in the tπchloroacetic acid precipitable radioactivity relative to the control Alternatively the tRNA synthetase catalysed partial PPi/ATP exchange reaction which measures the formation of radiolabelled ATP from PPi can be used to detect tryptophanyl tRNA synthetase inhibitors (Calender R & Berg P, 1966, Biochemistry, 5, 1681 - 1690)

SEQUENCE LISTING

(1) GENERAL INFORMATION

(ι) APPLICANT: Gentry, Danile

Greenwood, Claire Lawlor, Elizabeth

(ii) TITLE OF THE INVENTION: Novel trpS

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: SmithKline Beecham Corporation

(B) STREET: 709 Swedeland Road

(C) CITY: King of Prussia

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19406-0939

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 12-SEP-1997

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 9619072.3

(B) FILING DATE: 12-SEP-1996

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Gimmi, Edward R

(B) REGISTRATION NUMBER: 38,891

(C) REFERENCE/DOCKET NUMBER: P31624-1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 610-270-4478

(B) TELEFAX: 610-270-5090

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1026 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

ATGACTAAAC CCATTATTTT AACAGGAGAC CGTCCAACAG GAAAATTGCA TATTGGACAT 60

TATGTTGGAA GTCTCAAAAA TCGAGTATTA TTACAGGAAG AGGATAAGTA TGATATGTTT 120

GTG TCTTGG CTGACCAACA AGCCTTGACA GATCATGCCA AAGATCCTCA AACCATTGTA 180

GAGTCTATCG GAAATGTGGC TTTGGATTAT CTTGCAGTTG GATTGGATCC AAATAAGTCA 240

ACTATTTTTA TTCAAAGCCA GATTCCAGAG TTGGCTGAGT TGTCTATGTA TTATATGAAT 300

CTAGTTTCGT TAGCACGTTT GGAGCGAAAT CCAACAGTCA AGACAGAGAT TTCTCAGAAA 360

GGATTTGGAG AAAGCATTCC GACAGGATTC TTGGTCTATC CAATCGCTCA AGCAGCTGAT 42C

ATCACAGCTT TCAAGGCTAA TTATGTTCCT GTTGGGACAG ATCAGAAACC AATGATTGAG 480

CAAACTCGTG AAATTGTTCG TTCTTTTAAC AATGCATATA ACTGTGATGT CTTGGTAGAG 540

CCGGAAGGTA TTTATCCAGA AAATGAGAGA GCAGGGCGTT TGCCTGGTTT AGATGGAAAT 600

GCTAAAATGT CTAAATCACT AAATAATGGT ATTTATTTAG CTGATGATGC GGATACTTTG 660

CGTAAAAAAG TAATGAGTAT GTATACAGAT CCAGATCATA TCCGCGTTGA GGATCCAGGT 720

AAGATTGAGG GAAATATGGT TTTCCATTAT CTAGATGTTT TTGGTCGTCC AGAAGATGCT 780

CAAGAAATTG CTGATATGAA AGAACGTTAT CAACGAGGTG GTCTTGGTGA TGTGAAGACC 840

AAGCGTTATC TACTTGAAAT ATTAGAACGT GAACTGGGTC CTATTCGTGA GCGCCGTATT 900

GAATTTGCTA AGGATATGGG AGAAGTTTAT AATATGATTC AAAAAGGTAG TGAAAGAGCG 960

CGTGAAGTTG CGGGTCAAAC CCTATCTGAG GTAAAAGGGG CAATGGGACT TCATTACTTT 1020

AACTAA 1026

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 341 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Thr Lys Pro He He Leu Thr Gly Asp Arg Pro Thr Gly Lys Leu

1 5 10 15

His He Gly His Tyr Val Gly Ser Leu Lys Asn Arg Val Leu Leu Gin

20 25 30

Glu Glu Asp Lys Tyr Asp Met Phe Val Phe Leu Ala Asp Gin Gin Ala

35 40 45

Leu Thr Asp His Ala Lys Asp Pro Gin Thr He Val Glu Ser He Gly

50 55 60

Asn Val Ala Leu Asp Tyr Leu Ala Val Gly Leu Asp Pro Asn Lys Ser 65 70 75 80

Thr He Phe He Gin Ser Gin He Pro Glu Leu Ala Glu Leu Ser Met

85 90 95

Tyr Tyr Met Asn Leu Val Ser Leu Ala Arg Leu Glu Arg Asn Pro Thr

100 105 110

Val Lys Thr Glu He Ser Gin Lys Gly Phe Gly Glu Ser He Pro Thr

115 120 125

Gly Phe Leu Val Tyr Pro He Ala Gin Ala Ala Asp He Thr Ala Phe

130 135 140

Lys Ala Asn Tyr Val Pro Val Gly Thr Asp Gin Lys Pro Met He Glu 145 150 155 160

Gin Thr Arg Glu He Val Arg Ser Phe Asn Asn Ala Tyr Asn Cys Asp

165 170 175

Val Leu Val Glu Pro Glu Gly He Tyr Pro Glu Asn Glu Arg Ala Gly

180 185 190

Arg Leu Pro Gly Leu Asp Gly Asn Ala Lys Met Ser Lys Ser Leu Asn

195 200 205

Asn Gly He Tyr Leu Ala Asp Asp Ala Asp Thr Leu Arg Lys Lys Val

210 215 220

Met Ser Met Tyr Thr Asp Pro Asp His He Arg Val Glu Asp Pro Gly 225 230 235 240

Lys He Glu Gly Asn Met Val Phe His Tyr Leu Asp Val Phe Gly Arg

245 250 255

Pro Glu Asp Ala Gin Glu He Ala Asp Met Lys Glu Arg Tyr Gin Arg

260 265 270

Gly Gly Leu Gly Asp Val Lys Thr Lys Arg Tyr Leu Leu Glu He Leu

275 280 285

Glu Arg Glu Leu Gly Pro He Arg Glu Arg Arg He Glu Phe Ala Lys

290 295 300

Asp Met Gly Glu Val Tyr Asn Met He Gin Lys Gly Ser Glu Arg Ala 305 310 315 320

Arg Glu Val Ala Gly Gin Thr Leu Ser Glu Val Lys Gly Ala Met Gly

325 330 335

Leu His Tyr Phe Asn 340

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 594 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AAGGCTAATT ATGTTCCTGT TGGGACAGAT CAGAAACCAA TGATTGAGCA AACTCGTGAA 60

ATTGTTCGTT CTTTTAACAA TGCATATAAC TGTGATGTCT TGGTAGAGCC GGAAGGTATT 120

TATCCAGAAA ATGAGAGAGC AGGGCGTTTG CCTGGTTTAG ATGGAAATGC TAAAATGTCT 180

AAATCACTAA ATAATGGTAT TTATTTAGCT GATGATGCGG ATACTTTGCG TAAAAAAGTA 240

ATGAGTATGT ATACAGATCC AGATCATATC CGCGTTGAGG ATCCAGGTAA GATTGAGGGA 300

AATATGGTTT TCCATTATCT AGATGTTTTT GGTCGTCCAG AAGATGCTCA AGAAATTGCT 360

GATATGAAAG AACGTTATCA ACGAGGTGGT CTTGGTGATG TGAAGACCAA GCGTTATCTA 420

CTTGAAATAT TAGAACGTGA ACTGGGTCCT ATTCGTGAGC GCCGTATTGA ATTTGCTAAG 480

GATATGGGAG AAGTTTATAA TATGATTCAA AAAGGTAGTG AAAGAGCGCG TGAAGTTGCG 540

GGTCAAACCC TATCTGAGGT AAAAGGGGCA ATGGGACTTC AT ACTTTAA CTAA 594

(2) INFORMATION FOR SEQ ID NO: :

(I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 197 amino acids (3) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Lys Ala Asn Tyr Val Pro Val Gly Thr Asp Gin Lys Pro Met He Glu

1 5 10 15

Gin Thr Arg Glu He Val Arg Ser Phe Asn Asn Ala Tyr Asn Cys Asp

20 25 30

Val Leu Val Glu Pro Glu Gly He Tyr Pro Glu Asn Glu Arg Ala Gly

35 40 45

Arg Leu Pro Gly Leu Asp Gly Asn Ala Lys Met Ser Lys Ser Leu Asn 50 55 60

Asn Gly He Tyr Leu Ala Asp Asp Ala Asp Thr Leu Arg Lys Lys Val 65 70 75 80

Met Ser Met Tyr Thr Asp Pro Asp His He Arg Val Glu Asp Pro Gly

85 90 95

Lys He Glu Gly Asn Met Val Phe His Tyr Leu Asp Val Phe Gly Arg

100 105 110

Pro Glu Asp Ala Gin Glu He Ala Asp Met Lys Glu Arg Tyr Gin Arg

115 120 125

Gly Gly Leu Gly Asp Val Lys Thr Lys Arg Tyr Leu Leu Glu He Leu

130 135 140

Glu Arg Glu Leu Gly Pro He Arg Glu Arg Arg He ^" Glu Phe Ala Lys 145 150 155 160

Asp Met Gly Glu Val Tyr Asn Met He Gin Lys Gly Ser Glu Arg Ala

165 170 175

Arg Glu Val Ala Gly Gin Thr Leu Ser Glu Val Lys Gly Ala Met Gly

180 185 190

Leu His Tyr Phe Asn 195

(2) INFORllATION FOR SEQ ID NO:5:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 :

AAGGCTAATT ATGTTCCTGT TGGG 24

(2) INFORMATION FOR SEQ ID NO: 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

TTAGTTAAAG TAATGAAGTC CCAT 24