Title of Invention	AN ISOLATED POLYPEPTIDE AND APPLICATIONS THEREOF
Abstract	Disclosed herein is an isolated polypeptide which is a Group I signa subunit of Mycabacterium tuberculosis RNA polymerase, or a functionally equivalent modified form thereof.

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FORM 3 A THE PATENTS ACT, 1970 ComMPLETE SPECIFICATION (SEE SECTION - 10) OVEL PROCESS FOR PRODUCING NOVEL BACTERIAL ENZYME SUBUNITS' ME, M/S. ASTRA RESEARCH CENTRE INDIA, A REGISTERED INDIAN SOCIETY, OF 18TH CROSS, MALLESWARAM, BANGALORE - 560 003, KARNATAKA STATE, INDIA. THE INVENTORS OF THE PRESENT INVENTION ARE DR. MEENAKSHI BAL6ANESH AND MR. UMENDAR K. SHARMA. THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES AND ASCERTAINS THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED. BACKBROUND * Th« primary event involved in gene expreasion is the transcription of the specific DNA to a RNA Molecule. This transcription is achieved by a DNA dependent RNA polymerase or RNA polyaerase for brevity. The catalytically active RNA polyeerase holoenzyee is structurally coeplex and is coeprised of 0^2^ P , p and the subunits. RNA synthesis froe specific proMoters is catalysed by the enzyee in the presence of the sigaa Bubunit. The sigea subunit has been assigned the indirect role of conferring on the core enzyee the ability to select a start site and initiate RNA synthesis. Physical eeasureeents have shotm that the sigea subunit induces conforeational transition upon binding to core enzyee. Binding constants of IMA core enzyee Mith sigea shoM a difference of lO — iO orders of eagnitude. The functional characterisation of the different subunits of the RNA polyeerase indicate the B subunit to be involved in the catalysis, the B subunit in the DNA binding Mhile the a subunits function has yet to be delineated. Mutations in the sigea subimit effecting its association and ability to confer DNA sequence specificity to the enzyee are known to be lethal to the cell. AnothoNT interesting characteristic of the sigma subunits identified and sequenced froe different organisnis allows then to be classified into tMo broad categariesi The Broup Z sigea have also been referred to the sig«a-70 class or the house keeping sigea group. The sigea subunits belonging to this group recognise sieilar DMA sequences for proaoter activity Mhich is reflected in certain regions of the protein being highly conserved. The Broup II sigea differ in their sequences and even though shoM certain hoeologous sequences based on sieilarity of function, they aro involved in conferring cm the core RNA polyeerase the ability to recognise a variety of sequences (specific for the sigea) Mhich results in the regulation of the expression of the gene as a factor dependent on the availability of the specific sigea protein. The essential requiresent of the association of the sigea subunit to confer specificity to the core RNA polyeerase enzyme eakes the process of association a suitable target for drug design. Obviously the inhibition of the association prcwcess would require the identification of the prieary structures of the sigea Buribunits involved in trwriscription. The availability of the sequences and structure itould then enable the screening, identification or designing of molecules coepeting with the sigea subunit for binding to the core enzyme. These molecules/compound which prevent sigma binding to the core enzyme could then be developed into effective thereupetic agents. The sigma subunits identified the -far have very limited hcmology to eukaryotic transcription factors thus making the sigma's highly specific drug targets. Mycobacterium tuberculosis is a major pulmonary pathogen which is characterised by its very show growth rate. As a pathogen it gains access to the alveolar macrophages where it multiplies within the phagosome, finally lysing the cells and being disseminated through the blood stream not only to other areas of the lung but also to extrapulmonary tissues. Thus the pathogen multiplies in atleast two entirely different enviornments which would involve the utilisation of different nutrients and a variety of passible hostile factors | a successful invasion would thus involve the co-ordinated expression of new sets of genes. This regulation would resemble different physiological stages as best exemplified by Bacillus in which the expression of genes specific for different stages are transcribed by RNA polymerases associating with different sigma factors. This again the possibility of targetting not only the house keeping sigma of n.tuberculosis but also sigma subunits specific for the different stages of multiplication. 1) IDENTIFICATION OF DNA SEQUENCES IN MYCOBACTERIUM TUBERCULOSIS HonoLoeous TO THE SIGMA 70 OENE « chromosomal DNA from M.tuberculosis H37Rv, H37Ra and M.smegmatis were made following standaurd protocols, restricted with different restriction enzymes and the DNA fragments resolved on a 1% agarose gel electrophoresis (Fig.la of the draMings acconpanying the provisional specification). The resolved DNA was then transferred onto nylon aeebranes following standard procedures and the eeebranes subjectcwl to Southern analysis. To detect honologouB frageents the eeebranes were probed with a radioactively labelled DNA frageent generated by PC^. The PCR gencN^ated probe was a SOO bp DNA fragaent aaplified from the chroMosoee of H.tuberculosis using degenerate forward and reverse prieers designed based C3n the highly conserved amino acid sequence motiffs of E.coli and B.subtilis sigma 70 proteins. The amino acid sequence used to design primers is highlighted in Fig.2 of the drawings accompanying the provisional specification. Analysis of the Southern hybridisation experiment revealed the presence of atleast three hybridising fragments of approximately 4.2, 2.4 and 0.9 kb in the Sal 1 digested DNA (fig lb of the drawings accompany!ng the provisional specification). The pattern of hybridisation was identical in both the strains H.tuberculosis H37Ra and H37Rv. In the case of the Sal 1 digested/hybridised DNA of H.smegmatis only two hybridising fragments of 4.2 and 2.4 were detected. The hybridisation pattern with the EcoRl and Pvu 11 restricted/hybridised DNA was not clear probably due to the size of fragments generated. However based on the patterns obtained with the Sal 1 restricted DNA it could be concluded that there were multiple DNA fragments with homology to the known sigma 70 genes. Siailar Southern hybridisation experiment to identify the pattern of hoMologous genes in a 4 clinical isolates of H.tt^erculosis revealed an identical pattern indicating the presence of sieilar gene in other virulent isolates of M.tuberculosi s. 2> CLONINB OF THE HYBRIDISING DMA FRABHENTS REPRESENTINB PUTATIVE SISHA 70 HOnDLOQQUS s CLONING OF THE 4.2 Kb SAL 1 HYBRIDISING FRAGMENT f n.tuberculosis H37Rv DNA Mns restricted Mith Sal 1 and fragments ramolved by preparative agarose gel electrophoresis. The agarose gel corresponding to 4.0 to 5.0 kb size region Mas cut and the DNA extracted fro« the gel following standard protocols. This size fractionated DNA Mas then ligated to the cloning vector pBR 329 at its Sal 1 site. The ligated DNA Mas then transforeed into E.coli DH5a and the transformants of the sub—library screened by colony blotting using the PCR derived (described under identifications) probe following described protocols. Several hybridising colonies Mere identified and the analysis of the plasaid profile of these clones revealed thee to harbour a Sal 1 insert of 4.2 kb Mhich hybridised in southern blotting experiaents Mith the PCR probe confiraing the cloning of the honologous 4.2 kb frageent. CLONINB OF THE 2.2 Kb 8AL1 HYBRIDISINB FRA6MENT s A lauabda gtli library (obtained fro* WHO) of the chromosoMal DNA of n.tuberculosis Erdean strain Mas screened using the saee probe as described above folloMing standard procedures. One lambda gtll phage with a 4.2 kb EccAl insert was identified and confireed to hybridise utith the PCR probe. Restriction analysis of the EcoRl insert revealed it to have an internal 2.2 Kb Sal 1 frageent which hybridised with the saee probe confirming this clone to harbour the 2.2 Kb Sal 1 frageent identified earlier by Southern hybridisation. This 4.2 kb EcoRl frageent was excised as an EcoRl fragment from the lambda gt 11 DIM and subcloned into the cloning vector pBR 329 to obtain the recombinant plasmid pARC SigA. The restriction pattern of the two hybridising fragments being different the putative sigma 70 homologue on the 2.2 kb Sal 1 fragment (included within the 4.2 kb EcoR 1 insert of the lambda gt 11 phage) was designated M.tb Sigma A while the homologue on the 4.2 kb Sal 1 fragment was designated M.tb Sigma B. A schematic representation of the recoaribinant plasmids harbouring the Sig A gene is shown in fig 3 of the drawings accompanying the provisional specification and the Sig B gene is shown in Fig 4 of the drawings accompanying the provisional specification. In the accoapanying drawings i Fig 1 ropresents the DMA sequence of Sigea A and the saMe is referred to as Seq. ID No. 1 hereinafter. Fig 2 represents the protein sequence of Sigea A and the saee is identified as Seq. ID i4o. 2. Fig 3 represents the DNA sequence of Sigea B and the sa«e is refer-red to as Seq. ID No. 3 hereinafter. Fig 4 represents the protein sequence of Sigea B and the saae is identified as Seq. ID No. 4. The coding sequence of the Sig A gene Mas found to have an internal Sal 1 site Mhich could explain the hybridisation of the 0.9 kb frageents in the southern experiaents. NUCLEOTIDE SEQUENCE OF THE M.tb.Sig A AND Sig B 6ENES s Sig A I The DNA sequence of EcoRV — EcoRi fragtaent encompassing the entire SigA gene utas stubcloned into appropriate iil3 vectors and both strands of the gene sequenced by the dideoxy ecrthcxJ. The sequence obtained is shoitn in Fig.5 of the drawings accoepanying the provisional specification. An ORF of 1578 nucleotides coding for a protein of S26 aaiino acids was predicted from the DNA sequence. The derived amino acid sequence of the Sig A showed 60X identity with the E.coli Sigea 70 and 70X identity with the HrdB sequence of Streptoeyces coelicolar. The overall antoey of the Sig A sequence is compatible with that seen with the alignecnt aanng Sigma 70 proteins of different organisms. This consists of a highly conserved C-terminal half while the N-terminal half generally shows lesser hcMKilogy. The two regions are linked by a stretch of amino acids which vary in length and are found to be generally unigque for the protein. The Sig A sequence also has a similar structure where the unconserved central stretch correspond to amino acids in length. There is limited homology within the N-terminal half as compared to the Sigma 70 of E.coli but shows greater resemblance to that of the Sigma 70 homologue HrdB of S.coelicolor. The highly conserved motiffs of Region 3.1, 3.2, 4.1 and 4.2 proposed to be involved in DMA binding are were found to be near identical in the M.tb Sig A sequence also. The N—terminal start of the protein has been tentatively assigned based on homologous motiffs of the S.coelicolor HrdB sequence. The sequence similarity abled assignment of the Sig A to the Broup 1 sigma but definitely shows distinct differences especially a unique and lengthty N—terminal stretch of amino acids which may be essential for the transcription of promoter sequences of H.tuberculosis. Sig B I The nucleotide sequence of the Sig B (Fig 6 of the drawings accompanying the provisional specification) gene identific»s the gene to be nucleotides encoding a protein of amino acids. Alignnent of the aaiino acid sequence of the Sig B gene Mith other sigflia 70 proteinm places the Sig B gene into the Qroup 1 family of sigea 70 proteins. The overall structure of the protein folloMS the mamm pattern as described for Sig A. HoMever the Sig B sequence has only 60X hoMology Hith the Sig A sequence as there are considerable differences not only Mithin the unconserved regions of the protein but also Mithin the putative DMA binding regions of the Sig B protein. These characteristics suggest that the Sig B protein eay play a distinct function in the physiology of the organism. The N-ter«inal start of the protein has bemn tentatively identified based on the presence of the first Methionine of the ORF. HE CLAIH I 1. An isolatoNd polyptipticle which i» a Qroup I siQ«a mubunit of HycobmrtariuM tudswrculosis RNA polyiMMraBe« or a functionally equivalent Modified fore thereof. 2. A polypeptide according to claie 1 Mhich aeino acid sequence is identical to, or substantially sieilar to, SEQ ID NO i 2 or 4 in the Sequence Listing. 3. An isolated nucleic acid molecule Mhich has a nucleotide sequence coding for a polypeptide according to claie 1 or 2. 4. An isolated nucleic acid Molecule selected from t (a> DNA molecules comprising a nucleotide sequence as shown in SEQ ID NO I 1 or SEQ ID NO s 3 encoding a Broup I sigma subunit of HycobacteriuM tuberculosis RNA polyeerase } fb) nucleic acid Molecules coeprising a nucleotide sequence capidile of hybridizing to a nucleotide sequecne complementary the polypeptide coding region of a DNA molecule as defined in (a) and Mhich codes for a polypeptide Mhich is a Group I sigma subunit of HycobacteriuM tuberculosis or a functionally equivalent modified form thereof! (c> nucleic acid molecules coeprising a nucleic acid sequence Mhich is degenerate, as a result of the genetic code, to a nucleitide sequence as defined in and which codes for a polypeptide which is a Broup I sigMa subunit of Nycobacterium tuberculosis or a functionally equivalent modified form thereof. 5. A vector which comprises a nucleic acid molecule according to claie 3 or 4. 6. A vector according to claim 5 Mhich is the plasmid vector pARC BITS (NCIHB 4073B) or pARC 8176 (NCIHB 40739>. 7. A vector according to claim S Mhich im an expression vector capable of mediating the expression of a polypeptide according to claim 1 or 2. B. A host cell harbouring a verctor according to any one of claims 5 to 7. 9. A process for production of a polypeptide according to claim i or 2 Mhich comprises culturing a host cell according to claim B transformed Mith an expression vector according to claim 7 under conditions Mhereby said polypeptide is produced and recovering said polypeptide. 10. A method of assaying for compounds Mhich have the ability to inhibit the association of a sigma subunit Mith a Mycobacterium tuberculosis core RNA polymerase, said method comprising contacting a compound to be tested for said inhibition ability Mith a polypeptide according to claim 1 or claim 2 and a Hycobacterium tuberculosis core RNA polymerase| and (ii) detecting Mhether the said polypeptide associates Mith the said core RNA polymerase to form a RNA polymerase holoenzyme. 11. A Method according to claim lO wherein polypeptide which are associates to core RNA polymerase and/or polypeptides which are not associated to core RNA polymerase are detected by chromatography such as gel filtration. 12. A method according to claim 10 wherein RNA polymerase holoenzyme is detected by immunoprecipitation, using an antibody binding to RNA polymerase holoenzyme. 13. A method of assaying for compounds which have the ability to inhibit sigma subunit~dependent transcription by a Mycobacterium tuberculosis RNA polymerase, said method comprising (i> contacting a compound to be tested for said inhibition ability with a polypeptide according to claim 1 or 2, a Mycobacterium tuberculosis core RNA polymerase, and a DNA having a coding sequence operably—linked to a promoter sequence capable of recognition by said core RNA polymerase when bound to said polypeptide, said contacting being carried out under conditions suitable for transcription of said coding sequence when Mycobacterium tuberculosis RNA polymerase is bound to said promoter, and {ii> detecting formation of mRNA corresponding to said coding sequence. 14. A method of determining the protein structure of a Mycobacterium tuberculosis RNA polymerase sigma subunit, charicterised in that a polypeptide according to claim 1 or claim 2 is utilized in X-ray crystallography.

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293-mas-1995 abstract-duplicate.pdf

293-mas-1995 claims-duplicate.pdf

293-mas-1995 description (complete)-duplicate.pdf

293-mas-1995 drawings-duplicate.pdf

293-mas-95 abstract.pdf

293-mas-95 claims.pdf

293-mas-95 correspondence-others.pdf

293-mas-95 correspondence-po.pdf

293-mas-95 description (complete).pdf

293-mas-95 description (provisional).pdf

293-mas-95 drawings.pdf

293-mas-95 form-1.pdf

293-mas-95 form-19.pdf

293-mas-95 form-26.pdf

293-mas-95 form-3.pdf

293-mas-95 form-5.pdf

293-mas-95 form-6.pdf

293-mas-95 others.pdf

293-mas-95 pct.pdf