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WO1994026765A1 - Use of genes of m. tuberculosis, m. bovis and m. smegmatis which confer isoniazid resistance - Google Patents

Use of genes of m. tuberculosis, m. bovis and m. smegmatis which confer isoniazid resistance Download PDF

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Publication number
WO1994026765A1
WO1994026765A1 PCT/US1994/005398 US9405398W WO9426765A1 WO 1994026765 A1 WO1994026765 A1 WO 1994026765A1 US 9405398 W US9405398 W US 9405398W WO 9426765 A1 WO9426765 A1 WO 9426765A1
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tuberculosis
nucleic acid
ala
isoniazid
enzyme
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PCT/US1994/005398
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French (fr)
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William R. Jacobs
Asesh Banerjee
Desmond Collins
Theresa Mary Wilson
Geoffrey William De Lisle
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Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University
Agresearch
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Priority to AU69496/94A priority Critical patent/AU6949694A/en
Publication of WO1994026765A1 publication Critical patent/WO1994026765A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)

Definitions

  • polynucleotide includes genes, expressed mRNA, proteins, promoters, enhancers and enzymes.
  • This invention relates to the identification, cloning sequencing and characterization of M. tuberculosis. M. bovis and M. sme ⁇ matis polynucleotides which determine whether or not tuberculosis mycobacteria (i.e. M. tuberculosis. M. bovis or M. africanum) will be resistant to the anti-tuberculosis drugs isoniazid and ethianomide.
  • polynucleotides which include genes, and the enzymes they encode may be used to produce probes capable of identifying the nucleic acids in mycobacteria which encode isoniazid resistance, which probes can be used in the treatment and prevention of tuberculosis, to assess the susceptibility of various mycobacterial strains to isoniazid, and to determine whether various antibiotic drugs are effective against the M. tuberculosis complex.
  • This invention further relates to diagnostic kits containing said probes.
  • bovis pS5 mRNA can be produced, which compounds may be used to eliminate isoniazid resistant or isoniazid sensitive mycobacteria. Further, the polynucleotides of this invention may be used to generate vaccines for tuberculosis or novel recombinant BCG vaccines.
  • Tuberculosis (which includes infection caused by M. tuberculosis, M. bovis and . africanum) remains the largest cause of human death in the world from a single infectious disease, and is responsible for one in four avoidable adult deaths in developing countries. In addition, in 1990, there was a 10% increase in the incidence of tuberculosis in the United States. Further, . bovis causes tuberculosis in a wide range of animals, and is a major cause of animal suffering and economic loss in animal industries. isoniazid-resistant isolates appear to be catalase negative. Previous studies have shown that low-level isoniazid resistance correlates better with the co-acquisition of ethionamide resistance than with loss of catalase activity.
  • Drug resistance can often be mediated by an accumulation of mutations in the polynucleotide (gene) which encodes the target. These mutations result in amino acid substitutions which result in reduced binding of drugs to their target enzymes.
  • rifampicin resistance is often mediated by mutations in the gene encoding the ⁇ ' subunit of RNA polymerase, or trimethoprim resistance can be mediated by mutations in dihydrofolate reductase.
  • trimethoprim resistance can be mediated by mutations in dihydrofolate reductase.
  • isoniazid blocks mycolic acid biosynthesis in the M. tuberculosis complex.
  • the inhibition of mycolic acid biosynthesis in cell-free extracts of mycobacteria has confirmed that isoniazid specifically inhibits mycolic acid biosynthesis.
  • the target enzymes had not yet been identified prior hereto.
  • tuberculosis cells but appears n °t to be the target of action of isoniazid.
  • isoniazid-resistance can be accounted for by the loss of catalase activity, in some studies only 10% of the enzymes themselves, could then be used to eliminate isoniazid resistance of various strains of the M. tuberculosis complex.
  • inhA which encodes the enzyme (InhA) comprising the target of action of M. tuberculosis, M. avium and M. smegmatis for isoniazid
  • pS5 polynucleotide
  • the DNA sequences of these polynucleotides can be used to prepare anti-DNA or anti-mRNA oligonucleotides which are administered and which inhibit the mRNA activity of the polynucleotides, thereby preventing expression of the enzymes and eliminating isoniazid resistance so as to treat and prevent tuberculosis infection.
  • These DNA sequences can be used to produce oligonucleotide probes capable of identifying the nucleic acids in tuberculosis mycobacteria which encode isoniazid resistance, which probes may be used in diagnostic kits.
  • the polynucleotides of this invention can be used to assess the susceptibility of various strains of the M. tuberculosis complex in clinical samples to the antibiotic isoniazid.
  • the chromosomal DNA of the inhA polynucleotide (gene) of M. tuberculosis or the DNA of the pS5 polynucleotide (gene) of M. bovis from a clinical sample is isolated.
  • Oligonucleotides are then prepared using the DNA sequences.
  • the region of the gene from the clinical sample which is identified by the oligonucleotides is amplified, for example using PCR, and then single strand conformation polymorphism analysis is performed to determine whether a mutation gene exists in the tuberculosis from the clinical sample, the presence of a mutation indicating that the tuberculosis in the clinical sample is resistant to isoniazid.
  • the polynucleotides of this invention can also be used to determine whether various antibiotic drugs are effective against tuberculosis.
  • the polynucleotides which encode the enzymes are overexpressed so as to obtain said enzymes, which enzymes are then combined with a purified reagent to obtain a mycolic acid.
  • the mycolic acid is assayed to measure its level of biosynthesis, and then a drug is combined with the mycolic acid synthesis system to determine whether the drug blocks the biosynthetic activity of the system. If the biosynthesis of mycolic acid is blocked, then the drug is effective against tuberculosis.
  • Figure 1 represents minimal inhibitory concentrations of K. smeqmatis transformed with various cosmid clones from M. tuberculosis, M. smeqmatis, M. bovis and M. avium;
  • Figure 2 represents the DNA sequences of isoniazid resistant polynucleotides from M. tuberculosis, M. bovis and M. smeqmatis.
  • M. smeqmatis sequence the single point mutation that is different (in comparison to the isoniazid sensitive strain) is the key feature which determines that this polynucleotide is isoniazid resistant
  • Figure 3 represents the subcloning strategy used to demonstrate that the isoniazid resistance phenotype is conferred by the inhA open reading frame;
  • Figure 4 represents an analysis comparing the inhA open reading frame to the envM open reading frames of E. Coli and S. typhimurium. The close homology therebetween suggests that inhA is involved in lipid biosynthesis;
  • Figure 5 represents the percent inhibition of cell-free extracts derived from M. smeqmatis transformants containing various recombinant inhA plasmids in the presence of isoniazid;
  • Figure 6 represents an allelic exchange experiment
  • Figure 7 is comprised of Figure 7A, Figure 7B and Figure 7C, and represents the nucleic acid sequence of the
  • Figure 8 is comprised of Figure 8A, Figure 8B and
  • Figure 9 is comprised of Figure 9A, Figure 9B,
  • Figure 9C and Figure 9D represents the amino acid sequence of the pS5 M. bovis operon
  • Figure 10 represents the amino acid sequence of a fragment of the pS5 operon encoded by nucleic acid residues 1256-2062;
  • Figure 11 represents the amino acid sequence of a fragment of the pS5 operon encoded by nucleic acid residues 494-1234;
  • Figure 12 represents the nucleic acid sequence of the pS5 M. bovis operon
  • Figure 13 represents the vector map of pYUB18.
  • This invention is directed to polynucleotides which encode the enzymes of M. tuberculosis, M. bovis, M. avium and M. smegmatis which are the targets of action of isoniazid. Mutations of these polynucleotides confer isoniazid resistance onto mycobacteria.
  • polynucleotides In order to enable the elimination of isoniazid resistance conferred by these polynucleotides (inhA for M. tuberculosis and pS5 for M. bovis) , the polynucleotides have been identified, isolated, cloned, sequenced and characterized. The DNA sequences of the polynucleotides are used to develop treatments for tuberculosis and other mycobacterial infections, and can be used to determine whether various strains of the M. tuberculosis complex in clinical samples are resistant to isoniazid. In addition, these polynucleotides are used to assess whether antibiotic drugs are effective against the M. tuberculosis complex. The inventors have employed a genetic strategy to identify the targets of action of the M.
  • FIG. 1 represents the minimal inhibitory concentrations of M. smegmatis formed with the various cosmid clones.
  • a 3 kb BamHI DNA fragment from the M. smegmatis cosmid that retained its ability to confer isoniazid-resistance was used as a probe for Southern analysis and found to strongly hybridize to all of 11 different mycobacterial species tested, demonstrating that the inhA gene is highly conserved among mycobacteria.
  • the DNA sequences for the DNA fragments that conferred isoniazid-resistance were determined for DNAs from the isoniazid-sensitive strains of M. smegmatis and M. tuberculosis, and isoniazid-resistant strains of M. smegmatis and M. bovis.
  • the inhA genes are preceded by another open reading frame that shares 40% identity with acetyl CoA reductases.
  • the inhA genes are positioned within operons with the ORF1
  • the inhA gene has its own promoter.
  • ORF1 has been designated mabA, a gene which is involved in mycolic acid biosynthesis.
  • Figure 6 represents the allelic exchange experiment
  • Polynucleotides from M. smegmatis and M. tuberculosis (inhA) which encode the target of action for isoniazid and ethionamide have been identified, isolated, cloned, seguenced and characterized.
  • the nucleic acid sequences for these polynucleotides are shown below as well as in Figures 7 and 8, respectively.
  • GCGCGATCGA CTTCATCCCG GACAAGCGGG TCGGCACGGT CGAGGAGGTC GCGGGCGCGG 780 TCAGCTTCCT GGCCTCGGAG GACGCCTCCT ACATCGCGGG CGCGGTCATC CCCGTCGACG 840
  • CTGCACTTCG GCGGGGTGTC ACCGATCAAC GGCATCAACC GGGACCTGAT CGTCGCGATC 1920 GAGGCCGAAC TCGCCCGACG CGGCCGCAAC CTTCCGGTCT ACTTCGGCAA CCGCAACTGG 1980
  • GCAGCAGTGT CAACGGCGCA CCGTGCACGC CGGCCTGCTC GGCGTGACCC GCCCCGGGCG 2760
  • GCCATACGCG CCGAGCGCAC CACCCGCGTG AGGGGGCGCA GCGCCGAGTC GGCGATCTGA 2880
  • ACCTCCGACG AACTCTGCAG ACCGCTCGGG ATCAGACCCG CACTCACCGC GATGATGGCG 2940 TCGACATGGG CGGCGTTCTC CAGCACCCGC ACAGCCCGGG TCGGCGCGTG GTCGGGGACG 3000
  • GAAGGTCATC AACGCCAACC TCACCGGGGC GTTCCGGGTG GCTCAACGGG CATCGCGCAG 600
  • M. bovis for isoniazid has been identified, isolated, cloned, seguenced and characterized.
  • the amino acid sequence for this gene is shown below, as well as in Figure 9.
  • CGCTCTTCCC AGACTTGCAG CCCCGGGGCA CGGCGGCGGT TGGTGTCGAT GATCGCGGCG 120 GGAAGATCCG CGTCGATCCA CTTGGCGCCA TGGAAGGCAG AAGCCGAGTA GCCGGCCAGC 180
  • GGCGTCAATA CACCCGCAGC CAGGGCCTCG CTGCCCAGAA AGGGATCCGT CATGGTCGAA 360
  • GTGTGCTGAG TCACACCGAC AAACGTCACG AGCGTAACCC CAGTGCGAAA GTTCCCGCCG 420 GAAATCGCAG CCACGTTACG CTCGTGGACA TACCGATTTC GGCCCGGCCG CGGCGAGACG 480
  • CAG CAT GCA GCG CAA CAA ATT CGG TCG AAT GAT ATT CAT AGG TTC GGT 915 Ser Met Gin Arg Asn Lys Phe Gly Arg Met He Phe He Gly Ser Val 130 135 140
  • fragments of the pS5 operon may be responsible for conferring isoniazid resistance:
  • the pS5 operon of M. bovis has the following nucleic acid seguence:
  • SEQ ID NO: 6 GTTCGCTCCG GCGCGGTCAC GCGCATGCCC TCGATGACGC AGATCTCGTC GGGCTCGATG 60
  • the polynucleotides of the invention may be obtained by expressing a DNA sequence coding said polynucleotides in a host cell or organism.
  • a DNA molecule such as the nucleic acid sequence from residues 1256-2062, or 494-1234 of Figure 12, can be expressed in a host cell or organism.
  • promoters which comprise DNA molecules which consist of part or all of the nucleic acid sequence from residues 1-493 of Figure 12, can be used in the cloning and/or expression of a nucleic acid sequence.
  • DNA expression vectors containing polynucleotides of the invention can be prepared by transforming host cells capable of expressing the polynucleotides of the invention or fragments thereof with a DNA sequence encoding the polynucleotides of the invention. The transformed host cells are then cultured, and the expressed polynucleotide is recovered.
  • polynucleotides and fragments of polynucleotides of the invention can be prepared several ways. For example, they can prepared by isolating the polynucleotides from a natural source, or by synthesis using recombinant DNA techniques. In addition, variants of the polynucleotides of the invention can be prepared by utilizing site-specific mutagenesis of the DNA encoding the native amino acid sequences (see Adelman et al., DNA, Vol. 2, p. 183 (1983)). If recombinant DNA techniques are used, DNA encoding the polynucleotide must be obtained. This DNA can be isolated from mycobacteria. Alternatively, the DNA may be produced as intron free cDNA using conventional techniques. In addition, the DNA can be produced in the form of synthetic oligonucleotides.
  • the DNA is obtained, it is treated so that it is suitable for insertion into appropriate cloning and/or expression vectors.
  • the DNA is cleaved utilizing restriction enzymes.
  • the nucleic acid is then recovered.
  • the DNA is then tailored using conventional techniques, such as treatment with polymerase 1, phenol and chloroform.
  • the DNA is then extracted and precipitated by ethanol.
  • religation is performed by providing equimolar amounts of the desired components, appropriately tailored for correct matching and treatment with an appropriate ligase, such as T. DNA ligase.
  • Suitable cloning vectors may be constructed according to standard techniques, or may be selected from the large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used for expressing the polynucleotide-encoding DNA, useful cloning vectors will generally have the ability to self replicate, will possess a single target for any particular restriction endonuclease and will carry genes for a readily selectable marker such as isoniazid resistance. Two major types of cloning vectors which may be used are plasmids and bacterial viruses. Examples of such vectors include pUC18, pl8, Mpl9, pBR322, pf*!39, ColEl, and pCRl from E. coli.
  • a wide range of host plasmids include RP4, phage DNA's such as lambda and M13, and shuttle vectors such as pSA3 and pAT28.
  • phage DNA's such as lambda and M13
  • shuttle vectors such as pSA3 and pAT28.
  • the most suitable cloning vectors are pBluesript Ilks (Stratagene), and pYUB18, as shown in Figure 13.
  • the cloning vector must incorporate an expression control sequence.
  • a typical expression control sequence can be described as having a promoter region, a 5' untranslated region, a polypeptide coding sequence, a 3' untranslated region and a transcription termination region.
  • a promoter which may be used comprises all or part of the nucleic acid sequence from residues 1-493 shown in Figure 12.
  • Hosts which may be used in this invention include bacteria, yeasts, fungi, insects and animal and plant cells.
  • Procaryatic hosts are generally preferred.
  • Mycobacterial and bacterial hosts including E. coli and M. smec ⁇ tatis, are particularly suitable. Of the mycobacterial hosts,
  • M. smeornatis mc 155 is preferred.
  • the exogenous protein is then isolated using routine methods such as freeze-thaw extraction. Purification is then performed utilizing conventional techniques.
  • a DNA molecule comprising all or part of the nucleic acid sequences of the polynucleotides of the invention may be used as probes for identifying nucleic acids which code for polynucleotides associated with isoniazid resistance.
  • a probe may be labelled, for example, with radioactive isotopes, including 32P and 33P labels.
  • the probes of the invention are capable of hybridizing to the genetic elements associated with isoniazid resistance.
  • the probes of the invention are specific for sense-strand and anti-sense strand of the DNA which encodes for the isoniazid resistance gene.
  • the probe may be the entire nucleotide sequence depicted in Figure 12. More useful probes may be from 100-1000 base pairs. However, shorter probes are preferred.
  • diagnostic kits for use in detecting the presence of polynucleotides which confer isoniazid resistance in a clinical sample.
  • the diagnostic kits of the invention include nucleic acid probes, as discussed hereinabove, which may be labelled.
  • the diagnostic kits of the invention may also include a lysing agent, a denaturing solution, a neutralizing solution, an alkaline fixation solution and a saline citrate.
  • Clinical samples may be tested to determine whether the samples contain polynucleotides which confer isoniazid resistance.
  • Clinical samples are tested with labelled probes under conditions such that the probes will hybridize to the isoniazid resistance-associated genes.
  • hybrid DNA is detected in the sample. These procedures are performed using standard techniques.
  • polynucleotides which are associated with isoniazid resistance are amplified in a clinical sample. After amplification, the detection of the presence or absence of the amplified DNA in the sample indicates whether the sample contains polynucleotides which confer isoniazid resistance.
  • antibodies can be raised which are immunoreactive with epitopes on the polynucleotides of the invention which confer isoniazid resistance. These antibodies can be used in diagnostic immunoassays, and can be passively administered to prevent and treat tuberculosis and other mycobacterial infection and disease.
  • M. bovis isoniazid resistant strains In order to select M. bovis isoniazid resistant strains, a virulent wild-type New Zealand strain of M. bovis was cloned by four serial passages using a combination of liquid tween albumin broth (TAB) and 7H10 pyruvate agar media. A single colony of M. bovis was inoculated into TAB and incubated until visible growth was apparent. An appropriate dilution of this TAB was then inoculated onto solid media to obtain discrete colonies. After incubation, a single colony was inoculated into TAB and the process was repeated. The G4 strain was obtained after four cloning cycles.
  • TAB liquid tween albumin broth
  • the isoniazid resistant strains were obtained by inoculating G4 into a series of TABs containing different concentrations of isoniazid and then incubated. Of the TABs with luxuriant growth, the one with the highest concentration of isoniazid, was inoculated onto solid media containing isoniazid. A colony from this inoculation was used to inoculate a TAB containing isoniazid and incubated. When visible growth was apparent, this was used to inoculate a series of TABs containing varying concentrations of isoniazid. This selection procedure was repeated to obtain a series of clones of M. bovis with increasing resistance to isoniazid. The last strain selected, G4/100, is resistant to 100 ⁇ g/ml of isoniazid.
  • a cosmid library of G4/100 was made in the shuttle vector pYUB18 (as shown in Figure 13). This vector replicates independently in E. coli and various mycobacterial species. It contains a selectable kanamycin gene and a cos site.
  • the cosmid library was made by performing the following steps: (a) Partial Sau3AI digestion of purified chromosomal
  • M. smegmatis organisms were selected by growth on media containing kanamycin. Approximately 1200 kanamycin resistant clones were patched onto media containing isoniazid. Four isoniazid resistant clones were identified.
  • M. smeqmatis cultures of M. smeqmatis (5 ml) were incubated with cycloserine and ampicillin for 3 hours before harvest. The cells were pelleted and resuspended in 0.25 ml of 40 mM Tris-acetate, 2 mM EDTA, pH 7.9. To this, 0.5 ml of lysing solution was added (50 MM Tris, 3% SDS) and the solution was mixed for 30 minutes. The sample was heated to 60°C for 20 minutes, cooled for 10 minutes and the DNA was extracted by adding 0.8 ml of phenol (containing 50 mM NaCI). This was centrifuged and the upper layer containing the DNA was removed.
  • lysing solution 50 MM Tris, 3% SDS
  • pS5 The smallest plasmid obtained which conferred an isoniazid resistance phenotype on M. sme ⁇ matis was 2.3 Kb in size and was designated pS5.
  • pS5 was cloned into the vector pBluesript II KS+ (Stratagene, CA) .
  • This vector contains the T3 and T7 promoters which were used for the sequencing. Sequencing was carried out using the dsDNA cycle sequencing system from GIBCO BRL, Life Technologies, according to the manufacturer's instructions.
  • radioactively labelled nucleotide was either [ ⁇ - P] ATP or [ ⁇ - 33P] ATP (Amersham) .
  • the sequencing program used was
  • GCG Sequence Analysis Software Package
  • the enzyme was first isolated by pelleting a culture of M. bovis and resuspending in 50 mM potassium phosphate buffer, pH 7. This was added to a tube containing 0.5 g zirconium beads (Biospecs Products) and vortexed for 5 minutes. The sample was centrifuged briefly and the supernatant was diluted to 4 ml with 50 mM potassium phosphate buffer and then filter sterilized through 0.22 ⁇ m filters.
  • the catalase activity was assayed by incubating an aliquot of the above sample with 3 ⁇ M H,0_ in a total volume of 3 ml for 5 minutes. The reaction was stopped by adding 1.5 ml of titanium tetrachloride reagent (1.5 mg/ml
  • the G4 strain and the other virulent M. bovis strains contained similar levels of catalase activity. No catalase activity was detected in the G4/100 strain.
  • G4(S5) was electroporated into G4 to produce G4(S5).
  • G4(S5) grew on media containing a level of isoniazid that prevented growth of G4.
  • G4(S5) was also shown to have catalase activity similar to that of G4.
  • these polynucleotides can be used in the treatment of M. tuberculosis, M. avium, M. smegmatis, M. bovis and other mycobacterial infection.
  • One method of treating mycobacterial infection such as tuberculosis utilizing these polynucleotides is preparing anti-DNA or anti-RNA oligonucleotides which can be used to inhibit mRNA activity of the inhA operon of M. tuberculosis or M. bovis.
  • These oligonucleotides can be prepared utilizing the wild-type DNA sequence of the inhA operon of M. tuberculosis or the pS5 operon of M.
  • oligonucleotides can then be administered, either alone or in combination with other compositions, to treat mycobacterial infection, including tuberculosis.
  • These oligonucleotides can be administered orally, enterally, subcutaneously, intraperitoneally or intravenously.
  • the DNA sequences of these polynucleotides can also be used to assess the susceptibility of various strains of the M. tuberculosis complex in a clinical sample to isoniazid. In order to perform this, first the chromosomal DNA of the M. tuberculosis complex from a clinical sample must be isolated.
  • Oligonucleotides are prepared, for example using an oligonucleotide synthesizer, utilizing the wild-type inhA polynucleotide DNA sequence of M. tuberculosis depicted in Figure 8 or the DNA sequence of M. bovis depicted in Figure 12. Regions of the inhA polynucleo ide of M. tuberculosis or the PS5 polynucleotide of M. bovis from the clinical sample which are identified by the oligonucleotides are then amplified (for example by using polymerase chain reaction (PCR)) to obtain double stranded DNA. Next, single strand conformation polymorphism analysis is performed in order to determine whether a mutated gene exists in the M. tuberculosis complex organisms from the clinical sample. If a mutation exists, this indicates that the M. tuberculosis complex organisms from the clinical sample are resistant to isoniazid.
  • PCR polymerase chain reaction
  • PCR or other type of amplification is performed after substitution of half of the dCTP by 0.5 ⁇ l of
  • Electrophoresis is then performed at room temperature and constant power (6W for a 50 x 32 x 0.4 cm gel) overnight. Gels are then dried and exposed for autoradiography overnight.
  • An example of using single strand conformation polymorphism is described by Telenti et al . in "Detection Of Rifampicin-Resistance Mutations In Mycobacterium Tuberculosis", Vol. 341, pages 647-650 (March, 1993), which is incorporated herein by referenced.
  • the polynucleotide sequences can also be used to determine whether a drug is effective against tuberculosis. This is performed by overexpre ⁇ sing the M. tuberculosis inhA polynucleotide or the M. bovis pS5 polynucleotide (i.e., a gene which encodes the target of action enzyme for isoniazid) so as to obtain the enzyme. The enzyme is then combined with a purified reagent, such as a fatty acid or NADP, to obtain a mycolic acid. The mycolic acid is then assayed to measure its level of biosynthesis, such as by thin layer chromatography or spectrophotometry.
  • a purified reagent such as a fatty acid or NADP
  • a drug is combined with the system used to produce mycolic acid to determine whether the drug blocks mycolic acid biosynthesis. If there is a blockage, this indicates that the drug is effective against tuberculosis.
  • Drugs which may be tested for their effectiveness against tuberculosis by this method include isoniazid, ethionamide, rifampicin, streptomycin, ethambutol, ciprofloxacin, novobiocin and cyanide. Further, the polynucleotide which encodes an enzyme involved in mycolic acid biosynthesis can be used for treating tuberculosis.
  • the gene mabA which is depicted in Figure 7 from nucleic acid residue 96 to nucleic acid residue 863, and in Figure 8, from nucleic acid residue 224 to nucleic acid residue 967, has been isolated by the inventors and identified as a gene which encodes an enzyme involved in mycolic acid biosynthesis of tuberculosis.
  • a compound which blocks the biochemical activity of the enzyme encoded by the mabA gene is administered. The compound blocks the enzyme activity, thereby preventing mycolic acid biosynthesis. This causes the tuberculosis to organisms die.
  • compounds which block the activity of enzymes encoded by the polynucleotides can be prepared. This is performed by overexpressing the enzyme and purifying the enzyme, and then performing X-ray crystallography on the purified enzyme to obtain the molecular structure of the enzyme. Next, compounds are created which have a similar molecular structure to the enzyme. The compounds are then combined with the enzyme and attached thereto so as to block the biochemical activity of the enzyme. Since the enzyme is blocked, it is unable to confer isoniazid resistance on tuberculosis organisms. Further, tuberculosis-specific purified mycolic acid compounds can be produced by adding the enzyme encoded by the polynucleotide to the chemical reaction which produces mycolic acids.
  • polynucleotide DNA sequences can be used to produce or improve tuberculosis vaccines.
  • M. tuberculosis complex strains that have become isoniazid resistant often have reduced virulence and can be administered as vaccines.
  • mutated genes of M. tuberculosis and M. bovis can be added to BCG or tuberculosis vaccines to provide attenuated mutant tuberculosis vaccines.
  • These vaccines can be used to treat and prevent a wide variety of diseases, including tuberculosis, AIDS, leprosy, polio, malaria and tetanus.
  • GCGCGATCGA CTTCATCCCG GACAAGCGGG TCGGCACGGT CGAGGAGGTC GCGGGCGCGG 780 TCAGCTTCCT GGCCTCGGAG GACGCCTCCT ACATCGCGGG CGCGGTCATC CCCGTCGACG 840
  • CTGCACTTCG GCGGGGTGTC ACCGATCAAC GGCATCAACC GGGACCTGAT CGTCGCGATC 1920
  • GCAGCAGTGT CAACGGCGCA CCGTGCACGC CGGCCTGCTC GGCGTGACCC GCCCCGGGCG 2760
  • GCCATACGCG CCGAGCGCAC CACCCGCGTG AGGGGGCGCA GCGCCGAGTC GGCGATCTGA 2880
  • ACCTCCGACG AACTCTGCAG ACCGCTCGGG ATCAGACCCG CACTCACCGC GATGATGGCG 2940 TCGACATGGG CGGCGTTCTC CAGCACCCGC ACAGCCCGGG TCGGCGCGTG GTCGGGGACG 3000
  • GAAGGTCATC AACGCCAACC TCACCGGGGC GTTCCGGGTG GCTCAACGGG CATCGCGCAG 600
  • GGCGTCAATA CACCCGCAGC CAGGGCCTCG CTGCCCAGAA AGGGATCCGT CATGGTCGAA 360 GTGTGCTGAG TCACACCGAC AAACGTCACG AGCGTAACCC CAGTGCGAAA GTTCCCGCCG 420
  • CAG CAT GCA GCG CAA CAA ATT CGG TCG AAT GAT ATT CAT AGG TTC GGT 915 Ser Met Gin Arg Asn Lys Phe Gly Arg Met He Phe He Gly Ser Val 130 135 140 CTC CGG CAG CTG GGG CAT CGG CAA CCA GGC CAA CTA CGC AGC CTC CAA 963 Ser Gly Ser Trp Gly He Gly Asn Gin Ala Asn Tyr Ala Ala Ser Lys 145 150 155

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

This invention relates to the identification, cloning, sequencing and characterization of polynucleotides which determine mycobacterial resistance to the antibiotic isoniazid. These polynucleotides encode the expression of an enzyme which is the target of action of M. tuberculosis, M. smegmatis, M. bovis and M. avium for isoniazid. The polynucleotides have been DNA sequenced, and can be used in the treatment and prevention of mycobacterial infection, including tuberculosis. In addition, the polynucleotides can be used to determine the susceptibility or resistance of various strains of M. tuberculosis to various antibiotic drugs.

Description

USE OF GENES OF M. TUBERCULOSIS, M. BOVIS AND M. SMEGMATIS WHICH CONFER ISONIAZID RESISTANCE
Statement of Government Interest This invention was made with government support under
NIH Grant No. A126170 and National Cooperative Drug Discovery Group Grant No. UO1A130189. As such, the government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATIONS This Application is a Continuation-In-Part of
Application Serial No. 08/062,409 filed May 14, 1993, entitled USE OF GENES OF M. TUBERCULOSIS AND M. SMEGMATIS WHICH CONFER ISONIAZID RESISTANCE TO TREAT TUBERCULOSIS AND TO ASSESS DRUG RESISTANCE.
FIELD OF THE INVENTION
As used herein, the term "polynucleotide" includes genes, expressed mRNA, proteins, promoters, enhancers and enzymes. This invention relates to the identification, cloning sequencing and characterization of M. tuberculosis. M. bovis and M. smeαmatis polynucleotides which determine whether or not tuberculosis mycobacteria (i.e. M. tuberculosis. M. bovis or M. africanum) will be resistant to the anti-tuberculosis drugs isoniazid and ethianomide. These polynucleotides, which include genes, and the enzymes they encode may be used to produce probes capable of identifying the nucleic acids in mycobacteria which encode isoniazid resistance, which probes can be used in the treatment and prevention of tuberculosis, to assess the susceptibility of various mycobacterial strains to isoniazid, and to determine whether various antibiotic drugs are effective against the M. tuberculosis complex. This invention further relates to diagnostic kits containing said probes. In addition, compounds which block the biochemical activity of the enzyme or expression of M. tuberculosis inhA mRNA and/or . bovis pS5 mRNA can be produced, which compounds may be used to eliminate isoniazid resistant or isoniazid sensitive mycobacteria. Further, the polynucleotides of this invention may be used to generate vaccines for tuberculosis or novel recombinant BCG vaccines.
BACKGROUND OF THE INVENTION Tuberculosis (which includes infection caused by M. tuberculosis, M. bovis and . africanum) remains the largest cause of human death in the world from a single infectious disease, and is responsible for one in four avoidable adult deaths in developing countries. In addition, in 1990, there was a 10% increase in the incidence of tuberculosis in the United States. Further, . bovis causes tuberculosis in a wide range of animals, and is a major cause of animal suffering and economic loss in animal industries. isoniazid-resistant isolates appear to be catalase negative. Previous studies have shown that low-level isoniazid resistance correlates better with the co-acquisition of ethionamide resistance than with loss of catalase activity. Drug resistance can often be mediated by an accumulation of mutations in the polynucleotide (gene) which encodes the target. These mutations result in amino acid substitutions which result in reduced binding of drugs to their target enzymes. For example, rifampicin resistance is often mediated by mutations in the gene encoding the β' subunit of RNA polymerase, or trimethoprim resistance can be mediated by mutations in dihydrofolate reductase. However, the nature of isoniazid activity on organisms of the M. tuberculosis complex, and the precise targets of action in these organisms, have not yet been identified prior to this invention.
It has been suggested that isoniazid blocks mycolic acid biosynthesis in the M. tuberculosis complex. The inhibition of mycolic acid biosynthesis in cell-free extracts of mycobacteria has confirmed that isoniazid specifically inhibits mycolic acid biosynthesis. However, the target enzymes had not yet been identified prior hereto.
Because such a high percentage of the . tuberculosis complex strains are resistant to isoniazid, a great need has existed to determine the targets of action of the M. tuberculosis complex for isoniazid. The genes which encode the enzymes which are the targets of action, as well as the Infection with drug-sensitive strains of the M. tuberculosis complex (which includes infection caused by M. tuberculosis, M. africanum and K . bovis) can be effectively cured with certain antibiotics, including isoniazid, rifampicin, and pyrazinamide. Isoniazid was first reported to be active against M. tuberculosis in 1952, when it was shown to have a high specific activity against M. tuberculosis, M. bovis and BCG with considerably less activity against other mycobacteria. However, resistance to isoniazid was first reported in 1953, and in recent years has been as high as 15-35% in many U.S. cities.
A fraction of isoniazid-resistant strains have been shown to be associated with a loss of catalase activity. The catalase gene (katG) was recently cloned and deletions of this gene were shown to be correlated with isoniazid resistance in certain M. tuberculosis isolates. Furthermore, transfer of the M. tuberculosis katG gene to isoniazid-resistant M. smegmatis strains has resulted in the acquisition of isoniazid sensitivity, suggesting that the presence of the catalase activity results in the exquisite sensitivity of M. tuberculosis to isoniazid. Catalase thus appears to modify isoniazid to an activated intermediate or result in the accumulation of isoniazid in . tuberculosis cells, but appears n°t to be the target of action of isoniazid. Although isoniazid-resistance can be accounted for by the loss of catalase activity, in some studies only 10% of the enzymes themselves, could then be used to eliminate isoniazid resistance of various strains of the M. tuberculosis complex.
It is therefore an object of this invention to identify, isolate, characterize and sequence the polynucleotides which include the genes which encode the enzymes which are the targets of action of the M. tuberculosis complex for isoniazid.
It is another object of this invention to provide methods of preventing and treating M. tuberculosis and M. bovis infection utilizing the DNA sequences of the genes which encode the target of action enzymes of the M. tuberculosis complex for isoniazid.
It is a further object of this invention to provide a method of assessing the isoniazid susceptibility of various strains of the . tuberculosis complex from clinical samples.
It is a still further object of this invention to provide a method of determining whether various antibiotic drugs are effective against the M. tuberculosis complex.
It is yet another object of this invention to provide a method of producing compounds capable of blocking the activity of the isoniazid target action enzymes of the
M. tuberculosis complex, thereby eliminating isoniazid resistance. i
SUMMARY OF THE INVENTION This invention is directed to the polynucleotide
(inhA) which encodes the enzyme (InhA) comprising the target of action of M. tuberculosis, M. avium and M. smegmatis for isoniazid, and to the polynucleotide (pS5) which encodes the enzyme comprising the target of action of M. bovis for isoniazid. These polynucleotides have been identified, isolated, cloned, DNA sequenced and characterized. The DNA sequences of these polynucleotides can be used to prepare anti-DNA or anti-mRNA oligonucleotides which are administered and which inhibit the mRNA activity of the polynucleotides, thereby preventing expression of the enzymes and eliminating isoniazid resistance so as to treat and prevent tuberculosis infection. These DNA sequences can be used to produce oligonucleotide probes capable of identifying the nucleic acids in tuberculosis mycobacteria which encode isoniazid resistance, which probes may be used in diagnostic kits. The polynucleotides of this invention can be used to assess the susceptibility of various strains of the M. tuberculosis complex in clinical samples to the antibiotic isoniazid. To perform this, the chromosomal DNA of the inhA polynucleotide (gene) of M. tuberculosis or the DNA of the pS5 polynucleotide (gene) of M. bovis from a clinical sample is isolated. Oligonucleotides are then prepared using the DNA sequences. The region of the gene from the clinical sample which is identified by the oligonucleotides is amplified, for example using PCR, and then single strand conformation polymorphism analysis is performed to determine whether a mutation gene exists in the tuberculosis from the clinical sample, the presence of a mutation indicating that the tuberculosis in the clinical sample is resistant to isoniazid.
The polynucleotides of this invention can also be used to determine whether various antibiotic drugs are effective against tuberculosis. To perform this, the polynucleotides which encode the enzymes are overexpressed so as to obtain said enzymes, which enzymes are then combined with a purified reagent to obtain a mycolic acid. Next, the mycolic acid is assayed to measure its level of biosynthesis, and then a drug is combined with the mycolic acid synthesis system to determine whether the drug blocks the biosynthetic activity of the system. If the biosynthesis of mycolic acid is blocked, then the drug is effective against tuberculosis.
BRIEF DESCRIPTION OF THE DRAWINGS The above brief description, as well as further objects and features of the present invention, will be more fully understood by reference to the following detailed description of the presently preferred albeit illustrative, embodiment of the present invention when taken in conjunction with the accompanying drawings wherein:
Figure 1 represents minimal inhibitory concentrations of K. smeqmatis transformed with various cosmid clones from M. tuberculosis, M. smeqmatis, M. bovis and M. avium;
Figure 2 represents the DNA sequences of isoniazid resistant polynucleotides from M. tuberculosis, M. bovis and M. smeqmatis. On the M. smeqmatis sequence, the single point mutation that is different (in comparison to the isoniazid sensitive strain) is the key feature which determines that this polynucleotide is isoniazid resistant; Figure 3 represents the subcloning strategy used to demonstrate that the isoniazid resistance phenotype is conferred by the inhA open reading frame;
Figure 4 represents an analysis comparing the inhA open reading frame to the envM open reading frames of E. Coli and S. typhimurium. The close homology therebetween suggests that inhA is involved in lipid biosynthesis;
Figure 5 represents the percent inhibition of cell-free extracts derived from M. smeqmatis transformants containing various recombinant inhA plasmids in the presence of isoniazid;
Figure 6 represents an allelic exchange experiment
2 demonstrating that the point mutation in the mc 651 inhA polynucleotide results in isoniazid resistance;
Figure 7 is comprised of Figure 7A, Figure 7B and Figure 7C, and represents the nucleic acid sequence of the
M. smeqmatis inhA operon;
Figure 8 is comprised of Figure 8A, Figure 8B and
Figure 8C, and represents the nucleic acid sequence of the
M. tuberculosis inhA operon; Figure 9 is comprised of Figure 9A, Figure 9B,
Figure 9C and Figure 9D, and represents the amino acid sequence of the pS5 M. bovis operon; Figure 10 represents the amino acid sequence of a fragment of the pS5 operon encoded by nucleic acid residues 1256-2062;
Figure 11 represents the amino acid sequence of a fragment of the pS5 operon encoded by nucleic acid residues 494-1234;
Figure 12 represents the nucleic acid sequence of the pS5 M. bovis operon; and
Figure 13 represents the vector map of pYUB18.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to polynucleotides which encode the enzymes of M. tuberculosis, M. bovis, M. avium and M. smegmatis which are the targets of action of isoniazid. Mutations of these polynucleotides confer isoniazid resistance onto mycobacteria.
In order to enable the elimination of isoniazid resistance conferred by these polynucleotides (inhA for M. tuberculosis and pS5 for M. bovis) , the polynucleotides have been identified, isolated, cloned, sequenced and characterized. The DNA sequences of the polynucleotides are used to develop treatments for tuberculosis and other mycobacterial infections, and can be used to determine whether various strains of the M. tuberculosis complex in clinical samples are resistant to isoniazid. In addition, these polynucleotides are used to assess whether antibiotic drugs are effective against the M. tuberculosis complex. The inventors have employed a genetic strategy to identify the targets of action of the M. tuberculosis complex for isoniazid. To perform this, genomic libraries were constructed in shuttle cosmid vectors from isoniazid-resistant mutants of M. smegmatis and M. bovis. Upon transferring of the libraries into isoniazid sensitive M. smeqmatis strains, clones were identified that would consistently confer isoniazid-resistance. Figure 1 represents the minimal inhibitory concentrations of M. smegmatis formed with the various cosmid clones.
It was determined that transformation of libraries from isoniazid-sensitive strains of K . smegmatis, M. tuberculosis and BCG, as well as M. avium, yielded clones that conferred the isoniazid-resistance phenotype, thereby suggesting that overexpression of a putative target gene, termed inhA, on a multi-copy plasmid conferred an isoniazid-resistance phenotype. Hence, it was concluded that the inhA gene product is the target of action of M. tuberculosis for isoniazid. All of these cosmids also had the ability to confer ethionamide resistance to M. smeσmatis as well. A 3 kb BamHI DNA fragment from the M. smegmatis cosmid that retained its ability to confer isoniazid-resistance was used as a probe for Southern analysis and found to strongly hybridize to all of 11 different mycobacterial species tested, demonstrating that the inhA gene is highly conserved among mycobacteria. The DNA sequences for the DNA fragments that conferred isoniazid-resistance were determined for DNAs from the isoniazid-sensitive strains of M. smegmatis and M. tuberculosis, and isoniazid-resistant strains of M. smegmatis and M. bovis. The DNA sequences of resistant genes from M. tuberculosis. M. smegmatis and M. bovis are shown in Figure 2. Sequence analysis revealed that there are two open reading frames (ORFs) encoding proteins of 25 kDa and 29 kDa, respectively, in all three species. Subcloning analyses, as shown in Figure 3, demonstrated that ORF encoding the protein of 29 kDa from M. smegmatis was responsible for the isoniazid-resistance phenotype, and was named the inhA gene. The M. tuberculosis and the M. smegmatis inhA gene products show 38% and 40% homologies to the envM gene of S. tvphimurium (See Figure 4). In the M. smegmatis, M. tuberculosis. and M. bovis genomes, the inhA genes are preceded by another open reading frame that shares 40% identity with acetyl CoA reductases. In the M. bovis and the M. tuberculosis genomes, it appears that the inhA genes are positioned within operons with the ORF1, whereas in the M. smegmatis genome, the inhA gene has its own promoter. ORF1 has been designated mabA, a gene which is involved in mycolic acid biosynthesis.
The similarities of the inhA ORF and the ORF1 to lipid biosynthetic genes are consistent with the hypothesis that isoniazid inhibits an enzyme involved in mycolic acid biosynthesis. Cell free extracts containing the inhA clones all showed marked resistance to isoniazid inhibition compared to the wild-type control. Figure 5 represents the percent inhibition of the cell-free extracts derived from M. smegmatis transformants containing various recombinant inhA plasmids in the presence of isoniazid.
Sequence analysis of the inhA gene from the
2 isoniazid-resistant mutant of M. smegmatis, mc 651, compared to the isoniazid-sensitive parent, mc 2155, revealed the presence of a single base pair difference that resulted the amino acid substitution of an alanine for a serine at position
94 of the InhA protein. This is shown in Figure 4. In order to test if this difference causes the isoniazid-resistance phenotype in mc 2651, it was necessary to perform an allelic exchange on the M. smegmatis chromosome. Using a shuttle Pacl-cosmid mutagenesis system, it was possible to transform mc 2651 with a cloned 45 kb DNA fragment from mc2155 that had been tagged with a kanamycin-resistance gene.
Figure 6 represents the allelic exchange experiment
2 demonstrating that the point mutation in the mc 651 inhA gene results in isoniazid resistance. Kanamycin-resistant transformants were screened for their isoniazid sensitivity. The observation that isoniazid-sensitivity co-transformed with the kanamycin-resistance gene in 70% of the transformants strongly supports the hypothesis that serine to alanine substitution at position 94 of InhA results in the isoniazid-resistance phenotype. This hypothesis is further
strengthened by the observation that the same gene from an isoniazid-resistant M. bovis strain has the analogous mutation when compared to the inhA genes from isoniazid-sensitive M. bovis and M. tuberculosis strains which are themselves identical.
Polynucleotides from M. smegmatis and M. tuberculosis (inhA) which encode the target of action for isoniazid and ethionamide have been identified, isolated, cloned, seguenced and characterized. The nucleic acid sequences for these polynucleotides are shown below as well as in Figures 7 and 8, respectively.
Sequence of M. smeqmatis inhA operon SEQ ID NO: 1: GGATCCGCCG CACGGGGAGC CCCGAGGCGA TTTCTGGCTG GACCGGCCAA CACGTTAAGT 60 TGACGGGCGA AGACGCAGGA CGCGAGGAAC AGAGGATGAC TGTGACTGAC AATCCGGCCG 120
ACACCGCGGG CGAGGCCACT GCAGGCCGCC CGGCGTTCGT CTCCCGTTCG GTGCTGGTGA 180
CCGGTGGTAA CCGCGGCATC GGCCTGGCGA TCGCGCGACG GCTGGCCGCC GACGGGCACA 2 0
AGGTGGCCGT CACCCACCGC GGTTCCGGTG CACCCGACGA CCTGTTCGGT GTTCAATGTG 300
ACGTCACCGA CAGCGCTGGT GTCGACCGCG CCTTCAAAGA GGTCGAGGAG CACCAGGGCC 360 CGGTCGAGGT GCTGGTGGCC AACGCAGGCA TCTCCAAGGA CGCATTCCTC ATGCGCATGA 420
CCGAGGAGCG GTTCGAAGAG GTCATCAACA CCAACCTCAC GGGCGCGTTC CGGTGCGCCC 480
AGCGGGCGTC GCGCACCATG CAGCGCAAGC GGTTCGGGCG CATCATCTTC ATCGGGTCGG 540
TCTCGGGCAT GTGGGGGATC GGCAATCAGG CCAACTACGC GGCCGCCAAG GCGGGCCTGA 600
TCGGCATGGC CCGCTCGATC TCCCGTGAGC TGGACAAGGC GGGCGTCACC GCGAACGTGT 660 TGCCCCCCGG TTACATCGAC ACCGAGATGA CCCGGGCGCT CGACGAGCGC ATCCAGGGGG 720
GCGCGATCGA CTTCATCCCG GACAAGCGGG TCGGCACGGT CGAGGAGGTC GCGGGCGCGG 780 TCAGCTTCCT GGCCTCGGAG GACGCCTCCT ACATCGCGGG CGCGGTCATC CCCGTCGACG 840
GCGGTATGGG CATGGGCCAC TAGTCAAAAG CCCGGACACA CAAGATTTCT CGCTCACAAG 900
GAGTCACCAA ATGACAGGCC TACTCGAAGG CAAGCGCATC CTCGTCACGG GGATCATCAC 960
CGATTCGTCG ATCGCGTTCC ACATCGCCAA GGTCGCCCAG GAGGCCGGCG CCGAACTGGT 1020 GCTGACCGGT TTCGACCGCC TGAAGTTGGT CAAGCGCATC GCCGACCGCC TGCCCAAGCC 1080
GGCCCCGCTG CTGGAACTCG ACGTGCAGAA CGAGGAGCAC CTGTCGACTC TGGCCGACCG 1140
GATCACCGCC GAGATCGGTG AGGGCAACAA GATCGACGGT GTGGTGCACG CGATCGGGTT 1200
CATGCCGCAG AGCGGTATGG GCATCAACCC GTTCTTCGAC GCGCCGTACG AGGATGTGTC 1260
CAAGGGCATC CACATCTCGG CGTACTCGTA CGCCTCGCTC GCCAAAGCCG TTCTGCCGAT 1320 CATGAATCCG GGCGGCGGCA TCGTCGGCAT GGACTTCGAC CCCACGCGCG CGATGCCGGC 1380
CTACAACTGG ATGACCGTCG CCAAGAGCGC GCTCGAATCG GTCAACCGGT TCGTCGCGCG 1440
TGAGGCGGGC AAGGTGGGCG TGCGCTCGAA TCTCGTTGCG GCAGGACCGA TCCGCACGCT 1500
GGCGATGAGC GCAATCGTGG GCGGTGCGCT GGGCGACGAG GCCGGCCAGC AGATGCAGCT 1560
GCTCGAAGAG GGCTGGGATC AGCGCGCGCC GCTGGGCTGG AACATGAAGG ACCCGACGCC 1620 CGTCGCCAAG ACCGTGTGCG CACTGCTGTC GGACTGGCTG CCGGCCACCA CCGGCACCGT 1680
GATCTACGCC GACGGCGGCG CCAGCACGCA GCTGTTGTGA TACCGCCGTG TCGTATGACG 1740
CCTTGCTACT GCTGTCGTTC GACGGGCCGG AACTCCCGAG CAGGTGATGC CGTTCTTGGA 1800
GAACTCACCA GGGGCCGCGG AATCCCCAGG GAGCGGCTGG AATCGGTGGC CGAGCACTAT 1860
CTGCACTTCG GCGGGGTGTC ACCGATCAAC GGCATCAACC GGGACCTGAT CGTCGCGATC 1920 GAGGCCGAAC TCGCCCGACG CGGCCGCAAC CTTCCGGTCT ACTTCGGCAA CCGCAACTGG 1980
GAGCCGTACG TCGAAGACAC TGTCAAGGCG ATGTCCGACA ACGGAATCCG TCGTGCGGCG 2040
GTGTTCGCGA CCTCGGCGTG GGGTGGGTAC TCGGGATGCG CCCAGTACCA GGAGGACATC 2100
GCGCGTGGCC GGGCCGCCGC CGGGCCCGAG GCGCCGGAGC TGGTCAAGCT GCGCCAGTAT 2160
TTCGACCACC CGCTGTTCGT CGAGATGTTC GCCGACGCCG TCGCCGACGC CGCGGCCACC 2220 CTGCCCGAGG AACTGCGGGA CGAAGCGCGG CTGGTGTTCA CCGCCCACTC CATCCCGCTG 2280
CGTGCCGCGT CGCGTTGCGG TGCAGATCTC TACGAGCGGC AGGTGGGTTA CGCCGCGCGG 2340
CTGGTCGCGG CCGCAGCCGG GTACCGCGAA TACGACCAGG TATGGCAGTC CCGGTCCGGC 2400
CCGCCGCAGG TGCCGTGGCT CGAACCCGAC GTCGGAGATC ACCTTGAGGC GTTGGCGCGC 2460
AACGGCACCA GGGCGGTCAT CGTGTGTCCC CTCGGCTTCG TCGCCGACCA CATCGAGGTG 2520
GTGTGGGATC TGGACAACGA ACTGGCCGAG CAGGCCGCCG AGGCAGGCAT CGCGTTCGCG 2580
CGTGCCGCCA CGCCCAACTC CCAGCCACGT TTTGCCCAAC TTGTCGTCGA CCTGATCGAC 2640 GAAATGCTGC ACGGACTTCC GCCACGCCGG GTCGAGGGGC CCGATCCGTG CCCGCCTACG 2700
GCAGCAGTGT CAACGGCGCA CCGTGCACGC CGGCCTGCTC GGCGTGACCC GCCCCGGGCG 2760
CAGCGAGTCG GGCCGGGCGA TCAAGAACGC CAGGCGGAAT GCAGGATCGC CTCGAGTGCG 2820
GCCATACGCG CCGAGCGCAC CACCCGCGTG AGGGGGCGCA GCGCCGAGTC GGCGATCTGA 2880
ACCTCCGACG AACTCTGCAG ACCGCTCGGG ATCAGACCCG CACTCACCGC GATGATGGCG 2940 TCGACATGGG CGGCGTTCTC CAGCACCCGC ACAGCCCGGG TCGGCGCGTG GTCGGGGACG 3000
CGGTGCGCGC GCCCGGCGGC GAGGATCTGC TCGACCATCC CGCGCGGATC C 3051
Sequence of M. tuberculosis inhA operon
SEQ ID NO : 2 :
AGCGCGACAT ACCTGCTGCG CAATTCGTAG GGCGTCAATA CACCCGCAGC CAGGGCCTCG 60 CTGCCCAGAA AGGGATCCGT CATGGTCGAA GTGTGCTGAG TCACACCGAC AAACGTCACG 120
AGCGTAACCC CAGTGCGAAA GTTCCCGCCG GAAATCGCAG CCACGTTACG CTCGTGGACA 180
TACCGATTTC GGCCCGGCCG CGGCGAGACG ATAGGTTGTC GGGGTGACTG CCACAGCCAC 240
TGAAGGGGCC AAACCCCCAT TCGTATCCCG TTCAGTCCTG GTTACCGGAG GAAACCGGGG 300
GATCGGGCTG GCGATCGCAC AGCGGCTGGC TGCCGACGGC CACAAGGTGG CCGTCACCCA 360 CCGTGGATCC GGAGCGCCAA AGGGGCTGTT TGGCGTCGAA TGTGACGTCA CCGACAGCGA 420
CGCCGTCGAT CGCGCCTTCA CGGCGGTAGA AGAGCACCAG GGTCCGGTCG AGGTGCTGGT 480
GTCCAACGCC GGCCTATCCG CGGACGCATT CCTCATGCGG ATGACCGAGG AAAAGTTCGA 540
GAAGGTCATC AACGCCAACC TCACCGGGGC GTTCCGGGTG GCTCAACGGG CATCGCGCAG 600
CATGCAGCGC AACAAATTCG GTCGAATGAT ATTCATAGGT TCGGTCTCCG GCAGCTGGGG 660 CATCGGCAAC CAGGCCAACT ACGCAGCCTC CAAGGCCGGA GTGATTGGCA TGGCCCGCTC 720
GATCGCCCGC GAGCTGTCGA AGGCAAACGT GACCGCGAAT GTGGTGGCCC CGGGCTACAT 780
CGACACCGAT ATGACCCGCG CGCTGGATGA GCGGATTCAG CAGGGGGCGC TGCAATTTAT 840
CCCAGCGAAG CGGGTCGGCA CCCCCGCCGA GGTCGCCGGG GTGGTCAGCT TCCTGGCTTC 900
CGAGGATGCG AGCTATATCT CCGGTGCGGT CATCCCGGTC GACGGCGGCA TGGGTATGGG 960 CCACTGACAC AACACAAGGA CGCACATGAC AGGACTGCTG GACGGCAAAC GGATTCTGGT 1020
TAGCGGAATC ATCACCGACT CGTCGATCGC GTTTCACATC GCACGGGTAG CCCAGGAGCA 1080
GGGCGCCCAG CTGGTGCTCA CCGGGTTCGA CCGGCTGCGG CTGATTCAGC GCATCACCGA 1140
CCGGCTGCCG GCAAAGGCCC CGCTGCTCGA ACTCGACGTG CAAAACGAGG AGCACCTGGC 1200
CAGCTTGGCC GGCCGGGTGA CCGAGGCGAT CGGGGCGGGC AACAAGCTCG ACGGGGTGGT 1260 GCATTCGATT GGGTTCATGC CGCAGACCGG GATGGGCATC AACCCGTTCT TCGACGCGCC 1320
CTACGCGGAT GTGTCCAAGG GCATCCACAT CTCGGCGTAT TCGTATGCTT CGATGGCCAA 1380
GGCGCTGCTG CCGATCATGA ACCCCGGAGG TTCCATCGTC GGCATGGACT TCGACCCGAG 1440
CCGGGCGATG CCGGCCTACA ACTGGATGAC GGTCGCCAAG AGCGCGTTGG AGTCGGTCAA 1500
CAGGTTCGTG GCGCGCGAGG CCGGCAAGTA CGGTGTGCGT TCGAATCTCG TTGGCGCAGG 1560 CCCTATCCGG ACGCTGGCGA TGAGTGCGAT CGTCGGCGGT GCGCTCGGCG AAGAGGCCGG 1620
CGCCCAGATC CAGCTGCTCG AGGAGGGCTG GGATCAGCGC GCTCCGATCG GCTGGAACAT 1680
GAAGGATGCG ACGCCGGTCG CCAAGACGGT GTGCGCGCTG CTGTCTGACT GGCTGCCGGC 1740
GACCACGGGT GACATCATCT ACGCCGACGG CGGCGCGCAC ACCCAATTGC TCTAGAACGC 1800
ATGCAATTTG ATGCCGTCCT GCTGCTGTCG TTCGGCGGAC CGGAAGGGCC CGAGCAGGTG 1860 CGCCCGTTCC TGGAGAACGT TACCCGGGGC CGCGGTGTGC CTGCCGAACG GTTGGACGCG 1920
GTGGCCGAGC ACTACCTGCA TTTCGGTGGG GTATCACCGA TCAATGGCAT TAATCGCACA 1980
CTGATCGCGG AGCTGGAGGC GCAGCAAGAA CTGCCGGTGT ACTTCGGTAA CCGCAACTGG 2040
GAGCCGTATG TAGAAGATGC CGTTACGGCC ATGCGCGACA ACGGTGTCCG GCGTGCAGCG 2100
GTCTTTGCGA CATCTGCGTG GAGCGGTTAC TCGAGCTGCA CACAGTACGT GGAGGACATC 2160 GCGCGGCCCC CCGCGCGGCC GGGCGCGACG CGCCTGAACT GGTAAAACTG CGGCCCTACT 2220
TCGACCATCC GCTGTTCGTC GAGATGTTCG CCGACGCCAT CACCGCGGCC GCCGCAACCG 2280
TGCGCGGTGA TGCCCGGCTG GTGTTCACCG CGCATTCGAT CCCGACGGCC GCCGACCGCC 2340
GCTGTGGCCC CAACCTCTAC AGCCGCCAAG TCGCCTACGC CACAAGGCTG GTCGCGGCCG 2400
CTGCCGGATA CTGCGACTTT GACCTGGCCT GGCAGTCGAG ATCGGGCCCG CCGCAGGTGC 2460
CCTGGCTGGA GCCAGACGTT ACCGACCAGC TCACCGGTCT GGCTGGGGCC GGCATCAACG 2520 CGGTGATCGT GTGTCCCATT GGATTCGTCG CCGACCATAT CGAGGTGGTG TGGGATCTCG 2580
ACCACGAGTT GCGATTACAA GCCGAGGCAG CGGGCATCGC GTACGCCCGG GCCAGCACCC 2640
CCAATGCCGA CCCGCGGTTC GCTCGACTAG CCAGAGGTTT GATCGACGAA CTCCGTTACG 2700
GCCGTATACC TGCGCGGGTG AGTGGCCCCG ATCCGGTGCC GGGCTGTCTG TCCAGCATCA 2760
ACGGCCAGCC ATGCCGTCCG CCGCACTGCG TGGCTAGCGT CAGTCCGGCC AGGCCGAGTG 2820 CAGGATCGCC GTGACCGCGG ACATCCGGGC CGAGCGCACC ACGGCGGTCA ACGGTCTCAA 2880
CGCATCGGTG GCACGCTGAG CGTCCGACAA CGACTGCGTT CCGATCGGCA ATCGACTCAG 2940
CCCGGCACTG ACCGCGATGA TCGCATCGAC GTGCGCGGCA TTCTCGAGCA CCCGCAATGC 3000
GCGCGATGGC GCGTGGTCGG GAACCCGGTG TTGCCGTGAC GATTCGAGCA ACTGCTCGAC 3060
GAGGCCACGG GGCTTGGCGA CGTCGCTAGA TCCCAGTCCG ATGGTGCTCA AGGCTTCGGC 3120
Biochemical evidence performed on recombinant clones containing the inhA gene suggests that this gene encodes the enzyme involved in mycolic acid biosynthesis. Overexpression of the inhA gene will facilitate studies to identify the precise substrates and products catalyzed by this enzyme. The pS5 operon, which is the target of action of
M. bovis for isoniazid, has been identified, isolated, cloned, seguenced and characterized. The amino acid sequence for this gene is shown below, as well as in Figure 9.
Amino acid seguence of pS5
SEQ ID NO: 3:
GTTCGCTCCG GCGCGGTCAC GCGCATGCCC TCGATGACGC AGATCTCGTC GGGCTCGATG 60
CGCTCTTCCC AGACTTGCAG CCCCGGGGCA CGGCGGCGGT TGGTGTCGAT GATCGCGGCG 120 GGAAGATCCG CGTCGATCCA CTTGGCGCCA TGGAAGGCAG AAGCCGAGTA GCCGGCCAGC 180
ACGCCGCGGC GGCGCGAGCG CAGCCACAGC GCTTTTGCAC GCAATTGCGC GGTCAGTTCC 240
ACACCCTGCG GCACGTACAC GTCTTTATGT AGCGCGACAT ACCTGCTGCG CAATTCGTAG 300
GGCGTCAATA CACCCGCAGC CAGGGCCTCG CTGCCCAGAA AGGGATCCGT CATGGTCGAA 360
GTGTGCTGAG TCACACCGAC AAACGTCACG AGCGTAACCC CAGTGCGAAA GTTCCCGCCG 420 GAAATCGCAG CCACGTTACG CTCGTGGACA TACCGATTTC GGCCCGGCCG CGGCGAGACG 480
ATAGGTTGTC GG GGT GAC TGC CAC AGC CAC TGA AGG GGC CAA ACC CCC ATT 531 Val Thr Ala Thr Ala Thr Glu Gly Ala Lys Pro Pro Phe 5 10
CGT ATC CCG TTC AGT CCT GGT TAC CGG AGG AAA CCG GGG GAT CGG GCT 579 Val Ser Arg Ser Val Leu Val Thr Gly Gly Asn Arg Gly He Gly Leu 15 20 25 GGC GAT CGC ACA GCG GCT GGC TGC CGA CGG CCA CAA GGT GGC CGT CAC 627 Ala He Ala Gin Arg Leu Ala Ala Asp Gly His Lys Val Ala Val Thr 30 35 40 45
CCA CCG TGG ATC CGG AGC GCC AAA GGG GCT GTT TGG CGT CGA ATG TGA 675 His Arg Gly Ser Gly Ala Pro Lys Gly Leu Phe Gly Val Glu Cys Asp 50 55 60
CGT CAC CGA CAG CGA CGC CGT CGA TCG CGC CTT CAC GGC GGT AGA AGA 723 Val Thr Asp Ser Asp Ala Val Asp Arg Ala Phe Thr Ala Val Glu Glu
65 70 75
GCA CCA GGG TCC GGT CGA GGT GCT GGT GTC CAA CGC CGG CCT ATC CGC 771 His Gin Gly Pro Val Glu Val Leu Val Ser Asn Ala Gly Leu Ser Ala 80 85 90
GGA CGC ATT CCT CAT GCG GAT GAC CGA GGA AAA GTT CGA GAA GGT CAT 819 Asp Ala Phe Leu Met Arg Met Thr Glu Glu Lys Phe Glu Lys Val He 95 100 105 CAA CGC CAA CCT CAC CGG GGC GTT CCG GGT GGC TCA ACG GGC ATC GCG 867 Asn Ala Asn Leu Thr Gly Aia Phe Arg Val Ala Gin Arg Ala Ser Arg 110 115 120 125
CAG CAT GCA GCG CAA CAA ATT CGG TCG AAT GAT ATT CAT AGG TTC GGT 915 Ser Met Gin Arg Asn Lys Phe Gly Arg Met He Phe He Gly Ser Val 130 135 140
CTC CGG CAG CTG GGG CAT CGG CAA CCA GGC CAA CTA CGC AGC CTC CAA 963 Ser Gly Ser Trp Gly He Gly Asn Gin Ala Asn Tyr Ala Ala Ser Lys 145 150 155
GGC CGG AGT GAT TGG CAT GGC CCG CTC GAT CGC CCG CGA GCT GTC GAA 1011 Ala Gly Val He Gly Met Ala Arg Ser He Ala Arg Glu Leu Ser Lys 160 165 170
GGC AAA CGT GAC CGC GAA TGT GGT GGC CCC GGG CTA CAT CGA CAC CGA 1059 Ala Asn Val Thr Ala Asn Val Val Ala Pro Gly Tyr He Asp Thr Asp 175 180 185
TAT GAC CCG CGC GCT GGA TGA GCG GAT TCA GCA GGG GGC GCT GCA ATT 1107 Met Thr Arg Ala Leu Asp Glu Arg He Gin Gin Gly Ala Leu Gin Phe 190 195 200 205
TAT CCC AGC GAA GCG GGT CGG CAC CCC CGC CGA GGT CGC CGG GGT GGT 1155 He Pro Ala Lys Arg Val Gly Thr Pro Ala Glu Val Ala Gly Val Val 210 215 220
CAG CTT CCT GGC TTC CGA GGA TGC GAG CTA TAT CTC CGG TGC GGT CAT 1203 Ser Phe Leu Ala Ser Glu Aso Ala Ser Tyr He Ser Gly Ala Val He 225 230 235 CCC GGT CGA CGG CGG CAT GGG TAT GGG CCA CTG ACA CAA CAC AAG GAC 1251 Pro Val Asp Gly Gly Met Gly Met Gly His 240 245
GCA CAT GAC AGG ACT GCT GGA CGG CAA ACG GAT TCT GGT TAG CGG AAT 1299 Met Thr Gly Leu Leu Asp Gly Lys Arg He Leu Val Ser Gly He 250 255 260
CAT CAC CGA CTC GTC GAT CGC GTT TCA CAT CGC ACG GGT AGC CCA GGA 1347 He Thr Asp Ser Ser He Ala Phe His He Ala Arg Val Ala Gin Glu 265 270 275
GCA GGG CGC CCA GCT GGT GCT CAC CGG GTT CGA CCG GCT GCG GCT GAT 1395 Gin Gly Ala Gin Leu Val Leu Thr Gly Phe Asp Arg Leu Arg Leu He 280 285 290
TCA GCG CAT CAC CGA CCG GCT GCC GGC AAA GGC CCC GCT GCT CGA ACT 1443 Gin Arg He Thr Asp Arg Leu Pro Ala Lys Ala Pro Leu Leu Glu Leu 295 300 305 310 CGA CGT GCA AAA CGA GGA GCA CCT GGC CAG CTT GGC CGG CCG GGT GAC 1491 ASD Val Gin Asn Glu Glu His Leu Ala Ser Leu Ala Gly Arg Val Thr 315 320 325
CGA GGC GAT CGG GGC GGG CAA CAA GCT CGA CGG GGT GGT GCA TGC GAT 1539 Glu Ala He Gly Ala Gly Asn Lys Leu Asp Gly Val Val His Ala He 330 335 340
TGG GTT CAT GCC GCA GAC CGG GAT GGG CAT CAA CCC GTT CTT CGA CGC 1587 Gly Phe Met Pro Gin Thr Gly Met Gly He Asn Pro Phe Phe Asp Ala 345 350 355
GCC CTA CGC GGA TGT GTC CAA GGG CAT CCA CAT CTC GGC GTA TTC GTA 1635 Pro Tyr Ala Asp Val Ser Lys Gly He His He Ser Ala Tyr Ser Tyr 360 365 370
TGC TTC GAT GGC CAA GGC GCT GCT GCC GAT CAT GAA CCC CGG AGG TTC 1683 Ala Ser Met Ala Lys Ala Leu Leu Pro He Met Asn Pro Gly Gly Ser 375 380 385 390
CAT CGT CGG CAT GGA CTT CGA CCC GAG CCG GGC GAT GCC GGC CTA CAA 1731 He Val Gly Met Asp Phe Asp Pro Ser Arg Ala Met Pro Ala Tyr Asn
395 400 405
CTG GAT GAC GGT CGC CAA GAG CGC GTT GGA GTC GGT CAA CAG GTT CGT 1779 Trp Met Thr Val Ala Lys Ser Ala Leu Glu Ser Val Asn Arg Phe Val 410 415 420
GGC GCG CGA GGC CGG CAA GTA CGG TGT GCG TTC GAA TCT CGT TGC CGC 1827 Ala Arg Glu Ala Gly Lys Tyr Gly Val Arg Ser Asn Leu Val Ala Ala 425 430 435 AGG CCC TAT CCG GAC GCT GGC GAT GAG TGC GAT CGT CGG CGG TGC GCT 1875 Gly Pro He Arg Thr Leu Ala Met Ser Ala He Val Gly Gly Ala Leu 440 445 450
CGG CGA GGA GGC CGG CGC CCA GAT CCA GCT GCT CGA GGA GGG CTG GGA 1923 Gly Glu Glu Ala Gly Ala Gin He Gin Leu Leu Glu Glu Gly Trp Asp 455 460 465 470
TCA GCG CGC TCC GAT CGG CTG GAA CAT GAA GGA TGC GAC GCC GGT CGC 1971 Gin Arc Ala Pro He Gly Trp Asn Met Lys Asp Ala Thr Pro Val Ala
475 480 485
CAA GAC GGT GTG CGC GCT GCT GTC TGA CTG GCT GCC GGC GAC CAC GGG 2019 Lys Thr Val Cys Ala Leu Leu Ser Asp Trp Leu Pro Ala Thr Thr Gly 490 495 500
TGA CAT CAT CTA CGC CGA CGG CGG CGC GCA CAC CCA ATT GCT CTA GAA 2067 Asp He He Tyr Ala ASΌ Gly Gly Ala His Thr Gin Leu Leu 505 510 515 CGCATGCAAT TTGATGCCGT CCTGCTGCTG TCGTTCGGCG GACCGGAAGG GCCCGAGCAG 2127
GTGCGGCCGT TCCTGG.AG.AA CGTTACCCGG GGCCGCGGTG TGCCTGCCGA ACGGTTGGAC 2187
GCGGTGGCCG AGCACTACCT GCATTTCGGT GGGGTATCAC CGATC 2232
In addition, the following fragments of the pS5 operon may be responsible for conferring isoniazid resistance:
SEQ ID NO: 4:
Met Thr Gly Leu Leu Asp Gly Lys Arg He Leu Val Ser Gly He He 16 Thr Asp Ser Ser He Ala Phe His He Ala Arg Val Ala Gin Glu Gin 32
Gly Ala Gin Leu Val Leu Thr Gly Phe Asp Arg Leu Arg Leu He Gin 48
Arg He Thr Asp Arg Leu Pro Ala Lys Ala Pro Leu Leu Glu Leu Asp 64
Val Gin Asn Glu Glu His Leu Ala Ser Leu Ala Gly Arg Val Thr Glu 80
Ala He Gly Ala Gly Asn Lys Leu Asp Gly Val Val His Ala He Gly 96 Phe Met Pro Gin Thr Gly Met Gly He Asn Pro Phe Phe Asp Ala Pro 112
Tyr Ala Asp Val Ser Lys Gly He His He Ser Ala Tyr Ser Tyr Ala 128
Ser Met Ala Lys Ala Leu Leu Pro He Met Asn Pro Gly Gly Ser He 144
Val Gly Met Asp Phe Asp Pro Ser Arg Ala Met Pro Ala Tyr Asn Trp 160
Met Thr Val Ala Lys Ser Ala Leu Glu Ser Val Asn Arg Phe Val Ala 176 Arg Glu Ala Gly Lys Tyr Gly Val Arg Ser Asn Leu Val Ala Ala Gly 192
Pro He Arg Thr Leu Ala Met Ser Ala He Val Gly Gly Ala Leu Gly 208
Glu Glu Ala Gly Ala Gin He Gin Leu Leu Glu Glu Gly Trp Asp Gin 224
Arg Ala Pro He Gly Trp Asn Met Lys Asp Ala Thr Pro Val Ala Lys 240
Thr Val Cys Ala Leu Leu Ser Asp Trp Leu Pro Ala Thr Thr Gly Asp 256 He He Tyr Ala Asp Gly Gly Ala His Thr Gin Leu Leu 269
SEQ ID NO: 5:
Val Thr Ala Thr Ala Thr Glu Gly Ala Lys Pro Pro Phe Val Ser Arg 16
Ser Val Leu Val Thr Gly Gly Asn Arg Gly He Gly Leu Ala He Ala 32
Gin Arg Leu Ala Ala Asp Gly His Lys Val Ala Val Thr His Arg Gly 48 Ser Gly Ala Pro Lys Gly Leu Phe Gly Val Glu Cys Asp Val Thr Asp 64
Ser Asp Ala Val Asp Arg Ala Phe Thr Ala Val Glu Glu His Gin Gly 80
Pro Val Glu Val Leu Val Ser Asn Ala Gly Leu Ser Ala Asp Ala Phe 96
Leu Met Arg Met Thr Glu Glu Lys Phe Glu Lys Val He Asn Ala Asn 112
Leu Thr Gly Ala Phe Arg Val Ala Gin Arg Ala Ser Arg Ser Met Gin 128
Arg Asn Lys Phe Gly Arg Met He Phe He Gly Ser Val Ser Gly Ser 144 Trp Gly He Gly Asn Gin Ala Asn Tyr Ala Ala Ser Lys Ala Gly Val 160
He Gly Met Ala Arg Ser He Ala Arg Glu Leu Ser Lys Ala Asn Val 176
Thr Ala Asn Val Val Ala Pro Gly Tyr He Asp Thr Asp Met Thr Arg 192
Ala Leu Asp Glu Arg He Gin Gin Gly Ala Leu Gin Phe He Pro Ala 208
Lys Arg Val Gly Thr Pro Ala Glu Val Ala Gly Val Val Ser Phe Leu 224 Ala Ser Glu Asp Ala Ser Tyr He Ser Gly Ala Val He Pro Val Asp 240
Gly Gly Met Gly Met Gly His 247
The pS5 operon of M. bovis has the following nucleic acid seguence:
SEQ ID NO: 6: GTTCGCTCCG GCGCGGTCAC GCGCATGCCC TCGATGACGC AGATCTCGTC GGGCTCGATG 60
CGCTCTTCCC AGACTTGCAG CCCCGGGGCA CGGCGGCGGT TGGTGTCGAT GATCGCGGCG 120
GGAAGATCCG CGTCGATCCA CTTGGCGCCA TGGAAGGCAG AAGCCGAGTA GCCGGCCAGC 180
ACGCCGCGGC GGCGCGAGCG CAGCCACAGC GCTTTTGCAC GCAATTGCGC GGTCAGTTCC 240
ACACCCTGCG GCACGTACAC GTCTTTATGT AGCGCGACAT ACCTGCTGCG CAATTCGTAG 300 GGCGTCAATA CACCCGCAGC CAGGGCCTCG CTGCCCAGAA AGGGATCCGT CATGGTCGAA 360
GTGTGCTGAG TCACACCGAC AAACGTCACG AGCGTAACCC CAGTGCGAAA GTTCCCGCCG 420
GAAATCGCAG CCACGTTACG CTCGTGGACA TACCGATTTC GGCCCGGCCG CGGCGAGACG 480
ATAGGTTGTC GGGGTGACTG CCACAGCCAC TGAAGGGGCC AAACCCCCAT TCGTATCCCG 540
TTCAGTCCTG GTTACCGGAG GAAACCGGGG GATCGGGCTG GCGATCGCAC AGCGGCTGGC 600 TGCCGACGGC CACAAGGTGG CCGTCACCCA CCGTGGATCC GGAGCGCCAA AGGGGCTGTT 660
TGGCGTCGAA TGTGACGTCA CCGACAGCGA CGCCGTCGAT CGCGCCTTCA CGGCGGTAGA 720
AGAGCACCAG GGTCCGGTCG AGGTGCTGGT GTCCAACGCC GGCCTATCCG CGGACGCATT 780
CCTCATGCGG ATGACCGAGG AAAAGTTCGA GAAGGTCATC AACGCCAACC TCACCGGGGC 840
GTTCCGGGTG GCTCAACGGG CATCGCGCAG CATGCAGCGC AACAAATTCG GTCGAATGAT 900
ATTCATAGGT TCGGTCTCCG GCAGCTGGGG CATCGGCAAC CAGGCCAACT ACGCAGCCTC 960 CAAGGCCGGA GTGATTGGCA TGGCCCGCTC GATCGCCCGC GAGCTGTCGA AGGCAAACGT 1020
GACCGCGAAT GTGGTGGCCC CGGGCTACAT CGACACCGAT ATGACCCGCG CGCTGGATGA 1080
GCGGATTCAG CAGGGGGCGC TGCAATTTAT CCCAGCGAAG CGGGTCGGCA CCCCCGCCGA 1 140
GGTCGCCGGG GTGGTCAGCT TCCTGGCTTC CGAGGATGCG AGCTATATCT CCGGTGCGGT 1200
CATCCCGGTC GACGGCGGCA TGGGTATGGG CCACTGACAC AACACAAGGA CGCACATGAC 1260 AGGACTGCTG GACGGCAAAC GGATTCTGGT TAGCGGAATC ATCACCGACT CGTCGATCGC 1320
GTTTCACATC GCACGGGTAG CCCAGGAGCA GGGCGCCCAG CTGGTGCTCA CCGGGTTCGA 1380
CCGGCTGCGG CTGATTCAGC GCATCACCGA CCGGCTGCCG GCAAAGGCCC CGCTGCTCGA 1440
ACTCGACGTG CAAAACGAGG AGCACCTGGC CAGCTTGGCC GGCCGGGTGA CCGAGGCGAT 1500
CGGGGCGGGC AACAAGCTCG ACGGGGTGGT GCATGCGATT GGGTTCATGC CGCAGACCGG 1560 GATGGGCATC AACCCGTTCT TCGACGCGCC CTACGCGGAT GTGTCCAAGG GCATCCACAT 1620
CTCGGCGTAT TCGTATGCTT CGATGGCCAA GGCGCTGCTG CCGATCATGA ACCCCGGAGG 1680
TTCCATCGTC GGCATGGACT TCGACCCGAG CCGGGCGATG CCGGCCTACA ACTGGATGAC 1740
GGTCGCCAAG AGCGCGTTGG AGTCGGTCAA CAGGTTCGTG GCGCGCGAGG CCGGCAAGTA 1800
CGGTGTGCGT TCGAATCTCG TTGCCGCAGG CCCTATCCGG ACGCTGGCGA TGAGTGCGAT 1860 CGTCGGCGGT GCGCTCGGCG AGGAGGCCGG CGCCCAGATC CAGCTGCTCG AGGAGGGCTG 1920
GGATCAGCGC GCTCCGATCG GCTGGAACAT GAAGGATGCG ACGCCGGTCG CCAAGACGGT 1980
GTGCGCGCTG CTGTCTGACT GGCTGCCGGC GACCACGGGT GACATCATCT ACGCCGACGG 2040
CGGCGCGCAC ACCCAATTGC TCTAGAACGC ATGCAATTTG ATGCCGTCCT GCTGCTGTCG 2100
TTCGGCGGAC CGGAAGGGCC CGAGCAGGTG CGGCCGTTCC TGGAGAACGT TACCCGGGGC 2160 CGCGGTGTGC CTGCCGAACG GTTGGACGCG GTGGCCGAGC ACTACCTGCA TTTCGGTGGG 2220
GTATCACCGA TC 2232 The polynucleotides of the invention may be obtained by expressing a DNA sequence coding said polynucleotides in a host cell or organism. For example, a DNA molecule, such as the nucleic acid sequence from residues 1256-2062, or 494-1234 of Figure 12, can be expressed in a host cell or organism. In addition, promoters, which comprise DNA molecules which consist of part or all of the nucleic acid sequence from residues 1-493 of Figure 12, can be used in the cloning and/or expression of a nucleic acid sequence. DNA expression vectors containing polynucleotides of the invention can be prepared by transforming host cells capable of expressing the polynucleotides of the invention or fragments thereof with a DNA sequence encoding the polynucleotides of the invention. The transformed host cells are then cultured, and the expressed polynucleotide is recovered.
The polynucleotides and fragments of polynucleotides of the invention can be prepared several ways. For example, they can prepared by isolating the polynucleotides from a natural source, or by synthesis using recombinant DNA techniques. In addition, variants of the polynucleotides of the invention can be prepared by utilizing site-specific mutagenesis of the DNA encoding the native amino acid sequences (see Adelman et al., DNA, Vol. 2, p. 183 (1983)). If recombinant DNA techniques are used, DNA encoding the polynucleotide must be obtained. This DNA can be isolated from mycobacteria. Alternatively, the DNA may be produced as intron free cDNA using conventional techniques. In addition, the DNA can be produced in the form of synthetic oligonucleotides.
Once the DNA is obtained, it is treated so that it is suitable for insertion into appropriate cloning and/or expression vectors. The DNA is cleaved utilizing restriction enzymes. The nucleic acid is then recovered. The DNA is then tailored using conventional techniques, such as treatment with polymerase 1, phenol and chloroform. The DNA is then extracted and precipitated by ethanol. Next, religation is performed by providing equimolar amounts of the desired components, appropriately tailored for correct matching and treatment with an appropriate ligase, such as T. DNA ligase.
Suitable cloning vectors may be constructed according to standard techniques, or may be selected from the large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used for expressing the polynucleotide-encoding DNA, useful cloning vectors will generally have the ability to self replicate, will possess a single target for any particular restriction endonuclease and will carry genes for a readily selectable marker such as isoniazid resistance. Two major types of cloning vectors which may be used are plasmids and bacterial viruses. Examples of such vectors include pUC18, pl8, Mpl9, pBR322, pf*!39, ColEl, and pCRl from E. coli. A wide range of host plasmids include RP4, phage DNA's such as lambda and M13, and shuttle vectors such as pSA3 and pAT28. To date, the most suitable cloning vectors are pBluesript Ilks (Stratagene), and pYUB18, as shown in Figure 13.
For the expression of the polynucleotides of the invention in the host, the cloning vector must incorporate an expression control sequence. A typical expression control sequence can be described as having a promoter region, a 5' untranslated region, a polypeptide coding sequence, a 3' untranslated region and a transcription termination region. By way of example, a promoter which may be used comprises all or part of the nucleic acid sequence from residues 1-493 shown in Figure 12.
Hosts which may be used in this invention include bacteria, yeasts, fungi, insects and animal and plant cells. Procaryatic hosts are generally preferred. Mycobacterial and bacterial hosts, including E. coli and M. smecπτtatis, are particularly suitable. Of the mycobacterial hosts,
2
M. smeornatis mc 155 is preferred. After the host cells are cultured, the exogenous protein is then isolated using routine methods such as freeze-thaw extraction. Purification is then performed utilizing conventional techniques.
A DNA molecule comprising all or part of the nucleic acid sequences of the polynucleotides of the invention may be used as probes for identifying nucleic acids which code for polynucleotides associated with isoniazid resistance. A probe may be labelled, for example, with radioactive isotopes, including 32P and 33P labels. The probes of the invention are capable of hybridizing to the genetic elements associated with isoniazid resistance. Preferably, the probes of the invention are specific for sense-strand and anti-sense strand of the DNA which encodes for the isoniazid resistance gene. By way of example, the probe may be the entire nucleotide sequence depicted in Figure 12. More useful probes may be from 100-1000 base pairs. However, shorter probes are preferred. Such probes have the advantage of being stable over time and are therefore suitable for use in diagnostic kits. This invention is further directed to diagnostic kits for use in detecting the presence of polynucleotides which confer isoniazid resistance in a clinical sample. The diagnostic kits of the invention include nucleic acid probes, as discussed hereinabove, which may be labelled. The diagnostic kits of the invention may also include a lysing agent, a denaturing solution, a neutralizing solution, an alkaline fixation solution and a saline citrate.
Clinical samples may be tested to determine whether the samples contain polynucleotides which confer isoniazid resistance. Clinical samples are tested with labelled probes under conditions such that the probes will hybridize to the isoniazid resistance-associated genes. Next, hybrid DNA is detected in the sample. These procedures are performed using standard techniques. Alternatively, polynucleotides which are associated with isoniazid resistance are amplified in a clinical sample. After amplification, the detection of the presence or absence of the amplified DNA in the sample indicates whether the sample contains polynucleotides which confer isoniazid resistance.
Further, antibodies can be raised which are immunoreactive with epitopes on the polynucleotides of the invention which confer isoniazid resistance. These antibodies can be used in diagnostic immunoassays, and can be passively administered to prevent and treat tuberculosis and other mycobacterial infection and disease.
EXAMPLE
In order to select M. bovis isoniazid resistant strains, a virulent wild-type New Zealand strain of M. bovis was cloned by four serial passages using a combination of liquid tween albumin broth (TAB) and 7H10 pyruvate agar media. A single colony of M. bovis was inoculated into TAB and incubated until visible growth was apparent. An appropriate dilution of this TAB was then inoculated onto solid media to obtain discrete colonies. After incubation, a single colony was inoculated into TAB and the process was repeated. The G4 strain was obtained after four cloning cycles. The isoniazid resistant strains were obtained by inoculating G4 into a series of TABs containing different concentrations of isoniazid and then incubated. Of the TABs with luxuriant growth, the one with the highest concentration of isoniazid, was inoculated onto solid media containing isoniazid. A colony from this inoculation was used to inoculate a TAB containing isoniazid and incubated. When visible growth was apparent, this was used to inoculate a series of TABs containing varying concentrations of isoniazid. This selection procedure was repeated to obtain a series of clones of M. bovis with increasing resistance to isoniazid. The last strain selected, G4/100, is resistant to 100 μg/ml of isoniazid.
In order to produce a cosmid library and resistant clones, a cosmid library of G4/100 was made in the shuttle vector pYUB18 (as shown in Figure 13). This vector replicates independently in E. coli and various mycobacterial species. It contains a selectable kanamycin gene and a cos site.
The cosmid library was made by performing the following steps: (a) Partial Sau3AI digestion of purified chromosomal
DNA from G4/100;
(b) Sucrose gradient purification of 30-50 kb fragments;
(c) Ligation of the fragments to linearized pYtJBlδ; (d) Packaging of cosmids into λ phage using a commercial kit (Gigapack Gold Stratagene); (e) Transfection of the cosmids into an E. coli host: Approximately 5000 colonies were obtained; and (f) Pooling and amplifying the colonies using standard plasmid preparation techniques. The cosmid library was then transformed into
2
M. smegmatis strain mc 155 by electroporation. Transformed
M. smegmatis organisms were selected by growth on media containing kanamycin. Approximately 1200 kanamycin resistant clones were patched onto media containing isoniazid. Four isoniazid resistant clones were identified.
In order to perform plasmid extraction, cultures of M. smeqmatis (5 ml) were incubated with cycloserine and ampicillin for 3 hours before harvest. The cells were pelleted and resuspended in 0.25 ml of 40 mM Tris-acetate, 2 mM EDTA, pH 7.9. To this, 0.5 ml of lysing solution was added (50 MM Tris, 3% SDS) and the solution was mixed for 30 minutes. The sample was heated to 60°C for 20 minutes, cooled for 10 minutes and the DNA was extracted by adding 0.8 ml of phenol (containing 50 mM NaCI). This was centrifuged and the upper layer containing the DNA was removed. An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the DNA extract, the sample was centrifuged, and the supernatant containing the DNA was removed. To precipitate the DNA, a half volume of 7.5 M ammonium acetate was added, incubated on ice for 30 minutes and then centrifuged for 30 minutes. The DNA was resuspended in 10 mM Tris, 1 mM EDTA.
The smallest plasmid obtained which conferred an isoniazid resistance phenotype on M. smeσmatis was 2.3 Kb in size and was designated pS5. The nucleic acid sequence for pS5 and the amino acid sequence from two large open reading frames that it encodes are shown in Figure 9. In order to sequence the polynucleotide, pS5 was cloned into the vector pBluesript II KS+ (Stratagene, CA) .
This vector contains the T3 and T7 promoters which were used for the sequencing. Sequencing was carried out using the dsDNA cycle sequencing system from GIBCO BRL, Life Technologies, according to the manufacturer's instructions. The
32 radioactively labelled nucleotide was either [γ- P] ATP or [γ- 33P] ATP (Amersham) . The sequencing program used was
GCG (Sequence Analysis Software Package) . In order to assay for catalase assay, the enzyme was first isolated by pelleting a culture of M. bovis and resuspending in 50 mM potassium phosphate buffer, pH 7. This was added to a tube containing 0.5 g zirconium beads (Biospecs Products) and vortexed for 5 minutes. The sample was centrifuged briefly and the supernatant was diluted to 4 ml with 50 mM potassium phosphate buffer and then filter sterilized through 0.22 μm filters.
The catalase activity was assayed by incubating an aliquot of the above sample with 3 μM H,0_ in a total volume of 3 ml for 5 minutes. The reaction was stopped by adding 1.5 ml of titanium tetrachloride reagent (1.5 mg/ml
TiCl4. in 4.5 M H2_SO4. ) . The absorbance was read at 410 nm and the catalase activity was calculated using a standard curve of the amount of H-O- versus wavelength at 410 nm, and the activity was expressed as μmol/min/mg protein. The catalase activity of G4/100, G4 and another virulent . bovis strains were also determined by this method.
The G4 strain and the other virulent M. bovis strains contained similar levels of catalase activity. No catalase activity was detected in the G4/100 strain.
To demonstrate that the development of isoniazid resistance is not due entirely to loss of catalase activity, pS5 was electroporated into G4 to produce G4(S5). G4(S5) grew on media containing a level of isoniazid that prevented growth of G4. Using the method described above, G4(S5) was also shown to have catalase activity similar to that of G4.
Since the sequences of these polynucleotides have been determined, these polynucleotides can be used in the treatment of M. tuberculosis, M. avium, M. smegmatis, M. bovis and other mycobacterial infection. One method of treating mycobacterial infection such as tuberculosis utilizing these polynucleotides is preparing anti-DNA or anti-RNA oligonucleotides which can be used to inhibit mRNA activity of the inhA operon of M. tuberculosis or M. bovis. These oligonucleotides can be prepared utilizing the wild-type DNA sequence of the inhA operon of M. tuberculosis or the pS5 operon of M. bovis. These oligonucleotides can then be administered, either alone or in combination with other compositions, to treat mycobacterial infection, including tuberculosis. These oligonucleotides can be administered orally, enterally, subcutaneously, intraperitoneally or intravenously. The DNA sequences of these polynucleotides can also be used to assess the susceptibility of various strains of the M. tuberculosis complex in a clinical sample to isoniazid. In order to perform this, first the chromosomal DNA of the M. tuberculosis complex from a clinical sample must be isolated. Oligonucleotides are prepared, for example using an oligonucleotide synthesizer, utilizing the wild-type inhA polynucleotide DNA sequence of M. tuberculosis depicted in Figure 8 or the DNA sequence of M. bovis depicted in Figure 12. Regions of the inhA polynucleo ide of M. tuberculosis or the PS5 polynucleotide of M. bovis from the clinical sample which are identified by the oligonucleotides are then amplified (for example by using polymerase chain reaction (PCR)) to obtain double stranded DNA. Next, single strand conformation polymorphism analysis is performed in order to determine whether a mutated gene exists in the M. tuberculosis complex organisms from the clinical sample. If a mutation exists, this indicates that the M. tuberculosis complex organisms from the clinical sample are resistant to isoniazid.
In order to perform single strand conformation polymorphism analysis, PCR or other type of amplification is performed after substitution of half of the dCTP by 0.5μl of
32 P-α-dCTP or other chemiluminescent substrates per reaction to generate a radiolabelled 157 bp product. After amplification, 25 μL of PCR product is mixed with 100 μL of dilution buffer (10 mmol/L edetic acid, 0.1% sodium dodecyl sulphate) . A 3 μL aliquot of diluted product is then mixed with 3 μL of sequencing loading buffer (Sequenase kit), heated for ten minutes at 94°C, cooled on ice and loaded onto a non-denaturing sequencing format 0.5% MDE gel (Hydrolink, AT Biochem, Malvern, Pennsylvania). Electrophoresis is then performed at room temperature and constant power (6W for a 50 x 32 x 0.4 cm gel) overnight. Gels are then dried and exposed for autoradiography overnight. An example of using single strand conformation polymorphism is described by Telenti et al . in "Detection Of Rifampicin-Resistance Mutations In Mycobacterium Tuberculosis", Vol. 341, pages 647-650 (March, 1993), which is incorporated herein by referenced.
The polynucleotide sequences can also be used to determine whether a drug is effective against tuberculosis. This is performed by overexpreεsing the M. tuberculosis inhA polynucleotide or the M. bovis pS5 polynucleotide (i.e., a gene which encodes the target of action enzyme for isoniazid) so as to obtain the enzyme. The enzyme is then combined with a purified reagent, such as a fatty acid or NADP, to obtain a mycolic acid. The mycolic acid is then assayed to measure its level of biosynthesis, such as by thin layer chromatography or spectrophotometry. Next, a drug is combined with the system used to produce mycolic acid to determine whether the drug blocks mycolic acid biosynthesis. If there is a blockage, this indicates that the drug is effective against tuberculosis. Drugs which may be tested for their effectiveness against tuberculosis by this method include isoniazid, ethionamide, rifampicin, streptomycin, ethambutol, ciprofloxacin, novobiocin and cyanide. Further, the polynucleotide which encodes an enzyme involved in mycolic acid biosynthesis can be used for treating tuberculosis. By way of example, the gene mabA, which is depicted in Figure 7 from nucleic acid residue 96 to nucleic acid residue 863, and in Figure 8, from nucleic acid residue 224 to nucleic acid residue 967, has been isolated by the inventors and identified as a gene which encodes an enzyme involved in mycolic acid biosynthesis of tuberculosis. In order to treat tuberculosis, a compound which blocks the biochemical activity of the enzyme encoded by the mabA gene, is administered. The compound blocks the enzyme activity, thereby preventing mycolic acid biosynthesis. This causes the tuberculosis to organisms die.
In addition, utilizing the determined sequences of the polynucleotides of the invention, compounds which block the activity of enzymes encoded by the polynucleotides can be prepared. This is performed by overexpressing the enzyme and purifying the enzyme, and then performing X-ray crystallography on the purified enzyme to obtain the molecular structure of the enzyme. Next, compounds are created which have a similar molecular structure to the enzyme. The compounds are then combined with the enzyme and attached thereto so as to block the biochemical activity of the enzyme. Since the enzyme is blocked, it is unable to confer isoniazid resistance on tuberculosis organisms. Further, tuberculosis-specific purified mycolic acid compounds can be produced by adding the enzyme encoded by the polynucleotide to the chemical reaction which produces mycolic acids.
Finally, the polynucleotide DNA sequences can be used to produce or improve tuberculosis vaccines. For example, M. tuberculosis complex strains that have become isoniazid resistant often have reduced virulence and can be administered as vaccines. In addition, mutated genes of M. tuberculosis and M. bovis can be added to BCG or tuberculosis vaccines to provide attenuated mutant tuberculosis vaccines. These vaccines can be used to treat and prevent a wide variety of diseases, including tuberculosis, AIDS, leprosy, polio, malaria and tetanus.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs et al
(ii) TITLE OF INVENTION: USE OF GENES OF M TUBERCULOSIS, M BOVIS AND M SMEGMATIS WHICH
CONFER ISONIAZID RESISTANCE TO TREAT TUBERCULOSIS AND TO ASSESS DRUG RESISTANCE
(iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Amster, Rothstein & Ebenstein
(B) STREET: 90 Park Avenue
(C) CITY: New York
(D) STATE: New York
(E) COUNTRY: USA
(F) ZIP: 10016
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch 1.44 Mb storage diskette (B> COMPUTER: IBM PC Compatible (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: Word Processor (ASCII)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: Not yet assigned
(B) FILING DATE: Not yet assigned
(C) CLASSIFICATION: Not yet assigned
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION SERIAL NO: 08/062,409
(B) FILING DATE: May 14, 1993
(C) TITLE: USE OF GENES OF M TUBERCULOSIS AND M SMEGMATIS WHICH CONFER ISONIAZID
RESISTANCE TO TREAT TUBERCULOSIS AND TO ASSESS DRUG RESISTANCE
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Pasqualini, Patricia A. (B) REGISTRATION NUMBER: 34,894
(C) REFERENCE/DOCKET NUMBER: 96700/264
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 697-5995
(B) TELEFAX: (212) 286-0854 or 286-0082 (C) TELEX: TWX 710-5B1-4766
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3051
(B) TYPE: nucleic acid 5 (C) STRANDEDNESS: single
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (A) DESCRIPTION:
(iii) HYPOTHETICAL: No
10 (iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: inhA operon
(A) ORGANISM: M smegmatis
(B) STRAIN:
15 (C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE: 20 (H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE: M smegmatis
(viii) POSITION IN GENOME:
(A) CHROMOSOME SEGMENT: 25 (B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
30 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION: None
(A) AUTHORS:
__ (3) TITLE:
"-•-- (C) JOURNAL:
(D) VOLUME:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
40 (I) FILING DATE:
(J) PUBLICATION D.ATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GGATCCGCCG CACGGGGAGC CCCGAGGCGA TTTCTGGCTG GACCGGCCAA CACGTTAAGT 60
TGACGGGCGA AGACGCAGGA CGCGAGGAAC AGAGGATGAC TGTGACTGAC AATCCGGCCG 120
ACACCGCGGG CGAGGCCACT GCAGGCCGCC CGGCGTTCGT CTCCCGTTCG GTGCTGGTGA 180 CCGGTGGTAA CCGCGGCATC GGCCTGGCGA TCGCGCGACG GCTGGCCGCC GACGGGCACA 240
AGGTGGCCGT CACCCACCGC GGTTCCGGTG CACCCGACGA CCTGTTCGGT GTTCAATGTG 300
ACGTCACCGA CAGCGCTGGT GTCGACCGCG CCTTCAAAGA GGTCGAGGAG CACCAGGGCC 360
CGGTCGAGGT GCTGGTGGCC AACGCAGGCA TCTCCAAGGA CGCATTCCTC ATGCGCATGA 420
CCGAGGAGCG GTTCGAAGAG GTCATCAACA CCAACCTCAC GGGCGCGTTC CGGTGCGCCC 480 AGCGGGCGTC GCGCACCATG CAGCGCAAGC GGTTCGGGCG CATCATCTTC ATCGGGTCGG 540
TCTCGGGCAT GTGGGGGATC GGCAATCAGG CCAACTACGC GGCCGCCAAG GCGGGCCTGA 600
TCGGCATGGC CCGCTCGATC TCCCGTGAGC TGGACAAGGC GGGCGTCACC GCGAACGTGT 660
TGCCCCCCGG TTACATCGAC ACCGAGATGA CCCGGGCGCT CGACGAGCGC ATCCAGGGGG 720
GCGCGATCGA CTTCATCCCG GACAAGCGGG TCGGCACGGT CGAGGAGGTC GCGGGCGCGG 780 TCAGCTTCCT GGCCTCGGAG GACGCCTCCT ACATCGCGGG CGCGGTCATC CCCGTCGACG 840
GCGGTATGGG CATGGGCCAC TAGTCAAAAG CCCGGACACA CAAGATTTCT CGCTCACAAG 900
GAGTCACCAA ATGACAGGCC TACTCGAAGG CAAGCGCATC CTCGTCACGG GGATCATCAC 960
CGATTCGTCG ATCGCGTTCC ACATCGCCAA GGTCGCCCAG GAGGCCGGCG CCGAACTGGT 1020
GCTGACCGGT TTCGACCGCC TGAAGTTGGT CAAGCGCATC GCCGACCGCC TGCCCAAGCC 1080 GGCCCCGCTG CTGGAACTCG ACGTGCAGAA CGAGGAGCAC CTGTCGACTC TGGCCGACCG 1140
GATCACCGCC GAGATCGGTG AGGGCAACAA GATCGACGGT GTGGTGCACG CGATCGGGTT 1200
CATGCCGCAG AGCGGTATGG GCATCAACCC GTTCTTCGAC GCGCCGTACG AGGATGTGTC 1260
CAAGGGCATC CACATCTCGG CGTACTCGTA CGCCTCGCTC GCCAAAGCCG TTCTGCCGAT 1320
CATGAATCCG GGCGGCGGCA TCGTCGGCAT GGACTTCGAC CCCACGCGCG CGATGCCGGC 1380 CTACAACTGG ATGACCGTCG CCAAGAGCGC GCTCGAATCG GTCAACCGGT TCGTCGCGCG 1440
TG GGCGGGC AAGGTGGGCG TGCGCTCGAA TCTCGTTGCG GCAGGACCGA TCCGCACGCT 1500
GGCGATGAGC GCAATCGTGG GCGGTGCGCT GGGCGACGAG GCCGGCCAGC AGATGCAGCT 1560
GCTCGAAGAG GGCTGGGATC AGCGCGCGCC GCTGGGCTGG AACATGAAGG ACCCGACGCC 1620
CGTCGCCAAG ACCGTGTGCG CACTGCTGTC GGACTGGCTG CCGGCCACCA CCGGCACCGT 1680
GATCTACGCC GACGGCGGCG CCAGCACGCA GCTGTTGTGA TACCGCCGTG TCGTATGACG 1740 CCTTGCTACT GCTGTCGTTC GACGGGCCGG AACTCCCGAG CAGGTGATGC CGTTCTTGGA 1800
GAACTCACCA GGGGCCGCGG AATCCCCAGG GAGCGGCTGG AATCGGTGGC CGAGCACTAT 1860
CTGCACTTCG GCGGGGTGTC ACCGATCAAC GGCATCAACC GGGACCTGAT CGTCGCGATC 1920
GAGGCCGAAC TCGCCCGACG CGGCCGCAAC CTTCCGGTCT ACTTCGGCAA CCGCAACTGG 1980
GAGCCGTACG TCGAAGACAC TGTCAAGGCG ATGTCCGACA ACGGAATCCG TCGTGCGGCG 2040 GTGTTCGCGA CCTCGGCGTG GGGTGGGTAC TCGGGATGCG CCCAGTACCA GGAGGACATC 2100
GCGCGTGGCC GGGCCGCCGC CGGGCCCGAG GCGCCGGAGC TGGTCAAGCT GCGCCAGTAT 2160
TTCGACCACC CGCTGTTCGT CGAGATGTTC GCCGACGCCG TCGCCGACGC CGCGGCCACC 2220
CTGCCCGAGG AACTGCGGGA CGAAGCGCGG CTGGTGTTCA CCGCCCACTC CATCCCGCTG 2280
CGTGCCGCGT CGCGTTGCGG TGCAGATCTC TACGAGCGGC AGGTGGGTTA CGCCGCGCGG 2340 CTGGTCGCGG CCGCAGCCGG GTACCGCGAA TACGACCAGG TATGGCAGTC CCGGTCCGGC 2400
CCGCCGCAGG TGCCGTGGCT CGAACCCGAC GTCGGAGATC ACCTTGAGGC GTTGGCGCGC 2460
AACGGCACCA GGGCGGTCAT CGTGTGTCCC CTCGGCTTCG TCGCCGACCA CATCGAGGTG 2520
GTGTGGGATC TGGACAACGA ACTGGCCGAG CAGGCCGCCG AGGCAGGCAT CGCGTTCGCG 2580
CGTGCCGCCA CGCCCAACTC CCAGCCACGT TTTGCCCAAC TTGTCGTCGA CCTGATCGAC 2640 GAAATGCTGC ACGGACTTCC GCCACGCCGG GTCGAGGGGC CCGATCCGTG CCCGCCTACG 2700
GCAGCAGTGT CAACGGCGCA CCGTGCACGC CGGCCTGCTC GGCGTGACCC GCCCCGGGCG 2760
CAGCGAGTCG GGCCGGGCGA TCAAGAACGC CAGGCGGAAT GCAGGATCGC CTCGAGTGCG 2820
GCCATACGCG CCGAGCGCAC CACCCGCGTG AGGGGGCGCA GCGCCGAGTC GGCGATCTGA 2880
ACCTCCGACG AACTCTGCAG ACCGCTCGGG ATCAGACCCG CACTCACCGC GATGATGGCG 2940 TCGACATGGG CGGCGTTCTC CAGCACCCGC ACAGCCCGGG TCGGCGCGTG GTCGGGGACG 3000
CGGTGCGCGC GCCCGGCGGC GAGGATCTGC TCGACCATCC CGCGCGGATC C 3051 (3) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3120
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (A) DESCRIPTION:
(iii) HYPOTHETICAL: No
(iv) ANT I -SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: inhA operon
(A) ORGANISM: M tuberculosis
(B) STRAIN: (C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE: (H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE: M tuberculosis
(viii) POSITION IN GENOME:
(A) CHROMOSOME SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION: None
(A) AUTHORS :
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(F) PAGES :
(G) DATE:
(H) DOCUMENT NUMBER
(I) FILING DATE:
( J ) PUBLICATION DATE : ( K ) RELEVANT RESIDUES :
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :
AGCGCGACAT ACCTGCTGCG CAATTCGTAG GGCGTCAATA CACCCGCAGC CAGGGCCTCG 60 CTGCCCAGAA AGGGATCCGT CATGGTCGAA GTGTGCTGAG TCACACCGAC AAACGTCACG 120
AGCGTAACCC CAGTGCGAAA GTTCCCGCCG GAAATCGCAG CCACGTTACG CTCGTGGACA 180
TACCGATTTC GGCCCGGCCG CGGCGAGACG ATAGGTTGTC GGGGTGACTG CCACAGCCAC 240
TGAAGGGGCC AAACCCCCAT TCGTATCCCG TTCAGTCCTG GTTACCGGAG GAAACCGGGG 300
GATCGGGCTG GCGATCGCAC AGCGGCTGGC TGCCGACGGC CACAAGGTGG CCGTCACCCA 360 CCGTGGATCC GGAGCGCCAA AGGGGCTGTT TGGCGTCGAA TGTGACGTCA CCGACAGCGA 420
CGCCGTCGAT CGCGCCTTCA CGGCGGTAGA AGAGCACCAG GGTCCGGTCG AGGTGCTGGT 480
GTCCAACGCC GGCCTATCCG CGGACGCATT CCTCATGCGG ATGACCGAGG AAAAGTTCGA 5 0
GAAGGTCATC AACGCCAACC TCACCGGGGC GTTCCGGGTG GCTCAACGGG CATCGCGCAG 600
CATGCAGCGC AACAAATTCG GTCGAATGAT ATTCATAGGT TCGGTCTCCG GCAGCTGGGG 660 CATCGGCAAC CAGGCCAACT ACGCAGCCTC CAAGGCCGGA GTGATTGGCA TGGCCCGCTC 720
GATCGCCCGC GAGCTGTCGA AGGCAAACGT GACCGCGAAT GTGGTGGCCC CGGGCTACAT 780
CGACACCGAT ATGACCCGCG CGCTGGATGA GCGGATTCAG CAGGGGGCGC TGCAATT AT 840
CCCAGCGAAG CGGGTCGGCA CCCCCGCCGA GGTCGCCGGG GTGGTCAGCT TCCTGGCTTC 900
CGAGGATGCG AGCTATATCT CCGGTGCGGT CATCCCGGTC GACGGCGGCA TGGGTATGGG 960 CCACTGACAC AACACAAGGA CGCACATGAC AGGACTGCTG GACGGCAAAC GGATTCTGGT 1020
TAGCGGAATC ATCACCGACT CGTCGATCGC GTTTCACATC GCACGGGTAG CCCAGGAGCA 1080
GGGCGCCCAG CTGGTGCTCA CCGGGTTCGA CCGGCTGCGG CTGATTCAGC GCATCACCGA 1140
CCGGCTGCCG GCAAAGGCCC CGCTGCTCGA ACTCGACGTG CAAAACGAGG AGCACCTGGC 1200
CAGCTTGGCC GGCCGGGTGA CCGAGGCGAT CGGGGCGGGC AACAAGCTCG ACGGGGTGGT 1260 GCATTCGATT GGGTTCATGC CGCAGACCGG GATGGGCATC AACCCGTTCT TCGACGCGCC 1320
CTACGCGGAT GTGTCCAAGG GCATCCACAT CTCGGCGTAT TCGTATGCTT CGATGGCCAA 1380
GGCGCTGCTG CCGATCAΪGA ACCCCGGAGG TTCCATCGTC GGCATGGACT TCGACCCGAG 1440
CCGGGCGATG CCGGCCTACA ACTGGATGAC GGTCGCCAAG AGCGCGTTGG AGTCGGTCAA 1500
CAGGTTCGTG GCGCGCGAGG CCGGCAAGTA CGGTGTGCGT TCGAATCTCG TTGGCGCAGG 1560
CCCTATCCGG ACGCTGGCGA TGAGTGCGAT CGTCGGCGGT GCGCTCGGCG AAGAGGCCGG 1620
CGCCCAGATC CAGCTGCTCG AGGAGGGCTG GGATCAGCGC GCTCCGATCG GCTGGAACAT 1680 GAAGGATGCG ACGCCGGTCG CCAAGACGGT GTGCGCGCTG CTGTCTGACT GGCTGCCGGC 1740
GACCACGGGT GACATCATCT ACGCCGACGG CGGCGCGCAC ACCCAATTGC TCTAGAACGC 1800
ATGCAATTTG ATGCCGTCCT GCTGCTGTCG TTCGGCGGAC CGGAAGGGCC CGAGCAGGTG 1860
CGCCCGTTCC TGGAGAACGT TACCCGGGGC CGCGGTGTGC CTGCCGAACG GTTGGACGCG 1920
GTGGCCGAGC ACTACCTGCA TTTCGGTGGG GTATCACCGA TCAATGGCAT TAATCGCACA 1980 CTGATCGCGG AGCTGGAGGC GCAGCAAGAA CTGCCGGTGT ACTTCGGTAA CCGCAACTGG 2040
GAGCCGTATG TAGAAGATGC CGTTACGGCC ATGCGCGACA ACGGTGTCCG GCGTGCAGCG 2100
GTCTTTGCGA CATCTGCGTG GAGCGGTTAC TCGAGCTGCA CACAGTACGT GGAGGACATC 2160
GCGCGGCCCC CCGCGCGGCC GGGCGCGACG CGCCTGAACT GGTAAAACTG CGGCCCTACT 2220
TCGACCATCC GCTGTTCGTC GAGATGTTCG CCGACGCCAT CACCGCGGCC GCCGCAACCG 2280 TGCGCGGTGA TGCCCGGCTG GTGTTCACCG CGCATTCGAT CCCGACGGCC GCCGACCGCC 2340
GCTGTGGCCC CAACCTCTAC AGCCGCCAAG TCGCCTACGC CACAAGGCTG GTCGCGGCCG 2400
CTGCCGGATA CTGCGACTTT GACCTGGCCT GGCAGTCGAG ATCGGGCCCG CCGCAGGTGC 2460
CCTGGCTGGA GCCAGACGTT ACCGACCAGC TCACCGGTCT GGCTGGGGCC GGCATCAACG 2520
CGGTGATCGT GTGTCCCATT GGATTCGTCG CCGACCATAT CGAGGTGGTG TGGGATCTCG 2580 ACCACGAGTT GCGATTACAA GCCGAGGCAG CGGGCATCGC GTACGCCCGG GCCAGCACCC 2640
CCAATGCCGA CCCGCGGTTC GCTCGACTAG CCAGAGGTTT GATCGACGAA CTCCGTTACG 2700
GCCGTATACC TGCGCGGGTG AGTGGCCCCG ATCCGGTGCC GGGCTGTCTG TCCAGCATCA 2760
ACGGCCAGCC ATGCCGTCCG CCGCACTGCG TGGCTAGCGT CAGTCCGGCC AGGCCGAGTG 2820
CAGGATCGCC GTGACCGCGG ACATCCGGGC CGAGCGCACC ACGGCGGTCA ACGGTCTCAA 2880 CGCATCGGTG GCACGCTGAG CGTCCGACAA CGACTGCGTT CCGATCGGCA ATCGACTCAG 2940
CCCGGCACTG ACCGCG.ATGA TCGCATCGAC GTGCGCGGCA TTCTCGAGCA CCCGCAATGC 3000 GCGCGATGGC GCGTGGTCGG GAACCCGGTG TTGCCGTGAC GATTCGAGCA ACTGCTCGAC 3060 GAGGCCACGG GGCTTGGCGA CGTCGCTAGA TCCCAGTCCG ATGGTGCTCA AGGCTTCGGC 3120
(4) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2232
(B) TYPE: amino acid translation
(C) STRANDEDNESS: single
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (A) DESCRIPTION:
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: pS5 ODeron (A) ORGANISM: M bovis
(B) STRAIN: G4/100
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE: (F) TISSUE TYPE:
(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:
(vii) IMMEDIATE SOURCE: M bovis (viii) POSITION IN GENOME:
(A) CHROMOSOME SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE: (A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION: None (A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER: (I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GTTCGCTCCG GCGCGGTCAC GCGCATGCCC TCGATGACGC AGATCTCGTC GGGCTCGATG 60 CGCTCTTCCC AGACTTGCAG CCCCGGGGCA CGGCGGCGGT TGGTGTCGAT GATCGCGGCG 120
GGAAGATCCG CGTCGATCCA CTTGGCGCCA TGGAAGGCAG AAGCCGAGTA GCCGGCCAGC 180
ACGCCGCGGC GGCGCGAGCG CAGCCACAGC GCTTTTGCAC GCAATTGCGC GGTCAGTTCC 240
ACACCCTGCG GCACGTACAC GTCTTTATGT AGCGCGACAT ACCTGCTGCG CAATTCGTAG 300
GGCGTCAATA CACCCGCAGC CAGGGCCTCG CTGCCCAGAA AGGGATCCGT CATGGTCGAA 360 GTGTGCTGAG TCACACCGAC AAACGTCACG AGCGTAACCC CAGTGCGAAA GTTCCCGCCG 420
GAAATCGCAG CCACGTTACG CTCGTGGACA TACCGATTTC GGCCCGGCCG CGGCGAGACG 480
ATAGGTTGTC GG GGT GAC TGC CAC AGC CAC TGA AGG GGC CAA ACC CCC ATT 531 Val Thr Ala Thr Ala Thr Glu Gly Ala Lys Pro Pro Phe 5 10
CGT ATC CCG TTC AGT CCT GGT TAC CGG AGG AAA CCG GGG GAT CGG GCT 579 Val Ser Arg Ser Val Leu Val Thr Gly Gly Asn Arg Gly He Gly Leu 15 20 25
GGC GAT CGC ACA GCG GCT GGC TGC CGA CGG CCA CAA GGT GGC CGT CAC 627 Ala He Ala Gin Arg Leu Ala Ala Asp Gly His Lys Val Ala Val Thr 30 35 40 45
CCA CCG TGG ATC CGG AGC GCC AAA GGG GCT GTT TGG CGT CGA ATG TGA 675 His Arg Gly Ser Gly Ala Pro Lys Gly Leu Phe Gly Val Glu Cys Asp 50 55 60 CGT CAC CGA CAG CGA CGC CGT CGA TCG CGC CTT CAC GGC GGT AGA AGA 723 Val Thr Asp Ser Asp Ala Val Asp Arg Ala Phe Thr Ala Val Glu Glu 65 70 75
GCA CCA GGG TCC GGT CGA GGT GCT GGT GTC CAA CGC CGG CCT ATC CGC 771 His Gin Gly Pro Val Glu Val Leu Val Ser Asn Ala Gly Leu Ser Ala 80 85 90
GGA CGC ATT CCT CAT GCG GAT GAC CGA GGA AAA GTT CGA GAA GGT CAT 819 Asp Aid Phe Leu Met Arg Met Thr Glu Glu Lys Phe Glu Lys Val He 95 100 105
CAA CGC CAA CCT CAC CGG GGC GTT CCG GGT GGC TCA ACG GGC ATC GCG 867 Asn Ala Asn Leu Thr Gly Ala Phe Arg Val Ala Gin Arg Ala Ser Arg 110 115 120 125
CAG CAT GCA GCG CAA CAA ATT CGG TCG AAT GAT ATT CAT AGG TTC GGT 915 Ser Met Gin Arg Asn Lys Phe Gly Arg Met He Phe He Gly Ser Val 130 135 140 CTC CGG CAG CTG GGG CAT CGG CAA CCA GGC CAA CTA CGC AGC CTC CAA 963 Ser Gly Ser Trp Gly He Gly Asn Gin Ala Asn Tyr Ala Ala Ser Lys 145 150 155
GGC CGG AGT GAT TGG CAT GGC CCG CTC GAT CGC CCG CGA GCT GTC GAA 1011 Ala Gly Val He Gly Met Ala Arg Ser He Ala Arg Glu Leu Ser Lys 160 165 170
GGC AAA CGT GAC CGC GAA TGT GGT GGC CCC GGG CTA CAT CGA CAC CGA 1059 Ala Asn Val Thr Ala Asn Val Val Ala Pro Gly Tyr He Asp Thr Asp 175 180 185
TAT GAC CCG CGC GCT GGA TGA GCG GAT TCA GCA GGG GGC GCT GCA ATT 1107 Met Thr Arg Ala Leu Asp Glu Arg He Gin Gin Gly Ala Leu Gin Phe 190 195 200 205'
TAT CCC AGC GAA GCG GGT CGG CAC CCC CGC CGA GGT CGC CGG GGT GGT 1155 He Pro Ala Lys Arg Val Gly Thr Pro Ala Glu Val Ala Gly Val Val 210 215 220 CAG CTT CCT GGC TTC CGA GGA TGC GAG CTA TAT CTC CGG TGC GGT CAT 1203 Ser Phe Leu Ala Ser Glu Aso Ala Ser Tyr He Ser Gly Ala Val He 225 230 235
CCC GGT CGA CGG CGG CAT GGG TAT GGG CCA CTG ACA CAA CAC AAG GAC 1251 Pro Val Asp Gly Gly Met Gly Met Gly His 240 245
GCA CAT GAC AGG ACT GCT GGA CGG CAA ACG GAT TCT GGT TAG CGG AAT 1299 Met Thr Gly Leu Leu AΞD Gly Lys Arg He Leu Val Ser Gly He 250 255 260
CAT CAC CGA CTC GTC GAT CGC GTT TCA CAT CGC ACG GGT AGC CCA GGA 1347 He Thr Asp Ser Ser He Ala Phe His He Ala Arg Val Ala Gin Glu 265 270 275
GCA GGG CGC CCA GCT GGT GCT CAC CGG GTT CGA CCG GCT GCG GCT GAT 1395 Gin Gly Ala Gin Leu Val Leu Thr Gly Phe Asp Arg Leu Arg Leu He 280 285 290 TCA GCG CAT CAC CGA CCG GCT GCC GGC AAA GCC CCC GCT GCT CGA ACT 1443 Gin Arg He Thr Asp Arg Leu Pro Ala Lys Ala Pro Leu Leu Glu Leu 295 300 305 310 CGA CGT GCA AAA CGA GGA GCA CCT GGC CAG CTT GGC CGG CCG GGT GAC 1491 Asp Val Gin Asn Glu Glu His Leu Ala Ser Leu Ala Gly Arg Val Thr 315 320 325
CGA GGC GAT CGG GGC GGG CAA CAA GCT CGA CGG GGT GGT GCA TGC GAT 1539 Glu Ala He Gly Ala Gly Asn Lys Leu Asp Gly Val Val His Ala He 330 335 340 TGG GTT CAT GCC GCA GAC CGG GAT GGG CAT CAA CCC GTT CTT CGA CGC 1587
Gly Phe Met Pro Gin Thr Gly Met Gly He Asn Pro Phe Phe Asp Ala 345 350 355
GCC CTA CGC GGA TGT GTC CAA GGG CAT CCA CAT CTC GGC GTA TTC GTA 1635 Pro Tyr Ala Asp Val Ser Lys Gly He His He Ser Ala Tyr Ser Tyr 360 365 370
TGC TTC GAT GGC CAA GGC GCT GCT GCC GAT CAT GAA CCC CGG AGG TTC 1683 Ala Ser Met Ala Lys Ala Leu Leu Pro He Met Asn Pro Gly Gly Ser 375 380 385 390
CAT CGT CGG CAT GGA CTT CGA CCC GAG CCG GGC GAT GCC GGC CTA CAA 1731 He Val Gly Met Asp Phe Asp Pro Ser Arg Ala Met Pro Ala Tyr' Asn 395 400 405
CTG GAT GAC GGT CGC CAA GAG CGC GTT GGA GTC GGT CAA CAG GTT CGT 1779 Trp Met Thr Val Ala Lys Ser Ala Leu Glu Ser Val Asn Arg Phe Val 410 415 420 GGC GCG CGA GGC CGG CAA GTA CGG TGT GCG TTC GAA TCT CGT TGC CGC 1827 Ala Arg Glu Ala Gly Lys Tyr Gly Val Arg Ser Asn Leu Val Ala Ala 425 430 435
AGG CCC TAT CCG GAC GCT GGC GAT GAG TGC GAT CGT CGG CGG TGC GCT 1875 Gly Pro He Arg Thr Leu Ala Met Ser Ala He Val Gly Gly Ala Leu 440 445 450
CGG CGA GGA GGC CGG CGC CCA GAT CCA GCT GCT CGA GGA GGG CTG GGA 1923 Gly Glu Glu Ala Gly Ala Gin He Gin Leu Leu Glu Glu Gly Trp Asp 455 460 465 470
TCA GCG CGC TCC GAT CGG CTG GAA CAT GAA GGA TGC GAC GCC GGT CGC 1971 Gin Arg Ala Pro He Gly Trp Asn Met Lys Asp Ala Thr Pro Val Ala 475 480 485
CAA GAC GGT GTG CGC GCT GCT GTC TGA CTG GCT GCC GGC GAC CAC GGG 2019 Lys Thr Val Cys Ala Leu Leu Ser Asp Trp Leu Pro Ala Thr Thr Gly 490 495 500 TGA CAT CAT CTA CGC CGA CGG CGG CGC GCA CAC CCA ATT GCT CTA GAA 2067 ASD He He Tyr Ala Asp Gly Gly Ala His Thr Gin Leu Leu 505 510 515 CGCATGCAAT TTGATGCCGT CCTGCTGCTG TCGTTCGGCG GACCGGAAGG GCCCGAGCAG 2127
GTGCGGCCGT TCCTGGAGAA CGTTACCCGG GGCCGCGGTG TGCCTGCCGA ACGGTTGGAC 2187
GCGGTGGCCG AGCACTACCT GCATTTCGGT GGGGTATCAC CGATC 2232 (5) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: both (ii) MOLECULE TYPE: protein
(A) DESCRIPTION: product of inhA gene
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: pS5 operon
(A) ORGANISM: M bovis
(B) STRAIN: G4/100
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE: (E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE: (vii) IMMEDIATE SOURCE: M bovis
(viii) POSITION IN GENOME:
(A) CHROMOSOME SEGMENT:
(B) MAP POSITION:
(C) UNITS: (ιχ) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: (x) PUBLICATION INFORMATION: None
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER: (I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Thr Gly Leu Leu Asp Gly Lys Arg He Leu Val Ser Gly He He 16 Thr Asp Ser Ser He Ala Phe His He Ala Arg Val Ala Gin Glu Gin 32
Gly Ala Gin Leu Val Leu Thr Gly Phe Asp Arg Leu Arg Leu He Gin 48
Arg He Thr Asp Arg Leu Pro Ala Lys Ala Pro Leu Leu Glu Leu Asp 64
Val Gin Asn Glu Glu His Leu Ala Ser Leu Ala Gly Arg Val Thr Glu 80
Ala He Gly Ala Gly Asn Lys Leu Asp Gly Val Val His Ala He Gly 96 Phe Met Pro Gin Thr Gly Met Gly He Asn Pro Phe Phe Asp Ala Pro 112
Tyr Ala Asp Val Ser Lys Gly He His He Ser Ala Tyr Ser Tyr Ala 128
Ser Met Ala Lys Ala Leu Leu Pro He Met Asn Pro Gly Gly Ser He 144
Val Gly Met Asp Phe Asp Pro Ser Arg Ala Met Pro Ala Tyr Asn Trp 160
Met Thr Val Ala Lys Ser Ala Leu Glu Ser Val Asn Arg Phe Val Ala 176 Arg Glu Ala Gly Lys Tyr Gly Val Arg Ser Asn Leu Val Ala Ala Gly 192
Pro He Arg Thr Leu Ala Met Ser Ala He Val Gly Gly Ala Leu Gly 208
Glu Glu Ala Gly Ala Gin He Gin Leu Leu Glu Glu Gly Trp Asp Gin 224
Arg Ala Pro He Gly Trp Asn Met Lys Asp Ala Thr Pro Val Ala Lys 240
Thr Val Cys Ala Leu Leu Ser Asp Trp Leu Pro Ala Thr Thr Gly Asp 256 He He Tyr Ala Asp Gly Gly Ala His Thr Gin Leu Leu 269
(6) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
( A ) LENGTH : 247
( B ) TYPE : a .ino ac id
( C ) STRMOΞDNESS : s ingle
( D ) TOPOLOGY : both
(ii) MOLECULE TYPE: protein
(A) DESCRIPTION: ORF1 of pS5
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(V) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: pS5 operon
(A) ORGANISM: M bovis
(B) STRAIN: G4/100
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE: M bovis
(viii) POSITION IN GENOME:
(A) CHROMOSOME SEGMENT:
(B) MAP POSITION: (C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD: (D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION: None
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL: (D) VOLUME:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE: (j) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Val Thr Ala Thr Ala Thr Glu Gly Ala Lys Pro Pro Phe Val Ser Arg 16 Ser Val Leu Val Thr Gly Gly Asn Arg Gly He Gly Leu Ala He Ala 32
Gin Arg Leu Ala Ala Asp Gly His Lys Val Ala Val Thr His Arg Gly 48 Ser Gly Ala Pro Lys Gly Leu Phe Gly Val Glu Cys Asp Val Thr Asp 64
Ser Asp Ala Val Asp Arg Ala Phe Thr Ala Val Glu Glu His Gin Gly 80
Pro Val Glu Val Leu Val Ser Asn Ala Gly Leu Ser Ala Asp Ala Phe 96
Leu Met Arg Met Thr Glu Glu Lys Phe Glu Lys Val He Asn Ala Asn 112 Leu Thr Gly Ala Phe Arg Val Ala Gin Arg Ala Ser Arg Ser Met Gin 128
Arg Asn Lys Phe Gly Arg Met He Phe He Gly Ser Val Ser Gly Ser 144
Trp Gly He Gly Asn Gin Ala Asn Tyr Ala Ala Ser Lys Ala Gly Val 160
He Gly Met Ala Arg Ser He Ala Arg Glu Leu Ser Lys Ala Asn Val 176
Thr Ala Asn Val Val Ala Pro Gly Tyr He Asp Thr Asp Met Thr Arg 192 Ala Leu Asp Glu Arg He Gin Gin Gly Ala Leu Gin Phe He Pro Ala 208
Lys Arg Val Gly Thr Pro Ala Glu Val Ala Gly Val Val Ser Phe Leu 224
Ala Ser Glu Asp Ala Ser Tyr He Ser Gly Ala Val He Pro Val Asp 240
Gly Gly Met Gly Met Gly His 247
(7) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2232
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: both (ϋ) MOLECULE TYPE: DNA (A) DESCRIPTION:
(iii) HYPOTHETICAL: No
(iv) ANTI-SENSE: No
(v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: pS5 operon
(A) ORGANISM: M bovis
(B) STRAIN: G4/100
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE: (E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:
(vii) IMMEDIATE SOURCE: M bovis
(vi i i ) POSITION IN GENOME :
(A) CHROMOSOME SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION: None
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : GTTCGCTCCG GCGCGGTCAC GCGCATGCCC TCGATGACGC AGATCTCGTC GGGCTCGATG 60 CGCTCTTCCC AGACTTGCAG CCCCGGGGCA CGGCGGCGGT TGGTGTCGAT GATCGCGGCG 120 GGAAGATCCG CGTCGATCCA CTTGGCGCCA TGGAAGGCAG AAGCCGAGTA GCCGGCCAGC 180 ACGCCGCGGC GGCGCGAGCG CAGCCACAGC GCTTTTGCAC GCAATTGCGC GGTCAGTTCC 240 ACACCCTGCG GCACGTACAC GTCTTTATGT AGCGCGACAT ACCTGCTGCG CAATTCGTAG 300 GGCGTCAATA CACCCGCAGC CAGGGCCTCG CTGCCCAGAA AGGGATCCGT CATGGTCGAA 360 GTGTGCTGAG TCACACCGAC AAACGTCACG AGCGTAACCC CAGTGCGAAA GTTCCCGCCG 420 GAAATCGCAG CCACGTTACG CTCGTGGACA TACCGATTTC GGCCCGGCCG CGGCGAGACG 480 ATAGGTTGTC GGGGTGACTG CCACAGCCAC TGAAGGGGCC AAACCCCCAT TCGTATCCCG 540 TTCAGTCCTG GTTACCGGAG GAAACCGGGG G.ATCGGGCTG GCGATCGCAC AGCGGCTGGC 600 TGCCGACGGC C.-.C-AAGGTGG CCGTCACCCA CCGTGGATCC GGAGCGCCAA AGGGGCTGTT 660
TGGCGTCGAA TGTGACGTCA CCGACAGCGA CGCCGTCGAT CGCGCCTTCA CGGCGGTAGA 720
AGAGCACCAG GGTCCGGTCG AGGTGCTGGT GTCCAACGCC GGCCTATCCG CGGACGCATT 780
CCTCATGCGG ATGACCGAGG AAAAGTTCGA GAAGGTCATC AACGCCAACC TCACCGGGGC 840
GTTCCGGGTG GCTCAACGGG CATCGCGCAG CATGCAGCGC AACAAATTCG GTCGAATGAT 900 ATTCATAGGT TCGGTCTCCG GCAGCTGGGG CATCGGCAAC CAGGCCAACT ACGCAGCCTC 960
CAAGGCCGGA GTGATTGGCA TGGCCCGCTC GATCGCCCGC GAGCTGTCGA AGGCAAACGT 1020
GACCGCGAAT GTGGTGGCCC CGGGCTACAT CGACACCGAT ATGACCCGCG CGCTGGATGA 1080
GCGGATTCAG CAGGGGGCGC TGCAATTTAT CCCAGCGAAG CGGGTCGGCA CCCCCGCCGA 1140
GGTCGCCGGG GTGGTCAGCT TCCTGGCTTC CGAGGATGCG AGCTATATCT CCGGTGCGGT 1200 CATCCCGGTC GACGGCGGCA TGGGTATGGG CCACTGACAC AACACAAGGA CGCACATGAC 1260
AGGACTGCTG GACGGCAAAC GGATTCTGGT TAGCGGAATC ATCACCGACT CGTCGATCGC 1320
GTTTCACATC GCACGGGTAG CCCAGGAGCA GGGCGCCCAG CTGGTGCTCA CCGGGTTCGA 1380
CCGGCTGCGG CTGATTCAGC GCATCACCGA CCGGCTGCCG GCAAAGGCCC CGCTGCTCGA 1440
ACTCGACGTG CAAAACGAGG AGCACCTGGC CAGCTTGGCC GGCCGGGTGA CCGAGGCGAT 1500 CGGGGCGGGC AACAAGCTCG ACGGGGTGGT GCATGCGATT GGGTTCATGC CGCAGACCGG 1560
GATGGGCATC AACCCGTTCT TCGACGCGCC CTACGCGGAT GTGTCCAAGG GCATCCACAT 1620
CTCGGCGTAT TCGTATGCTT CGATGGCCAA GGCGCTGCTG CCGATCATGA ACCCCGGAGG 1680
TTCCATCGTC GGCATGGACT TCGACCCGAG CCGGGCGATG CCGGCCTACA ACTGGATGAC 1740
GGTCGCCAAG AGCGCGTTGG AGTCGGTCAA CAGGTTCGTG GCGCGCGAGG CCGGCAAGTA 1800 CGGTGTGCGT TCGAATCTCG TTGCCGCAGG CCCTATCCGG ACGCTGGCGA TGAGTGCGAT 1860
CGTCGGCGGT GCGCTCGGCG AGGAGGCCGG CGCCCAGATC CAGCTGCTCG AGGAGGGCTG 1920
GGATCAGCGC GCTCCGATCG GCTGGAACAT GAAGGATGCG ACGCCGGTCG CCAAGACGGT 1980
GTGCGCGCTG CTGTCTGACT GGCTGCCGGC GACCACGGGT GACATCATCT ACGCCGACGG 2040
CGGCGCGCAC ACCCAATTGC TCTAGAACGC ATGCAATTTG ATGCCGTCCT GCTGCTGTCG 2100 TTCGGCGGAC CGGAAGGGCC CGAGCAGGTG CGGCCGTTCC TGGAGAACGT TACCCGGGGC 2160
CGCGGTGTGC CTGCCGAACG GTTGGACGCG GTGGCCGAGC ACTACCTGCA TTTCGGTGGG 2220
GT.ATCήCCGA TC 2232

Claims

WE CLAIM
1. A polynucleotide which encodes the enzyme which is the target of action for isoniazid.
2. A polynucleotide which encodes the enzyme which is the target of action of M. tuberculosis for isoniazid, said polynucleotide having the nucleic acid sequence depicted in Figure 8.
3. A polynucleotide which encodes the enzyme which is the target of action of M. avium for isoniazid, said polynucleotide having the nucleic acid sequence depicted in Figure 8.
4. A polynucleotide which encodes the enzyme which is the target of action of M. smegmatis for isoniazid, said polynucleotide having the nucleic acid sequence depicted in Figure 7.
5. A polynucleotide which encodes the enzyme which is the target of action of M. smegmatis for isoniazid, said polynucleotide having the nucleic acid sequence depicted in Figure 2.
6. A polynucleotide which encodes the enzyme which is the target of action of M. bovis for isoniazid, said polynucleotide having the amino acid sequence depicted in Figure 9.
7. A polynucleotide fragment which encodes the enzyme which is the target of action of M. bovis for isoniazid, said polynucleotide having the amino acid sequence depicted in Figure 10.
8. A polynucleotide fragment which encodes the enzyme which is the target of action of M. bovis for isoniazid, said polynucleotide having the amino acid sequence depicted in Figure 11.
9. A polynucleotide which encodes the enzyme which is the target of action of M. bovis for isoniazid, said polynucleotide having the nucleic acid sequence depicted in Figure 12.
10. The polynucleotide of Claim 9 wherein said polynucleotide has the nucleic acid sequence depicted in Figure 12, from residue 1256 to residue 2062.
11. The polynucleotide of Claim 9 wherein said polynucleotide has the nucleic acid sequence depicted in Figure
12, from residue 494 to residue 1234.
12. An oligonucleotide probe capable of identifying the nucleic acids which encode isoniazid resistance of a tuberculosis mycobacteria.
13. The oligonucleotide probe of Claim 12 wherein the tuberculosis mycobacteria is selected from the ..group consisting of M. tuberculosis. M. bovis. M. smegmatis and M. avium.
14. The oligonucleotide probe of Claim 12, having the nucleic acid sequence depicted in Figure 7.
15. The oligonucleotide probe of Claim 12, having the nucleic acid sequence depicted in Figure 8.
16. The oligonucleotide probe of Claim 12, having the nucleic acid sequence depicted in Figure 12.
17. A diagnostic kit for detecting the presence of isoniazid resistance-associated genes in a clinical sample wherein said diagnostic kit contains an oligonucleotide probe capable of identifying the nucleic acids which encode isoniazid resistance of a mycobacteria, and wherein said oligonucleotide probe is labelled.
18. The diagnostic kit of Claim 17 wherein said oligonucleotide probe has the nucleic acid sequence depicted in
Figure 7.
19. The diagnostic kit of Claim 17 wherein said oligonucleotide probe has the nucleic acid sequence depicted in Figure 8.
20. The diagnostic kit of Claim 17 wherein said oligonucleotide probe has the nucleic acid sequence depicted in Figure 12.
21.The diagnostic kit of Claim 17 wherein said oligonucleotide probe is labelled with a radioactive isotope selected from the group consisting of 32P and 33P labels.
22. The diagnostic kit of Claim 17 which further includes:
(a) a lysing agent:
(b) a denaturing solution; (c) a neutralizing solution;
(d) an alkaline fixation solution; and (e) saline citrate.
23. A method of treating M. tuberculosis. M. avium or M. bovis infection comprising:
(a) preparing anti-DNA or anti-RNA oligonucleotides capable of inhibiting the mRNA activity of the inhA operon of M. tuberculosis or the mRNA activity of the pS5 operon of M. bovis. utilizing the wild-type DNA sequence of the inhA operon of M. tuberculosis or the mutant DNA sequence of the PS5 operon of M. bovis. respectively; and
(b) administering a pharmaceutically-effective amount of said oligonucleotides, either alone or in combination with other compositions.
24. The method of Claim 23 wherein the wild-type inhA operon has the nucleic acid sequence depicted in Figure 8.
25. The method of Claim 23 wherein the operon of M. bovis has the nucleic acid sequence depicted in Figure 12.
26. The method of Claim 23 wherein the oligonucleotides are administered orally, enterally, subcutaneously, intraperitoneally or intravenously.
27. A method of assessing the susceptibility of a strain of M. tuberculosis or M. bovis in a clinical sample to isoniazid comprising:
(a) isolating the chromosomal DNA of M. tuberculosis or M. bovis from a clinical sample; (b) preparing oligonucleotides utilizing the wild-type inhA operon DNA sequence of
M. tuberculosis or the pS5 operon DNA sequence of
M. bovis. respectively; (c) amplifying the region of the inhA operon of the . tuberculosis or the pS5 operon of the M. bovis from the clinical sample which has been identified by the oligonucleotides so as to obtain double stranded DNA; and (d) determining whether a mutated inhA operon exists in the M. tuberculosis of the clinical sample, or a mutated pS5 operon exists in the M. bovis of the clinical sample, the presence of a mutation indicating that said M. tuberculosis or said ' M. bovis is resistant to isoniazid.
28. The method of Claim 27 wherein the oligonucleotides are prepared with an oligonucleotide synthesizer.
29. The method of Claim 27 wherein the amplification is performed using PCR.
30. The method of Claim 29 wherein determining whether a mutation exists is performed by single strand conformation polymorphism analysis.
31. The method of Claim 27 wherein the wild-type inhA operon DNA has the nucleic acid sequence depicted in Figure 8.
32. The method of Claim 27 wherein the pS5 operon DNA has the nucleic acid sequence depicted in Figure 12.
33. A method of determining whether a drug is effective against tuberculosis comprising:
(a) overexpressing the . tuberculosis inhA operon or the M. bovis pS5 operon, which operon encodes the enzyme which is the target of action for isoniazid so as to obtain said enzyme;
(b) combining said enzyme with a purified reagent to obtain a mycolic acid;
(c) assaying said mycolic acid in a system to measure its level of biosynthesis; and
(d) combining a drug with said mycolic acid synthesis system to determine whether said drug blocks the biosynthetic activity of the system, a blockage indicating that said drug is effective against tuberculosis.
34. The method of Claim 33 wherein the operon which encodes the enzyme has the nucleic acid sequence depicted in Figure 8 or in Figure 12.
35. The method of Claim 33 wherein the purified reagent is a fatty acid or NADP.
36. The method of Claim 33 wherein the assay used to measure biosynthesis is thin layer chromatography or spectrophotometry.
37. The method of Claim 33 wherein the drug is selected from the group consisting of isoniazid, ethionamide, rifampicin, streptomycin, ethambutol, ciprofloxacin, novobiocin and cyanide.
38. A method of producing a compound which blocks the activity of InhA enzyme or pS5 enzyme comprising:
(a) overexpressing InhA or pS5 enzyme;
(b) purifying said overexpressed enzyme; (c) performing X-ray crystallography on said purified enzyme so as to obtain the molecular structure of said enzyme; (d) creating a compound with a similar molecular structure to said enzyme; and (e) combining said compound and said enzyme so as to block the biochemical activity of said enzyme.
39. A method of producing a tuberculosis-specific purified mycolic acid comprising adding the InhA enzyme encoded by the inhA operon of M. tuberculosis or the pS5 enzyme encoded by the pS5 operon of M. bovis to the chemical reaction which produces mycolic acid.
40. The method of Claim 39 wherein the inhA operon of M. tuberculosis has the nucleic acid sequence depicted in Figure 8 or wherein the pS5 operon of M. bovis has the nucleic acid sequence depicted in Figure 12.
41. A vaccine useful in the treatment and prevention of tuberculosis comprising the administration of a tuberculosis strain which contains a mutated inhA gene or a mutated pS5 gene.
42. A method of producing a recombinant BCG vaccine useful in the treatment and prevention of diseases, including, tuberculosis, AIDS, leprosy, polio, malaria and tetanus and having attenuated mutants of BCG or M. tuberculosis comprising adding a mutated inhA gene of M. tuberculosis or a mutated pS5 gene of M. bovis to a BCG or M. tuberculosis vaccine.
43. A polynucleotide which encodes the gene mabA.
44. A polynucleotide which encodes an enzyme involved in mycolic acid biosynthesis.
45. The polynucleotide of Claim 44 having the nucleic acid sequence depicted in Figure 7, from nucleic acid residue 96 to nucleic acid residue 863, or the nucleic acid seguence depicted in Figure 8, from nucleic acid residue 224 to nucleic acid residue 967.
46. A method of treating M. tuberculosis comprising the administration of a compound which blocks the mycolic acid biosynthesis activity of the enzyme encoded by the gene mabA.
47. The method of Claim 46 wherein the mabA gene has the nucleic acid sequence depicted in Figure 7, from nucleic acid residue 96 to nucleic acid residue 863, or the nucleic acid sequence depicted in Figure 8, from nucleic acid residue 224 to nucleic acid residue 967.
48. Antibody which is immunoreactive with polynucleotides which encode the enzyme which is the target of action for isoniazid.
49. A method of preventing and treating mycobacterial infection, including tuberculosis, comprising passively administering antibody which is immunoreactive with polynucleotides which encode the enzyme which is the target of action for isoniazid.
50. A vector system which expresses a polynucleotide which encodes the enzyme which is the target of action for isoniazid.
PCT/US1994/005398 1993-05-13 1994-05-13 Use of genes of m. tuberculosis, m. bovis and m. smegmatis which confer isoniazid resistance WO1994026765A1 (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6335436B1 (en) 1995-02-10 2002-01-01 Hybridon, Inc. Oligonucleotides active against drug-resistant bacteria
WO1999061625A1 (en) * 1998-05-27 1999-12-02 Isis Innovation Limited Mycobacterial n-acetyltransferases
FR2837836A1 (en) * 2002-03-29 2003-10-03 Centre Nat Rech Scient USE OF THE MYCOBACTERIUM TUBERCULOSIS Maba (FABG1) PROTEIN FOR THE DESIGN AND SCREENING OF ANTIBIOTICS
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WO2003082911A3 (en) * 2002-03-29 2004-04-01 Centre Nat Rech Scient Use of the protein maba (fabg1) of mycobacterium tuberculosis for designing and screening antibiotics

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