Abstract:
A novel mutation in the gyrase A gene involved in quinolon resistance phenotype acquisition of tubercle bacillus is identified and utilized to provide a method and kit for determining quinolon resistance of tubercle bacillus.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    The present invention relates to a method and kit for determining quinolon resistance of srtains of tubercle bacilli ( Mycobacterium tuberculosis ), more specifically, a method and kit for determining quinolon resistance by detecting a novel mutation in the gyrase A gene, which imparts the quinolon resistance phenotype to tubercle bacilli.  
           [0003]    2. Description of the Related Art  
           [0004]    Drug resistance phenotype acquisition mechanisms of bacteria include (1) such change of the cell wall structure that permeation of a drug into a cell should be blocked, (2) acquisition of a gene coding for an enzyme that decomposes or inactivates a drug, (3) change of a compound in a cell as a target of drug and so forth. Acquisition of these phenotypes are acheived by transmission among bacteria via transposable genetic elements such as plasmid and transposon, change of chromosomes or the like.  
           [0005]    Acquisition of resistance phenotype to drugs (hereinafter, referred to as “antituberculous drug”) by  Mycobacterium tuberculosis  (hereinafter, also referred to as “tubercle bacilli”) is attributable to a spontaneous chromosomal mutation (A. Telenti, Tuberculosis, 1997, 18 (1): 55-64). For example, the mechanism of resistance of tubercle bacilli to rifampicin, which is an antituberculous drug, is resulted from nucleotide substitution occurring in the gene encoding the RNA polymerase β subunit protein, which is a target substance of rifampicin (A Telenti et al., Lancet, 1993, 341: 647-650). Furthermore, nucleotide substitution in the rpsl and rrs genes or the like induces streptomycin resistance (Y. Suzuki, et al., J. Applied. Microbiol., 1997, 83: 634-640). Thus, mechanisms for acquisition of resistance phenotype by a mutation in a gene involved in each target molecule exist for some antituberculous drugs.  
           [0006]    Accordingly, most of drug resistance phenotype acquisition mechanisms of tubercle bacilli are explained based on spontaneous mutations.  
           [0007]    In  Escherichia coli,  bacteria belonging to the genus Staphylococcus and so forth, quinolon resistance is acquired by introduction of a nucleotide mutation into a gene encoding the DNA gyrase activity, topoisomerase IV activity or a membrane integral protein involved in uptake and excretion of a substance into and from a cell (Cambau E., et al., Res. Microbiol., 1996, 147: 52-59; Ferrero L., et al., Antimicrob. Agents Chemother., 1995, 39: 1554-1558) or the like. One of the target compounds of quinolon is gyrase A, and a mutation in the gyrase A gene greatly contributes to resistance to clinically important quinolon molecular species (ciprofloxacin, ofloxacin etc., Cambau E., et al., J. Infect. Dis., 1994, 170: 479-483).  
           [0008]    There have also been reported several mutations in the gyrase A gene involved in acquisition of quinolon resistance phenotype in tubercle bacilli (Antimicrob. Agents Chemother., 1996, 40 (8), 1768-1774; J. Infect. Dis., 1996, 174, 1127-1130; Eur. J. Clin. Microbiol. Infect. Dis., 1997, 16, 395-398; J. Infect. Dis., 2000, 182, 517-525).  
         SUMMARY OF THE INVENTION  
         [0009]    An object of the present invention is to provide a method and kit for determining quinolon resistance of tubercle bacilli by identifying a novel mutation in the gyrase A gene involved in acquisition of quinolon resistance phenotype in tubercle bacilli and utilizing the mutation.  
           [0010]    The inventors of the present invention assiduously studied in order to achieve the aforementioned object. As a result, they found a novel mutation imparting a quinolon resistance phenotype in the gyrase A gene sequence of tubercle bacilli and accomplished the present invention.  
           [0011]    That is, the present invention provides the followings.  
           [0012]    (1) A method for determining quinolon resistance of a  Mycobacterium tuberculosis  strain by detecting a mutation in a gyrase A gene of the bacterial strain, wherein the mutation is substitution of another amino acid residue for an amino acid residue corresponding to the 89th aspartic acid residue in an amino acid sequence encoded by the gyrase A gene.  
           [0013]    (2) The method according to (1), wherein the other amino acid residue is an asparagine residue.  
           [0014]    (3) The method according to (1) or (2), wherein the amino acid sequence encoded by the gyrase A gene is the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence of SEQ ID NO: 4 including substitution, deletion or insertion of one or several amino acid residues.  
           [0015]    (4) The method according to any one of (1) to (3), wherein the mutation is substitution of T for G that is the first nucleotide of a codon coding for an aspartic acid residue.  
           [0016]    (5) The method according to (4), wherein the mutation occurs at a position corresponding to the 530th nucleotide in the nucleotide sequence of SEQ ID NO: 3.  
           [0017]    (6) The method according to any one of (1) to (5), further comprising detecting a substitution of A for a base corresponding to G at the 545th position in the nucleotide sequence of SEQ ID NO: 3.  
           [0018]    (7) A kit for determining quinolon resistance of a  Mycobacterium tuberculosis  strain by detecting a mutation in a gyrase A gene of the bacterial strain, comprising an oligonucleotide for detecting a substitution of another amino acid residue for an amino acid residue corresponding to the 89th aspartic acid residue in an amino acid sequence encoded by the gyrase A gene.  
           [0019]    A novel mutation in the gyrase A gene involved in quinolon resistance phenotype acquisition by tubercle bacilli was identified by the present invention. By utilizing this mutation, a novel method and kit for determining quinolon resistance of tubercle bacilli are provided. 
       
    
    
     BRIEF EXPLANATION OF THE DRAWINGS  
       [0020]    [0020]FIG. 1 shows alignment of nucleotide sequences of gyrA genes from strains of tubercle bacilli resistant to quinolon. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0021]    Hereafter, the present invention will be explained in detail.  
         [0022]    The present invention provides a method for determining quinolon resistance of a tubercle bacillus strain by detecting a mutation in the gyrase A gene (hereinafter, referred to as “gyrA”) of the bacterial strain. Within the reported nucleotide sequence of the gyrA gene of wild tubercle bacillus strain (GenBank locus MSGGYRAB, accession L27512.1 GI: 1107467), the sequence of nucleotide numbers 2041-4923 is shown as SEQ ID NO: 3. Further, an amino acid sequence encoded by the nucleotide sequence is shown as SEQ ID NO: 4. The amino acid sequence is shown in both of SEQ ID NOS: 3 and 4.  
         [0023]    The method of the present invention is characterized in that the mutation in the aforementioned gyrA gene is a substitution of another amino acid residue for an amino acid residue corresponding to the 89th aspartic acid residue in the amino acid sequence encoded by the gyrA gene. FIG. 1 shows comparison of nucleotide sequences of a region around the mutation point. Further, a nucleotide sequence of the aforementioned region in the gyrA gene of a wild strain is shown as SEQ ID NO: 1. The amino acid sequence encoded by this sequence is shown as SEQ ID NO: 2. SEQ ID NO: 1 corresponds to the nucleotide numbers 511-560 in the nucleotide sequence including the gyrA gene shown as SEQ ID NO: 3. Further, the amino acid sequence of SEQ ID NO: 2 corresponds to the amino acid numbers 83-98 in the amino acid sequence encoded by the gyrA gene and shown as SEQ ID NO: 4.  
         [0024]    As the aforementioned other amino acid residue, an asparagine residue can be mentioned. As a mutation in the gyrA gene that induces a mutation for substituting an asparagine residue for the aspartic acid residue, substitution of T for G that is the first nucleotide of a codon coding for an aspartic acid residue can be mentioned. A specific example of this mutation is a mutation occurring at a position corresponding to the 530th nucleotide in the nucleotide sequence of the gyrA gene shown as SEQ ID NO: 3.  
         [0025]    The nucleotide or amino acid numbers in the nucleotide sequence or the amino acid sequence shown as SEQ ID NO: 3 or 4 may be changed due to a mutation in the gyrA gene occurring at a position that is not within a codon corresponding to the 89th aspartic acid residue. A gyrase A having such a mutation has an amino acid sequence including substitution, deletion or insertion of one or several amino acids in the amino acid sequence shown as SEQ ID NO: 4. The present invention can also be applied to a gene coding for the gyrase A including such a mutation so long as the mutation is involved in quinolon resistance. The expressions “amino acid residue corresponding to the 89th aspartic acid residue” and “position corresponding to the 530th nucleotide” refer to an amino acid residue or a nucleotide residue corresponding to such a position in the sequence shown as SEQ ID NO: 3 or 4 when the nucleotide numbers or amino acid numbers are changed as described above. For example, when the first amino acid residue of SEQ ID NO: 4 is deleted, an amino acid residue corresponding to the 89th aspartic acid residue means an aspartic acid residue that is the 88th amino acid residue from the N-terminus.  
         [0026]    In the present invention, known mutations in the gyrA gene involved in quinolon resistance and/or mutations yet to be found in future may be detected in addition to the substitution of an amino acid residue corresponding to the 89th aspartic acid residue. As such mutations, there can be mentioned substitution of A for a base corresponding to G at the 545th position in SEQ ID NO: 3.  
         [0027]    Mutations in the gyrA gene of tubercle bacillus can be detected in the same manner as in usual mutation detecting methods so long as a single nucleotide polymorphism can be detected. For example, there can be mentioned a method for detecting a mutation by directly determining the nucleotide sequence of the gyrA gene. Further, examples of a method for detecting a mutation utilizing PCR include methods such as the template-directed dye-terminator incorporation method (Chen, X. et al., Proc. Natl. Acad. Sci. USA, 20, 10756-10761 (1997)), the dye-labeled oligonucleotide ligation method (Chen, X. et al., Genome Res., 5, 549-556 (1998)), the molecular beacon method (Tyagi, S. et al., Nat. Biotechnol., 1, 49-53 (1998)), the dynamic allele-specific hybridization method (Howellm, W. M. et al., Nat. Biotechnol., 1, 87-88 (1998)) and so forth.  
         [0028]    Further, as methods that do not utilize PCR, there can be mentioned the padlock probe method developed by Mats Nilsson et al. in 1994 (Nilsson, M. et al., Science, 5181, 2085-2088 (1994)) and modified by Paul M. Lizardi et al. (Lizardi, P. et al., Nat. Genet., 3, 225-232 (1998)), the invader assay method (WO94/29482) developed by Victor Lyamichev and so forth.  
         [0029]    Furthermore, as other methods for detecting a single nucleotide polymorphism, the Luminex method utilizing flow cytometry and methods utilizing matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOFMS) are also known and can be used for the present invention.  
         [0030]    Further, microarray methods (WO95/11995, U.S. Pat. No. 5,837,832), which are widely used in recent years, such as the single base sequencing on oligomicroarray method (Drmanac, R. et al., Genomics, 2, 114-128 (1989)) can also be used. The microarray methods are techniques in which a large number of capture nucleic acids are immobilized on a solid phase, a labeled target nucleic acid is hybridized with the capture nucleic acids to be captured, and the presence or absence or existence ratio of the target nucleic acid is analyzed exhaustively.  
         [0031]    As an example of methods preferably used in the present invention, a method of detecting a target mutation in the gyrA gene by using an oligonucleotide immobilized on a substrate will be shown below.  
         [0032]    As the oligonucleotide immobilized on the substrate, used is an oligonucleotide having a nucleotide sequence of a region including the position of the aforementioned mutation in the gyrA gene (hereinafter, referred to as “capture oligo”).  
         [0033]    When the capture oligo is designed, it is usually preferred that a position corresponding to the aforementioned mutation included in the capture oligo should exist in the center portion of the capture oligo. When the capture oligo is too short, detection of the hybridization becomes difficult. When it is too long, inhibition of the hybridization due to a sequence unique to that type does not occur. Therefore, a length in the range of 10 to 24 nucleotides is preferred. Further, when there is a secondary structural failure that negatively affects the hybridization in a process of hybrid formation of the capture oligo and a target described later, a spacer or a nucleotide that does not form a hydrogen bond with any nucleotide can be introduced into the oligonucleotide sequence to avoid the aforementioned failure.  
         [0034]    Although DNA is usually used as the capture oligo, a peptide nucleic acid (PNA) may also be used. Since a hybrid formed by a peptide nucleic acid with a nucleic acid derived from a test human genome has a higher Tm (melting temperature) in comparison with that obtained by using an oligonucleotide, it is expected that a stable hybridization signal can be obtained. The peptide nucleic acid can be readily synthesized by using a usual peptide synthesizer.  
         [0035]    Synthesis of the oligonucleotide, preparation of chromosomal DNA, hybridization and PCR can be performed according to usual methods well known to those skilled in the art (refer to Maniatis, T. et al., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)). Further, the oligonucleotide can be synthesized by using a commercially available DNA synthesizer.  
         [0036]    Material of the substrate on which the oligonucleotide is immobilized is not particularly limited so long as the oligonucleotide can be stably immobilized thereon. However, for example, glass, synthetic resins such as polycarbonate and plastic can be mentioned. Although shape of the substrate is not also particularly limited, a plate-like or film-like substrate can be mentioned. The substrate preferably has a uniform and planar surface.  
         [0037]    The oligonucleotide can be immobilized on the substrate by using a method used in a usual hybridization method such as physical adsorption, electrical coupling and formation of a molecular covalent bond. For example, there can be mentioned a method using a substrate of which surface is coated with a compound having a carbodiimide group or an isocyanate group (Japanese Patent Laid-open Publication (Kokai) No. 08-023975). The substrate coated with a compound having a carbodiimide group or an isocyanate group on the surface thereof can be prepared by coating the substrate surface with a macromolecular compound having a carbodiimide group or an isocyanate group. The oligonucleotides can be immobilized with a covalent bond on the substrate by irradiating with a ultraviolet ray. Further, as a linker for bonding the carbodiimide group or isocyanate group and the oligonucleotide, a compound having an amino group or an imino group having high reactivity with the carbodiimide group or isocyanate group is used. In the case of imino group, the compound can be bonded with a carbodiimide group or an isocyanate group by polymerizing thymine to either one of the ends of capture oligo.  
         [0038]    The oligonucleotide can be immobilized on the substrate by, for example, spotting an oligonucleotide solution on the substrate by using a spotting machine. Usually, the oligonucleotide solution is preferably spotted in a substantially circular shape.  
         [0039]    Further, multiple kinds of oligonucleotide are each usually spotted at multiple positions on a single substrate, and these spots are preferably arranged in a grid pattern. When the spot size is 1000 μm in diameter, the total number of spots is preferably 1600 spots/cm 2  or less and 40×40 or less of spots are preferably provided when they are spotted in a square pattern. Further, when the spot size is 10 μm in diameter, the total number is preferably 400 spots/cm 2  or less, and 20×20 or less of spots are preferably provided when they are spotted in a square pattern. Further, when the vertical and horizontal sizes of the pattern are different, the numbers of the spots along the vertical and horizontal directions may be adjusted depending on shape of the pattern.  
         [0040]    The presence or absence of a specific mutation in the gyrA gene can be determined by hybridizing a nucleic acid fragment probe including a region corresponding to the position of the aforementioned mutation in the gyrA gene of chromosomal DNA of tubercle bacillus (referred to as “nucleic acid probe”) with a wild type capture oligo and a mutant capture oligo and examining with which type of capture oligo the probe hybridized. That is, to which drug the strain has resistance can be detected depending on the hybridization position on the substrate.  
         [0041]    The nucleic acid can be prepared from tubercle bacilli in the same manner as in a usual method for preparing nucleic acids from bacteria. For example, DNA can be prepared according to the method described in Maniatis, T. et al., “Molecular Cloning A Laboratory Manual, Second Editions”, Cold Spring Harbor Laboratory Press (1989).  
         [0042]    The nucleic acid probe can be prepared by amplifying a nucleic acid by using primers designed based on the nucleotide sequence of the capture oligo. Although DNA is usually used as the nucleic acid probe, RNA may also be used. As the method for amplifying a nucleic acid, there can be mentioned, for example, a method of amplifying a nucleic acid as DNA by polymerase chain reaction (PCR) and a method of amplifying a nucleic acid as RNA by the in vitro transcription method.  
         [0043]    The primers used in PCR are designed so that the nucleic acid probe should include a specific mutation site in the gyrA gene and sequences on both sides thereof. As such primers, there can be mentioned oligonucleotides having the nucleotide sequences shown as SEQ ID NOS: 5 and 6.  
         [0044]    If a primer is labeled beforehand, a labeled nucleic acid probe can be obtained. The nucleic acid probe may also be labeled during or after the nucleic acid amplification reaction. As a labeling substance, labeling substances similar to those used for a probe employed in usual hybridization such as a fluorescence substance or hapten can be used. Specific examples of the fluorescence substance include fluorescein (FITC), Rhodamine, phycoerythrin (PE), Texas Red, cyanine fluorescent dyes and so forth. Specific examples of the hapten include biotin, digoxigenin (Dig), dinitrophenyl (DNP) and so forth.  
         [0045]    The primers for preparing a nucleic acid probe can be included in a kit for determining quinolon resistance of tubercle bacilli together with a substrate on which the oligonucleotide is immobilized.  
         [0046]    The hybridization can be performed in the same manner as in usual nucleic acid hybridization. A specific method will be exemplified below.  
         [0047]    A nucleic acid probe is added to a mixed solution containing a salt solution such as SSC (standard saline citrate), a blocking solution containing sodium dodecylsulfate (SDS), bovine serum albumin (BSA) or the like and additives for promoting a hybridization reaction. When the target is a double strand, denaturation is performed by heating or the like. A nucleic acid probe solution is added onto a substrate in an amount of several μL and heated for several hours (usually 37-70° C.) to form a hybrid between an oligonucleotide immobilized on the substrate and the nucleic acid probe.  
         [0048]    A solution of 5×SSC or 3 M tetramethylammonium chloride is added onto the substrate and heated (usually 37-50° C.) to remove oligonucleotides that form a nonspecific hybrid or do not form specific hybrid from the substrate so that only specific hybrids should be selectively left on the substrate.  
         [0049]    The hybrids are detected by using the fluorescence substance or hapten introduced into the nucleic acid probe. When a hapten is used, a solution containing a conjugate of a protein that recognizes the hapten or protein that bonds to the hapten and alkaline phosphatase, horseradish peroxidase or the like (enzyme conjugate) is added onto the substrate and allowed to react at room temperature for several dozens of minutes. A nonspecific adsorption reaction of the enzyme conjugate and the substrate can be prevented by completely coating regions on the substrate with a protein such as BSA except for the regions in which the oligonucleotides are immobilized before a bonding reaction of the hapten and the enzyme conjugate is performed. This treatment can be performed by, after the oligonucleotides are immobilized, adding a solution of a protein such as BSA onto the substrate and leaving it at room temperature for several dozens of minutes. After the bonding reaction of the enzyme conjugate and the hapten of the nucleic acid probe is completed, only enzyme conjugates that bond to the hapten in the nucleic acid probe are left on the substrate by washing the substrate with an appropriate buffer containing a surfactant to remove enzyme conjugates that did not bond to the hapten.  
         [0050]    In order to visualize the hybrids, a compound that becomes insoluble only when only a bonding product of the hapten and the enzyme conjugate exists is added. The production of the insoluble compound is amplified by the enzymatic reaction, and thus the hybrids are visualized. As the compound used for this purpose, nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate, p-toluidine salt (BCIP) are used when the enzyme in the enzyme conjugate is alkaline phosphatase. When the enzyme is horseradish peroxidase, 3,3′,5,5′-tetramethylbenzidine (TMB) or the like can be used.  
         [0051]    Quinolon resistance of tubercle bacillius is determined based on the obtained results of the hybridization by examining pigmentation or fluorescent color development at positions at which the capture oligos are immobilized.  
         [0052]    The kit of the present invention is a kit for determining quinolon resistance of a tubercle bacillus strain by detecting a mutation in the gyrA gene of the bacterial strain, which includes an oligonucleotide for detecting a substitution of another amino acid residue for an amino acid residue corresponding to the 89th aspartic acid residue in the amino acid sequence encoded by the gyrase A gene. As the oligonucleotide, a capture oligo or a substrate on which the capture oligo is immobilized can be mentioned. Further, the kit of the present invention may include primers for preparing a nucleic acid probe, labeled nucleic acid probe, buffer, reagents for hybridization such as enzyme conjugate that recognizes hapten and so forth.  
         [0053]    As quinolon to which the present invention can be applied, there can be mentioned levofloxacin, ciprofloxacin, ofloxacin, gatifloxacin and so forth.  
       EXAMPLES  
       [0054]    Hereafter, the present invention will be explained more specifically with reference to the following example.  
         [0055]    &lt;1&gt; Isolation of Quinolon-resistant Strain of  Mycobacterium tuberculosis    
         [0056]    Sputum of a tuberculosis patient was applied on Ogawa medium and cultured in an incubator at 37° C. for 4 weeks. Appeared colonies were inoculated again on Ogawa medium containing 2.5 μg/mL of levofloxacin and cultured in an incubator at 37° C. for 4 weeks. The strains that formed colonies were isolated as quinolon-resistant strains.  
         [0057]    &lt;2&gt;Determination of Nucleotide Sequence of gyrA Gene of Quinolon-resistant Strain  
         [0058]    Genomic DNA was prepared from each of the quinolon-resistant strains isolated as described above by the method of Kent et al. (Kent, P. T., et al., Mycobacteriology, A Guide for the Level III Laboratory, p.31, U.S. Department of Health and Human Service, Public Health Service, Center for Disease Control, 1985). PCR was performed for the obtained genome by using primers having the nucleotide sequences shown as SEQ ID NOS: 5 and 6.  
         [0059]    rTaq (Takara Shuzo) was used for PCR. PCR was performed by a reaction at 98° C. for 1 minute, 35 cycles of reactions at 98° C. for 5 seconds, at 55° C. for 10 seconds and at 72° C. for 20 seconds, and a reaction at 72° C. for 1 minute. The amplification product was separated by 1% agarose gel electrophoresis, and a gel containing a 0.4 kbp band was excised. This gel piece was placed into a 1.5-mL micro tube and frozen at −135° C. for 15 minutes. Then, the gel was centrifuged at 15000 rpm for 10 minutes to obtain a supernatant. Resulting DNA solution was used as a template together with a primer shown as SEQ ID NO: 5 and Big Dye Terminator kit manufactured by ABI to perform a sequencing reaction. After completion of the reaction, the sequence was analyzed by using a genetic analyzer manufactured by ABI.  
         [0060]    [0060]FIG. 1 shows alignment of the obtained sequences. In FIG. 1, the number shown at the right end of each sequence represents the number of sequences that had the same mutation among the determined sequences. Among these mutations, substitution of A for G at the 530th position in the nucleotide sequence shown as SEQ ID NO: 3 is a novel mutation. Further, substitution of A for G at the 530th position and substitution of A for G at the 545th position constitute a novel combination of mutations. Other mutations were the same as the mutations already reported (Antimicrob. Agents Chemother., 1996, 40 (8), 1768-1774; J. Infect. Dis., 1996, 174, 1127-1130; Eur. J. Clin. Microbiol. Infect. Dis., 1997, 16, 395-398; J. Infect. Dis., 2000, 182, 517-525).  
     
       
       
         1 
         
           
             6  
           
           
             1  
             50  
             DNA  
             Mycobacterium tuberculosis  
             
               CDS  
               (2)..(49)  
             
           
            1 

c aac tac cac ccg cac ggc gac gcg tcg atc tac gac agc ctg gtg cgc     49 
  Asn Tyr His Pro His Gly Asp Ala Ser Ile Tyr Asp Ser Leu Val Arg 
    1               5                  10                  15 

a                                                                     50 

 
           
             2  
             16  
             PRT  
             Mycobacterium tuberculosis  
           
            2 

Asn Tyr His Pro His Gly Asp Ala Ser Ile Tyr Asp Ser Leu Val Arg 
  1               5                  10                  15 

 
           
             3  
             2884  
             DNA  
             Mycobacterium tuberculosis  
             
               CDS  
               (266)..(2782)  
             
           
            3 

aagatcaaca aggaagacgg cattcagcgg tacaagggtc taggtgaaat ggacgctaag     60 

gagttgtggg agaccaccat ggatccctcg gttcgtgtgt tgcgtcaagt gacgctggac    120 

gacgccgccg ccgccgacga gttgttctcc atcctgatgg gcgaggacgt cgacgcgcgg    180 

cgcagcttta tcacccgcaa cgccaaggat gttcggttcc tggatgtcta acgcaaccct    240 

gcgttcgatt gcaaacgagg aatag atg aca gac acg acg ttg ccg cct gac      292 
                            Met Thr Asp Thr Thr Leu Pro Pro Asp 
                              1               5 

gac tcg ctc gac cgg atc gaa ccg gtt gac atc gag cag gag atg cag      340 
Asp Ser Leu Asp Arg Ile Glu Pro Val Asp Ile Glu Gln Glu Met Gln 
 10                  15                  20                  25 

cgc agc tac atc gac tat gcg atg agc gtg atc gtc ggc cgc gcg ctg      388 
Arg Ser Tyr Ile Asp Tyr Ala Met Ser Val Ile Val Gly Arg Ala Leu 
                 30                  35                  40 

ccg gag gtg cgc gac ggg ctc aag ccc gtg cat cgc cgg gtg ctc tat      436 
Pro Glu Val Arg Asp Gly Leu Lys Pro Val His Arg Arg Val Leu Tyr 
             45                  50                  55 

gca atg ttc gat tcc ggc ttc cgc ccg gac cgc agc cac gcc aag tcg      484 
Ala Met Phe Asp Ser Gly Phe Arg Pro Asp Arg Ser His Ala Lys Ser 
         60                  65                  70 

gcc cgg tcg gtt gcc gag acc atg ggc aac tac cac ccg cac ggc gac      532 
Ala Arg Ser Val Ala Glu Thr Met Gly Asn Tyr His Pro His Gly Asp 
     75                  80                  85 

gcg tcg atc tac gac agc ctg gtg cgc atg gcc cag ccc tgg tcg ctg      580 
Ala Ser Ile Tyr Asp Ser Leu Val Arg Met Ala Gln Pro Trp Ser Leu 
 90                  95                 100                 105 

cgc tac ccg ctg gtg gac ggc cag ggc aac ttc ggc tcg cca ggc aat      628 
Arg Tyr Pro Leu Val Asp Gly Gln Gly Asn Phe Gly Ser Pro Gly Asn 
                110                 115                 120 

gac cca ccg gcg gcg atg agg tac acc gaa gcc cgg ctg acc ccg ttg      676 
Asp Pro Pro Ala Ala Met Arg Tyr Thr Glu Ala Arg Leu Thr Pro Leu 
            125                 130                 135 

gcg atg gag atg ctg agg gaa atc gac gag gag aca gtc gat ttc atc      724 
Ala Met Glu Met Leu Arg Glu Ile Asp Glu Glu Thr Val Asp Phe Ile 
        140                 145                 150 

cct aac tac gac ggc cgg gtg caa gag ccg acg gtg cta ccc agc cgg      772 
Pro Asn Tyr Asp Gly Arg Val Gln Glu Pro Thr Val Leu Pro Ser Arg 
    155                 160                 165 

ttc ccc aac ctg ctg gcc aac ggg tca ggc ggc atc gcg gtc ggc atg      820 
Phe Pro Asn Leu Leu Ala Asn Gly Ser Gly Gly Ile Ala Val Gly Met 
170                 175                 180                 185 

gca acc aat atc ccg ccg cac aac ctg cgt gag ctg gcc gac gcg gtg      868 
Ala Thr Asn Ile Pro Pro His Asn Leu Arg Glu Leu Ala Asp Ala Val 
                190                 195                 200 

ttc tgg gcg ctg gag aat cac gac gcc gac gaa gag gag acc ctg gcc      916 
Phe Trp Ala Leu Glu Asn His Asp Ala Asp Glu Glu Glu Thr Leu Ala 
            205                 210                 215 

gcg gtc atg ggg cgg gtt aaa ggc ccg gac ttc ccg acc gcc gga ctg      964 
Ala Val Met Gly Arg Val Lys Gly Pro Asp Phe Pro Thr Ala Gly Leu 
        220                 225                 230 

atc gtc gga tcc cag ggc acc gct gat gcc tac aaa act ggc cgc ggc     1012 
Ile Val Gly Ser Gln Gly Thr Ala Asp Ala Tyr Lys Thr Gly Arg Gly 
    235                 240                 245 

tcc att cga atg cgc gga gtt gtt gag gta gaa gag gat tcc cgc ggt     1060 
Ser Ile Arg Met Arg Gly Val Val Glu Val Glu Glu Asp Ser Arg Gly 
250                 255                 260                 265 

cgt acc tcg ctg gtg atc acc gag ttg ccg tat cag gtc aac cac gac     1108 
Arg Thr Ser Leu Val Ile Thr Glu Leu Pro Tyr Gln Val Asn His Asp 
                270                 275                 280 

aac ttc atc act tcg atc gcc gaa cag gtc cga gac ggc aag ctg gcc     1156 
Asn Phe Ile Thr Ser Ile Ala Glu Gln Val Arg Asp Gly Lys Leu Ala 
            285                 290                 295 

ggc att tcc aac att gag gac cag tct agc gat cgg gtc ggt tta cgc     1204 
Gly Ile Ser Asn Ile Glu Asp Gln Ser Ser Asp Arg Val Gly Leu Arg 
        300                 305                 310 

atc gtc atc gag atc aag cgc gat gcg gtg gcc aag gtg gtg atc aat     1252 
Ile Val Ile Glu Ile Lys Arg Asp Ala Val Ala Lys Val Val Ile Asn 
    315                 320                 325 

aac ctt tac aag cac acc cag ctg cag acc agc ttt ggc gcc aac atg     1300 
Asn Leu Tyr Lys His Thr Gln Leu Gln Thr Ser Phe Gly Ala Asn Met 
330                 335                 340                 345 

cta gcg atc gtc gac ggg gtg ccg cgc acg ctg cga ctg gac cag ctg     1348 
Leu Ala Ile Val Asp Gly Val Pro Arg Thr Leu Arg Leu Asp Gln Leu 
                350                 355                 360 

atc cgc tat tac gtt gac cac caa ctc gac gtc att gtg cgg cgc acc     1396 
Ile Arg Tyr Tyr Val Asp His Gln Leu Asp Val Ile Val Arg Arg Thr 
            365                 370                 375 

acc tac cgg ctg cgc aag gca aac gag cga gcc cac att ctg cgc ggc     1444 
Thr Tyr Arg Leu Arg Lys Ala Asn Glu Arg Ala His Ile Leu Arg Gly 
        380                 385                 390 

ctg gtt aaa gcg ctc gac gcg ctg gac gag gtc att gca ctg atc cgg     1492 
Leu Val Lys Ala Leu Asp Ala Leu Asp Glu Val Ile Ala Leu Ile Arg 
    395                 400                 405 

gcg tcg gag acc gtc gat atc gcc cgg gcc gga ctg atc gag ctg ctc     1540 
Ala Ser Glu Thr Val Asp Ile Ala Arg Ala Gly Leu Ile Glu Leu Leu 
410                 415                 420                 425 

gac atc gac gag atc cag gcc cag gca atc ctg gac atg cag ttg cgg     1588 
Asp Ile Asp Glu Ile Gln Ala Gln Ala Ile Leu Asp Met Gln Leu Arg 
                430                 435                 440 

cgc ctg gcc gca ctg gaa cgc cag cgc atc atc gac gac ctg gcc aaa     1636 
Arg Leu Ala Ala Leu Glu Arg Gln Arg Ile Ile Asp Asp Leu Ala Lys 
            445                 450                 455 

atc gag gcc gag atc gcc gat ctg gaa gac atc ctg gca aaa ccc gag     1684 
Ile Glu Ala Glu Ile Ala Asp Leu Glu Asp Ile Leu Ala Lys Pro Glu 
        460                 465                 470 

cgg cag cgt ggg atc gtg cgc gac gaa ctc gcc gaa atc gtg gac agg     1732 
Arg Gln Arg Gly Ile Val Arg Asp Glu Leu Ala Glu Ile Val Asp Arg 
    475                 480                 485 

cac ggc gac gac cgg cgt acc cgg atc atc gcg gcc gac gga gac gtc     1780 
His Gly Asp Asp Arg Arg Thr Arg Ile Ile Ala Ala Asp Gly Asp Val 
490                 495                 500                 505 

agc gac gag gat ttg atc gcc cgc gag gac gtc gtt gtc act atc acc     1828 
Ser Asp Glu Asp Leu Ile Ala Arg Glu Asp Val Val Val Thr Ile Thr 
                510                 515                 520 

gaa acg gga tac gcc aag cgc acc aag acc gat ctg tat cgc agc cag     1876 
Glu Thr Gly Tyr Ala Lys Arg Thr Lys Thr Asp Leu Tyr Arg Ser Gln 
            525                 530                 535 

aaa cgc ggc ggc aag ggc gtg cag ggt gcg ggg ttg aag cag gac gac     1924 
Lys Arg Gly Gly Lys Gly Val Gln Gly Ala Gly Leu Lys Gln Asp Asp 
        540                 545                 550 

atc gtc gcg cac ttc ttc gtg tgc tcc acc cac gat ttg atc ctg ttc     1972 
Ile Val Ala His Phe Phe Val Cys Ser Thr His Asp Leu Ile Leu Phe 
    555                 560                 565 

ttc acc acc cag gga cgg gtt tat cgg gcc aag gcc tac gac ttg ccc     2020 
Phe Thr Thr Gln Gly Arg Val Tyr Arg Ala Lys Ala Tyr Asp Leu Pro 
570                 575                 580                 585 

gag gcc tcc cgg acg gcg cgc ggg cag cac gtg gcc aac ctg tta gcc     2068 
Glu Ala Ser Arg Thr Ala Arg Gly Gln His Val Ala Asn Leu Leu Ala 
                590                 595                 600 

ttc cag ccc gag gaa cgc atc gcc cag gtc atc cag att cgc ggc tac     2116 
Phe Gln Pro Glu Glu Arg Ile Ala Gln Val Ile Gln Ile Arg Gly Tyr 
            605                 610                 615 

acc gac gcc ccg tac ctg gtg ctg gcc act cgc aac ggg ctg gtg aaa     2164 
Thr Asp Ala Pro Tyr Leu Val Leu Ala Thr Arg Asn Gly Leu Val Lys 
        620                 625                 630 

aag tcc aag ctg acc gac ttc gac tcc aat cgc tcg ggc gga atc gtg     2212 
Lys Ser Lys Leu Thr Asp Phe Asp Ser Asn Arg Ser Gly Gly Ile Val 
    635                 640                 645 

gcg gtc aac ctg cgc gac aac gac gag ctg gtc ggt gcg gtg ctg tgt     2260 
Ala Val Asn Leu Arg Asp Asn Asp Glu Leu Val Gly Ala Val Leu Cys 
650                 655                 660                 665 

tcg gcc ggc gac gac ctg ctg ctg gtc tcg gcc aac ggg cag tcc atc     2308 
Ser Ala Gly Asp Asp Leu Leu Leu Val Ser Ala Asn Gly Gln Ser Ile 
                670                 675                 680 

agg ttc tcg gcg acc gac gag gcg ctg cgg cca atg ggt cgt gcc acc     2356 
Arg Phe Ser Ala Thr Asp Glu Ala Leu Arg Pro Met Gly Arg Ala Thr 
            685                 690                 695 

tcg ggt gtg cag ggc atg cgg ttc aat atc gac gac cgg ctc gtg tcg     2404 
Ser Gly Val Gln Gly Met Arg Phe Asn Ile Asp Asp Arg Leu Val Ser 
        700                 705                 710 

ctg aac gtc gtg cgt gaa ggc acc tat ctg ctg gtg gcg acg tca ggg     2452 
Leu Asn Val Val Arg Glu Gly Thr Tyr Leu Leu Val Ala Thr Ser Gly 
    715                 720                 725 

ggc tat gcg aaa cgt acc gcg atc gag gaa tac ccg gta cag ggc cgc     2500 
Gly Tyr Ala Lys Arg Thr Ala Ile Glu Glu Tyr Pro Val Gln Gly Arg 
730                 735                 740                 745 

ggc ggt aaa ggt gtg ctg acg gtc atg tac gac cgc cgg cgc ggc agg     2548 
Gly Gly Lys Gly Val Leu Thr Val Met Tyr Asp Arg Arg Arg Gly Arg 
                750                 755                 760 

ttg gtt ggg gcg ttg att gtc gac gac gac agc gag ctg tat gcc gtc     2596 
Leu Val Gly Ala Leu Ile Val Asp Asp Asp Ser Glu Leu Tyr Ala Val 
            765                 770                 775 

act tcc ggc ggt ggc gtg atc cgc acc gcg gca cgc cag gtt cgc aag     2644 
Thr Ser Gly Gly Gly Val Ile Arg Thr Ala Ala Arg Gln Val Arg Lys 
        780                 785                 790 

gcg gga cgg cag acc aag ggt gtt cgg ttg atg aat ctg ggc gag ggc     2692 
Ala Gly Arg Gln Thr Lys Gly Val Arg Leu Met Asn Leu Gly Glu Gly 
    795                 800                 805 

gac aca ctg ttg gcc atc gcg cgc aac gcc gaa gaa agt ggc gac gat     2740 
Asp Thr Leu Leu Ala Ile Ala Arg Asn Ala Glu Glu Ser Gly Asp Asp 
810                 815                 820                 825 

aat gcc gtg gac gcc aac ggc gca gac cag acg ggc aat taa             2782 
Asn Ala Val Asp Ala Asn Gly Ala Asp Gln Thr Gly Asn 
                830                 835 

tcaggctcgc ccgacgacga tgcggatcgc gtagcgatct gaggaggaat cgggcagcta   2842 

ggctcggcag ccgggtacga gtgttaggag tcggggtgac tg                      2884 

 
           
             4  
             838  
             PRT  
             Mycobacterium tuberculosis  
           
            4 

Met Thr Asp Thr Thr Leu Pro Pro Asp Asp Ser Leu Asp Arg Ile Glu 
  1               5                  10                  15 

Pro Val Asp Ile Glu Gln Glu Met Gln Arg Ser Tyr Ile Asp Tyr Ala 
             20                  25                  30 

Met Ser Val Ile Val Gly Arg Ala Leu Pro Glu Val Arg Asp Gly Leu 
         35                  40                  45 

Lys Pro Val His Arg Arg Val Leu Tyr Ala Met Phe Asp Ser Gly Phe 
     50                  55                  60 

Arg Pro Asp Arg Ser His Ala Lys Ser Ala Arg Ser Val Ala Glu Thr 
 65                  70                  75                  80 

Met Gly Asn Tyr His Pro His Gly Asp Ala Ser Ile Tyr Asp Ser Leu 
                 85                  90                  95 

Val Arg Met Ala Gln Pro Trp Ser Leu Arg Tyr Pro Leu Val Asp Gly 
            100                 105                 110 

Gln Gly Asn Phe Gly Ser Pro Gly Asn Asp Pro Pro Ala Ala Met Arg 
        115                 120                 125 

Tyr Thr Glu Ala Arg Leu Thr Pro Leu Ala Met Glu Met Leu Arg Glu 
    130                 135                 140 

Ile Asp Glu Glu Thr Val Asp Phe Ile Pro Asn Tyr Asp Gly Arg Val 
145                 150                 155                 160 

Gln Glu Pro Thr Val Leu Pro Ser Arg Phe Pro Asn Leu Leu Ala Asn 
                165                 170                 175 

Gly Ser Gly Gly Ile Ala Val Gly Met Ala Thr Asn Ile Pro Pro His 
            180                 185                 190 

Asn Leu Arg Glu Leu Ala Asp Ala Val Phe Trp Ala Leu Glu Asn His 
        195                 200                 205 

Asp Ala Asp Glu Glu Glu Thr Leu Ala Ala Val Met Gly Arg Val Lys 
    210                 215                 220 

Gly Pro Asp Phe Pro Thr Ala Gly Leu Ile Val Gly Ser Gln Gly Thr 
225                 230                 235                 240 

Ala Asp Ala Tyr Lys Thr Gly Arg Gly Ser Ile Arg Met Arg Gly Val 
                245                 250                 255 

Val Glu Val Glu Glu Asp Ser Arg Gly Arg Thr Ser Leu Val Ile Thr 
            260                 265                 270 

Glu Leu Pro Tyr Gln Val Asn His Asp Asn Phe Ile Thr Ser Ile Ala 
        275                 280                 285 

Glu Gln Val Arg Asp Gly Lys Leu Ala Gly Ile Ser Asn Ile Glu Asp 
    290                 295                 300 

Gln Ser Ser Asp Arg Val Gly Leu Arg Ile Val Ile Glu Ile Lys Arg 
305                 310                 315                 320 

Asp Ala Val Ala Lys Val Val Ile Asn Asn Leu Tyr Lys His Thr Gln 
                325                 330                 335 

Leu Gln Thr Ser Phe Gly Ala Asn Met Leu Ala Ile Val Asp Gly Val 
            340                 345                 350 

Pro Arg Thr Leu Arg Leu Asp Gln Leu Ile Arg Tyr Tyr Val Asp His 
        355                 360                 365 

Gln Leu Asp Val Ile Val Arg Arg Thr Thr Tyr Arg Leu Arg Lys Ala 
    370                 375                 380 

Asn Glu Arg Ala His Ile Leu Arg Gly Leu Val Lys Ala Leu Asp Ala 
385                 390                 395                 400 

Leu Asp Glu Val Ile Ala Leu Ile Arg Ala Ser Glu Thr Val Asp Ile 
                405                 410                 415 

Ala Arg Ala Gly Leu Ile Glu Leu Leu Asp Ile Asp Glu Ile Gln Ala 
            420                 425                 430 

Gln Ala Ile Leu Asp Met Gln Leu Arg Arg Leu Ala Ala Leu Glu Arg 
        435                 440                 445 

Gln Arg Ile Ile Asp Asp Leu Ala Lys Ile Glu Ala Glu Ile Ala Asp 
    450                 455                 460 

Leu Glu Asp Ile Leu Ala Lys Pro Glu Arg Gln Arg Gly Ile Val Arg 
465                 470                 475                 480 

Asp Glu Leu Ala Glu Ile Val Asp Arg His Gly Asp Asp Arg Arg Thr 
                485                 490                 495 

Arg Ile Ile Ala Ala Asp Gly Asp Val Ser Asp Glu Asp Leu Ile Ala 
            500                 505                 510 

Arg Glu Asp Val Val Val Thr Ile Thr Glu Thr Gly Tyr Ala Lys Arg 
        515                 520                 525 

Thr Lys Thr Asp Leu Tyr Arg Ser Gln Lys Arg Gly Gly Lys Gly Val 
    530                 535                 540 

Gln Gly Ala Gly Leu Lys Gln Asp Asp Ile Val Ala His Phe Phe Val 
545                 550                 555                 560 

Cys Ser Thr His Asp Leu Ile Leu Phe Phe Thr Thr Gln Gly Arg Val 
                565                 570                 575 

Tyr Arg Ala Lys Ala Tyr Asp Leu Pro Glu Ala Ser Arg Thr Ala Arg 
            580                 585                 590 

Gly Gln His Val Ala Asn Leu Leu Ala Phe Gln Pro Glu Glu Arg Ile 
        595                 600                 605 

Ala Gln Val Ile Gln Ile Arg Gly Tyr Thr Asp Ala Pro Tyr Leu Val 
    610                 615                 620 

Leu Ala Thr Arg Asn Gly Leu Val Lys Lys Ser Lys Leu Thr Asp Phe 
625                 630                 635                 640 

Asp Ser Asn Arg Ser Gly Gly Ile Val Ala Val Asn Leu Arg Asp Asn 
                645                 650                 655 

Asp Glu Leu Val Gly Ala Val Leu Cys Ser Ala Gly Asp Asp Leu Leu 
            660                 665                 670 

Leu Val Ser Ala Asn Gly Gln Ser Ile Arg Phe Ser Ala Thr Asp Glu 
        675                 680                 685 

Ala Leu Arg Pro Met Gly Arg Ala Thr Ser Gly Val Gln Gly Met Arg 
    690                 695                 700 

Phe Asn Ile Asp Asp Arg Leu Val Ser Leu Asn Val Val Arg Glu Gly 
705                 710                 715                 720 

Thr Tyr Leu Leu Val Ala Thr Ser Gly Gly Tyr Ala Lys Arg Thr Ala 
                725                 730                 735 

Ile Glu Glu Tyr Pro Val Gln Gly Arg Gly Gly Lys Gly Val Leu Thr 
            740                 745                 750 

Val Met Tyr Asp Arg Arg Arg Gly Arg Leu Val Gly Ala Leu Ile Val 
        755                 760                 765 

Asp Asp Asp Ser Glu Leu Tyr Ala Val Thr Ser Gly Gly Gly Val Ile 
    770                 775                 780 

Arg Thr Ala Ala Arg Gln Val Arg Lys Ala Gly Arg Gln Thr Lys Gly 
785                 790                 795                 800 

Val Arg Leu Met Asn Leu Gly Glu Gly Asp Thr Leu Leu Ala Ile Ala 
                805                 810                 815 

Arg Asn Ala Glu Glu Ser Gly Asp Asp Asn Ala Val Asp Ala Asn Gly 
            820                 825                 830 

Ala Asp Gln Thr Gly Asn 
        835 

 
           
             5  
             23  
             DNA  
             Mycobacterium tuberculosis  
             
               CDS  
               (2)..(49)  
             
           
            5 

agcgcagcta catcgactat gcg                                             23 

 
           
             6  
             23  
             DNA  
             Mycobacterium tuberculosis  
             
               CDS  
               (2)..(49)  
             
           
            6 

cttcggtgta cctcatcgcc gcc                                             23