Chapter 16 : Toward Mapping and Sequencing the Genome of

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in

Toward Mapping and Sequencing the Genome of , Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555818357/9781555819101_Chap16-1.gif /docserver/preview/fulltext/10.1128/9781555818357/9781555819101_Chap16-2.gif


The aims of this chapter are to present the current status of the genome project, to summarize the main findings of that project, and to discuss its potential impact on future tuberculosis research. The chromosomal inserts carried by the latter clones can be excised by digestion with and are destined for systematic DNA sequence analysis. The rapid mapping of genes is achieved by hybridization of specific probes to cosmid grids, and since the distribution of cosmid clones within a contig is relatively uniform, genes can usually be positioned with a precision of 10 to 20 kb. Sequences are considered finished when both strands have been completely sequenced at an indel error rate of about 1/5,000 and when the sequence has been analyzed for genes and open reading frames (ORFs). Finished sequences are submitted electronically to GenBank and a mycobacterial mapping and sequence database, MycDB, based on the ACEDB software. Analysis of the TBC2 sequence revealed 23 putative genes, including 14 encoding polypeptides with homologies to other known proteins and 9 ORFs. The enzyme is highly homologous to PUR3. A putative transport protein, encoded by , shows weak homology to the ArsB proton pump, P-glycoprotein, and other transport proteins.

Citation: Cole S, Smith D. 1994. Toward Mapping and Sequencing the Genome of , p 227-238. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch16
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


Image of Figure 1
Figure 1

Integrated approach to mycobacterial genome mapping. The steps and approaches being used in analysis of the chromosome are shown in the form of a flow diagram. Genomic DNA is used for either PFGE analysis or cosmid cloning. The integrated map combines data from physical, contig, and gene mapping, and this map together with DNA sequences is stored in MycDB, a database dedicated to mycobacterial research.

Citation: Cole S, Smith D. 1994. Toward Mapping and Sequencing the Genome of , p 227-238. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch16
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 2
Figure 2

PFGE analysis of H37Rv and Ra. (A) DNA in agarose plugs from H37Ra was digested with I (lane 2) or I (lane 3), while H37Rv DNA was digested with I (lane 4) and separated by field inversion gel electrophoresis as described previously ( ). The agarose gel (1% in 0.66x Trisborate-EDTA [TBE]) was calibrated with yeast chromosomes (lane 1) or λ concatamers (not shown) and run for 48 h at constant voltage (240 V). (B) Similar DNA samples from H37Rv were digested with I (lane 2) and I (lane 3), and H37Ra DNA was digested with I (lane 4) or I (lane 5) and separated by contour-clamped homogeneous electric field gel electrophoresis as described previously ( ). The agarose gel (1.2% in 0.5 x TBE) was calibrated with 1 concatamer (lane 1) and run for 50 h at constant voltage (170 V). Fragments discussed in the text are indicated by arrowheads. The black arrow indicates the 480-kb I fragment, and the white arrows indicate 260- and 280-kb I fragments.

Citation: Cole S, Smith D. 1994. Toward Mapping and Sequencing the Genome of , p 227-238. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch16
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 3
Figure 3

Schematic diagram of the multiplex cosmid sequencing process. See text for explanation.

Citation: Cole S, Smith D. 1994. Toward Mapping and Sequencing the Genome of , p 227-238. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch16
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 4
Figure 4

Map of cosmid TBC2. A reference scale in kilobases is given below the map. The positions and orientations of genes are indicated on the map. Shaded arrows represent genes that have homology to other genes in public databases, and open arrows represent ORFs that do not have significant homologies to any known genes. Key to gene names: , pyruvate carboxylase; , 22-kDa putative cell cycle regulator; , homolog of a lipopolysaccharide operon gene; , phosphoribosylglycinamide formytransferase; , putative anion transporter; , putative UDP-sugar transferase; , rRNA methyl transferase; , putative methylase; , putative acetyl coenzyme A ligase; , homolog of the gene; , polyketide synthase (beta-keto reductase); , polyketide synthase (ketoacyl acyl carrier protein synthase); ,,, polyketide synthase (acyl transferase, dehydratase, enoyl reductase). See text for references.

Citation: Cole S, Smith D. 1994. Toward Mapping and Sequencing the Genome of , p 227-238. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch16
Permissions and Reprints Request Permissions
Download as Powerpoint


1. Andersen, Å. B.,, A. Worsaae,, and S. D. Chaparas. 1988. Isolation and characterization of recombinant lambda gtll bacteriophages expressing eight different mycobacterial antigens of potential immunological relevance. Infect. Immun. 56: 1344 1351.
2. Ansorge, W.,, B. S. Sproat,, J. Stegemann,, and C. J. Schwagc. 1986. Automated DNA sequencing; ultrasensitive detection of fluorescent bands during electrophoresis. Nucleic Acids Res. 15: 4593 4602.
3. Bergh, S.,, and S. T. Cole. MycDB, an integrated mycobacterial database. Submitted for publication.
4. Bloom, B. R.,, and C. J. L. Murray. 1992. Tuberculosis: commentary on a reemergent killer. Science 257: 1055 1064.
5. Canard, B.,, and S. T. Cole. 1989. Genome organization of the anaerobic pathogen Clostridium perfringens. Proc. Natl. Acad. Sci. USA 86: 6676 6680.
6. Canard, B.,, B. Saint-Joanis,, and S. T. Cole. 1992. Genomic diversity and organisation of virulence genes in the pathogenic anaerobe Clostridium perfringens. Mol. Microbiol. 6: 1421 1429.
7. Cheah, K. C, and P. A. Manning. 1993. Inactivation of the Escherichia coli B41 (O101:K99/F41) rfb gene encoding an 80-kDa polypeptide results in the synthesis of an antigenically altered lipopolysaccharide in E. coli K-12. Gene 123: 9 15.
8. Chen, C.-M.,, T. K. Misra,, S. Silver,, and B. P. Rosen. 1986. Nucleotide sequence of the structural genes for an anion pump: the plasmid-encoded arsenical resistance operon. J. Biol. Chem. 261: 15030 15038.
9. Church, G.,, and S. Kieffer-Higgins. 1988. Multiplex DNA sequencing. Science 240: 185 188.
10. Clark-Curtiss, J. E.,, W. R. Jacobs,, M. A. Docherty,, L. R. Ritchie,, and R. Curtiss III. 1985. Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. J. Bacteriol. 161: 1093 1102.
11. Cole, S. T.,, and I. Saint-Girons. Bacterial genomics. FEMS Microbiol. Rev., in press.
12. Coulson, A.,, J. Sulston,, S. Brenner,, and J. Kara. 1986. Toward a physical map of the genome of the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 83: 7821 7825.
13. Devereux, J.,, P. Haeberli,, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387 395.
14. Donadio, S.,, M. J. Staver,, J. B. McAlpine,, S. J. Swanson,, and L. Katz. 1991. Modular organization of genes required for complex polyketide biosynthesis. Science 252: 675 679.
15. Dooley, S. W.,, W. R. Jarvis,, W. J. Martone,, and D. E. Snider Jr.. 1992. Multi-drug resistant tuberculosis. Ann. Intern. Med. 117: 257 259.
16. Dunham, I.,, R. Durbin,, J. Thierry-Mieg,, and D. R. Bentley. 1993. Physical mapping projects and ACEDB. In M. J. Bishop (ed.), Guide to Human Genome Computing, in press. Academic Press, London.
17. Durbin, R.,, and J. Thierry-Mieg. 1991. A C. elegans database. Documentation, code, and data available from anonymous FTP servers at lirmm.lirmm.fr, cele.mrc-lmb.cam.ac.uk, and hcbi.nlm.nih.gov.
18. Ebole, D. J.,, and H. Zalkin. 1987. Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis. J. Biol. Chem. 262: 8274 8287.
19. Eiglmeier, K.,, N. Honore,, S. A. Woods,, B. Caudron,, and S. T. Cole. 1993. Use of an ordered cosmid library to deduce the genomic organisation of Mycobacterium leprae. Mol. Microbiol. 7: 197 206.
20. Eiglmeier, K.,, L. Pascopella,, N. Honore,, W. R. Jacobs Jr.,, and S. T. Cole. An ordered collection of shuttle cosmids representing the chromosome of Mycobacterium leprae: some practical applications. Unpublished data.
21. Endicott, J. A.,, F. Sarangi,, and V. Ling. 1992. Complete DNA sequences encoding the Chinese hamster P-glycoprotein gene family. DNA Sequence 2: 89 101.
22. Gill, D. R.,, G. F. Hatfull,, and G. P. C. Salmond. 1986. A new cell division operon in Escherichia coli. Mol. Gen. Genet. 205: 134 145.
23. Haydock, S. F.,, J. A. Dowson,, N. Dhillon,, G. A. Roberts,, J. Cortes,, and P. F. Leadlay. 1991. Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases. Mol. Gen. Genet. 230: 120 128.
24. Hector, J. S. R.,, Y. Pang,, G. H. Mazurek,, Y. Zhang,, A. A. Brown,, and R. J. Wallace Jr.. 1992. Large restriction fragment patterns of genomic Mycobacterium fortuitum DNA as strain-specific markers and their use in epidemiologic investigation of four nosocomial outbreaks. J. Clin. Microbiol. 30: 1250 1255.
25. Hermans, P. W. M.,, D. van Soolingen,, E. M. Bik,, P. E. W. de Haas,, J. W. Dale,, and J. D. A. van Embden. 1991. Insertion element IS 987 from Mycobacterium bovis BCG is located in a hot-spot region for insertion elements in Mycobacterium tuberculosis complex strains. Infect. Immun. 59: 2695 2705.
26. Honore 1, N.,, S. Chanteau,, F. Doucet-Populaire,, K. Eiglmeier,, T. Garnier,, C. Georges,, P. Launois,, P. Limpaiboon,, S. Newton,, K. Nyang,, P. del Portillo,, G. K. Ramesh,, T. Reddy,, J. P. Riedel,, N. Sittisombut,, S. Wu-Hunter,, and S. T. Cole. 1993. Nucleotide sequence of the first cosmid from the Mycobacterium leprae genome project: structure and function of the Rif-Str regions. Mol. Microbiol. 7: 207 214.
27. Ikeda, M.,, M. Wachi,, H. K. Jung,, F. Ishino,, and M. Matsuhashi. 1990. Nucleotide sequence involving murG and murC in the mra gene cluster region of Escherichia coli. Nucleic Acids Res. 18: 4014.
28. Jacobs, W. R. Jr.. 1993. Personal communication.
29. Jacobs, W. R. Jr.,, G. V. Kalpana,, J. D. Cirillo,, L. Pascopella,, S. B. Snapper,, R. A. Udani,, W. Jones,, R. G. Barletta,, and B. R. Bloom. 1991. Genetic systems for mycobacteria. Methods Enzymol. 204: 537 555.
30. Kieser, H.,, T. Kieser,, and D. Hopwood. 1992. A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome. J. Bacterial. 174: 5496 5507.
31. Kohara, Y.,, K. Akiyama,, and K. Isono. 1987. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50: 495 508.
32. Krawiec, S.,, and M. Riley. 1990. Organization of the bacterial chromosome. Microbiol. Rev. 54: 502 539.
33. Kwak, J. H.,, E. C. Choi,, and B. Weisblum. 1991. Transcriptional attenuation control of ermK, a ma-crolide-lincosamide-streptogramin B resistance determinant from Bacillus licheniformis. J. Bacteriol. 173: 4725 735.
34. Leblond, P.,, M. Redenbach,, and J. Cullum. 1993. Physical map of the Streptomyces lividans 66 chromosome and comparison with that of the related strain Streptomyces coelicolor A3(2). J. Bacteriol. 175: 3422 3429.
35. Levy-Frebault, V. V.,, M. F. Thorel,, A. Varnerot,, and B. Gicquel. 1989. DNA polymorphism in Mycobacterium paratuberculosis, "wood pigeon mycobacteria," and related mycobacteria analyzed by field inversion gel electrophoresis. J. Clin. Microbiol. 27: 2823 2826.
36. Lim, F.,, C. P. Morris,, F. Occhiodoro,, and J. C. Wallace. 1988. Sequence and domain structure of yeast pyruvate carboxylase. J. Biol. Chem. 263: 11493 11497.
37. Mathur, M.,, and P. E. Kolattukudy. 1992. Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongating multifunctional enzyme from Mycobacterium tuberculosis var. bovis bacillus Calmette-Guerin. J. Biol. Chem. 267: 19388 19395.
38. McAdam, R. A.,, P. W. M. Hermans,, D. van Soolingen,, Z. F. Zainuddin,, D. Catty,, J. D. A. van Embden,, and J. W. Dale. 1990. Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family. Mol. Microbiol. 4: 1607 1613.
39. Mintz, L.,, G. Gryan,, and G. M. Church. 1993. Unpublished data.
40. Philipp, W.,, S. Poulet,, L. Pascopella,, K. Eiglmeier,, B. Heym,, W. R. Jacobs Jr.,, and S. T. Cole. 1993. Genome organisation of Mycobacterium tuberculosis. Unpublished data.
41. Prober, J. M.,, G. L. Trainor,, R. J. Dam,, F. W. Hobbs,, C. W. Robertson,, R. J. Zagursky,, A. J. Cocuzza,, M. A. Jensen,, and K. Baumeister. 1987. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238: 336 341.
42. Ranes, M. G.,, J. Rauzier,, M. Lagranderie,, M. Gheorgiu,, and B. Gicquel. 1990. Functional analysis of pAL5000, a plasmid from Mycobacterium fortui-tum: construction of a "mini" mycobacterium-fis- cherichia coli shuttle vector. J. Bacteriol. 172: 2793 2797.
43. Rauzier, J.,, J. Moniz-Pereira,, and B. Gicquel-Sanzey. 1988. Complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum. Gene 71: 315 321.
44. Richterich, P.,, and G. M. Church. 1993. DNA sequencing by direct transfer electrophoresis and nonradioactive detection. Methods Enzymol. 218: 187 222.
45. Robison, K.,, and G. M. Church. 1993. Unpublished data.
46. Roncero, C.,, and M. J. Casadaban. 1992. Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus. J. Bacteriol. 174: 3250 3260.
47. Saiki, R. K.,, D. H. Gelfand,, S. Stoffel,, S. J. Scharf,, R. Higuchi,, G. T. Horn,, K. B. Mullis,, and H. A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487 491.
48. Schwartz, D. C.,, and C. R. Cantor. 1984. Separation of yeast chromosome-sized DNAs by pulsed-field gel electrophoresis. Cell 37: 67 75.
49. Shine, J.,, and L. Dalgarno. 1975. Terminal-sequence analysis of bacterial ribosomal RNA: correlation between the 3'-terminal-polypyrimidine sequence of 16S RNA and translational specificity of the ribosome. Eur. J. Biochem. 57: 221 230.
50. Smith, C. L.,, P. E. Warburton,, A. Gaal,, and C. R. Cantor,. 1986a. Analysis of genome organization and rearrangements by pulsed field gradient gel electrophoresis, p. 45 70. In J. K. Setlow, and A. Hollaender (ed.), Genetic Engineering. Plenum Publishing Corp., New York.
51. Smith, D. R.,, T. Avruch,, C. Butler,, N. Capparell,, S. Drill,, K. Falls,, H. Lee,, M. Rubenfield,, A. Smyth,, D. Torrey,, Q. Xu,, and J. Mao. 1993. Unpublished data.
52. Smith, L. M.,, J. Z. Sanders,, R. J. Kaiser,, P. Hughes,, C. Dodd,, C. R. Connell,, C. Heiner,, S. B. H. Kent,, and L. E. Hood. 1986b. Fluorescence detection in automated DNA sequence analysis. Nature (London) 321: 674 679.
53. Snapper, S. B.,, L. Lugosi,, A. Jekkel,, R. E. Melton,, T. Kieser,, B. R. Bloom,, and W. R. Jacobs. 1988. Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc. Natl. Acad. Sci. USA 85: 6987 6991.
54. Snapper, S. B.,, R. E. Melton,, S. Mustafa,, T. Kieser,, and W. R. Jacobs. 1990. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4: 1911 1919.
55. Snider, D. E. Jr.,, and W. L. Roper. 1992. The new tuberculosis. N. Engl. J. Med. 326: 703 705.
56. Sulston, J.,, F. Mallett,, R. Staden,, R. Durbin,, T. Horsnell,, and A. Coulson. 1988. Software for genome mapping by fingerprinting techniques. Comput. Appl. Biosci. 4: 125 132.
57. Thierry, D.,, A. Brisson-Noël,, V. Vincent-Lévy-Frébault,, S. Nguyen,, J. Guesdon,, and B. Gicquel. 1990a. Characterization of a Mycobacterium tuberculosis insertion sequence, ÏS6110, and its application in diagnosis. J. Clin. Microbiol. 28: 2668 2673.
58. Thierry, D.,, M. D. Cave,, K. D. Eisenach,, J. T. Crawford,, J. H. Bates,, B. Gicquel,, and J. L. Guesdon. 1990b. IS6/70, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res. 18: 188.
59. Varnerot, A.,, F. Clément,, M. Gheorghiu,, and V. Vincent-Lévy-Frébault. 1992. Pulsed field gel electrophoresis of representatives of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains. FEMS Microbiol. Lett. 98: 155 160.
60. Winter, N.,, M. Lagranderie,, J. Rauzier,, J. Timm,, C. Leclerc,, B. Guy,, M. P. Kieny,, M. Gheorghiu,, and B. Gicquel. 1991. Expression of heterologous genes in Mycobacterium bovis BCG: induction of a cellular response against HIV-1 Nef protein. Gene 109: 47 54.
61. Young, D.,, R. Lathigra,, R. Hendrix,, D. Sweetser,, and R. A. Young. 1988. Stress proteins are immune targets in leprosy and tuberculosis. Proc. Natl. Acad. Sci. USA 85: 4267 4270.
62. Young, D. B.,, and S. T. Cole. 1993. Leprosy, tuberculosis, and the new genetics. J. Bacteriol. 175: 1 6.
63. Young, R. A.,, B. R. Bloom,, C. Grosskinsky,, J. Ivanyi,, D. Thomas,, and R. W. Davis. 1985a. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proc. Natl. Acad. Sci. USA 82: 2583 2587.
64. Young, R. A.,, V. Mehra,, D. Sweetser,, T. Buchanan,, J. Clark-Curtiss,, R. W. Davis,, and B. R. Bloom. 1985b. Genes for the major protein antigens of the leprosy parasite Mycobacterium leprae. Nature (London) 316: 450 452.


Generic image for table
Table 1

Identities and sources of genetic markers mapped in

Citation: Cole S, Smith D. 1994. Toward Mapping and Sequencing the Genome of , p 227-238. In Bloom B (ed), Tuberculosis. ASM Press, Washington, DC. doi: 10.1128/9781555818357.ch16

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error