Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae

J. E. Clark-Curtiss, W. R. Jacobs, M. A. Docherty, L. R. Ritchie

Research output: Contribution to journalArticle

96 Citations (Scopus)

Abstract

Molecular analysis of DNA from Mycobacterium leprae, 'Mycobacterium lufu,' and Mycobacterium vaccae has demonstrated that the G+C (guanine plus cytosine) contents of the DNAs are 56, 61, and 65%, respectively, and that the genome sizes are 2.2 x 109, and 3.1 x 109, and 3.1 x 109 daltons, respectively. Because of the significant differences in both G+C content and genome size among M. leprae, 'M. lufu,' and M. vaccae DNAs, these species are not related, although hybridization experiments under nonstringent conditions, with two separate cloned M. leprae DNA inserts as probes, indicate that there are some conserved sequences among the DNAs. The G+C content of Dasypus novemcinctus (armadillo, the animal of choice for cultivating M. leprae) DNA was determined to be 36%. Genomic libraries potentially representing more than 99.99% of each genome were prepared by cloning into the cosmid vector. pHC79, in Escherichia coli K-12. A genomic library representing approximately 95% of the genome of M vaccae was prepared in pBR322. M. leprae DNA was subcloned from the pHC79::M leprae library into an expression vector, pYA626. This vector is a 3.8-kilobase derivative of pBR322 in which the promoter region of the asd (aspartate semialdehyde dehydrogenase) gene from Streptococcus mutans has been inserted in place of the EcoRI-to-PstI fragment of pBR322. Several (44% of those tested) pYA626::M, leprae recombinants and one pBR322::M. vaccae recombinant synthesized new polypeptides in minicells of E. coli, indicating that mycobacterial DNA can be expressed in E. coli K-12, although expression is probably dependent upon use of nonmycobacterial promotors recognized by the E. coli transcription-translation apparatus.

Original languageEnglish (US)
Pages (from-to)1093-1102
Number of pages10
JournalJournal of Bacteriology
Volume161
Issue number3
StatePublished - 1985
Externally publishedYes

Fingerprint

Mycobacterium leprae
Genomic Library
DNA
Base Composition
Escherichia coli
Genome Size
Mycobacterium
Aspartate-Semialdehyde Dehydrogenase
Genome
Armadillos
Cosmids
Streptococcus mutans
Conserved Sequence
Genetic Promoter Regions
Libraries
Organism Cloning
Peptides

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Clark-Curtiss, J. E., Jacobs, W. R., Docherty, M. A., & Ritchie, L. R. (1985). Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. Journal of Bacteriology, 161(3), 1093-1102.

Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. / Clark-Curtiss, J. E.; Jacobs, W. R.; Docherty, M. A.; Ritchie, L. R.

In: Journal of Bacteriology, Vol. 161, No. 3, 1985, p. 1093-1102.

Research output: Contribution to journalArticle

Clark-Curtiss, JE, Jacobs, WR, Docherty, MA & Ritchie, LR 1985, 'Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae', Journal of Bacteriology, vol. 161, no. 3, pp. 1093-1102.
Clark-Curtiss JE, Jacobs WR, Docherty MA, Ritchie LR. Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. Journal of Bacteriology. 1985;161(3):1093-1102.
Clark-Curtiss, J. E. ; Jacobs, W. R. ; Docherty, M. A. ; Ritchie, L. R. / Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. In: Journal of Bacteriology. 1985 ; Vol. 161, No. 3. pp. 1093-1102.
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abstract = "Molecular analysis of DNA from Mycobacterium leprae, 'Mycobacterium lufu,' and Mycobacterium vaccae has demonstrated that the G+C (guanine plus cytosine) contents of the DNAs are 56, 61, and 65{\%}, respectively, and that the genome sizes are 2.2 x 109, and 3.1 x 109, and 3.1 x 109 daltons, respectively. Because of the significant differences in both G+C content and genome size among M. leprae, 'M. lufu,' and M. vaccae DNAs, these species are not related, although hybridization experiments under nonstringent conditions, with two separate cloned M. leprae DNA inserts as probes, indicate that there are some conserved sequences among the DNAs. The G+C content of Dasypus novemcinctus (armadillo, the animal of choice for cultivating M. leprae) DNA was determined to be 36{\%}. Genomic libraries potentially representing more than 99.99{\%} of each genome were prepared by cloning into the cosmid vector. pHC79, in Escherichia coli K-12. A genomic library representing approximately 95{\%} of the genome of M vaccae was prepared in pBR322. M. leprae DNA was subcloned from the pHC79::M leprae library into an expression vector, pYA626. This vector is a 3.8-kilobase derivative of pBR322 in which the promoter region of the asd (aspartate semialdehyde dehydrogenase) gene from Streptococcus mutans has been inserted in place of the EcoRI-to-PstI fragment of pBR322. Several (44{\%} of those tested) pYA626::M, leprae recombinants and one pBR322::M. vaccae recombinant synthesized new polypeptides in minicells of E. coli, indicating that mycobacterial DNA can be expressed in E. coli K-12, although expression is probably dependent upon use of nonmycobacterial promotors recognized by the E. coli transcription-translation apparatus.",
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