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

Abstract

Molecular analysis of DNA from M. leprae, M. lufu and M. vaccae demonstrated that the G + C (guanine plus cytosine) contents of the DNA are 56, 61 and 65%, respectively, and that the genome sizes are 2.2 .times. 109, 3.1 .times. 109 daltons, respectively. Because of the significant differences in both G + C content and genome size among M. leprae, M. lufu and M. vaccae DNA, these species are not related, although hybridization experiments under nonstringent conditions, with 2 separate cloned M. leprae DNA inserts as probes, indicate that there are some conserved sequences among the DNA. 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 > 99.99% of each genome were prepared by cloning into the cosmid vector, pHC79, in Escherichia coli K-12. A genomic library representing .apprx. 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 2 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 promoters recognized by the E. coli transcription-translation apparatus.