| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1992: ¥600,000 (Direct Cost: ¥600,000)
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| Research Abstract |
Development of techniques for molecular biology of mycobacteria has long been considered to be difficult. During the experiments aimed to develop and establish molecular cloning systems in mycobacteria, the following results could be obtained :
1. A recombinant shuttle vector, pUT21 (19.2kb), capable of replicating in both E.coli and mycobacteria (M.bovis BCG and M.smegmatis) was constructed using the plasmids pACYC177 and pMF129 of E.coli and M.fortuitum, respectively. The recombinant plasmid could be introduced more frequently than a previously developed shuttle vector, pYT937, which has been constructed with a M.scrofulaceum plasmid (pMSC262) and pACYC177.
2. The efficiently transformable variants of M.smegmatis were isolated by subculturing the transformed cells 20 times followed by a curing treatment of the plasmid with ethidium bromide at 45ﾟC.
3. The genomic libraries of some species of mycobacteria were prepared using a E.coli cloning vector, pHSG298, and then introduced into E.coli recipient cells (K12 C600) by electroporation. However, no any transformants expressing the mycobacterial genes could be obtained, suggesting that the promotors of the mycobacterial structural genes would not be recognized in E.coli cells.
4. As a new phenotype of rapidly growing mycobacteria, extracellular hemolytic activity was demonstrated in culture supernatants of M.fortuitum, M.chelonae, and M.smegmatis. This new character seemed to be inadequate for cloning the gene since the active substance is not likely to be an enzyme or other type of protein.
Pathogenic role of the hemolytic activity has been remained to be studied.
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