| Research Abstract |
We constructed Mycobacterium-Escherichia coli shuttle vector.
pYT923, pMT923 can replicate in both of slow-grower M.bovis BCG and rapid-grower M.smegmatis J15cs. pYT923 carries kanamycin-resistance, and pMT923 carries chloramphenicol-resistance. We also constructed pYT937 and pMT933, which can replicate in only slow-grower M.bovis BCG.pYT937 carries kanamycin-resistance, and pMT933 carries chloramphenicol-resistance. pMT933 can coexist stably during 30 generations with a vector derived from pAL5000 in M.bovis BCG.This phenomenon suggests the different control mechanism of DNA replication of pYT937 and the vector derived from pAL5000, and the compatibility of these plasmids. This character is useful for genetic analysis. We analyzed the replication region in mycobacteria of these shuttle vectors. A1.6kb fragment which contains a repeat sequence rich region, and an open reading frame coding 29kd protein, need for replication in BCG.Moreover the relationship between the repeat sequence rich region, which is a putative ori region, and the open reading frame, which codes a rep protein, was important for replication in BCG.For replication in M.smegmatis, an upstream region in addition the 1.6kb fragment needed. The upstream region contained an open reading frame coding ca.29kd protein. The function of this protein is not known yet. As a probe this region, we tested a Southern hybridization experiment with chromosomes of M.scrofulaceum, M.kansasii, M.bovis BCG, M.tuberculosis, M.smegmatis, M.avium, M.intracellulare, M.phlei, M.gordonae, M.fortuitum. In those strains, only chromosomes of M.scrofulaceum indicated strong positive reaction. Chromosomes of M.kansasii indicated only weak reaction. The replication region of pMSC262 plasmid indicates high specificity for M.scrofulacueum. Using this vector, we tried the expression of HBs antigen ligated downstream kanamycin-resistant promoter in BCG.However, HBs antigen did not express in BCG.
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