1887

Abstract

A 9·2 kb cryptic plasmid, pMF1, was isolated from strain 110 and its restriction map constructed. A 42 kb dIII fragment of pMF1 was found to support replication in mycobacteria and this fragment was cloned and sequenced to characterize the replication elements of the plasmid. Computer analysis identified a putative Rep protein (362 amino acids) with high homology to the putative Rep protein of the plasmid pCLP and limited homology, mostly in the N-terminal region, to the Rep proteins of pLR7, pJAZ38 and pMSC262. A region containing a putative site was located upstream of the gene; this region displayed high homology at the nucleotide level with the predicted of pCLP and pJAZ38. A plasmid carrying the 42 kb dIII fragment and a kanamycin resistance marker, designated pBP4, was maintained as a single-copy plasmid in and was stably inherited in the absence of antibiotic selection. Plasmid pBP4 was incompatible with the pJAZ38 replicon but was compatible with the widely used pAL5000 replicon, indicating that among the mycobacterial vectors now available there are two incompatibility groups. Significantly, the plasmid was able to replicate in the pathogen , making it a useful tool for gene expression studies. To provide a choice of restriction sites and easy manipulation, a 21 kb fragment containing the minimal replication region was cloned to make the mycobacterial shuttle vector pBP10, which showed similar stability to pBP4.

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2000-02-01
2020-03-29
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