engleski

The structure of the chromosome ends in Streptomyces rimosus MV25

Physical mapping of the S. rimosus R6 chromosome showed it to be 8 Mb and linear. There are terminal inverted repeats of 550 kb including the terminal AseI-J fragment while the oxytetracycline (OTC) gene cluster lies some 600 kb from one end. In S. rimosus R6 there is also a linear plasmid pPZG101 of 387 kb in length with long terminal inverted repeats of about 180 kb. Mutants were found in which plasmid sequences had integrated into the chromosome but a free plasmid end was still present. In one of them (MV25) two AseI 'junction bands' could be seen. It was initially suggested that both ends of the chromosome might have been replaced by plasmid ends, but this is ruled out by the presence of at least one copy of the terminal AseI-J fragment in MV25. The relative intensity of the two AseI junction fragments varied in different DNA preparations from MV25 which suggested a mixed population. MV25 segregated white OTC-sensitive (10 ľg/ml) mutant colonies. Four mutants (MV25W1-4) were analysed using pulsed-field gel electrophoresis. All four showed an identical AseI pattern which differed from that of MV25 only in that the 690 kb junction band was missing; the 480 kb junction band was still present. A Southern blot was hybridised with cosmid pPZG25 which carries most of the OTC-cluster. The 690 kb fragment in MV25 hybridised, but the mutants did not showing deletion of the OTC cluster. Cosmids were isolated from the integration region in the parent, in MV25 and from pPZG101. This allowed precise characterisation of the integration event.

Streptomyces rimosus; linear chromosome; terminal inverted repeat

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