ASYMMETRICAL PROGRESSION OF REPLICATION FORKS JUST AFTER INITIATION ON MYCOPLASMA-CAPRICOLUM CHROMOSOME REVEALED BY 2-DIMENSIONAL GEL-ELECTROPHORESIS

Previously, we mapped the replication initiation site of the Mycoplasm a capricolum chromosome into a region containing the dnaA gene [M. Miy ata et al., 1993a. Nucleic Acids Res. 21, 4816-4823]. In this study, v arious regions including this functional domain were analyzed by two c omplementary two-dimensional (2D) gel electrophoretic methods. Sizes o f nascent strands in a 10.7-kb and a 5.6-kb region were examined by a neutral/alkaline (N/A) method. The shortest nascent strand was detecte d in an 875-bp region composed of the 3' end of the dnaA gene and its downstream non-coding sequence. The shortest nascent strand detected b ecame longer in an asymmetrical manner as position of the probe became further from the putative initiation site in both directions. The int ermediate forms of eight regions restricted at different sites were ex amined by a neutral/neutral (N/N) method. Bubble arcs were observed in four regions including the 875-bp region. The region containing the 8 75-bp region at about its center showed an asymmetrical are, although that containing the 875-bp region at its end showed a symmetrical are. These results show that the replication forks develop in the 875-bp r egion and proceed bidirectionally in an asymmetrical manner around the initiation site. The results of N/A analysis of the 5.6-kb region sho wed a shift of intensity in the nascent strand signal, which suggests an upshift of fork progression velocity. (C) 1997 Elsevier Science B.V .