Determination of genome size and a preliminary physical map an extreme alkaliphile Micrococcus sp. Y-1 by pulsed-field gel electrophoresis

Large restriction fragments of genomic DNA from Micrococcus sp. Y-1 were separated by pulsed-field gel electrophoresis (PFGE). Since Micrococcus sp. Y-1 has a G+C content of approximately 70%, restriction fragments were obtained by digesting chromosomal DNA with endonucleases which recognize A+T-rich sequences. Five enzymes, SspI, SpeI, XbaI, HpaI and EcoRI, were used for generation of distinctly separated fragments in the size range 100-500 kb. No site for DraI was detected. In contrast, sites for 8-base-recognizing enzymes, but not for NotI and SfiI, were frequent. The genome size of Micrococcus sp. Y-1 was determined from restriction fragments separated by PFGE, and was estimated to be approximately 4061 kb. Partial digestion experiments revealed the order of the six SspI fragments on the chromosome.ope