Restriction Mapping the Genome of Halobacterium halobium Strain NRC-1

To study DNA rearrangements in H. halobium, the complete genome of strain NRC-1 is being restriction mapped. As a first step, we have used pulsed-field gel electrophoresis and enzymes that cut infrequently. Pmel cuts the genome into six fragments. Two of these fragments correspond to the plasmid pNRC100 characterized previously. NRC-1 also has a 400 Kbp plasmid, pNRC200, which is closely related to pNRC100 but is uncut by PmeI. This leaves four PmeI fragments of total size 1,800 Kbp which constitute the chromosome. The chromosome is being mapped by three enzymes which cut infrequently, AflII, AseI and DraI. Southern blotting with junction clones and purified restriction fragments was of limited value because the ubiquitous ISH elements resulted in hybridization to many fragments not necessarily related. Blotting with anonymous unique probes was more helpful. But most information has been obtained from 2D gel electrophoresis using pairs of the enzymes. On the basis of these data, four contigs have been assembled which represent most of the chromosome. Their relationship is being determined.