Minimal replication origin of the 200-kilobase Halobacterium plasmid pNRC100.

摘要: We have identified the replication origin of pNRC100, a 200-kb plasmid Halobacterium halobium, by assaying for ability miniplasmids containing cloned fragments pNRC100 and mevinolin resistance selectable marker Haloferax volcanii. First, we showed pNGHCMEV1, which contains 19-kb HindIII-C fragment recovery DNA from mevinolin-resistant transformants H. halobium. The minimal approximately 3.9 kb was defined subcloning successively smaller regions pNGHCMEV1 in either halobium or same also recovered after transformation volcanii with library partial Sau3AI pNRC100. nucleotide sequence determined found to contain long open reading frame, named repH, transcribed away highly A+T-rich region. transcription start site primer extension analysis be 17 18 nucleotides 59 putative repH codon. predicted product gene, an acidic protein molecular weight 113,442, 24 27% identity gene products pHV2 p phi HL, suggesting that each is involved replication. One minireplicon, pNG11 delta 12, analyzed linker scanning mutagenesis, requirement Restoration frame one replication-defective 12 derivative introduction second small insertion resulted reversion proficiency. pNG11delta12 lost when entire region, about 550 bp long, deleted but not insertions deletions were introduced into this presence only 52 segment sufficient permit minireplicon at high frequency cells grown without selection, partitioning locus absent discuss possible roles region Images