Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAM beta 1.
The complete nucleotide (nt) sequence of a 5.1-kb EcoRI DNA restriction fragment carrying the replication region of the Streptococcus faecalis plasmid pAM beta 1 has been determined. Of the seven major open reading frames (ORF A-G) identified within this fragment, two (C and E) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that synthesis of the polypeptide (Mr 57,380) encoded by the largest ORF (E) was essential for replication. Deletion analysis indicated that the minimum unit of DNA required for replication resided on a 2.59-kb AccI-HpaI subfragment. ORF C resided outside of this fragment and encompassed an extensive region of directly repeated nt sequence. The encoded polypeptide (Mr 30,471) was therefore composed of large tracts of reiterated amino acid sequence (11 x VDP and 35 x TEP tripeptides) which probably caused the observed anomalous electrophoretic mobility of the synthesised protein (equivalent to 61 kDa). Deletion of a 416-bp segment of DNA between unique KpnI and StyI sites caused an increase in copy number, which correlated with the in vitro production of higher levels of ORF E polypeptide. Although homology was detected between the sequenced DNA, and the replicon of a closely related streptococcal plasmid (pSM19035), none was evident to any other characterised Gram+ plasmid.
Swinfield
T J
TJ
Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Salisbury, Wilts., U.K.
Oultram
J D
JD
Thompson
D E
DE
Brehm
J K
JK
Minton
N P
NP
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004261:DNA Replication; D015246:Deoxyribonuclease EcoRI; D013293:Enterococcus faecalis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D015246:Deoxyribonuclease EcoRI