MUTATIONS IN THE TRFA REPLICATION GENE OF THE BROAD-HOST-RANGE PLASMID RK2 RESULT IN ELEVATED PLASMID COPY NUMBERS

被引:65
作者
DURLAND, RH
TOUKDARIAN, A
FANG, F
HELSINKI, DR
机构
[1] UNIV CALIF SAN DIEGO,CTR MOLEC GENET,LA JOLLA,CA 92093
[2] UNIV CALIF SAN DIEGO,DEPT BIOL,LA JOLLA,CA 92093
关键词
D O I
10.1128/jb.172.7.3859-3867.1990
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Mutated forms of trfA, the replication protein gene of plasmid RK2, that support a minimal RK2 origin plasmid in Escherichia coli at copy numbers up to 23-fold higher than normal have been isolated. Six such high-copy-number (copy-up) mutations were mapped and sequenced. In each case, a single base transition led to an amino acid substitution in the TrfA protein primary sequence. The six mutations affected different residues of the protein and were located within a 69-base-pair region encoding 24 amino acids. Dominance tests showed that each of the mutants can be suppressed by wild-type trfA in trans, but suppression is highly dependent on the amount of wild-type protein produced. Excess mutant TrfA protein provided in trans significantly increased the copy number of RK2 and other self-replicating derivatives of RK2 that contain a wild-type trfA gene. These observations suggest that the mutations affect a regulatory activity of the TrfA replication protein that is a key factor in the control of initiation of RK2 replication.
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页码:3859 / 3867
页数:9
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