Mutational analysis of the bacteriophage phi X174 replication origin.
Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.
Baas
P D
PD
Institute of Molecular Biology and Medical Biotechnology, State University of Utrecht, The Netherlands.
D010584:Bacteriophage phi X 174 / Q000235:genetics* / Q000254:growth & development; D004261:DNA Replication; D004251:DNA Transposable Elements; D004278:DNA, Superhelical; D004279:DNA, Viral; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D014779:Virus Replication
D004251:DNA Transposable Elements; D004278:DNA, Superhelical; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides