Mutational analysis of an ssi region carried by plasmid pACYC184.
To investigate the functional contribution of some structural components of the signal that directs single-stranded initiation of DNA replication (ssi signal) carried by a 119-nt segment of plasmid pACYC184 (Bahk et al., 1988), we constructed mutants carrying one-base substitutions and insertions using oligodeoxyribonucleotide (oligo) directed mutagenesis. Two one-base substitution mutants were obtained. The mutants, M13 delta lac 184/Sp and M13 delta lac 184/Ev, carried an SplI site and an EcoRV site, respectively, created by base substitution. Three kinds of synthetic oligos, that is, a 10-bp EcoRI linker, an 8-bp ScaI linker and an 8-bp SmaI linker, were inserted into the SplI site of M13 delta lac 184/Sp, and into the EcoRV site of M13 delta lac 184/Ev. The SSI activity of each mutant examined indicated that the one-base substitutions had different effects on the SSI functions of the altered ssi signals. This fact suggests that some structural components within the 119-bp region make distinct contributions to the SSI function. Moreover, when the three kinds of synthetic linkers were inserted into the mutants M13 delta lac 184/Sp and M13 delta lac 184/Ev, each of the insertion mutations affected the rate of conversion of ss DNA to RFI in vivo and the growth of the recombinant phages in a distinct manner. Judging from the above results, the base composition and the length of a certain specific site were crucial for maintenance of the SSI functional activity, and structural components of the ssi signal contributed distinctly to the SSI function.
Bahk
J D
JD
Department of Agricultural Chemistry, Kyoto University, Japan.
Sakai
H
H
Komano
T
T
D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D015183:Restriction Mapping
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides