| Description |
An Escherichia coli mutant, ts-Jl1, was isolated following random insertional mutagenesis using phage lambda-Mu transposition. The mutant phenotype includes; inability to form colonies at 43°C and inability to propagate bacteriophage lambda at all temperatures. A lambda i434.£I-ts+Jl1 transducing phage was isolated on the basis of its ability to propagate on tS-Jll mutant bacteria. Using this transducing phage, I was able to show, through complementation and protein analyses, that the tql1 mutation is in the dnaB gene. Following southern blot analysis, I was able to localize the lambda-Mu insertion within the last 40 base pairs of the dnaB gene. The exact insertion event was identified through a new technique of DNA sequencing, which involves in vitro amplification of the DNA sequences containing the insertion junction. The insertion event in tS-Jl1 was mapped precisely between base pair number 1514 and 1515 of the dnaB gene. This result predicts that the mutant dnaB protein has lost its 6 terminal amino acids. The reading frame shifts into Mu-specific DNA sequences resulting in the addition of 20 extra amino acid residues. The E.coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage DNA replication. In tS-Jll, the carboxyl end of dnaB is altered, and the protein as a whole has lost its ability to function in host replication at elevated temperatures and lamda replication at all temperatures. My results demonstrate that the extreme carboxyl end sequence of the dnaB protein is required for productive interaction with the lambda P replication protein. |
