Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli
- 1 February 1983
- journal article
- research article
- Published by Elsevier BV in Journal of Molecular Biology
- Vol. 164 (2), 193-211
- https://doi.org/10.1016/0022-2836(83)90075-x
Abstract
The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184. The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid. In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene: the other orientation results in more than 60-fold overproduction of this protein. Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage λ+, are reversed by increased production of ssb-1 mutant protein. These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes. Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene. This observation has implications in the analysis of uvrA− mutant strains and will provide a means of transferring ssb− mutations from plasmids to the chromosome. On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes.This publication has 54 references indexed in Scilit:
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