Abstract
Previous attempts to clone the Escherichia coli polA+ gene onto a high copy number plasmid were unsuccessful. The apparent lethality of unregulated overproduction of DNA polymerase I can be eliminated by cutting at a BglII site 100 nucleotides upstream from the ATG start codon of the polA gene. This permitted the construction of plasmid pMP5 which contains both the coding sequence for DNA polymerase I and the lambda pL promoter for conditional control of polA gene expression. BglII cutting only damages but does not eliminate the polA promoter activity; the BglII site thus lies within the polA promoter region. Leakiness of the damaged polA promoter results in overproduction of DNA polymerase I even under conditions where pL is fully repressed. This overproduction is inhibitory of cell growth, as reflected in both growth rate and in the frequency of appearance of mutant plasmids which are nonproducers of DNA polymerase I. Transformation of plasmid pMP5 into E. coli N4830 yields strain ATL100 which under inducing conditions provides 138-fold amplification of DNA polymerase I. Optimization of growth and expression conditions are presented together with an optimized rapid polymerase purification scheme. In addition to providing a convenient source for preparation of DNA polymerase I, this work serves as the basis for a future detailed molecular genetic analysis of the polA gene product.
Highlights
Previous attempts to clone theEscherichia colipolA+ gene onto a high copy number plasmid were unsuccessful
The initial attemopft Kelley et al [2] yielded a recombinant X phage carrying thepolA gene which provided both asource of DNAfor polA gene sequence determination [3], and,throughinduction of the prophage, a means to increase the yield of pol I enzyme for further chemical studies [4].having the gene cloned inthephage genome complicates the purification of the amplified gene product because of the simultaneous amplification of the phage proteins andDNA
Their attempts to construct an equivalent plasmid containingpotlhAe+ gene were unsuccessful. They attributed the inabilityof the cell to maintain sucha plasmidto a detrimental effect resulting from overproduction of DNA polymerase I. Consistent with this notion, transcription from the polA promoter was not autogenously regulated [14], and greatly elevated levels of DNA polymerase I might be expected from multiplecopies of the polA gene
Summary
Previous attempts to clone theEscherichia colipolA+ gene onto a high copy number plasmid were unsuccessful. The apparent lethality of unregulated overproduction of DNA polymerase I can be eliminated by cutting at a BgZII site 100 nucleotides upstream from the ATG start codon of the polA gene This permitted the construction of plasmid pMP5 which contains both the coding sequence for DNA polymerase I and the X p~ promoter for conditional control of polA gene expression. DNApolymerase I, as codedfor by the polA gene, is a multifunctional DNA-binding protein whose chemistry and mechanism of action havebeen extensively characterized [1] This was accomplished despite the low levels at which the enzyme is expressed in wild type Escherichia coli cells. Her scientific contributions tothe study of polymerase and her vibrant personality are greatly missed by her colleagues
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
