PubMed · 232218
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J F Morrow. 1979. Recombinant DNA techniques.. https://doi.org/10.1016/0076-6879(79)68003-5
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We have developed a strategy for efficient sequence analysis of the genome of E. coli K-12 using insertions of a Tn5-derived mini-transposon into overlapping ordered lambda phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on a dnaBamber lacZamber E. coli strain. Insertion points every 0.5-1 kb were identified by 'analytical PCR' and segments between the transposon inserts and phage arms were amplified by 'preparative PCR' using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.
Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control. Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators. This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state. The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cI in trans. These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE. Finally, we also provide genetic evidence for impaired transcription of the cI gene from the CI-activated promoter, pM in the rpoA341 background.
A plasmid encoding a recombinant human O6-methylguanine-DNA methyltransferase (MGMT) fused to a fragment of the bacteriophage lambda N protein has been constructed. The fusion protein retained methyltransferase activity when expressed at high levels in E.coli and was purified to essential homogeneity by a simple procedure. Antisera raised against the purified fusion protein recognized MGMT in western blots of extracts of human cells. For most cell lines, there was a quantitative relation between the amount of immunologically detectable MGMT protein and enzyme activity. However, four cell lines contained detectable MGMT protein despite having no measurable methyltransferase activity. Additionally, a HeLa line contained considerably more immunoreactive MGMT protein than could be accounted for by its methyltransferase activity. Thus, some cells contain significant amounts of inactive MGMT. Preliminary characterization of the inactive protein in HeLaS3 cells indicated that it has some properties in common with MGMT methylated at the active cysteine residue.