Nonrandom orientation of transposon Tn5supF insertions in phage lambda.
Transposition of mini-transposon Tn5supF to phage lambda can be selected in two ways: (i) by plaque formation on a dnaB amber strain of Escherichia coli, which requires expression of the transposon-borne suppressor tRNA gene (supF) during lytic phage growth, or (ii) by lysogenization of a strain with amber mutations in tet and amp resistance genes, and selection of TcR ApR (Sup+) transductant colonies. Tn5supF insertions in several lambda clones were isolated and mapped using a polymerase chain reaction (PCR) amplification method. Among insertions selected during lytic growth, more than 90% were oriented such that supF could be transcribed from an upstream lambda promoter. In contrast, half of those selected by transduction were in each orientation. These results indicate that Tn5supF insertion occurs with equal frequency in each orientation. However, Tn5supF insertion phages in which transcription from the lambda and supF promoters would collide tend to be lost when supF is selected during lytic growth. The tendency to recover Tn5supF insertions in only one orientation is useful in a transposon- and crossover-PCR-based method for preparing templates for DNA sequencing.
Kersulyte
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Department of Molecular Microbiology, Washington University Medical School, St. Louis, MO 63110.
Krishnan
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Berg
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D001426:Bacterial Proteins / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics*; D004265:DNA Helicases; D004251:DNA Transposable Elements / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D053488:DnaB Helicases; D004926:Escherichia coli / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D012367:RNA, Viral; D012343:RNA, Transfer; D004265:DNA Helicases; D053488:DnaB Helicases