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A system of transposon mutagenesis for bacteriophage T4
Author(s) -
Woodworth Denise L.,
Kreuzer Kenneth N.
Publication year - 1992
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1992.tb00850.x
Subject(s) - transposable element , transposon mutagenesis , biology , sleeping beauty transposon system , tn3 transposon , transposition (logic) , genetics , insertional mutagenesis , bacteriophage mu , insert (composites) , transposase , p element , plasmid , bacteriophage , mutagenesis , insertion , genome , gene , mutant , escherichia coli , mechanical engineering , linguistics , philosophy , engineering
Summary We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24 , permitting a direct selection for transposition events into a gene 24‐deteted phage. The transposition occurred at a frequency of only 10 ‐7 per progeny phage, even though a dam ‐ host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon‐specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.