A system of transposon mutagenesis for bacteriophage T4.
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-deleted 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.
Woodworth
D L
DL
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Kreuzer
K N
KN
D001483:Base Sequence; D004251:DNA Transposable Elements; D004926:Escherichia coli; D015723:Gene Library; D005814:Genes, Viral; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D012641:Selection, Genetic; D013604:T-Phages / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics
D004251:DNA Transposable Elements; D015678:Viral Structural Proteins