Nonrandom orientation of transposon Tn 5supF insertions in phage lambda

Abstract

Transposition of mini-transposon Tn 5supF to phage λ 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 Tc RAp R (Sup +) transductant colonies. Tn 5supF insertions in several λ 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 λ promoter. In contrast, half of those selected by transduction were in each orientation. These results indicate that Tn 5supF insertion occurs with equal frequency in each orientation. However, Tn 5supF insertion phages in which transcription from the λ and supF promoters would collide tend to be lost when supF is selected during lytic growth. The tendency to recover Tn 5supF insertions in only one orientation is useful in a transposon- and crossover-PCR-based method for preparing templates for DNA sequencing.