Cloning of genes for bacteriophage T5 stable RNAs

Abstract

One EcoRI-generated fragment (440 basepairs) and two EcoRI/ HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo 32P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI ∗440 fragment contains the gene for tRNA 10 (tRNA Asp), the EcoRI/ HindIII ∗220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA His), and the EcoRI/ HindIII ∗960 fragment contains only a part of the gene for tRNA 9 (tRNA Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA Gln-tRNA His-RNA III-RNA I-…-tRNA Asp.