We describe a simple technique for isolating deletion mutants of phage λ and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of λ head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of λ wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type λ), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest λ chromosome packageable in a plaque-forming phage particle is 72-73% that of λ wild-type.
All Science Journal Classification (ASJC) codes
- Dand polA
- bacteriophage P1
- cloned foreign genes
- recombinant DNA