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Bacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56, denA and denB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA with EcoRI have been cloned using the vector plasmid pCR1. Clones containing T4 DNA were identified by hybridization with radioactive early and late T4 RNA. A simple marker rescue technique is described for the genetic identification of the cloned T4 fragments. Some of the T4-hybrid plasmids which contain entire T4 genes can complement temperature sensitive and amber mutants of T4.

ABSTRACT[3HJThymidine-labeled progeny DNA was isolated after infection of Escherichia coli with two different bacteriophage T4 mutants. These strands were isolated shortly after the initiation of DNA replication and hybridized to 15 different (EcoRI) T4 restriction fragments cloned in plasmid vectors. Uniformly labeled T4 [32P]DNA extracted from phage particles was cohybridized as a normalizing reference. The results obtained lead to the conclusions that, among the loci tested, initiation occurs predominantly in the area of genes 50-5 and less prominently in the area of genes 25-29. However, our data do not support the idea of initiation in the area of genes 4043. In contrast, this area displays the least replication among the genes tested.There has been some controversy concerning the number and location of origins used in the replication of T4 DNA. In prev'ious papers from this laboratory, physical/chemical (1) and electron microscopic (2) evidence was presented showing that the initiation of T4 DNA replication can occur at multiple but specific sites. The electron microscopic study showed that the areas expand bidirectionally, with the 3' end leading the 5' end by approximately 0.25 ,um (2). Evidence was also presented showing that at later times after infection the basic mode of DNA replication remains exponential (3). This is in contrast to the linear mode of replication that would be expected if a shift to the rolling circle model of replication occurs. In addition, it was demonstrated that DNA concatemers used in the production of phages are assembled by recombination (4). In contrast to the above, genetic evidence presented by others (5, 6) suggests that replication may initiate from a single origin located between genes 42 and 43 and proceed unidirectionally clockwise along the circular DNA template. It will now be demonstrated that, for the loci tested, replication initiates predominantly in the area of genes 50-5. A secondary area of initiation occurs in the area of genes 25-29. There were no differences observed between the two phages tested, T4 BOr and T4D imm sp rII-219. Although only one experiment using.each phage will be illustrated here, the experiments were performed seven times on T4 BO1r and twice on T4D imm sp rII-219. The patterns obtained in the CsCl fractionations, as well as the results of the hybridization experiments, were nearly identical for both phages in all the experiments. MATERIALS AND METHODSEscherichia coli strain B23 and T4 mutants T4BO1r (obtained from C. Thomas) and T4D imm sp rII-219 (obtained from A.H. Doermann) were used in these experiments. The Tris/ casamino acids/glucose medium, density (BrdUrd) and isotope labeling, and CsCl gradient analysis have been described (7).When used, [3H]thymidine was added as a "package", which upon dilution into the experimental medium resulted in a solution containing 5 ,ug of thymidine per ml, 500 ,uCi of [3H]-thymidine per ml (100 mCi/mg; 1 Ci = 3.7 X 1010 becquerels); 5 jig of 5-fluorodeoxyuridine per ml, 5 ug of adenine per ml, an...