Further Studies on Bacteriophage T4 DNA Synthesis in Sucrose-Plasmolyzed Cells

Author:

Stafford Mary E.1,Reddy G. Prem Veer1,Mathews Christopher K.1

Affiliation:

1. Department of Biochemistry, College of Medicine, University of Arizona, Tucson, Arizona 85724

Abstract

This paper describes several technical improvements in the sucrose-plasmolyzed cell system used in earlier experiments on DNA synthesis in situ with Escherichia coli infected by DNA-defective mutants of bacteriophage T4 (W. L. Collinsworth and C. K. Mathews, J. Virol. 13 :908-915, 1974). Using this system, which is based primarily on that of M. G. Wovcha et al. (Proc. Natl. Acad. Sci. U.S.A. 70 :2196-2200, 1973), we reinvestigated the properties of mutants bearing lesions in genes 1, 41, and 62, and we resolved some disagreements with data reported from that laboratory. We also asked whether the DNA-delay phenotype of T4 mutants is related to possible early leakage of DNA precursors from infected cells. Such cells display defective DNA synthesis in situ, even when ample DNA precursors are made available. Thus, the lesions associated with these mutations seem to manifest themselves at the level of macromolecular metabolism. Similarly, we examined an E. coli mutant defective in its ability to support T4 production, apparently because of a lesion affecting DNA synthesis (L. Simon et al., Nature [London] 252 :451-455). In the plasmolyzed cell system, reduced nucleotide incorporation is seen, indicating also that the genetic defect does not involve DNA precursor synthesis. The plasmolyzed cell system incorporates deoxynucleotide 5′-monophosphates into DNA severalfold more rapidly than the corresponding 5′-triphosphates. This is consistent with the idea that DNA precursor-synthesizing enzymes are functionally organized to shuttle substrates to their sites of utilization.

Publisher

American Society for Microbiology

Subject

Virology,Insect Science,Immunology,Microbiology

Reference22 articles.

1. Biochemistry of DNA-defective amber mutants of bacteriophage T4. IV. DNA synthesis in plasmolyzed cells;Collinsworth W. L.;J. Virol.,1974

2. Bacteriophage T4-directed DNA synthesis in toluene-treated cells;Dicou L.;J. Virol.,1973

3. The enzymology of virus-infected bacteria. X. A biochemicalgenetic study of the deoxynucleotide kinase induced by wild type and amber mutants ofphage T4;Duckworth D. H.;J. Biol. Chem.,1967

4. Membrane-associated proteins of T4-infected Escherichia coli;Huang W. M.;Virology,1975

5. Replication of T4 DNA in vitro. II. Assay system for and some properties of gene products required for T4 DNA replication;Imae Y.;J. Virol.,1976

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