Bacteriophage T7 is a relatively simple virus that has been well characterized physiologically and genetically. The nucleotide sequence of the T7 DNA molecule, 39,926 base pairs, is now completely known. The nucleotide sequence predicts 50 genes and mutations affecting all but six of them have been found. About 99% of the molecule has been cloned as specific, well defined fragments, which are being used for genetic and biochemical characterization of the virus. Thus, the T7 system is an excellent one in which to study the molecular details of DNA replication and related processes.
Our aim i s to understand all aspects of DNA metabolism during T7 infection, including entry of the DNA into the bacterial cell, protection from host nucleases, selective degradation of host DNA, replication and repair of T7 DNA, genetic recomination, and maturation and packaging into virions. Almost all of the proteins involved in these processes appear to be specified by T7 itself. We study the normal processes, and the changes induced in them by specific mutations, using techniques including radioactive labeling, sedimentation, gel electrophoresis, restriction analysis, filter hybridization and cloning. The interaction of infecting phages with cloned fragments of T7 DNA resident in the host is proving very informative. Examples of projects currently underway include the following: We have found that the mode of entry of T7 DNA into the cell plays an important part in overcoming host restriction, and we are analyzing how the entry process might be controlled and whether other aspects of DNA metabolism are coupled to DNA entry. We find that cloned origins of replication of T7 DNA serve as origins of replication in plasmids during T7 infection, and we are using this as an assay to define what elements of the nucleotide sequence are needed for initiation of replication. We have isolated conditional-defective mutants of the T7 single-stranded DNA-binding protein and are using them to analyze the role of this protein in DNA metabolism. We are exploring the possibility of using cloned fragments of T7 DNA to produce large amounts of specific T7 replication proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021872-12
Application #
3270753
Study Section
(MG)
Project Start
1978-02-01
Project End
1988-01-31
Budget Start
1986-02-01
Budget End
1987-01-31
Support Year
12
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Associated University-Brookhaven National Lab
Department
Type
DUNS #
City
Upton
State
NY
Country
United States
Zip Code
11973
Zhang, Xing; Studier, F William (2004) Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection. J Mol Biol 340:707-30
Cerritelli, M E; Studier, F W (1996) Purification and characterization of T7 head-tail connectors expressed from the cloned gene. J Mol Biol 258:299-307
Cerritelli, M E; Studier, F W (1996) Assembly of T7 capsids from independently expressed and purified head protein and scaffolding protein. J Mol Biol 258:286-98
Zhang, X; Studier, F W (1995) Isolation of transcriptionally active mutants of T7 RNA polymerase that do not support phage growth. J Mol Biol 250:156-68
Cheng, X; Zhang, X; Pflugrath, J W et al. (1994) The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. Proc Natl Acad Sci U S A 91:4034-8
Rosenberg, A H; Patel, S S; Johnson, K A et al. (1992) Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase. J Biol Chem 267:15005-12
Patel, S S; Rosenberg, A H; Studier, F W et al. (1992) Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation. J Biol Chem 267:15013-21
Dubendorff, J W; Studier, F W (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219:45-59
Dubendorff, J W; Studier, F W (1991) Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter. J Mol Biol 219:61-8
Studier, F W; Rosenberg, A H; Dunn, J J et al. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60-89

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