Bacteriophage T7 is a relatively simple, well characterized virus that infects E. coli. It contains a single molecule of linear DNA almost 40,000 base pair long whose complete nucleotide sequence is known. The nucleotide sequence predicts that there will be 56 T7 proteins, and mutations affecting almost all of them are available. Individual genes and genetic signals have been cloned and are being genetic and biochemical analysis and for manipulation of the virus. 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 T7 DNA into the cell; protection from host nucleases; selective degradation of host DNA; replication and repair of T7 DNA; genetic recombination; and maturation and packaging of the DNA into virions. These processes are studied in normal and mutant phage infections, using techniques that include radioactive labeling, sedimentation, gel electrophoresis, restriction analysis, filter hybridization, and cloning. T7 genes are expressed individually from clones, both to determine their effects on the host cell and to obtain large amounts of protein for biochemcial and structural analysis. A protein central to both RNA and DNA metabolism during T7 infection is T7 RNA polymerase, which recognizes a large promoter sequence not found in host DNA. This highly selective promoter recognition is used by T7 to direct both transcription and replication to its own DNA, and to ensure that its own DNA is packaged into phage particles. T7 RNA polymerase also forms a specific complex with T7 lysozyme, and interaction that shuts off late transcription and may have a role in replication, packaging and/or lysis. Genetic selections are being applied to the cloned gene in order to analyze further the functions and interactions of T7 RNA polymerase. Other aspects of T7 DNA metabolism currently being studied include the role of the T7 single-stranded DNA binding protein, the early steps in phage assembly, and DNA packaging.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021872-18
Application #
3270758
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-02-01
Project End
1994-01-31
Budget Start
1992-02-01
Budget End
1994-01-31
Support Year
18
Fiscal Year
1992
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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