Vaccinia virus has a genome of 185,000 bp that encodes approximately 200 polypeptides. These genes are expressed in a coordinated fashion so that some polypeptides are made before and others after DNA replication. One vaccinia virus gene that is expressed throughout the growth cycle was found to have two RNA start sites about 55 bp apart. The site nearest to the coding segment was used at early times in infection and the other was used at late times. In vitro mutagenesis studies revealed that there are two independent promoters and that the regulatory signals are located within 31 bp of each RNA start site. RNA polymerase subunits, synthesized in reticulocyte lysates programmed with early vaccinia virus mRNA, were immunoprecipitated by antibody prepared against the purified enzyme. The subunit genes were mapped by hybridization selection of mRNA to cloned DNA fragments prior to translation. The genes for 2 subunits of 147 kD and 22 kD were sequenced. The large subunit was shown to have considerable homology with the Beta' subunit of E. coli RNA polymerase. A soluble extract capable of selectively transcribing added early vaccinia virus genes was prepared by disrupting purified vaccinia virus particles. Correct initiation, termination and polyadenylation were demonstrated and the signals for each were defined by transcription of truncated templates. Termination was shown to occur about 50 bp downstream of the signal.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000307-04
Application #
4688456
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code
De Silva, Frank S; Paran, Nir; Moss, Bernard (2009) Products and substrate/template usage of vaccinia virus DNA primase. Virology 383:136-41
Hebben, Matthias; Brants, Jan; Birck, Catherine et al. (2007) High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara. Protein Expr Purif 56:269-78
Parrish, Susan; Resch, Wolfgang; Moss, Bernard (2007) Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression. Proc Natl Acad Sci U S A 104:2139-44
Katsafanas, George C; Moss, Bernard (2007) Colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions. Cell Host Microbe 2:221-8
Parrish, Susan; Moss, Bernard (2007) Characterization of a second vaccinia virus mRNA-decapping enzyme conserved in poxviruses. J Virol 81:12973-8
Garcia, Alonzo D; Otero, Joel; Lebowitz, Jacob et al. (2006) Quaternary structure and cleavage specificity of a poxvirus holliday junction resolvase. J Biol Chem 281:11618-26
Parrish, Susan; Moss, Bernard (2006) Characterization of a vaccinia virus mutant with a deletion of the D10R gene encoding a putative negative regulator of gene expression. J Virol 80:553-61
Domi, Arban; Moss, Bernard (2005) Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage lambda-based recombination. Nat Methods 2:95-7
De Silva, Frank S; Moss, Bernard (2005) Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors. Virol J 2:23
Katsafanas, George C; Moss, Bernard (2004) Vaccinia virus intermediate stage transcription is complemented by Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and cytoplasmic activation/proliferation-associated protein (p137) individually or as a heterodimer. J Biol Chem 279:52210-7

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