Molecular genetic analysis of vaccinia virus genes which confer resistance to alpha-amanitin Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/ms35td102

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  • Several laboratories have shown that the VV replication cycle is dependent on the host cell nucleus. The basic biological observation is that if the host cell nucleus is physically removed by cytochalasin B-mediated enucleation, functionally inactivated by UV-irradiation, or selectively blocked with a-amanitin, the results are the same: W is able to initiate infection and carry out macromolecular syntheses, but no infectious progeny virions are assembled. To elucidate the molecular mechanism of this virus-host cell interaction, one approach is to identify the viral gene(s) responsible for interaction with the host nucleus and subject them to a detailed molecular analyses. The genomic location of the gene(s) which provide an α-amanitin resistant phenotype to viral mutants have been mapped to Hindlll N/M region of the genome by the use of marker rescue techniques [E.C. Villarreal and D.E. Hruby (1986) Journal of Virology 57: 65-70] . In an attempt to understand the genetic organization of this part of the genome, the entire Hindlll N and part of the neighboring Hindlll M DNA fragments have been sequenced. The sequencing data revealed two complete leftward reading open reading frames (ORFs, N2 and Ml). In order to determine which of the two ORFs is responsible for confering resistance to a-amanitin, marker rescue analyses on a VV dual mutant (arts7) that has both temperature sensitive and resistant to α-amanitin was carried out. This showed that N2 ORF is the gene responsible for both phenotypes. To analyze regulatory sequences responsible for expression of the N2 and Ml genes, putative cis-acting regulatory sequences upstream from the N2 and Ml genes were isolated and abutted it to a bacterial reporter gene [chloramphenicol acetyl transferase (CAT)]. To assay for regulatory sequences, these promoter:CAT plasmids were initially used in transient expression protocols and subsequently these plasmids were used to make recombinant viruses.
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