Cloning of the protease/polymerase (3CD) coding sequences into a baculovirus expression plasmid vector

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Publication Type thesis
School or College School of Medicine
Department Biochemistry
Author Sheets, Rebecca Lynn
Title Cloning of the protease/polymerase (3CD) coding sequences into a baculovirus expression plasmid vector
Date 1987-12
Description Poliovirus, the prototypic member of the picornaviruses, replicates its RNA by a mechanism involving RNA-dependent RNA synthesis. The biochemistry of this reaction in not will-understood, but a few viral proteins have been shown to be involved. These include the viral RNA polymerase (3D), the genome linked protein (3B) or its precursor, and a protein of unknown function (2C). One approach to studying this process is to try to reconstitute the RNA replication reactions in vitro from purified components. For this purpose, a system for production of large amounts of viral proteins would be advantageous. Viral proteins thought to be involved in RNA replication could thus be purified individually, analyzed biochemically, and used in the reconstitution system. This thesis describes the cloning of the coding sequences for the viral protease/polymerase (3CD) into a plasmid that may subsequently be used to create a recombinant baculovirus. Such recombinant viruses have been shown previously to direct the synthesis of large quantities of active proteins upon infection of cultured insect cells that are permissive for replication of the baculovirus. I have constructed and obtained a clone of the protease/polymerase coding sequences in the proper orientation with respect to the baculoviral promoter. This could be use to create a recombinant virus that would express the polioviral protein 3CD. This protein should have inherent protease (3C) activity, and thus cleave itself from the polymerase (3D), allowing the purification of authentic polioviral polymerase. Thus, the polymerase could be obtained free of other proteins involved in poliovirus replication and could be sue to reconstitute the viral RNA replication reaction.
Type Text
Publisher University of Utah
Subject Genetics; Chemical Synthesis; Genetic Vectors; Microbial
Subject MESH Polioviruses; Peptide Hydrolases; Cloning, Molecular; RNA, Viral; DNA-Directed RNA Polymerases
Dissertation Institution University of Utah
Dissertation Name MS
Language eng
Relation is Version of Digital reproduction of "Cloning of the protease/polymerase (3CD) coding sequences into a baculovirus expression plasmid vector". Spencer S. Eccles Health Sciences Library.
Rights Management © Rebecca Lynn Sheets.
Format Medium application/pdf
Format Extent 491,716 bytes
Identifier undthes,3835
Source Original University of Utah Spencer S. Eccles Health Sciences Library (no longer available)
Master File Extent 491,811 bytes
ARK ark:/87278/s6n29zqh
Setname ir_etd
Date Created 2012-04-24
Date Modified 2021-05-06
ID 190779
Reference URL