Title: Translation of potyvirus RNA in a rabbit reticulocyte lysate
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00099249/00001
 Material Information
Title: Translation of potyvirus RNA in a rabbit reticulocyte lysate
Alternate Title: Potyvirus RNA in a rabbit reticulocyte lysate
Physical Description: xi, 92 leaves : ill. ; 28 cm.
Language: English
Creator: Dougherty, William George, 1952- ( Dissertant )
Aldrich, Henry C. ( Thesis advisor )
Hiebert, Ernest ( Thesis advisor )
Purcifull, Dan ( Reviewer )
Edwardson, John E. ( Reviewer )
Preston, James F. ( Reviewer )
Davis, Francis ( Reviewer )
Fry, Jack L. ( Degree grantor )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 1979
Copyright Date: 1979
Subjects / Keywords: Plant viruses   ( lcsh )
Viral genetics   ( lcsh )
Tobacco mosaic virus   ( lcsh )
Microbiology thesis Ph. D
RNA -- Synthesis   ( lcsh )
Dissertations, Academic -- Microbiology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Abstract: Potyviruses are filamentous plant viruses with a diameter of 12 nm and a length of 630 to 900 nm. The viral genome consists of a single stranded, non-segmented RNA, sedimenting around 39 S on sucrose density gradients, and with an estimated molecular weight of 3-0-3-5 x 10 . The capsid consists of protein monomers with an estimated size varying Li between 3-0-3-6 x 10 daltons (30-36 kd) . The cytoplasmic cylindrical inclusion associated with all potyvirus infections consists of nonstructural protein monomers with a size of 67 to 70 kd . Inclusions in the nucleus, detected only in certain potyviruses, have been shown to consist of two protein monomers with sizes of kS kd and 5^ kd . Cylindrical and nuclear inclusion characterization studies have shown the inclusions to be virus specific and host independent, but direct evidence for their genetic origin is still needed. The primary purpose of this thesis was to determine if the genetic information coding for these non-structural proteins res i des in the potyviral genome. To do this, viral RNA was isolated and translated in a rabbit reticulocyte cell-free system. Labeled products of the cell-free translation were analyzed and compared with authentic products, using sodium dodecyl sulfate polyacry lamide gel electrophoresis (SDS-PAGE), immunoprecipi tat ion, and peptide mapping. Tobacco etch virus (TEV) and pepper mottle virus (PeMV) , members of the potyvirus group, were investigated. Both TEV and PeMV form cylindrical inclusions, but nuclear inclusions have not been found in PeMV infections. The optimum conditions for cell-free translation of TEV- and PeMVRNA are reported. The distribution of in vitro products was dependent on RNA quality. Full length RNA resulted in the cell-free synthesis of a 87 kd product for TEV and a 78 kd product for PeMV. RNA with limited fragmentation produced a number of discrete products in the cell-free system while highly fragmented RNA did not act as a template. Four of the 6 discrete cell-free products identified reacted with anitsera to *t virus-specific proteins. The cell-free 30 kd product of TEV RNA and the 33 kd product of PeMV RNA were identified as capsid protein on the basis of co-migration with authentic capsid proteins during SDS-PAGE, immunoprecipi tation with the respective capsid protein antiserum and by the comparison of the proteolytic peptide pattern with that for authentic capsid protein. Two cell-free products of TEV co-migrating with authentic nuclear inclusion proteins during SDS-PAGE, were immunoprecipitated with antisera specific to the nuclear inclusion proteins, and gave a similar proteolytic peptide pattern as authentic nuclear inclusion proteins. A PeMV RNA cell-free product co-migrating with PeMV cylindrical inclusion protein was immunoprecipi tated with antiserum to PeMV cylindrical inclusion protein. A faint product, co-migrating with TF.V cylindrical inclusion protein, was detected in the immunoprecip i tat ion analysis of TEV cell-free products with TEV cylindrical inclusion antiserum. A number of cell-free products, which were consistently immunoprecipitated with more than one antiserum, were detected. These products were presumed to be products of gene readthroughs on the basis of serological reactions and on the basis of size. These readthroughs were useful in linking genes on the potyviral genome. This thesis provides direct evidence for the viral origin of the non-structural proteins (inclusions) associated with potyvirus infection. Six distinct potyviral gene products are synthesized in the cell-free system and are genetically linked on the genome. The proposed genetic map accounts for 35% of the coding capacity of the TEV RNA and 93% of the PeMV RNA.
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 87-91.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by William George Dougherty.
 Record Information
Bibliographic ID: UF00099249
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000014183
oclc - 06272880
notis - AAB7381


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