Degree Type

Dissertation

Date of Award

1993

Degree Name

Doctor of Philosophy

Department

Genetics, Development and Cell Biology

First Advisor

W. Allen Miller

Second Advisor

John H. Hill

Abstract

Barley yellow dwarf virus (BYDV) has a positive sense, 5.7kb RNA genome encoding at least six open reading frames (ORFs). BYDV uses variety of unusual translational strategies to express its genes. These include frameshifting in the polymerase (60K) ORF, leaky scanning at the AUGs of the overlapping coat protein (CP) and 17K ORFs, and readthrough of the CP stop codon;Full-length cDNA clone of BYDV-PAV has been constructed to understand functions of different viral genes and the roles of specific sequences in translation, replication and encapsidation. Translation in cell-free extracts using in vitro synthesized full-length transcripts resulted in expected 99 kDa frameshift product. These transcripts were biologically active. The 39K ORF at the 5' end of the genome overlaps by 13 nucleotides with a 60K ORF. The region of overlap contains a "shifty" heptanucleotide, followed by a highly structured region that may contain a pseudoknot. The 39K ORF stop codon immediately next to the shifty heptanucleotide sequence is necessary for in vivo frameshifting and viral replication. Deletion mutants indicate that the ORF1, ORF2 and ORF6 or 3[superscript]' terminal 1157 nucleotides are necessary for BYDV-PAV replication in protoplasts;The CP, 17K and 50K ORFs located at the 3' half of the genome are expressed from a tricistronic subgenomic RNA (sgRNA1), that is generated in infected cells. The 17K ORF is embedded in the sequence that encodes the CP ORF, in a different reading frame. The 17K ORF start codon is 43 bases downstream of the CP start codon. Ribosomes initiate translation at the start codons of both ORFs, giving rise to both proteins. Systematic mutagenesis was performed to show, in vitro and in vivo, that overlapping ORFs of the sgRNA1 are expressed by Kozak's leaky scanning mechanisms. A new ribosome pausing model is proposed to explain the unexpected observation that mutations that reduce translation initiation at the downstream 17K AUG also reduce initiation at the upstream CP AUG. Implications of these results on expression of luteovirus coat protein in transgenic plants are discussed;The 50K ORF is translated by readthrough of the CP stop codon, giving rise to 72 kDa polypeptide in vitro. The GUS reporter gene was inserted downstream of the CP stop codon in the full-length BYDV-PAV infectious clone to elucidate the mechanism of readthrough for the luteovirus class. Results indicate that the signals required in luteovirus class are more complex and different from the well-studied tobacco mosaic virus like group of readthrough. Use of readthrough strategy to express biologically active recombinant proteins are discussed.

DOI

https://doi.org/10.31274/rtd-180813-11064

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

S.P. Dinesh-Kumar

Language

en

Proquest ID

AAI9413969

File Format

application/pdf

File Size

192 pages

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