Holness, Claire Louise Lesley (1989) Isolation and characterisation of mutants of cowpea mosaic virus. PhD thesis, University of Warwick.

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Abstract

A nitrous acid-induced, temperature sensitive mutant of cowpea mosaic virus
(CPMV) known as 8-14, (Evans 1985, Virology 1985, 141, 275-282), was
characterised. The phenotypic defect in 8 -14 was shown not to affect translation
of the RNA or the first proteolytic cleavage of the B RNA-encoded polyprotein.
The defect is probably at the level of genome replication. The technique of two
dimensional RNA fingerprinting showed the mutant genome to be similar to the
parental wild-type but did not resolve the genetic alteration(s) specific for the
mutation.
The mechanism of CPMV translation was investigated by site-directed
mutagenesis of a full-length cDNA clone of CPMV M RNA from which infectious
RNA could be generated by in vitro transcription. The results obtained confirm
the AUG at position 161 is used to direct the synthesis of the 105K protein
in vitro. The detection of a 58K protein in infected protoplasts suggests that it
is also used in vivo. The synthesis of the 95K protein can be initiated from
either of the AUGs at positions 512 and 524. Synthesis of this protein is not
essential for CPMV replication in protoplasts.
Several deletion mutations were created in the M RNA cDNA clone in order to
determine the regions of M RNA essential for replication of M RNA. Analysis of
one mutant indicated that sequences between 1446 and 1620 are probably not
required for replicase recognition. However, the accumulation of this mutant in
protoplasts was reduced, presumably as a result of lack of encapsidation of the
RNA as this mutant is thought not to synthesise functional coat protein. Data
from several mutants showed that alterations of M RNA around nucleotides 161
and 189 prevent transcript accumulation in protoplasts possibly owing to a
severe reduction in replicability of the input RNA.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QH Natural history > QH301 Biology Q Science > QH Natural history > QH426 Genetics
Library of Congress Subject Headings (LCSH):	Virus diseases of plants , Viral genetics, Plant viruses -- Genetics
Official Date:	September 1989
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Evans, D. J. (David J.) ; Lomonossoff, G. P. ; Maule, A. J.
Extent:	x, 117 leaves : illustrations
Language:	eng

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