Isolation and characterisation of an Hsp90 homologue from the resurrection plant Xerophyta viscosa

Master Thesis

2002

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http://hdl.handle.net/11427/20194
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thesis_sci_2002_walford_sally_ann.pdf (3.63 MB)
Authors
Walford, Sally-Ann
Supervisors
Mundree, Sagadevan G
Thomson, Jennifer Ann
Farrant, Jill M
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University of Cape Town

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Department of Molecular and Cell Biology
Faculty
Faculty of Science
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Abstract
Prior to this study, a eDNA library of dehydrated Xerophyta viscosa was differentially screened and several genes were found to be upregulated during dehydration. One of these cDNAs was found to share a high degree of sequence identity with the ER-Iocated Hsp90 or Grp94 family of proteins (hereafter referred to as XVGrp94) and forms the basis of this work. The XVGrp94 eDNA was found to be truncated at the 5· terminus and a full length eDNA was isolated using SMART-RACE™ (§witching Mechanism gt 5' end of RNA Iranscript- Random ~mplification of Complementary .!;rids). This eDNA was sequenced and appeared to be a representative of the Hsp90 family of genes. The putative gene contained an ORF (Open Reading frame) potentially coding for an 812 amino acid protein with a calculated size of 92.83 kDa. It shares 85% homology with other Hsp90s from plants and it contains several characteristic features of these proteins. Additionally, it contains the ER (endoplasmic reticulum) targeting and retention signals. Southern blot analysis confirmed the presence of the gene in the X. viscosa genome possibly as a member of a family of closely related genes. Northern blot analysis revealed a transcript size of 2.8 kb, however, expression patterns of the transcript could not be established. Western blot analysis showed that the XVGrp94 concentration increased significantly in response to heat and dehydration, and a slight increase was observed in response to conditions of high salt, but no response was seen in response to high light, cold or exogenous ABA (abscisic acid) application. The XVGrp94 open reading frame was cloned into the pProEX HTa expression vector and expressed in E. coli, but purification of the recombinant protein was not successful.
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Bibliography: pages 128-163.

Keywords
Molecular and Cell Biology

Reference:

Walford, S. 2002. Isolation and characterisation of an Hsp90 homologue from the resurrection plant Xerophyta viscosa. University of Cape Town.

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