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|Title:||Isolation and characterization of calmodulin-binding heat shock proteins and cDNAs encoding calmodulin-binding proteins in cultured tobacco cells|
Heat shock proteins
|Abstract:||Calmodulin-binding heat shock proteins were characterized in cultured tobacco cells (Nicotinan tabacum L. cv Wisconsin-38). Analyses of 35S-labeled calmodulin-binding proteins (CaMBPs) purified by affinity chromatography and SDS-PAGE indicated that heat shock (38°C) enhanced/induced the synthesis of some CaMBPs while the synthesis of others was repressed during the heat shock response (HSR). The synthesis of CaMBPs with apparent molecular weights of 82, 78, 71, 68, 22, 20, 19.5 and 17 Kd was stimulated by heat shock. A procedure for the isolation of cDNA clones encoding CaMBPs was refined. Twenty five positive cDNA clones were isolated by screening a tobacco heat shock cDNA expression library with 35S-CaM as a ligand probe. These clones produced peptides exhibiting Ca2+-dependent, CaM-binding activity when assayed by gel overlay analysis. While most cloned mRNAs such as pTCB40 were unaffected by heat shock, two clones, pTCB60 and pTCB48 were heat shock-related. Analysis of Northern blot demonstrated that a 2.1 kb mRNA recognized by pTCB60 decreased by at least 70% in a 2 hour heat shock treatment. The expression of pTCB48 mRNA was induced by heat shock. This translationally active mRNA was detected after 15 minutes of heat shock and accumulated to maximum amounts after 1.5 hours. These results suggest that the ca2+/CaM second messenger system plays a role in tobacco heat shock response. The natures of CaM-binding domains were determined for pTCB48 and pTCB60. DNA sequences of these clones were determined and several deletion constructs from both the 5' and 3' ends of the inserts were constructed. The CaM-binding activities of the proteins generated from these deletion constructs were assayed by gel overlay analysis. These data combined with secondary structure analyses of deduced proteins localized the CaM-binding domains in the C-terminus of these proteins. The CaM-binding domain of TCB60 was a basic amphiphilic a-helix similar to that of several animal and human CaMBPs. No similar structure was found in the c-terminal region of TCB48 suggesting that an alternative structure is responsible for the CaM-binding.|
Thesis (Ph. D.)--University of Hawaii at Manoa, 1991.
Includes bibliographical references (leaves 103-130)
x, 130 leaves, bound ill. 29 cm
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|Appears in Collections:||Ph.D. - Botanical Sciences (Plant Physiology)|
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