Characterization of a cDNA encoding a novel heat-shock protein that binds to calmodulin.
A cDNA clone (pTCB48) encoding a calmodulin-binding protein was isolated by screening a lambda ZAPII cDNA expression library constructed from cell cultures of heat-shocked tobacco (Nicotiana tabacum L. cv Wisconsin-38) with metabolically labeled [35S]calmodulin. Calmodulin gel overlay analysis indicated that pTCB48 generated major peptides of 53, 36, and 22 kD and two minor peptides of 37 and 16 kD that bound calmodulin in a Ca(2+)-dependent manner. Deletion analysis of pTCB48 indicated that these and the minor calmodulin-binding proteins resulted from the insert. A probe made from the cDNA insert recognized two bands with sizes of 2.1 and 1.8 kb on northern blot analysis. Both species of RNAs were undetectable in the control and were induced after 15 min of heat-shock treatment at 38 degrees C. The intensity of the two bands reached maximum after 1.5 h of heat-shock treatment. The cDNA clone was not full length; however, the complete sequence was determined by 5' rapid amplification of cDNA ends using nested antisense primers. The full-length cDNA contains 1648 bp and a single open reading frame of 1347 bp and is expected to encode a protein of approximately 50 kD. No significant homology with other reported genes and proteins was found. Structural predictions, deletion analysis, and gel overlay analysis suggested that the calmodulin-binding domain was a basic amphiphilic alpha-helix near the C terminus of the protein. The strong induction of the mRNA for this protein suggests a role for Ca2+/calmodulin-mediated process in the heat-shock response.
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Department of Plant Molecular Physiology, University of Hawaii at Manoa, Honolulu 96822, USA.
Dharmasiri
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Harrington
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HM
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010947:Plants, Toxic; D011485:Protein Binding; D014026:Nicotiana / Q000166:cytology / Q000235:genetics* / Q000378:metabolism
D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D006360:Heat-Shock Proteins; D010940:Plant Proteins; C094900:TCB48 protein, Nicotiana tabacum