ISOLATION OF TOBACCO CDNA CLONES ENCODING CALMODULIN-BINDING PROTEINS AND CHARACTERIZATION OF A KNOWN CALMODULIN-BINDING DOMAIN

A cDNA library, constructed with the expression vector lambda ZAPII and poly(A) RNA purified from suspension cultured tobacco cells (Nicotiana tabacum, L., cv Wisconsin-38), was screened with [S-135]-labeled calmodulin as a ligand probe for the presence of calmodulin-binding proteins. Twenty-five independent positive clones were isolated from 10(6) recombinants. The insert sizes ranged from 0.8 to 5.3 kb. Gel overlay analysis of protein extracts indicated that all positive clones synthesized peptides that bound calmodulin in a Ca2+-dependent manner. Most fusion proteins were unstable in Escherichia coli with as many as 10 partially degraded peptides per clone retaining the ability to bind calmodulin. The largest calmodulin binding fusion proteins observed from individual clones ranged in approximate size from 28 to 100 kDa. Deletion analysis indicated that the calmodulin-binding proteins expressed by the clones resulted from the inserts. Clone pTCB60 was analyzed in detail and found to contain a 1572 bp insert with an open reading frame of 1184 bp. This sequence encoded a 393 residue peptide with a calculated molecular mass of 48.5 kDa including the beta-galactosidase fragment. Analysis of expression using a probe made from pTCB60 detected a 2.1 kb mRNA in northern blots of tobacco RNAs. This indicates that the pTCB60 is not full length. The mRNA level was reduced by at least 70% during a 2 h. heat shock treatment. Deletion mutants combined with predictions of secondary protein structure and gel overlay analysis defined a basic amphiphilic alpha-helix located in C-terminus as the calmodulin-binding domain in TCB60. No significant homology between pTCB60/TCB60 and other reported genes or proteins was found. Similarly, no functional motifs were found.