Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis.
A gene coding for a calmodulin was synthesized and expressed in Escherichia coli. The gene was produced by the enzymatic ligation of 61 chemically synthesized DNA fragments. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis by the replacement of specific gene segments with synthetic double-stranded DNA. An expression vector containing the calmodulin gene was used to transform E. coli. Purification and characterization of calmodulin (VU-1 calmodulin) expressed by these transformants showed that it lacks two posttranslational modifications: an amino-terminal blocking group and N epsilon, N epsilon, N epsilon-trimethyllysine at position 115. The cyclic nucleotide phosphodiesterase activator properties of VU-1, higher plant, and vertebrate calmodulins were not statistically different. However, VU-1 calmodulin was found to activate nicotinamide adenine dinucleotide (NAD) kinase to a maximal level that was at least 3-fold higher than that found with higher plant and vertebrate calmodulins. This higher level of activation is also characteristic of calmodulins from Dictyostelium discoideum and Chlamydomonas reinhardtii [Roberts, D. M., Burgess, W. H., & Watterson, D. M. (1984) Plant Physiol. 75, 796-798; Marshak, D. R., Clarke, M., Roberts, D. M., & Watterson, D. M. (1984) Biochemistry 23, 2891-2899]. The only common feature among Dictyostelium, Chlamydomonas, and VU-1 calmodulins not found in higher plant and vertebrate calmodulins is an unmethylated lysine at position 115. The results indicate that the lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000138:chemical synthesis*; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D007887:Fabaceae / Q000201:enzymology; D005784:Gene Amplification; D005796:Genes; D005818:Genetic Engineering; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology; D010946:Plants, Medicinal; D010957:Plasmids
D002147:Calmodulin; D009838:Oligodeoxyribonucleotides; D004247:DNA; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D004262:DNA Restriction Enzymes