[24] The use of synthetic oligodeoxyribonucleotides in the examination of calmodulin gene and protein structure and function

Publisher Summary This chapter discusses how synthetic oligonucleotides are used in studies of calmodulin and related calcium-binding proteins. The focus is on two specific applications of synthetic oligonucleotides: site-specific mutagenesis and detection of recombinant DNA vectors containing DNA inserts coding for calmodulin-like structures. The database of information on the phylogeny and ontogeny of calmodulin provides the necessary background for the initiation of studies utilizing a direct approach to structure and function that combines recombinant DNA, comparative enzymology, and protein chemistry techniques. The synthetic calmodulin gene was designed for mutagenesis by two approaches: cassette-based and M13-based mutagenesis. Mutagenesis is performed on a construction consisting of the synthetic calmodulin gene cloned in the EcoRI and BamHI sites of pUC8. Once the mutant calmodulin gene sequence is generated and verified, an expression vector containing the mutated gene sequences is constructed. Calmodulin mutant genes are expressed by cloning on the expression plasmid pKK223-3. The final purified product is characterized by a variety of analytical techniques including SDS-polyacrylamide gel electrophoresis, amino acid composition analyses of acid hydrolysates of the whole protein and selected peptide fractions, and limited amino acid sequence analyses.