[22] Calmodulin from Trypanosoma brucei: Immunological analysis and genomic organization

This chapter discusses that calmodulin from Trypanosoma brucei is distinct from vertebrate calmodulin in protein structure, antigenicity, and genomic organization. The following protocols are used to examine the calmodulin protein and gene(s) from African trypanosomes, flagellated protozoa that infect the bloodstream, lymph, central nervous system, and other interstitial spaces of the mammalian host. Identification of calmodulin coding sequences within the genome of T. brucei is a critical step toward understanding regulation of calmodulin content within the cell. A genomic study offers the advantages that the entire gene, including introns and flanking sequences, can be identified. This chapter describes how a genomic library is prepared, screened with the cDNA probe pCM109, and mapped by multiple endonuclease digestions. Antibody specificity is determined by competition assays in which rabbit serum is first pre-absorbed with test protein prior to its incubation with the microtiter plate. Competition assays are run by adding 1 /zg bovine brain calmodulin to each well of a polyvinyl chloride microtiter plate.