Three testis cDNAs have been isolated that share homology with a cDNA that encodes chicken slow muscle troponin c. Each cDNA hybridizes to a distinct RNA that is expressed during a specific stage of testis differentiation. A 700 nt cDNA crossreacts strongly with troponin c and recognizes 800 nt RNA whose level peaks in a round spermatid enriched fraction. This cDNA (TTcL3) hybridizes to an RNA from rat heart that comigrates with authentic troponin c RNA. Both the 900 nt and the 1200 nt cDNAs isolated crosshybridize strongly with each other or a chicken calmodulin cDNA but weakly with the chicken troponin c probe. These two cDNAs both hybridize to RNAs of the same size and follow the same developmental pattern recognized by the chicken calmodulin cDNA. These RNAs are highly enriched in a pachytene spermatocyte fraction and decrease significantly as round spermatids develop. The identities of the cDNAs will be established by sequencing each cDNA and comparing the nucleic acid and deduced protein sequence to available data banks. The cDNAs will be introduced into bacterial expression vectors to produce sufficient amounts of the encoded proteins for biochemical studies and provide a vector for later in vitro mutagenesis studies. Antibodies will be produced against either the entire purified protein(s) or synthetic peptides. Indirect immunofluorescence microscopy and/or immunocytochemistry will be used to localize the relevant proteins in testis cells. In situ RNA hybridization will be used to positively identify the cells in which RNAs are synthesized. The tissue specificity of expression of the RNA and protein will be established by Northern RNA hybridization and Western immunoblot and radioimmunoassay. The functional significance of the encoded proteins will be studied by assessing Ca2+ binding properties and interaction with other testis proteins by affinity chromatography and 125I-protein binding. The number of crosshybridizing genes will be determined and genomic cloning will be initiated to identify the promotors that specify expression in different stages of spermatogenesis. Determination of these critical sequences is relevant to both studies of fertility and contraception.
