Chicken calmodulin genes. A species comparison of cDNA sequences and isolation of a genomic clone

Abstract

A cDNA library, prepared from poly(A+) mRNA isolated from chicken brain, was screened for calmodulin sequences using the cloned full length structural gene from Electrophorus electricus as probe (Lagacé, L., Chandra, T., Woo, S.L.C., and Means, A.R. (1983) J. Biol. Chem. 258, 1684-1688). Fifteen positive signals were detected among 4500 recombinant clones from which two overlapping clones (pCB12 and pCB15) were selected for subsequent DNA sequencing. The combined unique sequences of the two cDNA clones yielded 1395 base pairs and contained the entire coding region for calmodulin, 94 base pairs of the 5'-nontranslated region, and the entire 3'-nontranslated region of 857 base pairs. The derived amino acid sequence of chicken calmodulin is identical with that of the bovine or human protein. Compared to the eel, there is a single conservative amino acid substitution at position 74 which is occupied by Arg in the chicken and Lys in the eel. The overall nucleotide homology between the amino acid coding regions of chicken and eel calmodulin mRNA is 79%. However, the 5'- and 3'-nontranslated regions of the chicken and eel mRNA for calmodulin are highly diverged with sequence homologies of 21 and 29%, respectively. The cDNA clones were used as probes to determine the size and distribution of calmodulin mRNA in a variety of chicken tissues. In all tissues examined, two species of mRNA for calmodulin were detected at 1600 and 1900 nucleotides. Both mRNAs occurred in the cytoplasm with an abundance ratio of 4:1 for the 1600 and 1900 species, respectively. The two mRNAs appear to result from differential processing of transcripts from a single calmodulin gene. Screening of a chicken genomic phage library using pCB12 as a probe yielded a single positive designated CL-1 which contains a DNA insert of 13.5 kilobase pairs. Partial sequencing of CL-1 has confirmed the presence of sequences which code for calmodulin. A comparison of the restriction maps of CL-1 and pCB12 and pCB15 indicates that CL-1 contains at least 3 intervening sequences.