Before defining the components responsible for regulation it is necessary to isolate and characterize each of the human calmodulin genes using both chromosome-specific libraries and a genomic library. This would allow analysis of the two or more active genes thus clarifying the distinguishing and common features between these genes particularly in the non-coding segments. Since there are at least 4 different calmodulin sequences in the human genome, it is possible that more than 2 bona fide genes exist. We have evidence now from sequencing data that a clone isolated from a chromosome 17 specific library is a pseudogene. The amino acid substitutions in this pseudogene are both radical and conservative. Knowledge of gene structure would permit examination of the arrangement and organization of exons and introns. The location of intervening sequences in the Drosophila, chicken and rat gene structures have been shown to be conserved. The human gene structure would reveal if this is an evolutionary feature of this gene. The second part of this study deals with the expression and regulation of the individual genes. It is not clear how and when the two or more active genes are transcribed. Oligonucleotide probes or gene fragments representing unique nucleotide sequences of each gene will be used. By employing polyadenylated RNA from various human cell lines or tissues in Northern blots, solution hybridization. RNA dot blots and primer extension assay, it will be possible to determine whether there is a difference in the expression of the two or more active genes. Tissue specificity, the level and size of transcripts will be elucidated. For the determination of possible regulatory cis-acting sequences, vectors containing a """"""""reporter"""""""" gene and driven by a human calmodulin gene promoter, or portions of it, will be allowed to transfect different types of human cells in culture.
| Pegues, J C; Friedberg, F (1990) Multiple mRNAs encoding human calmodulin. Biochem Biophys Res Commun 172:1145-9 |
