Abstract
Two mouse genomic libraries were probed in order to isolate the murine cardiac myosin heavy chain (MHC) genes. Two overlapping cosmid clones that encode the cardiac genes were isolated. One of them encompasses the entire alpha-cardiac MHC gene, its 5'-flanking region and approximately 10 kilobase pairs (kb) of the 3'-end of the beta-cardiac MHC gene which we determined is located approximately 4 kb upstream of the alpha-cardiac MHC gene. Four clones isolated from a bacteriophage library were found to overlap with the 5'-region of the cosmid clones, and sequence analysis confirmed that the 5'-end of the beta-cardiac MHC gene was contained within one of the clones. Primer extension and polymerase chain reaction (PCR) analyses were used to define the transcriptional start site and the 5'-organization of the alpha-cardiac MHC gene. This region exhibits greater than 90% homology to the corresponding nucleotides of the rat alpha-cardiac MHC gene. To assess the importance of the intergenic region in directing expression of the alpha-cardiac MHC gene, a fragment containing the 3'-end of the beta-cardiac gene and the 5'-end of the alpha-cardiac gene was linked to a chloramphenicol acetyltransferase gene and used to generate transgenic mice. Analyses of the chloramphenicol acetyltransferase (CAT) activity in two lines indicate that the intergenic region is sufficient to properly direct expression in a tissue-specific manner.
Highlights
The &cardiac gene and the 5’-end of the a-cardiac gene a (A,) and @I(@ A2)homodimers have been observed,with the was linked to a chloramphenicol acetyltransferase AI form being constitutively expressed and exogenous thyroid gene and used to generate transgenic mice
We describe the isolation of the murine cardiac MHC locus and the characterization of the intergenic region with respect to its ability to properly direct expression of the CAT gene in transgenic mice
As a first step toward analyzing the a-cardiac MHC pro- weak signals are due to high levels of expression in a restricted moter, a5.7-kb genomic DNA fragment encompassing part of fiber population.' Inthe mouse, ithas been shown that the @-cardiacMHC gene (-100 bp of the last intron and all expression of the a-cardiac gene is up-regulated immediately of the last exon), the intergenic region (-4 kb), and the 5'- after birth [26]
Summary
The &cardiac gene and the 5’-end of the a-cardiac gene a (A,) and @I(@ A2)homodimers have been observed,with the was linked to a chloramphenicol acetyltransferase AI form being constitutively expressed and exogenous thyroid gene and used to generate transgenic mice. In order to initiatethese types of studies for the cardiac myosins, we have screened two mouse genomic libraries for the MHC genes.
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