Isolation of the cDNA encoding rat skeletal muscle myosin light chain kinase. Sequence and tissue distribution.

A cDNA clone encoding skeletal muscle myosin light chain kinase (MLCK) was isolated from a rat skeletal muscle library using oligonucleotide probes. The total length of the rat skeletal muscle MLCK cDNA was 2823 base pairs with an open reading frame of 1830 base pairs. The deduced sequence of the 610-amino acid protein exhibited 96% amino acid identity to rabbit skeletal muscle MLCK in the carboxyl-terminal portion of the molecule, which contains the catalytic and the calmodulin-binding domains, and 58% identity in the amino-terminal region. Analysis of total rat mRNA revealed a single mRNA species of 3.4 kilobases that was unique to skeletal muscle. Further analysis of skeletal muscle tissue using fast-twitch glycolytic, fast-twitch oxidative glycolytic, and slow-twitch oxidative fibers isolated from rat leg revealed that the mRNA level for MLCK varied among the three fiber types. The results of kinase assays performed on the fibers showed that MLCK activity levels paralleled the MLCK mRNA levels found in each of the three types of skeletal muscle fibers studied. Fast-twitch oxidative glycolytic (gastrocnemius red) and slow-twitch oxidative (soleus) exhibited 60 and 13%, respectively, of the enzymatic activity present in fast-twitch glycolytic (gastrocnemius white) fibers.

Intron-mediated recombination may cause a deletion in an alpha 1 type I collagen chain in a lethal form of osteogenesis imperfecta.

To understand the nature of the mutation in type I collagen genes in cells from an infant with the perinatal lethal form of osteogenesis imperfecta (type II), we cloned and sequenced almost 2 kilobases of a normal alpha 1(I) collagen gene and the corresponding region of a mutant alpha 1(I) gene from cell strain CRL 1262. The mutant gene had undergone recombination between two non-homologous introns, which resulted in the loss of three exons coding for 84 amino acids in the triple-helical domain. The deletion predicted the loss of amino acid residues surrounding and including the methionine at the junction between the CNBr peptides alpha 1(I) CB8 and alpha 1(I) CB3, a result confirmed by analysis of the cleavage peptides from the product of the mutant gene. Although large deletions from collagen genes are uncommon causes of the osteogenesis imperfecta type II phenotype, analysis of the de novo change in gene structure in this cell strain suggests that similar rearrangements may have occurred during the evolution of the large collagen genes.

DNA and chromatin structure of the human alpha 1 (I) collagen gene.

The human alpha 1 (I) collagen gene and 48 kilobase pairs of flanking DNA have been isolated on two overlapping cosmids. The alpha 1 (I) gene is 18 kilobase pairs long and contains a single repetitive element of the Alu family; at least 15 repetitive elements are present in the flanking DNA. Analysis of chromatin structure in nuclei isolated from cultured fibroblasts demonstrated a single chromatin domain greater than 65 kilobase pairs in length that contained 9 DNase I-hypersensitive sites. The pattern of hypersensitive sites was also determined in nuclei derived from placental tissue. Five of the DNase I-hypersensitive sites were observed in both placental and fibroblast chromatin including one site near the 5' end and another near the 3' end of alpha 1 (I). An additional two sites located near the 3' end of the alpha 1 (I) gene in fibroblast chromatin are associated with the tissue-specific use of different polyadenylation sites. Two DNase I-hypersensitive sites found only in fibroblast chromatin and one site found only in placental chromatin were located more than 10 kilobase pairs away from the alpha 1 (I) gene and may be related to tissue-specific expression of other genes in the domain. However, the only abundant placental mRNAs from the 65-kilobase pair domain were those transcribed from the alpha 1 (I) gene. These findings suggest that physical linkage does not play a predominant role in controlling coordinate expression of collagen genes.