Human cardiac myosin heavy chain genes and their linkage in the genome.

Human myosin heavy chains are encoded by a multigene family consisting of at least 10 members. A gene-specific oligonucleotide has been used to isolate the human beta myosin heavy chain gene from a group of twelve nonoverlapping genomic clones. We have shown that this gene (which is expressed in both cardiac and skeletal muscle) is located 3.6kb upstream of the alpha cardiac myosin gene. We find that DNA sequences located upstream of rat and human alpha cardiac myosin heavy chain genes are very homologous over a 300bp region. Analogous regions of two other myosin genes expressed in different muscles (cardiac and skeletal) show no such homology to each other. While a human skeletal muscle myosin heavy chain gene cluster is located on chromosome 17, we show that the beta and alpha human cardiac myosin heavy chain genes are located on chromosome 14.     

The genes coding for the cardiac muscle actin, the skeletal muscle actin and the cytoplasmic beta-actin are located on three different mouse chromosomes.

The actins are a group of highly conserved proteins encoded by a multigene family. We have previously reported that the skeletal muscle actin gene is located on mouse chromosome 3, together with several other unidentified actin DNA sequences. We show here that the gene coding for the cardiac muscle actin, which is closely related to the skeletal muscle actin (1.1% amino acid replacements), is located on mouse chromosome 17. The gene coding for the cytoplasmic beta-actin is located on mouse chromosome 5. Thus, these three actin genes are located on three different chromosomes.     

Isolation and mapping of two porcine skeletal muscle myosin heavy chain isoforms

The present paper describes the isolation and linkage mapping of two isoforms of skeletal muscle myosin heavy chain in pig. Two partial cDNAs (pAZMY4 and pAZMY7), coding for the porcine myosin heavy chain-2B and -β respectively, have been isolated from a pig skeletal muscle cDNA library. Four RFLPs were detected with the putative porcine skeletal myosin heavy chain-2B probe (pAZMY4) and one RFLP was identified with the putative myosin heavy chain-β probe (pAZMY7). Two myosin heavy chain loci were mapped by linkage analysis performed with the five RFLPs against the PiGMaP linkage consortium ResPig database: the MYH1 locus, which identifies the fast skeletal muscle myosin heavy chain gene cluster, was located at the end of the map of porcine chromosome 12, while the MYH7 locus, which identifies the myosin heavy chain-α/-β gene cluster, was assigned to the long arm of porcine chromosome 7.     

Evolutionary implications of three novel members of the human sarcomeric myosin heavy chain gene family

Sarcomeric myosin heavy chain (MyHC) is the major contractile protein of striated muscle. Six tandemly linked skeletal MyHC genes on chromosome 17 and two cardiac MyHC genes on chromosome 14 have been previously described in the human genome. We report the identification of three novel human sarcomeric MyHC genes on chromosomes 3, 7, and 20, which are notable for their atypical size and intron-exon structure. Two of the encoded proteins are structurally most like the slow-beta MyHC, whereas the third one is closest to the adult fast IIb isoform. Data from pairwise comparisons of aligned coding sequences imply the existence of ancestral genomes with four sarcomeric genes before the emergence of a dedicated smooth muscle MyHC gene. To further address the evolutionary relationships of the distinct sarcomeric and nonsarcomeric rod sequences, we have identified and further annotated human genomic DNA sequences corresponding to 14 class-II MyHCs. An extensive analysis provides a timeline for intron gain and loss, gene contraction and expansion, and gene conversion among genes encoding class-II myosins. One of the novel human genes is found to have introns at positions shared only with the molluscan catchin/MyHC gene, providing evidence for the structure of a pre-Cambrian ancestral gene.     

Chromosomal assignment of two myosin alkali light-chain genes encoding the ventricular/slow skeletal muscle isoform and the atrial/fetal muscle isoform (MYL3, MYL4)

Summary In all eukaryotes, myosin plays a major role in the maintenance of cell shape and in cellular movement; in association with actin and other contractile proteins it is also a major structural component of the muscle sarcomere. Several isoforms of myosin alkali light chain have been identified, associated with different muscle types. We have recently localized the gene encoding the fast skeletal muscle alkali light-chain isoforms MLC1_F and MLC3_F (HGM symbol, MYL1) to human chromosome 2q32.1-qter (Cohen-Haguenauer 1988). We present here the chromosomal assignment of two loci encoding the ventricular muscle isoform MLC1_V (equivalent to the slow skeletal muscle isoform MLC1_Sb) and the atrial muscle isoform MLC1_A (equivalent to the fetal isoform MLC1_emb) using a panel of 25 independent man-rodent somatic cell hybrids. The MLC1_V gene (HGM symbol, MYL3) was mapped to human chromosome 3 using a human full-length cDNA probe that hybridizes to a single major human TaqI 2.8-kb fragment. The MLC1_A probe (HGM symbol, MYL4) was a 360-bp mouse cDNA fragment that gave a distinct signal with human DNA using low stringency conditions of hybridization and washings and after presaturation of the Southern blots with rodent DNA. A single PstI 7.8-kb fragment gives an intense signal, and its presence correlates with the presence of chromosome 17 among the hybrids. These data are in keeping with the localizations of the MLC1_V gene to mouse chromosome 9, and of the MLC1_A gene to mouse chromosome 11, which share some markers in common with human chromosomes 3 and 17 respectively.     

Human ventricular/slow twitch myosin alkali light chain gene characterization, sequence, and chromosomal location

The gene coding for the human ventricular/slow twitch myosin alkali light chain isoform was isolated and sequenced. It was found to contain a total of seven exons, the last of which is completely 3'-untranslated sequence. Comparison of this gene sequence with that of the various fast twitch skeletal isoform gene sequences revealed that the exon-intron arrangement is conserved within the myosin alkali light chain gene family. In fact the introns are in exactly the same positions within analogous codons. Comparison of the derived amino acid sequence from the human ventricular/slow twitch isoform gene with that of other isoform protein sequences indicated that the protein encoded by this gene is more homologous to the chicken cardiac isoform protein sequence than to any of the other protein sequences. These results indicate that the gene duplication which gave rise to the ventricular/slow twitch and fast twitch isoform genes must have occurred prior to the divergence of mammals and avians. We have also localized the human ventricular/slow twitch isoform gene to the short arm of human chromosome 3. Interestingly the corresponding mouse gene has been mapped to the distal region of mouse chromosome 9 which contains a conserved syntenic group of genes that map to the short arm of human chromosome 3.     

Mammalian skeletal muscle myosin light chain kinases. A comparison by antiserum cross-reactivity

Purified myosin light chain kinases from skeletal muscle are reported to be significantly smaller (Mr = 75,000-90,000) than the kinases purified from smooth muscle (Mr = 130,000-155,000). It has been suggested that the smaller kinases from striated muscle are proteolytic fragments of a larger enzyme which is homologous, if not identical, to myosin light chain kinase from smooth muscle. Therefore, we have used an antiserum to rabbit skeletal muscle myosin light chain kinase and Western blot analysis to compare the subunit molecular weight of the kinase in skeletal muscle extracts of several mammalian species. In rabbit skeletal muscle, the antiserum only recognized a polypeptide of Mr = 87,000, with no indication that this polypeptide was a proteolyzed fragment of a larger protein. The apparent molecular weights observed in different animal species were 75,000 (mouse), 83,000 (guinea pig), 82,000 (rat), 87,000 (rabbit), 100,000 (dog), and 108,000 (steer). The molecular weight of myosin light chain kinase was constant within an animal species, regardless of skeletal muscle fiber type. The antiserum inhibited the catalytic activity of skeletal muscle myosin light chain kinase. Similar antibody dilution curves for inhibition of myosin light chain kinase activity in extracts were observed for all animal species (rabbit, rat, mouse, guinea pig, dog, cat, steer, and chicken) and different fibers (slow twitch oxidative, fast twitch oxidative glycolytic, and fast twitch glycolytic) tested. The antiserum did not inhibit the activity of rabbit smooth muscle myosin light chain kinase. These results suggest that there may be at least two classes of muscle myosin light chain kinase represented in skeletal and smooth muscles, respectively.     

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.     

Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.     

Characterization of chicken skeletal muscle myosin light chain kinase. Evidence for muscle-specific isozymes

Myosin light chain kinase purified from chicken white skeletal muscle (Mr = 150,000) was significantly larger than both rabbit skeletal (Mr = 87,000) and chicken gizzard smooth (Mr = 130,000) muscle myosin light chain kinases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Km and Vmax values with rabbit or chicken skeletal, bovine cardiac, and chicken gizzard smooth muscle myosin P-light chains were very similar for the chicken and rabbit skeletal muscle myosin light chain kinases. In contrast, comparable Km and Vmax data for the chicken gizzard smooth muscle myosin light chain kinase showed that this enzyme was catalytically very different from the two skeletal muscle kinases. Affinity-purified antibodies to rabbit skeletal muscle myosin light chain kinase cross-reacted with chicken skeletal muscle myosin light chain kinase, but the titer of cross-reacting antibodies was approximately 20-fold less than the anti-rabbit skeletal muscle myosin light chain kinase titer. There was no detectable antibody cross-reactivity against chicken gizzard myosin light chain kinase. Proteolytic digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high performance liquid chromatography showed that these enzymes are structurally very different with few, if any, overlapping peptides. These data suggest that, although chicken skeletal muscle myosin light chain kinase is catalytically very similar to rabbit skeletal muscle myosin light chain kinase, the two enzymes have different primary sequences. The two skeletal muscle myosin light chain kinases appear to be more similar to each other than either is to chicken gizzard smooth muscle myosin light chain kinase.     

Characterization of diverse forms of myosin heavy chain expressed in adult human skeletal muscle.

In an attempt to define myosin heavy chain (MHC) gene organization and expression in adult human skeletal muscle, we have isolated and characterized genomic sequences corresponding to different human sarcomeric MHC genes (1). In this report, we present the complete DNA sequence of two different adult human skeletal muscle MHC cDNA clones, one of which encodes the entire light meromyosin (LMM) segment of MHC and represents the longest described MHC cDNA sequence. Additionally, both clones provide new sequence data from a 228 amino acid segment of the MHC tail for which no protein or DNA sequence has been previously available. One clone encodes a "fast" form of skeletal muscle MHC while the other clone most closely resembles a MHC form described in rat cardiac ventricles. We show that the 3' untranslated region of skeletal MHC cDNAs are homologous from widely separated species as are cardiac MHC cDNAs. However, there is no homology between the 3' untranslated region of cardiac and skeletal muscle MHCs. Isotype-specific preservation of MHC 3' untranslated sequences during evolution suggests a functional role for these regions.     

Identification of SNPs,mapping and analysis of allele frequencies in two candidate genes for meat production traits: the porcine myosin heavy chain 2B (MYH4) and the skeletal muscle myosin regulatory light chain 2 (HUMMLC2B)

Myosin is one of the most important skeletal muscle proteins. It is composed of myosin heavy chains and myosin light chains that exist with different isoforms coded by different genes. We studied the porcine myosin heavy chain 2B (MYH4) and the porcine skeletal muscle myosin regulatory light chain 2 (HUMMLC2B) genes. A single nucleotide polymorphism (SNP), identified for each gene, was used for linkage mapping of MYH4 and HUMMLC2B to porcine chromosome (Sscr) 12 and Sscr 3, respectively. The mapping of these two genes was confirmed by using a porcine-rodent radiation hybrid panel, even if for MYH4 the LOD score and the retention fraction were low. Allele frequencies at the two loci were studied in a sample of 307 unrelated pigs belonging to seven different pig breeds. Moreover the distribution of the alleles at these two loci was analysed in groups of pigs with extreme divergent (positive and negative) estimated breeding values (EBV) for four meat production traits that have undergone selection in Italian heavy pigs.     

A polymorphic human myosin heavy chain locus is linked to an anonymous single copy locus (D17S1) at 17p13

Restriction fragment length polymorphisms (RFLPs) have been developed for an adult human skeletal muscle myosin heavy chain gene which was previously mapped to the short arm of human chromosome 17. Using RFLP analysis of DNA from 140 individuals, we have found tight linkage (LOD score of 6.9) between this myosin heavy chain gene and the anonymous DNA probe, D17S1.     

Formation and properties of smooth muscle myosin 20-kDa light chain-skeletal muscle myosin hybrids and photocrosslinking from the maleimidylbenzophenone-labeled light chain to the heavy chain.

Experimental conditions which permit the exchange of smooth muscle 20-kDa light chain into skeletal muscle myosin are described. The hybridization does not result in the regulation of actin-activated ATPase activity of the hybrid myosin by smooth light chain phosphorylation. Further, the KCl dependence of the Mg-ATPase activity of the hybrid was similar to that of skeletal muscle myosin. The dephosphorylation of the smooth light chain in the hybrid did not induce a conformational change in the hybrid from the 6 S to the 10 S state, thereby indicating that the conformational transition is dependent also on the nature of the heavy chain subunit. Exchange of the smooth light chain premodified at its Cys-108 by photolabile 4-(N-maleimido)benzophenone and photolysis resulted in crosslinking to the heavy chain subunit. Immunopeptide mapping using a monoclonal antibody against residues 1-23 at the N-terminus of the skeletal muscle myosin heavy chain identified the location of the photocrosslinking site to be beyond 92 kDa away from the N-terminus.     

Mutation of the IIB myosin heavy chain gene results in muscle fiber loss and compensatory hypertrophy

The fast skeletal IIb gene is the source of most myosin heavy chain (MyHC) in adult mouse skeletal muscle. We have examined the effects of a null mutation in the IIb MyHC gene on the growth and morphology of mouse skeletal muscle. Loss in muscle mass of several head and hindlimb muscles correlated with amounts of IIb MyHC expressed in that muscle in wild types. Decreased mass was accompanied by decreases in mean fiber number, and immunological and ultrastructural studies revealed fiber pathology. However, mean cross-sectional area was increased in all fiber types, suggesting compensatory hypertrophy. Loss of muscle and body mass was not attributable to impaired chewing, and decreased food intake as a softer diet did not prevent the decrease in body mass. Thus loss of the major MyHC isoform produces fiber loss and fiber pathology reminiscent of muscle disease.     

Domain characterization of rabbit skeletal muscle myosin light chain kinase.

Myosin light chain kinase can be divided into three distinct structural domains, an amino-terminal "tail," of unknown function, a central catalytic core and a carboxy-terminal calmodulin-binding regulatory region. We have used a combination of deletion mutagenesis and monoclonal antibody epitope mapping to define these domains more closely. A 2.95-kilobase cDNA has been isolated that includes the entire coding sequence of rabbit skeletal muscle myosin light chain kinase (607 amino acids). This cDNA, expressed in COS cells encoded a Ca2+/calmodulin-dependent myosin light chain kinase with a specific activity similar to that of the enzyme purified from rabbit skeletal muscle. Serial carboxy-terminal deletions of the regulatory and catalytic domains were constructed and expressed in COS cells. The truncated kinases had no detectable myosin light chain kinase activity. Monoclonal antibodies which inhibit the activity of the enzyme competitively with respect to myosin light chain were found to bind between residues 235-319 and 165-173, amino-terminal of the previously defined catalytic core. Thus, residues that are either involved in substrate binding or in close proximity to a light chain binding site may be located more amino-terminal than the previously defined catalytic core.     

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.