Molecular genetic characterization of a developmentally regulated human perinatal myosin heavy chain

We have isolated a human cDNA which corresponds to a developmentally regulated sarcomeric myosin heavy chain. RNA hybridization and DNA sequence analysis indicate that this cDNA, called SMHCP, encodes a perinatal myosin heavy chain isoform. The nucleotide and deduced amino acid sequences of the 3.4-kb cDNA insert show strong homology with other sarcomeric myosin heavy chains. The strongest homology is to a previously described 970-bp cDNA encoding a rat perinatal isoform (Periasamy, M., D. F. Wieczorek, and B. Nadal-Ginard. 1984. J. Biol. Chem. 259:13573-13578). The homology between the analogous human and rat perinatal myosin heavy chain cDNAs is maintained through the highly isoform-specific final 20 carboxyl-terminal amino acids, as well as the 3' untranslated region. Ribonuclease protection studies show that the mRNA encoding this isoform is expressed at high levels in 21-wk fetal skeletal tissue and not in fetal cardiac muscle. In contrast to the rat perinatal isoform, which was not found to be expressed in adult hind-leg tissue, the gene encoding SMHCP continues to be expressed in adult human skeletal tissue, but at lower levels relative to fetal skeletal tissue.     

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.     

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.     

Partial characterization of the human beta-myosin heavy-chain gene which is expressed in heart and skeletal muscle

A human myosin heavy-chain gene, cloned in gamma Charon 4A phage (and as a clone designated lambda gMHC-1), was shown to code for a cardiac myosin heavy chain of the beta-type. The 5' end of the 14,200-base-pair genomic DNA clone is located in the head region of the myosin chain. The 3' end was shown to extent to the COOH terminus and includes the 3'-nontranslated sequence of the corresponding mRNA. The identification of lambda gMHC-1 as coding for a cardiac beta-myosin heavy chain was achieved by heteroduplex mapping using genomic cardiac myosin heavy-chain DNA of rabbit as a probe and, furthermore, by DNA sequence analysis of three selected subregions of the clones DNA including the 3'-nontranslated sequence. It was demonstrated by the S1 nuclease protection technique that the beta-myosin heavy-chain gene is transcribed in human heart muscle. In addition, we have found by the same technique that it is also expressed in human skeletal muscle.     

The complete sequence of the human beta-myosin heavy chain gene and a comparative analysis of its product

We have isolated and sequenced the gene and the cDNA coding for the human cardiac beta-myosin heavy chain (designated MYH7). The gene is 22,883 bp long. The 1935 amino acids of this protein (Mr223,111) are encoded by 38 exons. The 5' untranslated region (86 bp) is split by two introns. The 3' untranslated region is 114 bp long. Three Alu repeats were identified within the gene and a fourth one in the 3' flanking intergenic region. The molecular organization of this gene reflects the conservative pattern with respect to size, coding ratio, and number or position of introns characteristic of vertebrate sarcomeric myosin heavy chain genes. The protein sequence of the human beta-heavy chain was compared with corresponding (homologous) sequences of rabbit, rat, and hamster as well as with the (heterologous) embryonic heavy chain sequences of rat, chicken, and man. The results show that protein subregions responsible for basic functions of myosin heavy chains (nucleotide binding and actin binding) are very similar in homologous and heterologous heavy chains. Regions that differ in their primary sequences in heterologous heavy chains appear to be highly conserved within mammalian beta-myosin heavy chains. Constant and variable subregions of heavy chains are discussed in terms of functional significance and evolutionary relatedness.     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

Changes in myosin isozymes during development of chicken breast muscle

The patterns of myosin isozymes in embryonic and adult chicken pectoralis muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light chains and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, the predominant isozyme component in embryonic pectoralis myosin could be clearly distinguished from adult myosin isozymes. SDS-polyacrylamide gel electrophoresis indicated that the light chain composition of embryonic myosin was also different from that of adult myosin. The pattern of peptide fragments produced by myosin digestion with a-chymotrypsin differed significantly between embryonic and adult skeletal myosin. These results suggest that myosin in the embryonic pectoralis muscle is different in both light and heavy chain composition from myosin in the same adult tissue.     

The genes coding for the muscle contractile proteins, myosin heavy chain, myosin light chain 2, and skeletal muscle actin are located on three different mouse chromosomes.

The chromosomal distribution of murine genes expressed during differentiation of skeletal muscle cells was determined by Southern blot analysis of DNA from mouse-Chinese hamster hybrid cell lines containing incomplete subsets of mouse chromosomes. All detectable myosin heavy chain genes are located on chromosome 11. The gene for the myosin light chain 2 is located on chromosome 7. The skeletal muscle alpha-actin gene and several other actin genes, or pseudogenes, are located on chromosome 3. Additional actin DNA sequences are distributed on other mouse chromosomes.     

Amino-acid sequence of the short subfragment-2 in adult chicken skeletal muscle myosin

The amino-acid sequence of a short subfragment-2 in the amino-terminal portion of subfragment-2 (S-2) derived from adult chicken skeletal muscle myosin was completely determined. Peptides cleaved by cyanogen bromide and by lysyl endopeptidase of S-carboxymethylated S-2, and hydrolytic peptides obtained with trypsin or dilute acetic acid of larger CNBr fragments were isolated and sequenced. This region was composed of 257 amino-acid residues, and hydrophobic and charged residue repeat units were found highly conserved and with a periodicity in 7 or 28 residues. This sequence of the short S-2 fragment of chicken skeletal muscle myosin was compared with the sequence of chicken and rat embryonic skeletal muscle myosins, rabbit skeletal and rabbit cardiac muscle myosin (alpha-myosin heavy chain), and 95.3%, 86.8%, 89.9% and 94.2% sequence identities were observed, respectively.     

Embryonic myosin heavy chain as a differentiation marker of developing human skeletal muscle and rhabdomyosarcoma. A monoclonal antibody study

Hybridoma cell lines were obtained from the fusion of NS-O myeloma cells with spleen cells of mice immunized with bovine fetal skeletal myosin. A stable hybridoma clone, BF-G6, produced immunoglobulin G1 k antibodies reacting specifically with embryonic-type myosin heavy chains present in fetal but not in neonatal or adult human skeletal muscle, as determined by enzyme immunoassay and immunoblot analysis. Fetal but not adult skeletal muscle fibers were stained by this monoclonal antibody in indirect immunofluorescence assays; smooth muscle cells and cardiac muscle cells, as well as non-muscle cells were also unreactive. Solid tumors of infants and children were tested for reactivity with BF-G6 by immunofluorescence and immunoperoxidase staining. Embryonic myosin heavy chain was expressed in rhabdomyosarcomas but not in other types of tumor, except for Wilms' tumor. Rhabdomyosarcoma cells isolated from a bone marrow metastasis and grown in vitro for several months were also labelled by BF-G6. Embryonic myosin heavy chain can thus be used as a specific differentiation marker of normal and neoplastic skeletal muscle tissue.     

Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail

We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity.     

Two different forms of beta myosin heavy chain are expressed in human striated muscle

Summary We have found evidence for two beta-like myosin heavy chains in humans, one cardiac and one skeletal. The cDNA sequences of the cardiac beta myosin heavy chain cDNA clone pHMC3 and the skeletal beta-like myosin heavy chain cDNA clone pSMHCZ, were compared to each other. It was found that the 3 untranslated regions as well as 482 nucleotides specifying the carboxyl coding region, were 100% homologous. Further examination revealed that the skeletal clone pSMHCZ diverges from the human cardiac beta myosin heavy chain cDNA clone pHMC3 at the 5 end. We present evidence in this report which indicates that the cardiac beta myosin heavy chain mRNA is expressed in skeletal muscle tissues. The human cardiac beta myosin heavy chain cDNA clone, pHMC3, which codes for a portion of the light meromyosin section of the myosin heavy chain, was used as a probe for S1 nuclease mapping studies with RNA derived from cardiac tissue, smooth muscle and skeletal muscle tissues consisting of fast-twitch, slow-twitch and mixed fast- and slow-twitch muscle fibres. Two probes were used to examine the expression of the mRNA. One probe (406 nucleotides) constitutes the 3 untranslated region and a portion of the coding region of the beta cardiac myosin heavy chain cDNA clone, which is 100% homologous to pSMHCZ, the skeletal cDNA clone. The other constitutes the majority of the coding region (1017 nucleotides) of the cardiac clone pHMC3 in which the first 216 nucleotides from the labelled end are 100% homologous to the skeletal clone pSMHCZ. In the soleus muscle, which is rich in slow-twitch type I muscle fibres, the expression of the cardiac beta myosin heavy chain mRNA was very prominent. In gastrocnemius muscle, a mixed fibre muscle, the expression of this mRNA was detected to a lesser degree than that for the soleus muscle. In vastus lateralis and vastus medialis, which consist of predominantly type II, fast-twitch fibres, there were trace amounts of the cardiac beta myosin heavy chain mRNA. When expression of this mRNA was tested in smooth muscle tissue none could be detected.     

In vivo developmental modifications of the expression of genes encoding muscle-specific enzymes in rat

cDNA clones for rat muscle-type creatine kinase and glycogen phosphorylase and aldolase A were isolated from a rat muscle cDNA library. An additional clone recognizing an unidentified 2.7-kilobase pair mRNA species was also isolated. These cDNA clones were used as probes to investigate the expression of the corresponding mRNAs during muscle development. Two aldolase A mRNA species were detected, one of 1650 bases expressed in non-muscle tissues, fetal muscle, and adult slow-twitch muscle, the other of 1550 bases was highly specific of adult fast-twitch skeletal muscle differentiation. These aldolase A mRNAs were shown by primer extension to differ by their 5' ends. The accumulation of muscle-type phosphorylase and creatine kinase and muscle-specific aldolase A mRNA accumulation during muscle development seems to be a coordinate process occurring progressively from the 17th day of intrauterine life up to the 30th day after birth. In contrast, the 2.7-kilobase pair RNA species is maximally expressed at the 1st week after birth as is the neonatal form of myosin heavy chain mRNA.     

The genes and mRNA coding for the heavy chains of chick embryonic skeletal myosin

A size class of polysomes was isolated from chick embryonic leg skeletal muscle which synthesized almost exclusively a polypeptide chain with a molecular weight identical to the myosin heavy chain. The mRNA purified from these polysomes was shown to synthesize the 200,000 dalton polypeptide in the wheat germ cell-free translation system. At least 90% of the polypeptide had properties similar to the myosin heavy chain. Isoelectric focusing indicated that the myosin heavy chain synthesized in vitro contained two chains in equal amounts, as did purified embryonic leg skeletal muscle myosin. The kinetics of hybridization of the complementary DNA with an excess of the myosin heavy chain mRNA (MHC mRNA) indicated the presence of two different mRNA sequences. Reassociation of the cDNA to an excess of the DNA of the genome suggest that there is little, if any, reiteration of the myosin heavy chain genes.     

Construction of a human ventricular cDNA library and characterization of a beta myosin heavy chain cDNA clone

Summary We have constructed and characterized for the first time a complementary DNA (cDNA) clone, pHMC3, which codes for a cardiac myosin heavy chain mRNA from human heart. This clone contains a 1.7 kb DNA segment and specifies 543 amino acids of the carboxyl portion of the myosin heavy chain. The DNA sequence and encoded amino acid sequence were compared to the hamster alpha (pVHC1) and beta (pVHC2/pVHC3) cardiac myosin heavy chain cDNA and amino acid sequences and the rat cardiac myosin heavy chain sequences as well. The myosin heavy chain mRNAs are highly conserved and this is reflected in our cDNA clone. The pHMC3 clone is 87.9% homologous to the hamster alpha cDNA and 92.2% homologous to the hamster beta cDNA clones. The 3 untranslated region of pHMC3 is 64.1% homologous to the hamster beta clone while the hamster alpha myosin heavy chain shows only 25% homology to pHMC3 and exhibits extensive diversity. Similar results rere obtained when pHMC3 was compared to the rat cardiac myosin heavy chain cDNA sequences. The comparisons showed that pHMC3 is a beta cardiac myosin heavy chain cDNA clone.     

Structural and phylogenetic analysis of the chicken ventricular myosin heavy chain rod

Summary We have isolated and characterized five overlapping clones that encompass 3.2 kb and encode a part of the short subfragment 2, the hinge, and the light meromyosin regions of the myosin heavy chain rod as well as 143 bp of the 3 untranslated portion of the mRNA. Northern blot analysis showed expression of this mRNA mainly in ventricular muscle of the adult chicken heart, with trace levels detected in the atrium. Transient expression was seen in skeletal muscle during development and in regenerating skeletal muscle following freeze injury. To our knowledge, this is the first report of an avian ventricular myosin heavy chain sequence. Phylogenetic analysis indicated that this isoform is a distant homolog of other ventricular and skeletal muscle myosin heavy chains and represents a distinct member of the multigene family of sarcomeric myosin heavy chains. The ventricular myosin heavy chain of the chicken is either paralogous to its counterpart in other vertebrates or has diverged at a significantly higher rate.Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, IL60637, USA