Isolation and characterization of a previously unrecognized myosin heavy chain gene present in the Syrian hamster

A full length (25,000 base-pair) myosin heavy chain gene completely contained within a single cosmid clone was isolated from a Syrian hamster cosmid genomic library. Sequence comparison of the 3' untranslated region indicated the presence of a 75% homology with the rat embryonic myosin heavy chain gene. Extensive 5' flanking region regulatory element conservation was also found when the sequence was compared to the rat myosin heavy chain gene. S1 nuclease digestion analysis, however, indicated that the Syrian hamster myosin heavy chain gene exhibited expression in adult Syrian hamster ventricular tissue, as well as the adult vastus medialis, a fast twitch skeletal muscle. Expression also appears to be enhanced in myopathic relative to control hearts. This myosin heavy chain gene is neither the alpha nor beta cardiac myosin heavy chain gene, but is a unique, previously unrecognized, myosin heavy chain gene present in both myocardial and skeletal muscle tissues.     

Isolation and characterization of the complete human β-myosin heavy chain gene

Summary The entire gene coding for the human -myosin heavy chain has been isolated from genomic EMBL3A phage libraries by chromosomal walking starting from clone gMHC-1, reported earlier (Appelhans and Vosberg 1983). gMHC-1 has been shown to carry coding information for the C-terminal two-thirds of -myosin heavy chain, which is expressed in cardiac muscle and in slow skeletal muscle fibers (Lichter et al. 1986). Three DNA clones were identified as overlapping with gMHC-1 by restriction mapping and DNA sequencing. They span a 30-kb region in the genome. About 22 kb extend from the initiation codon ATG to the poly(A) addition site. The clones include about 4 kb of 5 flanking sequences upstream of the promoter. Comparisons of - and -myosin heavy chain sequences indicate that gene duplication of the cardiac myosin heavy chain isogenes preceded the mammalian species differentiation.     

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.     

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.     

Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene

We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.     

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.     

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.     

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.     

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.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

Single chicken cardiac myosin alkali light-chain gene generates two different mRNAs by alternative splicing of a complex exon

We have isolated and characterized two kinds of cDNA for the chicken cardiac myosin alkali light chain. The sequences of the two cDNAs are identical, except for a notable divergence in part of the 3' untranslated sequence. By analysis of isolated genomic clones, it was shown that the genomic sequences corresponding to the different sequences in the 3' untranslated regions of the two mRNAs were arranged within a limited part of a single stretch of DNA; also the two distinct 3' untranslated regions of the two mRNAs shared part of the last exon, which was 0.6 x 10(3) base-pairs long. There are two canonical acceptor sites available for RNA splicing in the last exon, the first being located at the 5' end of the exon, and the second at 370 base-pairs downstream from this end. Together with analysis by S1 nuclease mapping, the foregoing results lead us to conclude that, by the differential use of these two acceptor sites, a single gene generates two distinct mRNAs of 1.45 x 10(3) base-pairs and 1.1 x 10(3) base-pairs with or without the 5' half of the last exon. The two mRNAs appear to utilize the same modified poly(A) signal, AGTAAA, rather than the authentic AATAAA sequence present about 30 base-pairs downstream from the poly(A) attachment sites. This is probably because another consensus G + T-rich sequence is present at an appropriate distance from the AGTAAA sequence, but not from the AATAAA sequence. The gene for the cardiac myosin alkali light chain has proved to be expressed in ventricular muscle and in atrial and anterior latissimus dorsi muscles, the last of these being characteristic of slow skeletal muscle. In these muscles, two kinds of mRNA for the cardiac myosin alkali light chain, identical with those in ventricular muscle, were expressed and their relative amount in each tissue was almost the same as that in ventricular muscle.     

Isolation of a non-muscle myosin heavy chain gene from Acanthamoeba

We have isolated a non-muscle myosin heavy chain gene from Acanthamoeba castellanii using as a heterologous probe a sarcomeric myosin heavy chain gene from Caenorhabditis elegans. The amoeba genomic clone has been tentatively identified as containing a myosin II heavy chain gene based on hybridization to a 5300-nucleotide RNA species, hybrid selection of a mRNA encoding a 185-kDa polypeptide, specific immunoprecipitation of this polypeptide with antiserum to myosin II, and an exact match between the DNA sequence and a carboxyl-terminal myosin II peptide previously sequenced by protein chemical methods (C?té, G.P., Robinson, E.A., Appella, E., and Korn, E. D. (1984) J. Biol. Chem. 259, 12781-12787). We also sequenced a region of the gene whose deduced amino acid sequence shows strong homology with that region of muscle myosins which is thought to be involved in nucleotide binding. These results indicate that the amoeba genomic clone contains at least 90% of the coding information for the 185-kDa heavy chain polypeptide and that the bulk of the gene contains very little intron DNA. Genomic blots of amoeba DNA probed with a portion of this myosin gene indicate the presence of additional highly related sequences within the amoeba genome.     

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.     

Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1047-1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3'-carboxyl coding region and a portion of the 3'-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry 27, 3807-3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.     

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.     

A repressor region in the human beta-myosin heavy chain gene that has a partial position dependency.

Expression of the human beta-myosin heavy chain (beta MHC) gene was studied by transient assay in culture and in situ by direct injection of plasmids into adult rat hearts. In this report we describe a unique repressor region located -326/-309 (5'-TTGGTGGTCGTGGTCAGT-3') of the human beta MHC gene that is conserved among the rat, rabbit, and human beta MHC genes. This sequence conferred repression onto heterologous promoters when the sequence was located 5' but not 3' to the promoters. This partial positional dependency suggests that the factor may act by limiting the binding of enhancers, located more proximally, to their DNA binding sites.