Alkali myosin light chains in man are encoded by a multigene family that includes the adult skeletal muscle, the embryonic or atrial, and nonsarcomeric isoforms

A set of cDNA clones coding for alkali myosin light chains (AMLC) was isolated from fetal human skeletal muscle. Nucleotide sequence analysis and RNA expression patterns of individual clones revealed related sequences corresponding to (i) fast fiber type MLC1 and MLC3; (ii) the embryonic MLC that is also expressed in fetal ventricle and adult atrium (MLCemb); and (iii) a nonsarcomeric MLC isoform that is found in all nonmuscle cell types and smooth muscle. The AMLC gene family in man comprises unique copies for MLC1, MLC3 and MLCemb, and multiple copies for the nonsarcomeric MLC genes. The gene coding for MLC1 and MLC3 is located on human chromosome 2.     

Structure and sequence of the myosin alkali light chain gene expressed in adult cardiac atria and fetal striated muscle

Mammalian cardiac muscle contains two myosin alkali light chains which are the major isoforms present in either atrial (MLC1A) or ventricular (MLC1V) muscle, and which are different from the fast skeletal muscle isoforms (MLC1F and MLC3F). The atrial isoform is also expressed in fetal skeletal and fetal ventricular muscle, where this isoform is also described as the fetal isoform MLC1emb. We have previously isolated a cDNA clone encoding part of the mouse MLC1A/MLC1emb isoform and have used this clone to demonstrate the identity of MLC1A and MLC1emb in the mouse. To date no information on the amino acid sequence of this mammalian atrial/fetal isoform has been available. Here we present the complete structure and sequence of the mouse MLC1A/MLC1emb gene, together with the predicted amino acid sequence of this isoform. Comparison of the MLC1A/MLC1emb gene and polypeptide with those of MLC1F and MLC1V suggests that MLC1A/MLC1emb and MLC1V were generated from a common ancestral gene. The NH2-terminal region of MLC1A/MLC1emb, thought to be involved in the actomyosin interaction, shows conservation with MLC1V but not with MLC1F suggesting a shared functional domain in these cardiac isoforms. Comparison with the chicken embryonic MLC (L23) suggests that although MLC1A/MLC1emb and L23 show very different patterns of expression, both during development and in the adult, they probably represent the homologous gene in these two species.     

A common myosin light chain is expressed in chicken embryonic skeletal, cardiac, and smooth muscles and in brain continuously from embryo to adult

We have isolated cDNA clones of the mRNA for chick embryonic myosin light chain (MLC), L23, by cross-hybridization with chicken skeletal muscle MLC1 cDNA. The identification of the isolated cDNAs was carried out by in vitro translation of hybrid-selected mRNA. Sequence analysis of the cloned cDNAs revealed that the cDNA insert contained 832 nucleotides and predicted a polypeptide of 185 amino acids with a calculated molecular weight of 20,687. The deduced amino acid sequence for L23 showed high sequence similarities to those of adult alkali type MLCs from various tissues, indicating that L23 belongs to the alkali MLC group. Using the cloned cDNA as a hybridization probe, we have revealed by RNA blot analysis that the expression of L23 mRNA was regulated in temporal and tissue-specific manners. The L23 mRNA of 1.1 kilobases is transiently expressed in embryonic skeletal, cardiac, and smooth muscles of chickens. It is also found in the brain of chickens during all stages of development so far investigated. Only a single gene for L23 was detected by Southern blot of chick genomic DNA. We therefore suggest that L23 is expressed from a single gene in both embryonic muscles and brain.     

Nonmuscle and smooth muscle myosin light chain mRNAs are generated from a single gene by the tissue-specific alternative RNA splicing

We have isolated two cDNA clones for myosin alkali light chain (MLC) mRNA from two respective cDNA libraries of chick gizzard and fibroblast cells by cross-hybridization to the previously isolated cDNA of skeletal muscle MLC. Sequence analysis of the two cloned cDNAs revealed that both of them are homologous to but distinct from the cDNA sequence used as the probe so that they may be classified into members of the MLC family, that they are identical with each other in the 3' and 5' untranslated sequence as well as in the coding sequence with a notable exception of a 39-nucleotide insertion in the fibroblast cDNA, 26 nucleotides of which are used for encoding the C-terminal amino acid sequence, and, therefore, that they encode the identical 142-amino acid sequence with different C-terminals of nine amino acids, each specific for fibroblast and gizzard smooth muscle MLC. The position of the inserted block corresponds exactly to one of the exon-intron junctions in the other MLC genes whose structures have so far been elucidated. DNA blot analysis suggested that the two MLC mRNAs of gizzard (smooth muscle) and fibroblast cells (nonmuscle) are generated from a single gene, probably through alternative RNA splicing mechanisms. RNA blot analysis and S1 nuclease mapping analysis using RNA preparations from fibroblast and gizzard tissues showed that the fibroblast MLC mRNA is expressed predominantly in fibroblast cells, but not, or very scantily if at all, in the gizzard, whereas the reverse is true for the gizzard smooth muscle MLC mRNA.     

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.     

Differential regulation of the atrial isoforms of the myosin light chains during striated muscle development.

We have isolated a cDNA that encodes the human regulatory myosin light chain isoform predominant in adult atrial muscle. The cDNA contains an open reading frame of 175 amino acids and encodes a hydrophilic protein of a largely helical structure with two potential phosphorylation sites. The protein is different from any other regulatory myosin light chain so far described and is the product of a previously uncharacterized single copy gene. An isoform-specific probe was used to analyze the expression of this isoform in adult muscle and in cardiac and skeletal muscle development in vivo and in vitro. Parallel analysis of the corresponding human alkali myosin light chain (predominant in adult atrium) showed that both isoforms are expressed in early heart development, in both atrium and ventricle. Although the atrial alkali light chain is expressed throughout embryonic striated muscle development, the regulatory myosin light chain was not detected in skeletal myogenesis in vivo or in vitro. Thus the atrial isoforms are not universally or exclusively "paired" and can be independently regulated. We propose that the manner in which these particular isoforms fulfill the functional requirements of the muscle at different developmental times may have direct impact on their regulation.     

Isolation and sequencing of porcine fast skeletal muscle alkali myosin light chain 3 cDNA

Abstract

A cDNA coding for alkali myosin light chain 3 (MLC3_F) was isolated from a porcine skeletal muscle library. This clone has an insert of 859 bp encompassing the complete CDS (coding sequence) plus the 5’ and 3’ untranslated regions. Computer analysis showed that porcine MLC3_F cDNA is highly homologous to the corresponding cDNAs of human, rabbit and rat. Moreover, Northern analysis showed the presence of two bands that represent the mature mRNAs of the MLCl_F and MLC3_F isoforms according to data observed in other species.     

Mammalian nonsarcomeric myosin regulatory light chains are encoded by two differentially regulated and linked genes [published erratum appears in J Cell Biol 1990 Nov;111(5 Pt 1):2207]

The myosin 20,000-D regulatory light chain (RLC) has a central role in smooth muscle contraction. Previous work has suggested either the presence of two RLC isoforms, one specific for nonmuscle and one specific for smooth muscle, or the absence of a true smooth muscle-specific isoform, in which instance smooth muscle cells would use nonmuscle isoforms. To address this issue directly, we have isolated rat RLC cDNAs and corresponding genomic sequences of two smooth muscle RLC based on homology to the amino acid sequence of the chicken gizzard RLC. These cDNAs are highly homologous in their amino acid coding regions and contain unique 3'-untranslated regions. RNA analyses of rat tissue using these unique 3'-untranslated regions revealed that their expression is differentially regulated. However, one cDNA (RLC-B), predominantly a nonmuscle isoform, based on abundant expression in nonmuscle tissues including brain, spleen, and lung, is easily detected in smooth muscle tissues. The other cDNA (RLC-A; see Taubman, M., J. W. Grant, and B. Nadal-Ginard. 1987. J. Cell Biol. 104:1505-1513) was detected in a variety of nonmuscle, smooth muscle, and sarcomeric tissues. RNA analyses comparing expression of both RLC genes with the actin gene family and smooth muscle specific alpha-tropomyosin demonstrated that neither RLC gene was strictly smooth muscle specific. RNA analyses of cell lines demonstrated that both of the RLC genes are expressed in a variety of cell types. The complete genomic structure of RLC-A and close linkage to RLC-B is described.     

Functional activity of the two promoters of the myosin alkali light chain gene in primary muscle cell cultures: comparison with other muscle gene promoters and other culture systems.

Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters.     

cDNA sequence, tissue-specific expression, and chromosomal mapping of the human slow-twitch skeletal muscle isoform of troponin I

Troponin I (TnI) is a myofibrillar protein involved in the calcium-mediated regulation of striated muscle contraction. Three isoforms of TnI are known and each is expressed in a muscle fiber-type-specific manner. TnI-fast and TnI-slow are expressed exclusively in fast-twitch and slow-twitch skeletal muscle myofibers, respectively, while a third isoform, TnI-card, is expressed in both the atrium and the ventricle of the heart. An explanation of the myofiber-type-restricted expression of the troponin I multigene family will further aid in understanding how various types of striated muscle fibers are established. To initiate the study of TnI isoform gene expression, we have isolated a full-length cDNA representing the human slow-twitch skeletal muscle isoform of troponin I. Sequence comparisons demonstrate that the TnI-slow protein is highly conserved between species. Therefore, the cDNA was used as a probe to investigate the tissue-specific and developmental regulation of the TnI-slow gene in both rodent and human myogenic cells. TnI-slow message appears to be restricted to muscle tissue containing slow-twitch skeletal muscle myofibers. TnI-slow gene expression is induced in differentiated cultures of primary human muscle cells and several (but not all) myogenic cell lines. In addition, a human-specific probe prepared from the 3' untranslated region of the cDNA has been used to probe a panel of human/mouse somatic cell hybrid lines, resulting in the assignment of the human TnI-slow gene to the q12----qter region of chromosome 1. The locus is designated TNNI1.     

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.     

Characterization and differential expression of human vascular smooth muscle myosin light chain 2 isoform in nonmuscle cells

The 20-kDa regulatory myosin light chain (MLC), also known as MLC-2, plays an important role in the regulation of both smooth muscle and nonmuscle cell contractile activity. Phosphorylation of MLC-2 by the enzyme MLC kinase increases the actin-activated myosin ATPase activity and thereby regulates the contractile activity. We have isolated and characterized an MLC-2 cDNA corresponding to the human vascular smooth muscle MLC-2 isoform from a cDNA library derived from umbilical artery RNA. The translation of the in vitro synthesized mRNA, corresponding to the cDNA insert, in a rabbit reticulocyte lysate results in the synthesis of a 20,000-dalton protein that is immunoreactive with antibodies raised against purified chicken gizzard MLC-2. The derived amino acid sequence of the putative human smooth muscle MLC-2 shows only three amino acid differences when compared to chicken gizzard MLC-2. However, comparison with the human cardiac isoform reveals only 48% homology. Blot hybridizations and S1 nuclease analysis indicate that the human smooth muscle MLC-2 isoform is expressed restrictively in smooth muscle tissues such as colon and uterus and in some, but not all, nonmuscle cell lines. Previously reported MLC-2 cDNA from rat aortic smooth muscle cells in culture is ubiquitously expressed in all muscle and nonmuscle cells, and it was suggested that both smooth muscle and nonmuscle MLC-2 proteins are identical and are probably encoded by the same gene. In contrast, the human smooth muscle MLC-2 cDNA that we have characterized from an intact smooth muscle tissue is not expressed in skeletal and cardiac muscles and also in a number of nonmuscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and ontogenetic expression analysis of the myosin light chains from the fast white muscle of mandarin fish Siniperca chuatsi

Three full-length complementary DNA (cDNA) clones were isolated encoding the skeletal myosin light chain 1 (MLC1; 1237 bp), myosin light chain 2 (MLC2; 1206 bp) and myosin light chain 3 (MLC3; 1079 bp) from the fast white muscle cDNA library of mandarin fish Siniperca chuatsi. The sequence analysis indicated that MLC1 and MLC3 were not produced from differentially spliced messenger RNAs (mRNA) as reported in birds and rodents but were encoded by different genes. The MLC2 encodes 170 amino acids, which include four EF-hand (helix-loop-helix) structures. The primary structures of the Ca(2+)-binding domain were well conserved among the MLC2s of seven other fish species. The ontogenetic expression analysis by real-time PCR showed that the three light-chain mRNAs were first detected in the gastrula stage, and their expression increased from the tail bud stage to the larval stage. All three MLC mRNAs showed longitudinal expression variation in the fast white muscle of S. chuatsi, especially MLC1 which was highly expressed at the posterior area. Taken together, the study provides a better understanding about the MLC gene structure and their expression pattern in muscle development of S. chuatsi.     

Isolation and characterization of the gene for myosin light chain two of Drosophila melanogaster

A recombinant lambda-phage DNA clone containing Drosophila melanogaster sequences encoding the gene for myosin light chain (MLC) two has been isolated from a library of randomly sheared DNA. The Drosophila MLC2 gene is located in region 99E1-3 on the right arm of chromosome 3, several bands removed from the site reported for the other myosin light chain gene at 98B. The MLC2 sequence at 99E1-3 appears to encode all of the isoforms of Drosophila MLC2. The polypeptide encoded at 99E was identified as MLC2 by the following criteria: the in vitro translation product is identical in size to MLC2 isolated from Drosophila muscle, and on two-dimensional gels the in vitro translation product can be separated into two or more peptides that co-migrate with isoforms of larval and thoracic MLC2. RNA encoding the polypeptide was detected in embryos only after the onset of muscle differentiation and was also abundant in adult thoracic muscle. The nucleotide sequence of cDNA generated from late embryonic RNA would be translated to yield a protein sequence with multiple regions of homology to vertebrate MLC2. (There are shorter regions of homology to vertebrate MLC1). Like a number of vertebrate muscle proteins, Drosophila MLC2 has an acetylated amino-terminus.     

The myosin alkali light chains of mouse ventricular and slow skeletal muscle are indistinguishable and are encoded by the same gene

We have isolated a cDNA recombinant plasmid (pA29) identified as encoding part of the ventricular muscle myosin light chain MLC1v. This cDNA contains a 300-base pair fragment which under conditions of moderate stringency shows specific hybridization to MLC1v mRNA with no detectable cross-hybridization with the mRNAs encoding the fast skeletal muscle isoforms MLC1F and MLC3F, or the atrial muscle isoform MLC1A. Under these conditions hybridization is seen with an abundant mRNA present in slow skeletal muscle (soleus) which is indistinguishable from ventricular MLC1V mRNA on the basis of size and of thermal stability of hybrids formed with plasmid pA29. The mouse MLC1V and MLC1S proteins are found to co-migrate on two-dimensional gels. We therefore conclude that these isoforms are the same and are encoded by the same mRNA. Analysis of mouse DNA has identified a single region of the genome which hybridizes to this same fragment of pA29. This region has been isolated in a recombinant phage and has been shown to contain a single gene showing homology with MLC1V mRNA by R-loop analysis. We therefore conclude that MLC1V and MLC1S are encoded by a single gene. The pattern of segregation of a restriction fragment length polymorphism identified for this gene between Mus musculus and Mus spretus has been followed in an F1 backcross between these two mouse species. The results show the MLC1V/MLC1S gene to be closely linked to a marker at the distal end of mouse chromosome 9.