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.     

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.     

The human embryonic myosin alkali light chain gene: use of alternative promoters and 3'' non-coding regions.

Recently we have found evidence that the human embryonic myosin alkali light chain (MLC1 emb) gene has two functional promoters and that its mRNAs exhibit heterogeneity in their 3'untranslated regions (UTR). To study this more in detail we have isolated and characterized the human MLC1emb gene. We focussed in particular on 2 kilobases of 5'flanking region and the alternative 3'UTRs. RNA primer extension and S1 mapping analyses revealed that the MLC1emb gene can indeed be driven either by a proximal or a distal promoter, both in fetal and adult cardiac tissue. These MLC1emb RNAs can contain either the proximal or distal 3'UTR. In contrast to this, in fetal as well as adult masseter muscle MLC1emb mRNA is predominantly transcribed from the proximal promoter and contains mainly the distal 3'UTR. These results explain the known heterogeneity of MLC1emb mRNAs. Finally, we present evidence that the murine MLC1emb gene also contains a functional distal promoter element which has hitherto been undetected.     

Molecular cloning and characterization of human atrial and ventricular myosin alkali light chain cDNA clones

We have isolated essentially full-length cDNA clones for atrial (ALC1) and ventricular (VLC1) myosin alkali light chains from a human fetal heart cDNA library. Comparison of overall nucleotide sequences of ALC1 and VLC1 cDNA clones has revealed that, while these two inserts show significant DNA sequence homology (78.4%) with respect to their coding regions, the 5'- and 3'-untranslated regions are highly divergent. Our statistical analysis suggests that human ALC1 and VLC1 diverged approximately 300 million years ago, during the time of separation of birds and mammals. RNA blot analysis shows that ALC1 mRNA is expressed in fetal ventricular and fetal skeletal muscles as well as fetal and adult atrial muscles and VLC1 mRNA is expressed in adult slow skeletal muscle as well as fetal and adult ventricular muscles. Southern blot analysis indicates that each protein is encoded by a single gene. Finally, we show that VLC1 mRNA is induced in pressure-overloaded human atrium.     

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.     

Assignment of the human fast skeletal muscle myosin alkali light chains gene (MLC1F/MLC3F) to 2q 32.1-2qter

Summary A DNA probe derived from a mouse intronless pseudogene including coding regions for the myosin fast skeletal muscle alkali light chains, MLC1_F/MLC3_F (suggested HGM symbol, MYL1), was tested on a panel of 25 independent man-rodent somatic cell hybrids in order to assign the human MLC1_F/MLC3_F gene to a human chromosome. A 3.7-kb TaqI human fragment was found to correlate with the presence of chromosome 2 in the hybrids, characterized both by cytogenetic analysis and reference enzyme markers. A regional assignment to 2q32.1-qter was possible using hybrids whose human parental strains bore a reciprocal translocation t(X;2) (p22;q32.1). The fact that IDH1 and the MLC1_F/MLC3_F gene are closely linked on chromosome 1 in the mouse and map to the same region of human chromosome 2 in man indicates, that these chromosomes have a conserved region of homology between them and that the human 3.7-kb TaqI fragment corresponds indeed to a functional gene.     

Molecular cloning and characterization of mutant and wild-type human beta-actin genes.

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.     

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.     

Transition of myosin isozymes during development of human masseter muscle. Persistence of developmental isoforms during postnatal stage

Previous results have shown that the adult human masseter muscle contains myosin isoforms that are specific to early stages of development in trunk and limb muscles, i.e. embryonic and fetal (neonatal) myosin heavy chains (MHC) and embryonic myosin light chain (MLC1emb). We wanted to know if this specific pattern is the result of a late maturation or of a distinct evolution during development. We show here that the embryonic and the fetal MHC and the MLC1emb are expressed throughout perinatal and postnatal masseter development. Our results also demonstrate that MLC1emb accumulation increases considerably during the postnatal period. In addition, both the slow MLCs and the slow isoform of tropomyosin are expressed later in the masseter than quadriceps and the fast skeletal muscle isoform MLC3 is not detected during fetal and early postnatal development in the masseter whereas it is expressed throughout fetal development in the quadriceps. Our results thus confirm previous histochemical data and demonstrate that the masseter muscle displays a pattern of myosin and tropomyosin isoform transitions different to that previously described in trunk and limb muscles. This suggests that control of masseter muscle development involves mechanisms distinct from other body muscles, possibly as a result of either its craniofacial innervation or of a possibly different embryonic origin.