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.     

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.     

Three fast myosin heavy chains in adult rat skeletal muscle

A new fast myosin heavy chain isoform was electrophoretically detected in adult rat skeletal muscles. It was present at high levels in diaphragm and, therefore, designated as MHCIId. Appreciable amounts of MHCIId were detected in tongue musculature, the extraocular muscles, and in the deep red portions of various fast muscles. Its concentration in fast-twitch muscle was greatly increased by chronic stimulation.     

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.     

Expression of myosin light chains during fetal development of human skeletal muscle.

The expression of myosin light chains (MLCs) during the development of human skeletal muscle was investigated by using two different two-dimensional electrophoretic techniques. In both electrophoretic systems the predominant light chain 1 (LC1) expressed during the whole fetal period was found to co-migrate with the adult fast LC1 (LC1F). The main LC2 expressed during the whole fetal period was found to be different from the main fast LC2 (LC2F) and slow LC2 (LC2S) usually present in adult muscle, but co-migrated with a minor component often present in adult muscle. This fetal LC2 was phosphorylatable, and the phosphorylated form co-migrated with the main component of LC2F expressed in the adult. The adult fast LC3 appeared as early as week 20 of gestation, whereas the adult slow light chains (LC1S and LC2S) appeared only during the late fetal period. A minor component of LC1, previously described in humans as an 'embryonic LC' (LCemb.) [Strohman, Micou-Eastwood, Glass & Matsuda (1983) Science 221, 955-957], was only expressed in the early fetal period and was found to co-migrate with atrial LC1 (ALC1). We discuss the expression of these specific developmental forms of MLCs co-existing with immature myosin heavy chains during fetal life.     

Possible presence of the difference peptide in alkali light chain 1 of fish fast skeletal myosin

1. Presence of N-terminal peptide ("difference peptide") in alkali light chain 1 (A1) of fish fast skeletal myosin was examined by comparing two kinds of light chain-based myosin subfragment 1 (S1) isozymes from the yellowtail Seriola quinqueradiata. 2. On tryptic digestion, A1 was cleaved to a smaller fragment (mol. wt decrement by 2000) along with the cleavage of S1 heavy chain, while A2 was resistant to trypsin. Two-dimensional gel electrophoresis showed that A1 released a basic peptide by tryptic digestion. 3. Both S1 isozymes showed clear kinetic differences in actin-activated Mg-ATPase activity, suggesting a higher affinity of A1 for actin. Affinity of A2 for heavy chain was also estimated to be about 2-fold higher than that of A1, as judged by the model experiments in which rabbit S1 isozymes were hybridized with heterologous alkali light chains.     

2-D analysis of human skeletal muscle myosin light chains with immobilized pH gradients

Myosin light chains (LC) are a low molecular mass fraction non-covalently bound to the heavy chains. They are present in the myosin molecules and exhibit various degrees of polymorphism among the different species. By utilizing a highly-resolving 2-D technique, in narrow immobilized pH gradients, we have compared the LC forms of skeletal muscle in human and rabbit. Our findings: (1) both forms, LC1 and LC3, migrate in the two species with rather similar electrophoretic constants (both in terms of pI and Mr); (2) the LC2 forms of rabbit and humans exhibit the same Mr but quite different pI values, the rabbit forms being more acidic; (3) the chain LC2Sb is resolved into two spots in both rabbit and humans. In the former, the two bands have equal intensity, while in the latter the high pI component is clearly the most abundant.     

Effect of myosin alkali light chains on myosin subfragment 1 interaction with actin in solution and in ghost muscle fiber

At low ionic strength (7-25 mM) Mg2(+)-ATPase of myosin subfragment 1 (S1) isoforms containing alkali light chain A1 [S1(A1)] is activated by actin 1.5-2.5 times as strongly as Mg2(+)-ATPase of S1 isoforms containing alkali light chain A2[S1(A2)]. Data from analytical ultracentrifugation suggest that at low ionic strength in the absence of ATP in solution S1(A1) displays a higher affinity for F-actin than S1(A2). Such a higher affinity of S1(A1) for F-actin was also demonstrated by experiments, in which the interaction of S1 isoforms fluorescently labeled by 1.5-IAEDANS with F-actin of ghost fibers (single glycerinated muscle fibers containing F-actin but devoid of myosin) was studied. Using polarization microfluorimetry, it was shown that the interaction of both S1 isoforms with ghost fiber F-actin induces similar changes in the parameters of polarized tryptophan fluorescence. At the same time the mobility of the fluorescent probe, 1.5-IAEDANS, specifically attached to the SH-group of Cys-374 in the C-terminal region of action is markedly decreased by S1(A1) and is only slightly affected by S1(A2). The data obtained suggest that S1(A1) and S1(A2) interact with the C-terminal region of the actin molecule in different ways, i.e. S1(A1) is attached more firmly than S1(A2). This may be due to the existence of contacts between the alkali light chain of A1 of S1(A1) and the C-terminal region of actin as well as to the absence of such contacts in the case of S1(A2).     

Evolutionarily conserved promoter motifs and enhancer organization in the mouse gene encoding the IIB myosin heavy chain isoform expressed in adult fast skeletal muscle.

We have isolated the mouse gene for the MHC isoform expressed in adult type IIB (fast-contracting, glycolytic) skeletal muscle fibers, and determined the DNA sequence of the promoter region. This sequence represents the first example of a promoter for a gene encoding an adult-specific isoform of a mammalian skeletal MHC. The proximal 200 bp of the promoter contains several sequence motifs which are identical or very similar to homologous motifs found in the promoters of a family of chicken skeletal MHC genes. Of these, two novel AT-rich sequences may be important for regulation of the promoter. A second feature of the mouse IIB MHC promoter sequence concerns a number of sequence motifs located at ca. -1,000 bp which are organized in a similar fashion in the IIB MHC promoter and a homologous region of promoter of the mouse muscle creatine kinase gene.     

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.     

Studies on the alkali light chains of vertebrate skeletal muscle myosin. Effect of tyrosyl modification on the ability to reassociate to heavy chains

The structures of the alkali light chain subunits A1 and A2 have been studied by examining the effect of the conformationally sensitive reagent tetranitromethane, which reacts specifically with tyrosyl residues. Whereas reaction in the presence of 6 M guanidine hydrochloride results in modification of the three tyrosyl residues of both these light chains, only two tyrosyl residues are exposed to the reagent in the native conformations of these proteins. By gel chromatography of the CNBr-cleaved chains it was demonstrated that the two reactive tyrosyls are those located in the CB-1 and CB-3 segments and that these tyrosyl residues are modified simultaneously and not sequentially. The unreactive tyrosyl residue is in the CB-6 segment and is separated by two residues from the single cysteinyl residue of these chains. It is found that the modified light chains cannot be made to reassociate with the heavy chains by the NH4Cl hybridization procedure of Wagner and Weeds [J. Mol. Biol. 109, 455-470 (1977)] or by the thermal hybridization procedure [Burke and Sivaramakrishnan (1981) Biochemistry 20, 5908-5913]. Furthermore, reduction of the nitrotyrosyl groups to aminotyrosyl residues by sodium dithionite does not restore this effect. The data suggest that regions of the light chains at CB-1 and CB-3 are involved in the association to the heavy chains.     

Differences in the transient response of fast and slow skeletal muscle fibers. Correlations between complex modulus and myosin light chains

Sinusoidal analysis of the mechanochemical properties of skinned muscle fibers under conditions of maximal activation was applied to fibers from several rabbit skeletal muscles (psoas, tibialis anterior, extensor digitorum longus, diaphragm, soleus, semitendinosus). This investigation distinguished between two general classes of fibers, which on the basis of their myosin light chain complements could be classified as fast and slow. In fast fibers (e.g., psoas) we identified the presence of at least three exponential processes (A), (B), (C) of comparable magnitudes. In slow fibers (e.g., soleus) we identified the presence of at least four exponential processes (A)-(D) of very different magnitudes; magnitudes of processes (A) and (B) are very small compared with those of (C) and (D). The apparent rate constants are 8-29-fold slower in slow fibers. Because our sinusoidal characterization takes less than or equal to 22 s and does not involve chemical denaturation or other means of disruption of the myofilament lattice, it allows the different physiological classes of fibers to be characterized and then studied further by other techniques. The perfect correlation between physiological and molecular properties as assayed by gel electrophoresis after sinusoidal analysis demonstrates this and justifies its use in distinguishing between fiber types.     

Coexistence of cardiac-type and fast skeletal-type myosin light chains in embryonic chicken cardiac muscle

Myosin from embryonic chicken ventricle contained a light chain component which comigrated with fast skeletal myosin light chain 1 (Lf1) on two dimensional electrophoresis in addition to cardiac type light chains (Lc1 and Lc2). Immunoblot analysis showed that this minor light chain band reacted with anti-Lf1 antibody. Antigens binding with anti-Lc1 and anti-Lf1 antibodies were located on myofibrils in embryonic cardiac muscle cells in vivo and in vitro. From these observations, we conclude that a small amount of Lf1 exists in embryonic chicken cardiac muscle.     

Slow muscle myoblasts differentiating in vitro synthesize both slow and fast myosin light chains

Whether fast and slow skeletal muscles of the embryo develop from cells of a common origin or from two separate cellular origins is not known. Recent evidence suggests that prior to innervation all muscles of the embryo are of one type, the fast type, i.e., all synthesize fast but not slow myosin light chains. Innervation has been thought to play the central role in the shift of a fast to a slow muscle. Experiments reported here demonstrate that myoblasts from slow muscle regions of the embryo when isolated in tissue culture differentiate into myotubes which synthesize both fast and slow myosin light chains, and that innervation is not required to initiate slow myosin light-chain synthesis.     

Modification of the alkali light chains of skeletal myosin inhibits actin binding and adenosine triphosphate cleavage.

Heavy meromyosin treated with the ATP analog, 6,6'-dithiobis(inosinyl-5'-yl imidodiphosphate), (slppNHp)2, in the presence of adenyl-5'-yl imidodiphosphate at 0 degrees loses its EDTA-ATPase activity and actin binding ability in a parallel manner. Studies with myosin show that under the above conditions (slppNHp)2 reacts preferentially with the single cysteines of the alkali light chains (Mr = 20,700 and 16,500) suggesting a role for these subunits in regulating actin-myosin interaction and ATP cleavage.