Myosin Light Chain Gene Research Articles

Cardiac myosin heavy and light chain gene expression in hypertrophy and heart failure

Cardiac myosin heavy and light chain gene expression in hypertrophy and heart failure

Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during alpha-adrenergic stimulation of neonatal rat ventricular cells. Identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression.

To study the mechanisms which mediate the transcriptional activation of cardiac genes during alpha adrenergic stimulation, the present study examined the regulated expression of three cardiac genes, a ventricular embryonic gene (atrial natriuretic factor, ANF), a constitutively expressed contractile protein gene (cardiac MLC-2), and a cardiac sodium channel gene. alpha 1-Adrenergic stimulation activates the expression and release of ANF from neonatal ventricular cells. As assessed by RNase protection analyses, treatment with alpha-adrenergic agonists increases the steady-state levels of ANF mRNA by greater than 15-fold. However, a rat cardiac sodium channel gene mRNA is not induced, indicating that alpha-adrenergic stimulation does not lead to an increase in the expression of all cardiac genes. Studies employing a series of rat ANF luciferase and rat MLC-2 luciferase fusion genes identify 315- and 92-base pair cis regulatory sequences within an embryonic gene (ANF) and a constitutively expressed contractile protein gene (MLC-2), respectively, which mediate alpha-adrenergic-inducible gene expression. Transfection of various ANF luciferase reporters into neonatal rat ventricular cells demonstrated that upstream sequences which mediate tissue-specific expression (-3003 to -638) can be segregated from those responsible for inducibility. The lack of inducibility of a cardiac Na+ channel gene, and the segregation of ANF gene sequences which mediate cardiac specific from those which mediate inducible expression, provides further insight into the relationship between muscle-specific and inducible expression during cardiac myocyte hypertrophy. Based on these results, a testable model is proposed for the induction of embryonic cardiac genes and constitutively expressed contractile protein genes and the noninducibility of a subset of cardiac genes during alpha-adrenergic stimulation of neonatal rat ventricular cells.

Paired MyoD-binding sites regulate myosin light chain gene expression.

The muscle-specific enhancer element located downstream of the myosin light chain (MLC) locus encoding MLC1 and MLC3 contains three binding sites (A, B, and C) for the myogenic determination factor MyoD. A 173-base-pair region of the MLC gene enhancer, including these three sites, retains full enhancer function when transfected into muscle cells. Whereas mutation of either upstream MyoD binding site (A or B) has a mild effect on muscle-specific enhancer activity, mutation of the third MyoD binding site (C) substantially weakens the enhancer, both in muscle cells or in nonmuscle cells cotransfected with a MyoD, myogenin, or myf5 expression vector. Site C is necessary but insufficient, since double mutation of two MyoD binding sites (A plus B) abrogates enhancer activity. Thus, site C requires either site A or B for enhancer function. This study shows a hierarchy of function among the three MyoD binding sites in the MLC enhancer. We propose that a protein-DNA complex is formed with at least two of these sites (A and C or B and C) to effect activation of the locus encoding MLC1/3 during myogenesis.

A Novel Expression System for Production of a Labile Protein in Escherichia coli by infection with Cytosin-substituting T4 Phage.

A novel expression system was developed for the high level production of a labile protein in Escherichia coli. The regulatory signal of bacteriophage T4 uvsY gene was fused in frame with the coding region of human ventricular myosin alkali light chain (VLC1) gene. Expression from the regulatory signal was enhanced and continued in a lysis-inhibition state by infection with a cytosine-substituting T4 phage mutant. VLC1 protein was produced at a low level without infection because of its instability in the cells. Although the productivity was partly improved in a lon-deficient mutant without infection, it was improved about 100-fold with T4 phage infection. T4 phage produces protease inhibitor(s) (pin gene product) against proteases of host cell including the lon gene product (protease La).

Functional identification of the transcriptional regulatory elements within the promoter region of the human ventricular myosin alkali light chain gene.

We have identified and functionally characterized DNA sequences that regulate the expression of the human ventricular/slow twitch isoform of myosin alkali light chain (VLC1) gene. By using primer extension and S1 nuclease mapping techniques, we have shown that the VLC1 gene is transcribed from the identical site in the ventricular and slow twitch skeletal muscles. Comparison of the VLC1 sequences from +1 to -1296 in the genes for human and mouse showed that the 5'-proximal flanking region, up to about 220 nucleotides, was highly conserved (83% homology). To determine the location of sites that may be important for the function of the VLC1 promoter, a series of transient expression vectors containing progressive deletions of the VLC1 gene 5'-flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into myogenic and nonmyogenic cells. Deletion mutagenesis of sequences between -357 and +40 revealed the presence of positive and negative activity in all the cells tested. We demonstrated that the minimal promoter sequence required to generate muscle cell-specific expression is the region between -94 to -64 upstream from the cap site and a sequence element located between -107 and -94 was found to have a positive effect in both myogenic cells and nonmyogenic cells. These two proximal regions located between -107 and -64 appear to act together to determine the cell type-specific high level expression of the VLC1 gene in muscle cells. Competition gel retardation assays revealed that the CArG sequence located between -96 and -87 interacts specifically with nuclear extracts from myogenic and nonmyogenic cells and compete for binding with the CArG sequence present in the human cardiac alpha-actin gene and with the serum response element of the c-fos gene. These results strongly suggested that similar, if not identical, the CArG box binding proteins interact with the functionally different promoter element in the VLC1, cardiac alpha-actin, and c-fos genes.

Activation of the interferon system during myogenesis in vitro

Abstract Differentiation of skeletal muscle involves withdrawal of myoblasts from cell replication, fusion to form multinucleated myotubes, coordinate appearance of a variety of muscle-specific proteins and the disappearance of a set of other proteins responsible for cell growth. The possible activation of the interferon (IFN) system in this process was studied. Thus, the activity of two IFN-induced enzymes known to be part of the system – (2′-5′) oligoadenylate synthetase (2–5A synthetase) and double-stranded RNA-activated protein kinase as well as the expression of 2–5A synthetase coding genes were examined during myogenesis. It is demonstrated that the activity of the enyzmes is transiently increased in cultured myoblasts, reaching a peak activity on the 3rd day in culture and then declining to a basal level. This peak activity precedes both cell fusion and the appearance of muscle-specific proteins – acetylcholine receptors (AChR) and creatine kinase. The same kinetics of 2–5A synthetase activity was evident in myoblasts from chick, rat or mouse origin. The enzymatic product appears to be primarily the trimer form of 2–5A, rather than a set of oligomers observed in enzymatic reactions performed on IFN-treated cells, including muscle cultures. The kinetics of 2–5A synthetase gene expression revealed that the largest amount of specific RNA transcripts appeared on the 1st day after seeding, followed by a reduction thereafter. In addition, a decrease was also observed in expression of c-myc, a cell-growth-associated protooncogene. However, an increase towards the 2nd day of both AChR and myosin light chain gene expression was evident, indicating selective regulation of gene expression during myogenesis.

Transcriptional regulation of actin and myosin genes during differentiation of a mouse muscle cell line

Abstract During terminal differentiation of skeletal muscle cells in vitro there is a transition from a predominantly nonmuscle contractile protein phenotype to a sarcomeric contractile protein phenotype. In order to investigate whether this transition and subsequent changes in expression are primarily transcriptionally regulated, we have analysed the rate of transcription and level of corresponding RNA accumulation of actin and myosin light chain genes during differentiation of a mouse muscle cell line under different culture conditions (low-serum and serum-free). We have found by ‘nuclear run-on’ analysis, that the α-cardiac actin, α-skeletal actin, myosin light chain 1F/3F and embryonic myosin light chain genes are transcriptionally activated as myoblasts begin to fuse to form myotubes. In contrast the nonsarcomeric β-actin gene is transcribed at high levels in myoblasts and is transcriptionally down-regulated during differentiation. There is a sequential transition in transcription and RNA accumulation from predominantly α-cardiac to predominantly α-skeletal actin during subsequent myotube maturation, which reflects the pattern of expression found during development in vivo. A similar transition from embryonic to adult patterns of myosin light chain expression does not occur. RNA accumulation of actin and myosin light chains is regulated at both transcriptional and post-transcriptional levels. In our culture system the expression of myosin light chains 1F and 3F, which are encoded by a single gene, is uncoupled, 3F predominating. These data are discussed in the context of gene regulation mechanisms.

Regulation of the chicken embryonic myosin light-chain (L23) gene: existence of a common regulatory element shared by myosin alkali light-chain genes.

The transcriptional regulation of the chicken myosin alkali light-chain (MLC) L23 gene was analyzed. Two different types of cis-regulatory regions were identified: one was a silencerlike region located between 3.7 and 2.7 kilobases upstream of the mRNA initiation site, and the other was essential for the expression of L23 in skeletal muscle cells and was located between 106 and 91 base pairs upstream of the cap site. This 16-base-pair cis-acting element was designated as the MLC box since it is well conserved in various muscle-specific MLC promoter regions. The activity of the MLC box showed tissue specificity. To analyze the relationship between the nucleotide sequence and the activity of the MLC box precisely, mutation analysis was performed. The 16-base-pair sequence was indispensable for the active transcription of L23 gene, and the MLC box could function in either orientation. The inverted sequence of the MLC box was similar to the sequence of the alpha-actin CArG box. By using a gel mobility retardation assay, the nuclear protein(s) that binds to both MLC box and CArG box was detected with nuclear extract prepared from chicken embryonic breast muscle. These observations imply that a common factor regulates the coordinate expression of these contractile proteins in muscle differentiation.

Regulation of the Chicken Embryonic Myosin Light-Chain (L23) Gene: Existence of a Common Regulatory Element Shared by Myosin Alkali Light-Chain Genes

The transcriptional regulation of the chicken myosin alkali light-chain (MLC) L23 gene was analyzed. Two different types of cis-regulatory regions were identified: one was a silencerlike region located between 3.7 and 2.7 kilobases upstream of the mRNA initiation site, and the other was essential for the expression of L23 in skeletal muscle cells and was located between 106 and 91 base pairs upstream of the cap site. This 16-base-pair cis-acting element was designated as the MLC box since it is well conserved in various muscle-specific MLC promoter regions. The activity of the MLC box showed tissue specificity. To analyze the relationship between the nucleotide sequence and the activity of the MLC box precisely, mutation analysis was performed. The 16-base-pair sequence was indispensable for the active transcription of L23 gene, and the MLC box could function in either orientation. The inverted sequence of the MLC box was similar to the sequence of the alpha-actin CArG box. By using a gel mobility retardation assay, the nuclear protein(s) that binds to both MLC box and CArG box was detected with nuclear extract prepared from chicken embryonic breast muscle. These observations imply that a common factor regulates the coordinate expression of these contractile proteins in muscle differentiation.

Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12.

The Myf-6 gene, a novel member of the human gene family of muscle determination factors has been detected by its highly conserved sequence coding for a putative helix-loop-helix domain. This sequence motif is a common feature of all Myf factors and other regulatory proteins. The new Myf gene is located on human chromosome 12, approximately 6.5 Kb upstream of the Myf-5 locus in a closely linked cluster of myogenic determination genes. Myf-6 cDNAs were isolated from human and mouse skeletal muscle, the only tissue in which expression of the corresponding mRNA was observed. In contrast to human primary muscle cell cultures which express moderate levels of Myf-6 mRNA, most established rodent muscle cell lines completely lack this mRNA. Myogenic 10T1/2 cells, however, induced by the expression of either pEMSV-Myf-4 or pEMSV-Myf-5 activate their endogenous mouse Myf-6 gene. Constitutive expression of Myf-6 cDNA in C3H 10T1/2 fibroblasts establishes the muscle phenotype at a similar frequency to the previously characterized myogenic factors. Moreover, muscle-specific CAT reporter constructs containing either the human myosin light chain (MLC) enhancer or the promoter of the embryonic myosin light chain gene are activated in NIH 3T3 fibroblasts or in CV1 kidney cells by cotransfection of Myf-6 expression vehicles. This transcriptional activation occurs in the absence of any apparent conversion of the cellular phenotype of the recipient cells. Glutathione-S-transferase fusion proteins with Myf-3, Myf-4 or Myf-5 specifically bind to a MEF-like consensus sequence present in the human MLC enhancer and the MLC1 emb promoter. In contrast, the Myf-6 hybrid protein interacts weakly with the same sequences showing lower affinity and reduced specificity. Since co-expressed pEMSV-Myf-6, nevertheless, is able to activate transcription of the MLC-CAT reporter constructs in non-muscle tissue culture cells, the different DNA binding properties in vitro might suggest that transactivation of gene expression by Myf-6 involves distinct binding sites and/or additional protein factors.

A highly conserved enhancer downstream of the human MLC1/3 locus is a target for multiple myogenic determination factors

A potent muscle-specific enhancer element, originally described in the rat myosin light chain (MLC) 1/3 locus located downstream of the coding region, is found in an analogous position in the human MLC1/3 gene. When linked to a CAT reporter gene and transfected into muscle or non-muscle cells, the human MLC enhancer directs high levels of muscle-specific gene expression from homologous or heterologous promoters, irrespective of position or orientation relative to the CAT transcription unit. A significant degree of sequence homology (over 85%) in the 3'-flanking regions of the two MLC genes is restricted to a 200 bp sequence which lies approximately 1.5 kb downstream of the polyadenylation site in both species. The human enhancer sequence includes binding sites for human myogenic determination factors containing a common basic helix-loop-helix motif, and it can be trans-activated to varying degrees in non-muscle cells by these factors. This study establishes the MLC enhancer as an evolutionarily conserved, integral component of the MLC1/3 locus which constitutes a novel target for the action of myogenic determination factors.

Myosin light chain gene expression in developing and denervated fetal muscle in the mouse

We have investigated the accumulation of mRNA transcripts of the atrial (or embryonic) myosin light chain MLC1A (MLC1emb), and the two adult fast muscle myosin light chains (MLC1F and MLC3F) during fetal skeletal muscle development in the mouse. In 15-day fetal muscle, MLC1A is the predominant mRNA detectable, by 18 days MLC1F has become the major transcript and MLC3F mRNA is detectable for the first time. By 12 days after birth, MLC1A transcripts are undetectable and MLC1F and MLC3F are similar in abundance. In fetuses treated with beta-bungarotoxin and which therefore develop in the absence of functional nerve, MLC1A and MLC1F undergo normal transitions but MLC3F mRNA accumulation is significantly retarded. This demonstrates that these myosin light chain mRNAs accumulate with differing kinetics, and that MLC3F mRNA accumulation is nerve-dependent during fetal development. The results are discussed in terms of secondary muscle fibre formation, and in relation to the independent regulation of MLC1F and MLC3F mRNAs which are transcribed from the same gene.

Structure, organization, and expression of the rat cardiac myosin light chain-2 gene: Identification of a 250-base pair fragment which confers cardiac-specific expression

Abstract The present study characterized the structure, organization, and expression of the rat cardiac myosin light chain (MLC) -2 gene. The rat cardiac MLC-2 gene has seven exons which display complete conservation with the exon structure of the rat fast twitch skeletal MLC-2 gene. A 250-base pair (bp) sequence of the 5'-flanking region contains CArG motifs and additional cis elements, each greater than 10 bp in length, which were conserved in sequence and relative position with the chick cardiac MLC-2 gene. A series of MLC-2/luciferase fusion genes consisting of nested 5' deletions of the MLC-2 5'-flanking region were constructed and transfected into primary neonatal rat myocardial cells and a non-myocardial cell line (CV-1), demonstrating that this 250 bp of the MLC-2 5'-flanking region was sufficient to confer cardiac specific expression on a luciferase reporter gene. This study suggests the presence of important proximal regulatory sequences in the MLC-2 5'-flanking region which are capable of directing the cardiac specific expression of the rat cardiac myosin light chain-2 gene.

Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription.

A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.

Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription.

A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.

Co-localization to chromosome bands 99E1-3 of the Drosophila melanogaster myosin light chain-2 gene and a haplo-insufficient locus that affects flight behavior.

Using overlapping synthetic deficiencies, we find that a haplo-insufficient locus affecting flight behavior and the myosin light chain-2 gene co-map to the Drosophila melanogaster polytene chromosome interval 99D9-E1 to 99E2-3. From screening over 9000 EMS-treated chromosomes, we obtained alleles of two complementation groups that map to this same interval. One of these complementation groups lfm(3)99Eb, exhibits dominant flightless behavior; thus, flightless behavior of the deficiency is in all likelihood due to hemizygosity of this single locus. Rescue of flightless behavior by a duplication indicates that the single allele, E38, of the Ifm(3)99Eb complementation group is a hypomorph. Based upon its map position and a reduction in concentration of myosin light chain-2 mRNA in heterozygotes, we propose that Ifm(3)Eb(E38) is a mutant allele of the myosin light chain-2 gene. Our genetic analysis also resulted in the identification of four dominant flightless alleles of an unlinked locus, l(3)nc99Eb, that exhibits dominant lethal synergism with Ifm(3)99Eb.

Novel approaches to study myocardial hypertrophy towards development of anti‐hypertensive drugs: Expression of myosin light chain genes during cardiac hypertrophy

AbstractMyocardial hypertrophy as an adaptive response to various physiological and pathological conditions is characterized by major alterations in the cellular restructuring of the heart as well as by many biochemical and genetic alterations. Hence, the development of hypertrophy provides a unique opportunity to study the various mechanisms underlying the functions of the normal and diseased heart, as well as to understand the molecular mechanisms that operate to regulate cardiac specific gene expression. Both the spontaneously hypertensive rat strain (SHR) and the myocardial cell culture system developed by Paul Simpson have been used as model systems to study cardiac specific gene expression. We have isolated and characterized in detail the rat ventricular specific myosin light chain (MLC)‐1 and MLC‐2 cDNA clones. Using a sensitives S1 nuclease analysis we have shown that while no MLC‐1 mRNA of the ventricular type is detectable in the normal and SHR atrial muscle, there is a selective increase in ventricular MLC‐2 mRNa in both 6 week old (prehypertensive stage) and 18 week old (hypertensive) SHR heart atria. The myocardial cell culture system has been used to examine the effects of α‐adrenergic stimulation on the accumulation of contractile units and the expression of MLC‐2 gene. Stimulation of α‐adrenergic receptors with norepinephrine results in an increase in cellular MLC‐2 protein content as well as an increase in steady‐state level of MLC‐2 mRNA. This increase in MLC‐2 mRNA is mediated in part by an increase in transcription of the MLC‐2 gene. In order to characterize the regulatory elements of the MLC‐2 gene, we have characterized in detail the genomic clone corresponding to rat cardiac MLC‐2 cDNA. The results show a complete conservation of exon and intron organizations between cardiac and skeletal MLC‐2 genes. There is also a strong conservation of DNA sequence elements upstream to the transcription initiation site between rat cardiac and chicken cardiac MLC‐2 genes. Analysis of the cis‐ and trans‐acting factors that regulate MLC‐2 gene expression during cardiac hypertrophy should prove very useful in understanding the molecular mechanisms involved in hypertropic response.

Isolation of the chick myosin alkali light chain gene expressed in embryonic gizzard muscle and transitional expression of the light chain gene family in vivo

A chick embryonic myosin alkali light chain L23 gene that is expressed transiently at embryonic stages in chick skeletal, cardiac and smooth muscles and in brain continuously from embryo to adult stages, was isolated and characterized. Sequence analysis showed that the exonic sequence of this gene was identical with that of embryonic myosin light chain mRNA except for one base replacement. This gene is a single gene of 5200 bases, which is divided into seven exons by six introns, and the positions of inserts of all the introns are well-conserved as in the skeletal and cardiac muscle myosin alkali light chain genes. Therefore, this embryonic myosin light chain gene can be classified as a member of the myosin alkali light chain gene family, and these three genes may have originated from a common ancestral gene. Transcription of the embryonic light chain gene starts from the same initiation site 33 bases upstream from ATG in embryonic muscle tissues and brain. Comparison of the nucleotide sequence around the promotor region of the embryonic myosin light chain gene with the corresponding regions of the skeletal and cardiac myosin light chain genes showed that the 11-base consensus sequence (TCC A TTTTATAG) is present about 100 bases upstream from the transcription initiation site in each gene.

Regulatory myosin light-chain genes of Caenorhabditis elegans.

We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes.

Regulatory myosin light-chain genes of Caenorhabditis elegans.

We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes.