Protein-binding sites within the 5'' DNase I-hypersensitive region of the chicken alpha D-globin gene.

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.     

Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus.

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5'-rho-beta H-beta A-epsilon-3'). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of rho to just downstream of epsilon. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the rho-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of rho, beta H, and epsilon. (v) A site 3' of beta A and 5' of epsilon was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo II sites. These data are consistent with the hypothesis that, in vivo, topo II preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.     

Isolation and characterization of the chicken cardiac myosin light chain (L-2A) gene. Evidence for two additional N-terminal amino acids

The contractile proteins of striated muscle are encoded by multigene families and constitute an excellent system to investigate differentiation and developmental control of gene expression. Different forms of myosin light chains are expressed in skeletal muscle as well as in the myocard. To study the gene structure and molecular mechanisms underlying differential gene expression, the structural cardiac myosin light chain 2 (MLC-2A) gene was isolated from a chicken genomic DNA library. Restriction enzyme mapping, electron microscopic analysis, and partial sequencing revealed that the gene coding for the MLC mRNA of 700 nucleotides in length extends over 4.2 kilobases of DNA and is interrupted by 5 introns. Sequence analysis led to the detection of two codons for additional amino acids at the N terminus which were not reported to be present in the mature protein and are presumably removed post-translationally. These two amino acids, methionine and alanine, are coded on two separate exons split by the largest intron of the entire gene. Southern blot analysis of genomic chicken DNA indicates the presence of one MLC-2A gene per haploid chicken genome.     

A chicken embryo-specific myosin light chain (L23) appears to be identical with one of brain myosin light chains

A novel embryo-specific myosin light chain of 23 kDa molecular weight (L23) was found previously in embryonic chicken skeletal, cardiac, and smooth muscles (Takano-Ohmuro et al. (1985) J. Cell Biol. 100, 2025-2030). When we examined myosin in embryonic and adult brain by two-dimensional electrophoresis, 23 kDa myosin light chain present in brain (Burridge & Bray (1975) J. Mol. Biol. 99, 1-14) comigrated with L23. Two monoclonal antibodies, EL-64 and MT-185d, were applied to clarify the identity of the brain 23 kDa myosin light chain and the chicken embryonic muscle L23. The two antibodies recognize different antigenic determinants in the L23 molecule; the former antibody is specific for L23, whereas the latter recognizes the sequence common to fast skeletal muscle myosin light chains 1 and 3, and also L23. The immunoblots combined with two-dimensional gel electrophoresis showed that both EL-64 and MT-185d can bind to the brain 23 kDa myosin light chain as well as the chicken embryonic muscle L23. These results indicate that chicken brain and chicken embryonic muscles contain a common myosin light chain of 23 kDa molecular weight.     

Two-site phosphorylation of the phosphorylatable light chain (20-kDa light chain) of chicken gizzard myosin

When prepared under specified conditions chicken gizzard myosin was obtained which when incubated with ATP gave rise to a diphosphorylated as well as the monophosphorylated form of P light chain. Formation of the diphosphorylated light chain occurred more readily with these myosin preparations, but could also be obtained by prolonged incubation of the isolated whole light chain fraction with kinase preparations from rabbit skeletal and chicken gizzard muscles. Using isolated light chains as substrate the more readily formed monophosphorylated light chain contained serine phosphate while the diphosphorylated form contained serine and threonine phosphates.     

The nucleotide sequence of myosin light chain (L-2A) mRNA from embryonic chicken cardiac muscle tissue.

The nucleotide sequence of a cDNA clone (pML10) for chicken cardiac myosin light chain is described. The cDNA insert contains 613 nucleotides representing the entire coding sequence, with the exception of nine NH2-terminal amino acids, and the full 3'-non-coding region of 146 nucleotides. The missing 5' terminus of the mRNA, not represented in the clone pML10, was obtained by extension of the cDNA using a 43 nucleotide long internal EcoR1 fragment as a primer. The non-coding region contains several direct and inverted repeated sequences and the polyadenylation signal sequence AATAAA. The coding portion exhibits non-random usage of synonymous codons with a strong bias for codons ending in G and C.     

Restricted expression of cardiac myosin light chain 2A gene in muscular dystrophic condition

In order to understand the mechanism of defective myofibrilogenesis in muscular dystrophy, we have used the genomic cloned DNA specific for myosin light chain 2A (MLC 2A) to check its expression. The fusion of a partial digest of lambda LC5, containing the upstream sequence of MLC 2A gene with the expression vector of PSVOCAT has already been reported. Using this CAT-fused recombinant containing 1.6 kb of MLC 2A gene, we were able to detect the promoter activity in normal heart cells, H9C2 cell line whereas a restricted expression of MLC 2A gene was noticed in muscular dystrophic muscle cells from heart and skeletal. We have also measured the transient transfection efficiency by contransfecting with the plasmid LacZ. Simultaneous assay of beta-galactosidase and CAT in the cell extract was performed. With beta-galactosidase as control, we confirmed that the promoter activity of MLC 2A gene is inhibited in muscular dystrophy though there is a normal rate of transfection occurred.     

Myocardial functional correlates of cardiac myosin light chain 2 phosphorylation

In vitro and in situ studies have proposed a potentiation of submaximal force production after myosin light chain 2 (P-light chain) phosphorylation in mammalian striated muscle. The purpose of this study was to ascertain the relationship between the augmentation in left ventricular pressure development and cardiac myosin P-light chain phosphorylation at different times during and after submaximal treadmill exercise involving adult female Sprague-Dawley rats. In vivo hemodynamic measurements were monitored with an indwelling high-fidelity solid-state pressure transducer. Exercise heart rate, peak left ventricular (LV) pressure, and rate of LV pressure development/relaxation (LV +/- dP/dt) were significantly elevated compared with a normal sedentary group (P less than 0.001). Peak LV pressure remained significantly elevated throughout 20 min of postexercise recovery (P less than 0.01), and heart rate, LV end-diastolic pressure, and LV +/- dP/dt returned rapidly to preexercise values. Corresponding to these in vivo hemodynamic changes, increased levels of P-light chain phosphorylation were observed during both exercise (16%, P less than 0.01) and subsequent recovery periods (14%, P less than 0.02) compared with the NC group. A quasi-temporal relationship was observed between postexercise peak LV pressure potentiation and P-light chain phosphorylation. These results demonstrate that cardiac myosin P-light chain phosphorylation is associated, in part, with the augmentation of peak LV pressure observed during both exercise and recovery.     

Tissue-specific DNase I-sensitive sites of the maize P gene and their changes upon epimutation

A map has been developed of nuclease-hypersensitive sites of P-rr , the standard allele of the P -locus of Zea mays L. Using a traditional DNase I assay, eight such sites have been found that are specific for the expressing tissue and span a region of more than 25 kb of the P -locus, making it one of the largest plant genes yet described. The maps of the standard allele have also been compared with the recently described moderately stable P-pr allele, which arose from epimutation. Six of the eight sites exhibit the same tissue-specificity in P-pr plants, while two stay repressed as in non-expressing tissues of plants with the standard allele. Interestingly, the two repressed sites coincide with two hypermethylated restriction sites that have previously been correlated with the expression potential of the P-pr allele. On the other hand, four of the DNase I sites, coinciding with CpG islands that were not hypermethylated by the epimutation, also showed no differences in their sensitivity to DNase I between the standard allele and the P-pr allele. This suggests that the epimutation affects both site-specific methylation changes and a specific local chromatin structure of the P gene involved in its regulation.     

Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.     

Mapping single cysteine mutants of light chain 2 in chicken skeletal myosin.

The NH2- and COOH-terminal regions of the regulatory light chain (LC2) have been mapped to the head/rod junction by immunoelectron microscopy, using monoclonal and anti-fluorescyl antibodies as probes. In order to map the entire length of the light chain, we have engineered and purified mutants that contain a single cysteine residue at positions 2, 73, 94, 126, or 155. The single cysteine residues were labeled with either 5-iodoacetamido fluorescein or N-ethylmaleimide-biotin. We observed that the reactivity of Cys126 is far greater than that of Cys155, confirming that cysteine 126 is the fast-reacting thiol in wild-type light chain. The labeled light chains were exchanged into myosin stripped of its native LC2 by immunoaffinity chromatography, and the reconstituted myosin was reacted with anti-fluorescein antibody or avidin prior to rotary shadowing for electron microscopy. The position of the antibody or avidin was found to be near the head/rod junction for all mutants. These mapping studies, together with our finding that cysteines widely separated in the primary sequence can form multiple disulfide bonds (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 226, 19777-19785), support a model for LC2 as a flexible, globular molecule localized mainly in the vicinity of the head/rod junction of myosin.     

Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.