Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene.

We have isolated three overlapping genomic clones extending over 39 kilobases (kb), which encodes the rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene (SERCA2). S1 nuclease mapping and primer extension analysis of the 5' end of the cardiac/slow-twitch (SERCA2a) and smooth/non-muscle (SERCA2b) mRNAs showed that both transcripts are initiated from the same transcription initiation site, located 528 base pairs (bp) upstream of the translation initiation codon AUG. The putative promoter revealed a "TATA box" like element at -24 bp and a "CAAT box" at -78 bp relative to the cap site.

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing.

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions.

Characterization of a rat myosin alkali light chain gene expressed in ventricular and slow twitch skeletal muscles.

Mammalian cardiac muscle contains two myosin alkali light chains: 1) the atrial light chain (MLC1A), and 2) the ventricular light chain (MLC1V) predominantly expressed either in the atrium or in the ventricle. In this report we describe the isolation and characterization of the complete gene for rat MLC1V. The rat MLC1V gene is approximately 6.

Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis.

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M.

Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase.

We have isolated and sequenced full-length cDNA clones from a rabbit uterine library which encode the smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. These cDNAs resulted from an alternative splice of the cardiac/slow-twitch Ca2+-ATPase gene transcript, and encoded a protein identical to rabbit cardiac/slow-twitch Ca2+-ATPase except for the replacement of the carboxyl-terminal four amino acids with an extended and relatively hydrophobic sequence of 49 amino acids. This cDNA was virtually identical to the alternatively spliced product of the cardiac/slow-twitch Ca2+-ATPase gene recently identified in human kidney (Lytton, J.

Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone.

The sarcoplasmic reticulum (SR) and the contractile protein myosin play an important role in myocardial performance. Both of these systems exhibit plasticity--i.e.

Regulation of myosin heavy-chain gene expression during skeletal-muscle hypertrophy.

Changes in the myosin phenotype of differentiated muscle are a prominent feature of the adaptation of the tissue to a variety of physiological stimuli. In the present study the molecular basis of changes in the proportion of myosin isoenzymes in rat skeletal muscle which occur during compensatory hypertrophy caused by the combined removal of synergist muscles and spontaneous running exercise was investigated. The relative amounts of sarcomeric myosin heavy (MHC)- and light (MLC)-chain mRNAs in the plantaris (fast) and soleus (slow) muscles from rats was assessed with cDNA probes specific for different MHC and MLC genes.

Genetic and non-genetic control of myocardial calcium.

Some aspects of the genetic and non-genetic control of the amount and rate of calcium cycled during steady-state activation of papillary muscles from right ventricular rabbit myocardium are presented. Genetic reorganization of the intracellular structure of the myocardium is achieved by producing right ventricular pressure overload and thyrotoxic hypertrophy. The mechanical performance of the pressure overload heart is slowed while time to peak tension is increased.

Characterization of a mammalian smooth muscle myosin heavy chain cDNA clone and its expression in various smooth muscle types.

A cDNA clone, SMHC-29, encoding the light meromyosin of smooth muscle myosin heavy chain (MHC), was isolated from a rabbit uterus cDNA library constructed in phage lambda gt11. This smooth muscle MHC cDNA demonstrates significant nucleotide and amino acid sequence homologies with known sarcomeric MHC genes from rabbit, rat skeletal, and nematode body wall myosin, and even with nonmuscle MHC gene from a slime mold (Dictyostelium discoideum), suggesting that smooth muscle, striated muscle, and nonmuscle MHC genes diverged from a common ancestor. The deduced amino acid sequences of the smooth muscle light meromyosin show very similar periodic distributions of hydrophobic and charged residues as found for the light meromyosin of striated muscle MHCs together with a high potential for alpha-helical formation, indicating an alpha-helical coiled-coil structure for the smooth muscle light meromyosin sequences.

Complete nucleotide and encoded amino acid sequence of a mammalian myosin heavy chain gene. Evidence against intron-dependent evolution of the rod.

The complete nucleotide sequence and exon/intron structure of the rat embryonic skeletal muscle myosin heavy chain (MHC) gene has been determined. This gene comprises 24 X 10(3) bases of DNA and is split into 41 exons. The exons encode a 6035 nucleotide (nt) long mRNA consisting of 90 nt of 5' untranslated, 5820 nt of protein coding and 125 nt of 3' untranslated sequence.

Sarcomeric myosin heavy chain gene family: organization and pattern of expression.

The sarcomeric myosin heavy chains (MHCs), which exhibit different levels of ATPase activity, are encoded by a closely related multigene family from which seven members have been identified and characterized in the rat. The MHC genes appear to map to a single chromosome, and at least two of them, alpha- and beta-cardiac, are closely linked in the genome. Each of these genes is approximately 25 kilobases long, and their coding sequences are interrupted by 40 introns.

Characterization of cDNA and genomic sequences corresponding to an embryonic myosin heavy chain.

We report here the isolation and characterization of cDNA and genomic sequences corresponding to a rat embryonic myosin heavy chain (MHC) protein. This gene, which is present as a single copy in the rat genome, comprises about 25 kilobase pairs of DNA and contains approximately 80% intronic sequences. The embryonic MHC gene belongs to a highly conserved multigene family, and exhibits a high degree of nucleotide and amino acid sequence conservation with other sarcomeric MHC genes from nematode to man.

Myosin light-chain 1 and 3 gene has two structurally distinct and differentially regulated promoters evolving at different rates.

DNA fragments located 10 kilobases apart in the genome and containing, respectively, the first myosin light chain 1 (MLC1f) and the first myosin light chain 3 (MLC3f) specific exon of the rat myosin light chain 1 and 3 gene, together with several hundred base pairs of upstream flanking sequences, have been shown in runoff in vitro transcription assays to direct initiation of transcription at the cap sites of MLC1f and MLC3f mRNAs used in vivo. These results establish the presence of two separate, functional promoters within that gene. A comparison of the nucleotide sequence of the rat MLC1f/3f gene with the corresponding sequences from mouse and chicken shows that: the MLC1f promoter regions have been highly conserved up to position -150 from the cap site while the MLC3f promoter regions display a very poor degree of homology and even the absence or poor conservation of typical eucaryotic promoter elements such as TATA and CAT boxes; the exon/intron structure of this gene has been completely conserved in the three species; and corresponding exons, except for the regions encoding most of the 5' and 3' untranslated sequences, show greater than 75% homology while corresponding introns are similar in size but considerably divergent in sequence.

Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature.

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM.

Intron positions are conserved in the 5' end region of myosin heavy-chain genes.

We have determined the 5' end sequence of the rat embryonic skeletal muscle myosin heavy-chain (MHC) gene comprising the first three amino-terminal coding exons. Comparison with the corresponding regions of the rat ventricular alpha and the nematode Caenorhabditis elegans unc-54 MHC genes (Mahdavi, V., Chambers, A.

Characterization of a developmentally regulated perinatal myosin heavy-chain gene expressed in skeletal muscle.

A cDNA clone, labeled pFOD5, isolated from a fetal-rat skeletal-muscle cDNA library, has been characterized and found to contain sequences corresponding to a perinatal-specific skeletal myosin heavy-chain (MHC) mRNA. This MHC cDNA demonstrates a high degree of nucleotide- and amino acid-sequence conservation with other MHC genes, but its carboxyl-terminal peptide and 3'-untranslated region are highly divergent and specific for this gene. S1 nuclease mapping experiments have shown that the perinatal MHC gene represented by this cDNA clone is only transiently expressed during skeletal-muscle development.

Fast skeletal muscle myosin light chains 1 and 3 are produced from a single gene by a combined process of differential RNA transcription and splicing.

Fast myosin light chains (MLCf) 1 and 3 are proteins associated with the thick filament in vertebrate fast muscle fibers. MLC1f and MLC3f have complete sequence homology for the first 141 amino acids from their COO- end, but they differ in length and amino acid sequence at their NH+3 ends (MLC1f = 49 amino acids, MLC3f = 8 amino acids), and they are translated from different mRNAs. To elucidate the structural relationship between mammalian fast myosin light chain 1 and 3 isoforms, cDNA clones have been isolated from rat skeletal muscle and their primary nucleotide sequences were determined.

Cloning and characterization of cDNA sequences corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin.

A library of cDNA clones was constructed from adult rat skeletal muscle mRNA, from which a set of contractile protein clones was selected. These clones were identified by sequencing the cDNA inserts and comparing the derived amino acid sequences with published sequences of rabbit contractile proteins. In this manner, clones corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin were identified.

Structural effects in solvolytic reactions; carbon-13 NMR studies of carbocations: Effect of increasing electron demand on the carbon-13 NMR shifts in substituted tert-cumyl and 1-aryl-1-cyclopentyl carbocations-correlation of the data by a new set of substituent constants, sigma.

The cationic carbon substituent chemical shifts (DeltadeltaC(+)) for nine representative meta-substituted tert-cumyl carbocations are correlated satisfactorily by the sigma(m) (+) substituent constants (slope rho+ = -18.18, correlation coefficient r = 0.990).

Protein kinase associated with hnRNP from Hela cell nuclei. Partial purification and properties.

The protein kinase previously described as an endogenous activity present in ribonucleoprotein particles containing heterogenous RNA from HeLa cells (Blanchard et al, Eur. J. Biochem (1977), 79, 11-131) has been partially purified by a combination of chromatography on DEAE cellulose, phosphocellulose and Sephacryl S-200.