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Biomedical subjects

M E Buckingham

Publications and source records attributed to M E Buckingham.

At least 37 records · Page 2Linked to original sources
PubMed1991

Three linked myosin heavy chain genes clustered within 370 kb of each other show independent transcriptional and post-transcriptional regulation during differentiation of a mouse muscle cell line.

We have examined myosin heavy chain gene transcription in the mouse muscle cell line C2/7 under different culture conditions. Gene-specific probes for embryonic (MHCemb), perinatal (MHCpn), and adult (MHCIIB) MHC sequences were used in nuclear run-on experiments, and transcriptional levels compared with cytoplasmic RNA accumulation of the transcripts during muscle cell differentiation. Transcripts are not detectable in myoblasts. These three MHC genes are physically linked within 370 kb of each other. However, they are not activated coordinately, but show independent transcriptional regulation as muscle cells differentiate into myotubes and as myotubes mature in culture. Post-transcriptional mechanisms also regulate cytoplasmic RNA accumulation of these MHC genes.

Animals↗
PubMed1991

Carbonic anhydrase III, an early mesodermal marker, is expressed in embryonic mouse skeletal muscle and notochord.

Carbonic anhydrase III (CAIII) is an abundant soluble protein in adult mammalian slow twitch skeletal muscle fibers. It is thought to be an early marker for myogenesis based upon its high level of expression in myoblasts in vitro prior to fusion. Using in situ hybridization, we have studied the in vivo distribution of CAIII gene transcripts in mouse embryos and fetuses from 7.25 days to 17.5 days post coitum (p.c.). CAIII mRNAs are first detected in the myotomes of somites between 9.5 and 10.5 days p.c. (20-30 somites). At 15.5 days p.c., CAIII begins to be restricted to developing slow muscle fibers. By two weeks post partum (p.p.), CAIII mRNAs are detected mainly in slow muscle fibers. CAIII transcripts are detected at an earlier stage (7.25 days p.c.) in the developing notochord. CAIII is expressed at a much higher level in the notochord than it is in developing skeletal muscle. As the notochord forms the nucleus pulposus in fetal mice, CAIII mRNA levels remain very high. Expression of CAIII in the notochord is of interest in the context of skeletal myogenesis because the notochord is thought to play an important role in somite formation. In addition to the notochord, CAIII transcripts are detected prenatally in several other non-muscle tissues: in cells of the choroid plexus, endocardial cushion and ureter, and in adipocytes.

Adipose Tissue, Brown↗
PubMed1991

alpha-Actin proteins and gene transcripts are colocalized in embryonic mouse muscle.

The alpha-actins are among the earliest muscle-specific mRNAs to appear in developing cardiac and skeletal muscle. To determine if there is coexpression of the alpha-actin proteins at early stages of myogenesis, we have used an alpha-actin-specific polyclonal antibody and in situ hybridization with specific cRNA probes to cardiac and skeletal alpha-actin transcripts on serial slides of mouse embryo sections. As soon as we can detect alpha-actin mRNAs in embryonic striated muscle, we also detect the protein suggesting that alpha-actin transcripts are translated very rapidly after transcription during myogenesis. In skeletal muscle, this colocalization of alpha-actin mRNA and protein was observed both in the myotomes of somites and in developing muscles in the limbs. In cardiac muscle, alpha-actin transcripts and proteins are abundantly expressed as soon as a cardiac tube forms.

Actins↗
PubMed1991

Developmental regulation of creatine kinase gene expression by myogenic factors in embryonic mouse and chick skeletal muscle.

The B isoform of creatine kinase (BCK), which is expressed at a high level in embryonic neural tissues, is also expressed abundantly in developing striated muscle and is an early marker for skeletal myogenesis. Using isoform-specific 35S-labeled antisense cRNA probes for in situ hybridization, we have detected BCK mRNAs in embryonic mouse and chick myotomes, the first skeletal muscle masses to form in developing embryos. These transcripts are detectable as soon as myotomes are morphologically distinguishable. BCK is expressed at high levels in both skeletal and cardiac muscle in mouse and chick embryos. In the mouse, BCK transcript levels fall of rapidly in striated muscle shortly after the onset of MCK gene expression. The M isoform of creatine kinase (MCK), the striated muscle-specific isoform, is expressed later than BCK. In the mouse, BCK transcripts are expressed in myotomes at 8.5 days post coitum (p.c.), but MCK transcripts are not detected before 13 days p.c. In the chick, BCK mRNAs are present at Hamburger-Hamilton stage 13, but MCK mRNAs are not detected before stage 19. We have compared the patterns of expression of the CK genes with those of myogenic differentiation factor genes, which are thought to regulate skeletal muscle-specific gene expression. In the chick, both CMD1, first detected at stage 13, and myogenin, first detected at stage 15, are present prior to MCK, which begins to be expressed at stage 19. Unlike the mouse embryo, CMD1, the chick homologue of MyoD1, is expressed before chick myogenin. In the mouse, myogenin, first detected at 8.5 days p.c., is expressed at the same time as BCK in myotomes. Both myogenin and MyoD1, which begins to be detected two days later than myogenin, are expressed at least two days before MCK. It has been proposed that the myogenic factors, MyoD1 and myogenin, directly regulate MCK gene expression in the mouse by binding to its enhancer. However, our results show that MCK transcripts are not detected until well after MyoD1 and myogenin mRNAs are expressed, suggesting that these factors by themselves are not sufficient to initiate MCK gene expression.

Animals↗
PubMed1990

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

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 alpha-cardiac actin, alpha-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 beta-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 alpha-cardiac to predominantly alpha-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.

Actins↗
PubMed1989

Chronic stimulation-induced changes of myosin light chains at the mRNA and protein levels in rat fast-twitch muscle.

Transitions in the expression of the myosin light chains (LC) were investigated in fast-twitch muscles of the rat during chronic (10 h/day), low-frequency (10 Hz) stimulation. Changes were followed at the mRNA level by Northern blot analysis and in vitro translation, as well as at the protein level by electrophoresis under denaturing and nondenaturing conditions. In vivo synthesis of the light chains was assessed by measuring the incorporation of intramuscularly injected [35S]methionine. Chronic stimulation induced a transition in the isomyosin pattern with an increase of FM3, a concomitant decrease in FM1 and, after longer stimulation periods, the appearance of low concentrations of the slow isomyosin. These changes were accompanied by an elevated LC1f/LC3f ratio and increases in the amounts of both the LC1sb and, to a lesser degree, LC2s proteins. Alterations in the amounts of specific mRNAs were the same whether determined by Northern blot analysis or by in vitro translation of total RNA preparations from the same muscles. Generally, the changes in the relative concentrations of fast and slow light-chain proteins agreed with the changes detected at the mRNA level and the alterations in protein synthesis detected with the use of an in vivo labeling assay. An exception was the elevated tissue content of LC2s where no changes were detectable in the concentration of its mRNA as determined by in vitro translation or in vivo synthesis. The increase in LC2s protein may, therefore, have been due to reduced degradation. In addition, the decrease in LC3f was more pronounced at the protein level than at the mRNA level. This might indicate an increased turnover of LC3f or the existence of additional post-transcriptional regulations of LC3f expression.

Animals↗
PubMed1989

A developmental study of the abnormal expression of alpha-cardiac and alpha-skeletal actins in the striated muscle of a mutant mouse.

BALB/c mice possess a 5' duplication of the alpha-cardiac actin gene which is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue. This mutation therefore provides a potential tool for the study of the inter-relationship between the striated muscle actins. We have examined the expression of this actin gene pair throughout the development of skeletal and cardiac muscle in BALB/c mice. During embryonic and fetal development, the expression of these two genes is indistinguishable from that in normal mice, as determined by in situ hybridization. A quantitative postnatal study demonstrates that in the hearts of normal mice the level of alpha-cardiac actin mRNA declines, whereas that of alpha-skeletal actin increases. In mutant mice, these trends are exaggerated so that whereas normal mice have 95.8% alpha-cardiac mRNA and 4.2% alpha-skeletal mRNA in the adult heart, BALB/c mice have 52.4 and 47.6% of these mRNAs, respectively. This difference is also reflected at the protein level. In developing skeletal muscle, the expression of these genes follows kinetics similar to that observed in the heart with a decrease in the relative level of alpha-cardiac mRNA as the muscle matures. Cardiac actin mRNA levels are again lower in the mutant mouse, but here the effect is less striking because skeletal actin is the predominant isoform. These results are discussed in the context of the interaction between this actin gene pair in developing and adult striated muscle.

Actins↗
PubMed1989

The control of muscle gene expression: a review of molecular studies on the production and processing of primary transcripts.

Many muscle proteins exist as multiple isoforms. This diversity is generated by the presence of multigene families and by alternative splicing of individual genes. Examples are given of different modes of alternative splicing undergone by the primary transcripts of muscle genes, and preliminary studies on the mechanism are mentioned. The chromosomal organization of muscle genes is discussed briefly. Studies on their transcriptional regulation are reviewed first in terms of cis-acting sequences in the proximal promoter region and elsewhere in the vicinity of the gene, which are necessary for its expression, and, secondly, in terms of trans-acting transcriptional factors which interact with such sequences. Molecular regulation of splicing and of transcription is discussed mainly with reference to the muscle genes of mammals.

Animals↗
PubMed1989

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.

Animals↗
PubMed1988

Promoter analysis of myosin alkali light chain genes expressed in mouse striated muscle.

There are three principal myosin alkali light chain (MLC) genes expressed in mouse striated muscle. The skeletal muscle gene MLC1F/MLC3F, the ventricular muscle/slow skeletal muscle gene MLC1V(MLC1S), and the atrial muscle/foetal striated muscle gene MLC1A(MLC1emb). MLC1V and MLC1A are expressed in both cardiac and skeletal muscle, and we show here that these genes use a single site of initiation of transcription, and therefore the same proximal promoter region, in both muscle types, and in myogenic cell lines in culture. We have previously shown that for the MLC1F/MLC3F gene, 1200bp of upstream sequence from the MLC1F promoter is sufficient to allow tissue specific and developmentally regulated expression. We have therefore isolated, characterised, and sequenced over 1200bp upstream of each of the three MLC genes in order to look for elements which may be involved in their regulation. Detailed comparison of their promoter sequences, as well as those of the cardiac and skeletal muscle alpha-actin genes, reveals a number of common elements. Among these is an "MLC-sequence" (CCTTTTATAG) common to all MLC genes, including those of chick and rat, and a "cardiac sequence" common to the mouse MLC1A, MLC1V and alpha-cardiac actin genes expressed in the heart.

Amino Acid Sequence↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1986

A 5' duplication of the alpha-cardiac actin gene in BALB/c mice is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue.

We describe the structure and transcriptional activity of the 5' portion of the alpha-cardiac actin gene of BALB/c mice. Southern blotting and DNA sequencing reveal that the promoter and first three exons of the gene are present as perfect repeats in a direct duplication of 9.5 kbp situated immediately upstream of the gene. Both promoters are active in adult cardiac tissue. Transcripts from the partial gene duplication give rise to novel RNAs that are spliced correctly in the actin region and polyadenylated. The level of mature alpha-cardiac actin mRNA is only 16.5% that found in mice that do not possess the duplication. This is due, at least in part, to interference at the transcriptional level. Transcripts from the alpha-skeletal actin gene accumulate to abnormally high levels in the hearts of such mutant mice. This result suggests tight regulatory coupling for this actin gene pair.

Actins↗
PubMed1985

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.

Animals↗
PubMed1985

The same myosin alkali light chain gene is expressed in adult cardiac atria and in fetal skeletal muscle.

We have isolated from a cDNA library constructed using mouse cardiac mRNA sequences, a clone (pC6) homologous to part of the mRNA encoding the myosin alkali light chain MLC1A from adult mouse atria. This sequence also hybridizes to mRNA encoding the fetal light chain form MLC1emb expressed in both fused myotubes in culture and in 18 day fetal skeletal muscle. These mRNA sequences are indistinguishable from the MLC1A messenger both on the basis of size and of their thermal stability of hybridization. In vitro translation of mRNA selected by hybridization with pC6 results in a protein that comigrates with the fetal MLC1emb isoform, and two-dimensional gel electrophoresis of adult atrial and fetal skeletal muscle proteins shows MLC1A and MLC1emb to be indistinguishable in the mouse. Southern blot hybridization of clone pC6 to mouse genomic DNA and the analysis of restriction fragment length polymorphisms between different mouse species demonstrates the presence of a single hybridizing locus in the mouse genome. These data provide strong evidence that the atrial MLC1A and fetal skeletal MLC1emb isoform are encoded by the same gene and by the same mRNA and are thus identical proteins.

Animals↗
PubMed1983

Number and organization of actin-related sequences in the mouse genome.

Recombinant plasmids containing cDNA sequences complementary to the two mouse striated-muscle actin messenger RNAs (pAF81, pAM91) and to a non-muscle actin mRNA (pAL41) have been used to examine the number and organization of actin-related sequences in the mouse genome. A large number (greater than 20) of actin-related sequences are detected on Southern blots of restricted mouse DNA, the majority of which hybridize to both the 5' and 3' ends of the actin-coding sequence, even under conditions revealing only sequences greater than 80% homologous to the actin cDNA probes. More stringent washing of these blots indicates that the two striated muscle actins are each encoded by single genes, and that a non-muscle (beta or gamma) actin cDNA detects one homologous and two closely related sequences in mouse DNA. The segregation of the two striated-muscle actin genes in recombinant inbred mouse strains shows that these genes are not closely linked (greater than 1 centimorgan), and that the skeletal muscle actin gene is not linked to a non-muscle actin gene. Screening a bank of mouse genomic DNA, cloned in Charon 4A, indicates that the number of actin-related sequences in the mouse genome is much higher than 20. In particular, five phages have been isolated representing part of a sub-family of 20 to 50 similar but non-identical sequences, only weakly homologous to actin cDNA probes (probably a family of actin pseudogenes), which are the result of a recent amplification of a greater than 17 X 10(3) base region of mouse DNA.

Actins↗