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S J Burden

Publications and source records attributed to S J Burden.

At least 37 records · Page 2Linked to original sources
PubMed1993

Electrical activity-dependent regulation of the acetylcholine receptor delta-subunit gene, MyoD, and myogenin in primary myotubes.

Expression of the skeletal muscle acetylcholine receptor (AChR) is regulated by nerve-evoked muscle activity. Studies using transgenic mice have shown that this regulation is controlled largely by transcriptional mechanisms because responsiveness to electrical activity can be conferred by transgenes containing cis-acting sequences from the AChR subunit genes. The lack of a convenient muscle cell culture system for studying electrical activity-dependent gene regulation, however, has made it difficult to identify the important cis-acting sequences and to characterize an electrical activity-dependent signaling pathway. We developed a muscle culture system to study the mechanisms of electrical activity-dependent gene expression. Gene fusions between the murine AChR delta-subunit gene and the human growth hormone gene were transfected into primary myoblasts, and the amount of growth hormone secreted into the culture medium from either spontaneously electrically active or inactive myotube cultures was measured. We show that 181 bp of 5'-flanking DNA from the AChR delta-subunit gene are sufficient to confer electrical activity-dependent gene expression. In addition, we show that the rate of AChR delta-subunit gene expression differs among individual nuclei in a single myotube but that highly expressing nuclei are not necessarily colocalized with AChR clusters. We also show that expression of MyoD and myogenin are regulated by electrical activity in primary myotube cultures and that all nuclei within a myotube express similar levels of MyoD and similar levels of myogenin.

Animals↗
PubMed1993

Synapse-specific gene expression.

Skeletal myofibers are specialized at the site of contact between nerve and muscle, and recent studies indicate that mechanisms involved in the formation of this specialized region are similar to mechanisms used to establish specialized domains in other syncytial cells. In this review experiments are summarized that indicate that nuclei in the synaptic region of syncytial myofibers are transcriptionally distinct from nuclei in the remainder of the myofiber, and the steps used to establish synapse-specific gene expression are compared with the mechanisms used to regulate gene expression among subsets of nuclei in other syncytia, namely the syncytial blastoderm of Drosophila melanogaster and the syncytial germ line of Caenorhabditis elegans.

Animals↗
PubMed1993

Development of the neuromuscular synapse.

Major advances have occurred in our understanding of the signaling events involved in neuromuscular synapse formation. In particular, it has recently been shown that agrin is necessary for synapse formation, that acetylcholine receptor genes are specifically transcribed by synaptic nuclei in response to signals from the synaptic basal lamina, and that synaptic competition between motor neurons can occur by a Hebbian mechanism in cell culture.

Animals↗
PubMed1993

An E box mediates activation and repression of the acetylcholine receptor delta-subunit gene during myogenesis.

The genes encoding the skeletal muscle acetylcholine receptor (AChR) are induced during muscle development and are regulated subsequently by innervation. Because both the initiation and the subsequent regulation of AChR expression are controlled by transcriptional mechanisms, an understanding of the steps that regulate AChR expression following innervation is likely to require knowledge of the pathway that activates AChR genes during myogenesis. Thus, we sought to identify the cis-acting sequences that regulate expression of the AChR delta-subunit gene during muscle differentiation. We transfected muscle and nonmuscle cell lines with gene fusions between 5'-flanking DNA from the AChR delta-subunit gene and the human growth hormone gene, and we show here that 148 bp of 5'-flanking DNA from the AChR delta-subunit gene contains two regulatory elements that control muscle-specific gene expression. One element is an E box, which is important both for activation of the delta-subunit gene in myotubes and for its repression in myoblasts and nonmuscle cells. Mutation of this E box, which prevents binding of MyoD-E2A and myogenin-E2A heterodimers, decreases expression in myotubes and increases expression in myoblasts and nonmuscle cells. An E-box binding activity, which does not contain MyoD, myogenin, or E2A proteins, is present in muscle and nonmuscle cells and may be responsible for repressing the delta-subunit gene in myoblasts and nonmuscle cells. An enhancer, which lacks E boxes, is also required for expression of the delta-subunit gene but does not confer muscle-specific expression.

Animals↗
PubMed1992

Spatial restriction of AChR gene expression to subsynaptic nuclei.

Acetylcholine receptors (AChRs) and the mRNAs encoding the four AChR subunits are highly concentrated in the synaptic region of skeletal myofibers. The initial localization of AChRs to synaptic sites is triggered by the nerve and is caused, in part, by post-translational mechanisms that involve a redistribution of AChR protein in the myotube membrane. We have used transgenic mice that harbor a gene fusion between the murine AChR delta subunit gene and the human growth hormone gene to show that innervation also activates two independent transcriptional pathways that are important for establishing and maintaining this non-uniform distribution of AChR mRNA and protein. One pathway is triggered by signal(s) that are associated with myofiber depolarization, and these signals act to repress delta subunit gene expression in nuclei throughout the myofiber. Denervation of muscle removes this repression and causes activation of delta subunit gene expression in nuclei in non-synaptic regions of the myofiber. A second pathway is triggered by an unknown signal that is associated with the synaptic site, and this signal acts locally to activate delta subunit gene expression only in nuclei within the synaptic region. Synapse-specific expression, however, does not depend upon the continuous presence of the nerve, since transcriptional activation of the delta subunit gene in subsynaptic nuclei persists after denervation. Thus, the nuclei in the synaptic region of multinucleated skeletal myofibers are transcriptionally distinct from nuclei elsewhere in the myofiber, and this spatially restricted transcription pattern is presumably imposed initially by the nerve.

Animals↗
PubMed1992

Synaptic basal lamina contains a signal for synapse-specific transcription.

Nuclei in the synaptic region of multinucleated skeletal myofibers are transcriptionally distinct, since acetylcholine receptor genes are transcribed at a high rate by these nuclei, but not by nuclei elsewhere in the myofiber. Although this spatially restricted transcription pattern is presumably imposed by the motor nerve, the continuous presence of the nerve is not required, since synapse-specific transcription persists after denervation. These results suggest either that a transcriptional signal persists at synaptic sites after nerve terminals have degenerated, or that a transcriptional pattern in the myofiber, once established, is stable in the absence of a nerve-derived signal. To distinguish between these possibilities, we denervated muscle and damaged the myofibers and specialized cells located near synaptic sites, and then studied transcription of an acetylcholine receptor gene in myofibers that regenerated in their original basal lamina sheaths, but remained denervated. We show that synapse-specific transcription is re-induced in these regenerated myofibers, and we conclude that a signal for synapse-specific transcription is stably maintained in the synaptic basal lamina.

Animals↗
PubMed1991

Dystrophin is a component of the subsynaptic membrane.

A subsynaptic protein of Mr approximately 300 kD is a major component of Torpedo electric organ postsynaptic membranes and copurifies with the AChR and the 43-kD subsynaptic protein. mAbs against this protein react with neuromuscular synapses in higher vertebrates, but not at synapses in dystrophic muscle. The Torpedo 300-kD protein comigrates in SDS-PAGE with murine dystrophin and reacts with antibodies against murine dystrophin. The sequence of a partial cDNA isolated by screening an expression library with mAbs against the Torpedo 300-kD protein shows striking homology to mammalian dystrophin, and in particular to the b isoform of dystrophin. These results indicate that dystrophin is a component of the postsynaptic membrane at neuromuscular synapses and raise the possibility that loss of dystrophin from synapses in dystrophic muscle may have consequences that contribute to muscular dystrophy.

Amino Acid Sequence↗
PubMed1991

Multiple dystrophin isoforms are associated with the postsynaptic membrane of Torpedo electric organ.

We have shown previously that dystrophin is a component of postsynaptic membranes in Torpedo electric organ and is localized at mammalian neuromuscular synapses. In skeletal muscle, dystrophin is also detectable at the non-synaptic membrane of the myofiber, whereas in the electric organ, dystrophin is strictly localized to the postsynaptic membrane, and is not detectable in non-synaptic membranes. Multiple isoforms of dystrophin are present in skeletal muscle, and different isoforms could potentially be targetted to synaptic and non-synaptic membranes. We sought to determine whether the electric organ contains a single, or multiple isoforms of dystrophin, and we show here that the electric organ contains both a and b isoforms of dystrophin. Because dystrophin is found only at the postsynaptic membrane of the electric organ, we conclude that the two isoforms coexist in the postsynaptic membrane.

Animals↗
PubMed1989

Muscle-specific gene expression controlled by a regulatory element lacking a MyoD1-binding site.

Muscle-specific expression of the gene encoding the delta subunit of the acetylcholine receptor is controlled by a 54-base-pair region that does not contain a binding site for MyoD1, a protein involved in activation of the myogenic program. A MyoD1-binding site is present in the proximal promoter region of the gene encoding the delta-subunit, but is neither sufficient nor necessary for muscle-specific expression in transfected muscle cells.

Animals↗
PubMed1988

Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis.

The level of transcripts encoding the skeletal muscle acetylcholine receptor (AChR) was determined during embryonic development in Xenopus laevis. cDNAs encoding the alpha, gamma, and delta subunits of the Xenopus AChR were isolated from Xenopus embryo cDNA libraries using Torpedo AChR cDNAs as probes. The Xenopus AChR cDNAs have greater than 60% amino acid sequence homology to their Torpedo homologues and hybridize to transcripts that are restricted to the somites of developing embryos. Northern blot analysis demonstrates that a 2.3-kb transcript hybridizes to the alpha subunit cDNA, a 2.4-kb transcript hybridizes to the gamma subunit cDNA, and that two transcripts, of 1.9 and 2.5 kb, hybridize to the delta subunit cDNA. RNase protection assays demonstrate that transcripts encoding alpha, gamma, and delta subunits are coordinately expressed at late gastrula and that the amount of each transcript increases in parallel with muscle-specific actin mRNA during the ensuing 12 h. After the onset of muscle activity the level of actin mRNA per somite remains relatively constant, whereas the level of alpha subunit and delta subunit transcripts decrease fourfold per somite and the level of gamma subunit transcript decreases greater than 50-fold per somite. The decrease in amount of AChR transcripts per somite, however, occurs when embryos are paralyzed with local anaesthetic during their development. These results demonstrate that AChR transcripts in Xenopus are initially expressed coordinately, but that gamma subunit transcript levels are regulated differently than alpha and delta at later stages. Moreover, these results demonstrate that AChR transcript levels in Xenopus myotomal muscle cells are not responsive to electrical activity and suggest that AChR transcript levels are influenced by other regulatory controls.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of the mouse acetylcholine receptor delta subunit gene: identification of a 148-bp cis-acting region that confers myotube-specific expression.

We have isolated the gene encoding the delta subunit of the mouse skeletal muscle acetylcholine receptor (AChR) and have identified a 148-bp cis-acting region that controls cell type-specific and differentiation-dependent gene expression. The 5' flanking region of the delta subunit gene was fused to the protein-coding region of the chloramphenicol acetyltransferase (CAT) gene and gene fusions were transfected into C2 mouse skeletal muscle cells. Both transiently and stably transfected cells were assayed for CAT gene expression. Deletions from the 5' end of the mouse delta gene demonstrate that 148 bp of 5' flanking DNA is sufficient to confer cell type-specific and differentiation-dependent expression: CAT activity is present in transfected myotubes, but not in transfected 3T3 cells or 10T1/2 cells. Moreover, the level of CAT expression in myotubes transfected with constructs containing 148 bp of 5' flanking DNA from the delta subunit gene is identical to that in myotubes transfected with constructs containing 3.2 kb of 5' flanking DNA and similar to expression from the SV-40 early promoter. Increased CAT activity in myotubes is a result of an increased rate of transcription from the delta subunit promoter, since CAT RNA levels are also 35-fold more abundant in myotubes than myoblasts. In contrast, the SV-40 early promoter is similarly active in all cell types. Thus, 148 bp of 5' flanking DNA from the delta subunit gene contains all the information required for cell type-specific and differentiation-dependent expression of the AChR delta subunit.

Animals↗
PubMed1988

Regulation of transcript encoding the 43K subsynaptic protein during development and after denervation.

The postsynaptic membrane of vertebrate neuromuscular synapses is enriched in the four subunits of the acetylcholine receptor (AChR) and in a peripheral membrane protein of Mr = 43 x 10(3) (43K). Although AChRs are virtually restricted to the postsynaptic membrane of innervated adult muscle, developing and denervated adult muscle contain AChRs at nonsynaptic regions. These nonsynaptic AChRs accumulate because the level of mRNA encoding AChR subunits increases in response to a loss of muscle cell electrical activity. We have determined the level of mRNA encoding the 43K subsynaptic protein in developing muscle and in innervated and denervated adult muscle. We isolated a cDNA that encodes the entire protein-coding region of the 43K subsynaptic protein from Torpedo electric organ and used this cDNA to isolate a cDNA that encodes the 43K subsynaptic protein from Xenopus laevis. We used the Xenopus cDNA to measure the level of transcript encoding the 43K protein in embryonic muscle and in innervated and denervated adult muscle by RNase protection. The level of transcript encoding the 43K protein is low in innervated adult muscle and increases 25- to 30-fold after denervation. The level of transcript encoding the alpha subunit of the AChR increases to a similar extent after denervation. Moreover, during development, transcripts encoding the 43K protein and the alpha subunit are expressed initially at late gastrula and are present in similar quantities in embryonic muscle. These results demonstrate that transcripts encoding the 43K protein and AChR subunits appear coordinately during embryonic development and that the level of mRNA encoding the 43K protein is regulated by denervation.

Amino Acid Sequence↗
PubMed1987

300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses.

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.

Animals↗
PubMed1985

The subsynaptic 43-kDa protein is concentrated at developing nerve-muscle synapses in vitro.

A 43-kDa peripheral membrane protein is known to copurify with acetylcholine receptor (AcChoR)-rich membranes isolated from the electric organ of Torpedo californica. Immunoelectron microscopy and crosslinking studies have demonstrated that this 43-kDa protein is closely associated with the cytoplasmic domain(s) of the AcChoR and suggest that the 43-kDa protein could regulate the distribution of the AcChoR in the postsynaptic membrane. This paper demonstrates that this postsynaptic protein appears at developing neuromuscular synapses in Xenopus nerve/muscle cocultures as early as AcChoRs become clustered at synaptic sites. Moreover, this protein is concentrated at AcChoR clusters that occur on noninnervated muscle cells. The close spatial and temporal relationship of this subsynaptic protein and AcChoR clusters is consistent with a role for the 43-kDa protein in the formation and/or stabilization of AcChoR clusters.

Animals↗
PubMed1983

Crosslinking of proteins in acetylcholine receptor-rich membranes: association between the beta-subunit and the 43 kd subsynaptic protein.

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four subunits (alpha, beta, gamma, delta) of the acetylcholine receptor (AChR) and for polypeptides at 43 kd and 270 kd. Reaction of these membranes with 3H-N-ethylmaleimide (3H-NEM) demonstrates that most of the available free sulfhydryls reside on the 43 kd protein. Cross-linking reagents that contain NEM as one reactive group, and N-hydroxysuccinimide as the other, were used to study the topography of the 43 kd protein in AChR-rich membranes. Proteins from cross-linked membranes were resolved by SDS-PAGE and the composition of crosslinked products was determined by Western blots and monoclonal antibodies. A crosslinked product at 110 kd was labeled by a monoclonal antibody to the beta-subunit and by a monoclonal antibody to the 43 kd protein, but not by monoclonal antibodies to the alpha, gamma, or delta subunits. The 110 kd crosslink was not produced in the presence of 10 mM lithium diiodosalicylate, which dissociates the 43 kd protein from the membrane. Thus the 43 kd protein is intimately associated with the AChR and in close proximity to the beta-subunit.

Animals↗