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J P Merlie

Publications and source records attributed to J P Merlie.

At least 91 records · Page 5Linked to original sources
PubMed1988

Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase).

An oligonucleotide probe was used to isolate a clone encoding prostaglandin endoperoxide synthetase (cyclooxygenase, EC 1.14.99.1) from a sheep seminal vesicle cDNA library. The protein predicted from nucleic acid sequence contains 599 amino acids including a 23-amino acid signal sequence. Thus, the mature cyclooxygenase deduced from the cDNA compares favorably in molecular size to the 70-kDa protein determined by gel electrophoresis. A putative transmembrane region and potential carbohydrate addition sites for N-linked sugars can be inferred from the amino acid sequence. Significantly, sequence similarities exist between cyclooxygenase, myeloperoxidase, and several other heme-containing proteins. The putative glycosylation sites, transmembrane domain, and sequence similarities with functionally related enzymes have been incorporated into a model for the topology of cyclooxygenase in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1988

Native folding of an acetylcholine receptor alpha subunit expressed in the absence of other receptor subunits.

An alpha subunit cDNA of the mouse nicotinic acetylcholine receptor under transcriptional control of the Rous Sarcoma virus long terminal repeat was transfected into and expressed in a quail fibroblast cell line. The biosynthesis and post-translational modification of the alpha subunit protein made in this heterologous system have been studied using immunoprecipitation and ligand binding assays. The polypeptide is present at high steady-state levels and inserted in the correct transmembrane orientation. However, in the absence of assembly with other subunits the alpha subunit is confined to an intracellular membrane compartment and is not transported to the plasma membrane. Twenty percent of the newly synthesized alpha subunit acquired high affinity alpha bungarotoxin binding in a time-dependent process within 20 min of translation. Sucrose gradient fractionation demonstrated that both the polypeptide and toxin binding forms of the alpha subunit have a sedimentation coefficient of 5 s suggesting the absence of stable homo-oligomers. Quantitative binding assays demonstrated that the apparent affinity and rate of association of alpha bungarotoxin to the unassembled alpha subunit are greater than for native receptor. On the other hand, the affinities for the small ligands D-tubocurarine and gallamine are 10(3) lower than for native receptor; no detectable binding was observed for decamethonium, hexamethonium, or carbamylcholine. Thus, the acetylcholine receptor alpha subunit, independent of other subunits of the receptor, acquires a mature conformation and high affinity alpha bungarotoxin binding when expressed in a quail fibroblast cell line.

Animals↗
PubMed1988

Studies of acetylcholine receptor subunit gene expression: chromatin structural changes during myogenesis.

Myogenesis proceeds stepwise from pluripotential stem cell to differentiated myotube. The precise number of transitions that occur along the developmental pathway remains to be determined. We examined the myogenic pathway as modelled by mouse mesodermal stem cell and muscle cell lines for stage-specific alterations in the chromatin structure of the acetylcholine receptor delta and gamma subunit genes. We reasoned that such an analysis would allow us to observe either the primary events in the activation of these muscle-specific genes or processes secondary to the binding of muscle-specific regulatory proteins. We probed chromatin structure with DNase I (deoxyribonuclease I) and precisely mapped to the 5' ends of the delta and gamma genes DNase I hypersensitive (DH) sites whose induction is unique to each myogenic stage. Putative mesodermal stem cells have the simplest pattern of DH sites with no sites near the 5' ends of the delta and gamma genes, whereas differentiated myotubes express the most complex pattern; the myoblast pattern is intermediate and of two types. In muscle cell lines where differentiation must be induced the myoblasts have a simple pattern (one more site than stem cells); in muscle lines where differentiation is spontaneous the myoblasts express a complex pattern of DH sites (one less site than myotubes). Inducible myoblasts seem to be arrested in an earlier step in the myogenic pathway than spontaneously differentiating myoblasts. Thus, myogenic activation of acetylcholine receptor subunit genes appears to be a stepwise process that can be detected by chromatin structural changes specific to four distinct stages of muscle development: stem cell, early myoblast, late myoblast, and differentiated myotube.

Animals↗
PubMed1988

Acetylcholine receptor-associated 43K protein contains covalently bound myristate.

Torpedo electroplaque and vertebrate neuromuscular junctions contain high levels of a nonactin, 43,000-Mr peripheral membrane protein referred to as the 43K protein. 43K protein is associated with the cytoplasmic face of postsynaptic membranes at areas of high acetylcholine receptor density and has been implicated in the establishment and/or maintenance of these receptor clusters. Cloning of cDNAs encoding Torpedo 43K protein revealed that its amino terminus contains a consensus sequence sufficient for the covalent attachment of the rare fatty acid myristate. To examine whether 43K protein is, in fact, myristoylated, mouse muscle BC3H1 cells were metabolically labeled with either [35S]cysteine or [3H]myristate and immunoprecipitated with a monospecific antiserum raised against isolated Torpedo 43K protein. In cells incubated with either precursor, a single labeled species was specifically recovered that comigrated on SDS-PAGE with 43K protein purified from Torpedo electric organ. Approximately 95% of the 3H labeled material released from [3H]myristate-43K protein by acid methanolysis was extractable in organic solvents and eluted from a C18 reverse-phase HPLC column exclusively at the position of the methyl myristate internal standard. Thus, 43K protein contains authentic myristic acid rather than an amino or fatty acid metabolite of [3H]myristate. Myristate appears to be added to 43K protein cotranslationally and cannot be released from it by prolonged incubation in SDS, 2-mercaptoethanol, or hydroxylamine (pH 7.0 or 10.0), characteristics consistent with amino terminal myristoylation. Covalently linked myristate may be responsible for the high affinity of purified 43K protein for lipid bilayers despite the absence of a notably hydrophobic amino acid sequence.

Animals↗
PubMed1988

Stepwise activation of the mouse acetylcholine receptor delta- and gamma-subunit genes in clonal cell lines.

We used the DNase I-hypersensitive sites around the mouse acetylcholine receptor delta-subunit gene as a guide toward the cloning and sequencing of delta and gamma transcriptional regulatory regions and as a means to assess chromatin structural activation of the delta- and gamma-subunit genes during myogenesis. Genomic cloning of hypersensitive sites downstream of the delta-subunit gene revealed the presence of the gamma-subunit gene approximately 5 kilobases away; the hypersensitive sites mapped to the 5' end of the gamma-subunit gene. Sequence comparison of restriction fragments containing hypersensitive sites in analogous locations at the 5' ends of the delta- and gamma-subunit genes uncovered little overall homology between the two genomic fragments; however, an 11- of 13-base-pair match between the two sequences was found. Homologs to this sequence were also found to be present in the upstream regions of the chicken alpha- and mouse beta-subunit genes. By RNase protection and primer extension analyses, the delta-subunit gene transcription start site was mapped to 56 base pairs upstream of the initiator ATG codon. Clonal cell lines with various potentials to differentiate to the skeletal muscle phenotype were examined for their hypersensitive site pattern within the delta-gamma locus. Only remote hypersensitive sites flanking the locus appear in pluripotential mesodermal cells. A cell line of determined but inducible myoblasts expressed only one more intergenic site, while in permissively differentiating myoblasts hypersensitive sites were already present at the 5' ends of the delta and gamma genes prior to differentiation. Terminal differentiation resulted in an identical pattern of hypersensitive sites in all muscle cell lines examined so far, with an intergenic site near the gamma-subunit gene being the only site specific to the differentiated muscle phenotype.

Animals↗
PubMed1988

[Regulation of synaptogenesis in the adult skeletal muscle].

Several behaviors that axons exhibit during reinnervation of adult skeletal muscle demonstrate that they are guided by cues that the muscle provides. For example, axons form synapses on denervated but not on normally innervated muscle fibers; axons selectively reinnervate original synaptic sites on denervated muscle fibers; regenerating axons become specialized for synaptic transmission only in regions where they contact muscle fibers; and motor axons prefer to reinnervate muscles derived from matching levels of the body's rostro-caudal axis. This chapter describes these phenomena and summarizes progress toward identifying the soluble, membrane-bound, and extracellular matrix molecules that underly them.

Animals↗
PubMed1987

Formation of the alpha-bungarotoxin binding site and assembly of the nicotinic acetylcholine receptor subunits occur in the endoplasmic reticulum.

During the process by which newly synthesized subunits of the nicotinic acetylcholine receptor (stoichiometry = alpha 2 beta gamma delta) mature and acquire the properties of the fully functional cell surface receptor, they undergo numerous covalent and noncovalent modifications. Using ligand-mediated and subunit-specific immunoprecipitation, four forms in the maturation of the alpha subunit can be detected: the primary translation product; alpha subunit that can bind alpha-bungarotoxin; alpha subunit assembled with the other subunits; and surface receptor. The alpha subunit acquires the ability to bind alpha-bungarotoxin with a t1/2 of approximately 40 min after translation and becomes assembled with a t1/2 of 80 min after translation. Using metabolic labeling and sucrose gradient fractionation, we have determined the subcellular location of alpha subunit when it acquires the ability to bind alpha-bungarotoxin and when it is assembled. Golgi membranes were identified across the gradient by the enzymatic activities UDP-galactose:N-acetylglucosamine galactosyltransferase and alpha-mannosidase. Endoplasmic reticulum membranes were identified by the enzymatic activity glucose-6-phosphatase and by the presence of newly synthesized alpha and beta subunits. Pulse-labeled alpha subunit that bound alpha-bungarotoxin was first detected co-migrating in the gradient with the glucose-6-phosphatase activity. Therefore, the capacity to bind alpha-bungarotoxin was acquired while the alpha subunit was in the endoplasmic reticulum. Assembled alpha subunit was detected by immunoprecipitating with an anti-beta subunit-specific monoclonal antibody. By this method, assembled receptor was first detected 15 min after translation in both the endoplasmic and Golgi portions of the gradient. To validate this method of detecting assembled receptor, we determined the sedimentation coefficient of the receptor subunits in the endoplasmic reticulum. Both unassembled subunits with sedimentation coefficients of 5 S and assembled receptor with a sedimentation coefficient of 9 S were recovered from the endoplasmic reticulum portion of the gradient. Thus, our data concerning the subcellular site of assembly are consistent with assembly occurring in the endoplasmic reticulum followed by rapid transport to the Golgi.

Binding Sites↗
PubMed1987

cDNAs for the postsynaptic 43-kDa protein of Torpedo electric organ encode two proteins with different carboxyl termini.

Postsynaptic membranes isolated from Torpedo electric organ are highly enriched in the nicotinic acetylcholine receptor and a nonreceptor protein of 43 kDa; the distribution of the 43-kDa protein and the receptor is coextensive in the electrical membrane. As a first step in understanding the regulation of 43-kDa protein expression, we have isolated and characterized 43-kDa protein cDNAs. A lambda gt11 cDNA library was constructed from Torpedo californica electric organ mRNA and screened with a pool of 26-mer oligonucleotides encoding a short tryptic fragment of the 43-kDa synaptic protein. Positive clones were purified and sequenced; the amino acid sequences were deduced, and they matched chemically determined protein sequences of the 43-kDa protein. Two distinct classes of cDNAs were obtained; one class encoded a 43-kDa protein of 389 amino acids with a calculated molecular mass of 43,988 daltons, and another class encoded a second 43-kDa protein containing 23 additional amino acids at the C terminus. Therefore, it appears that two 43-kDa proteins with different carboxyl termini are encoded by separate mRNAs. Consistent with this idea, blot hybridization analysis revealed multiple polyadenylylated 43-kDa mRNAs in electric organ. One polyadenylylated mRNA of approximately equal to 2.0 kilobases in length was apparent in both embryonic day-11 chick muscle and the mouse muscle cell line BC3H1.

Amino Acid Sequence↗
PubMed1987

Regulation of phosphorylation of nicotinic acetylcholine receptors in mouse BC3H1 myocytes.

By using 32P-labeling methods and performing immunoprecipitations with specific antibodies, we have found that three subunits of the nicotinic acetylcholine receptor are phosphorylated in mouse skeletal muscle cells. In nonstimulated cells, the molar ratios of phosphate estimated in alpha, beta, and delta subunits were 0.02, 0.05, and 0.5, respectively. All three subunits contained predominantly phosphoserine with some phosphothreonine; the beta subunit also contained phosphotyrosine. Incubating cells with agents that stimulate cAMP-dependent pathways (isoproterenol, forskolin, 8-Br-cAMP) increased the phosphorylation of the delta subunit by 50%, but phosphate labeling of the beta subunit was depressed by a third. In contrast, when cells were incubated with the divalent cation ionophores A-23187 or ionomycin, phosphorylation of both the delta and beta subunits increased. The results indicate that acetylcholine receptors are phosphorylated to significant levels in skeletal muscle cells and that cAMP-dependent and Ca2+-dependent pathways exist for controlling the phosphorylation state of the receptor subunits.

Animals↗
PubMed1986

A universal oligonucleotide probe for acetylcholine receptor genes. Selection and sequencing of cDNA clones for the mouse muscle beta subunit.

A region of 25 nucleotides is highly conserved in genes coding for the alpha, beta, gamma, and delta subunits of the nicotinic acetylcholine receptor (AChR) of human, mouse, calf, chicken, and Torpedo. Based on this observation, a 2-fold degenerate oligonucleotide was synthesized and used as a probe to screen a cDNA library made from a mouse myogenic cell line. Clones coding for the beta, gamma, and delta subunits were identified by the probe. The protein sequence deduced from the beta subunit clones codes for a precursor polypeptide of 501 amino acids with a calculated molecular weight of 56,930 daltons, which includes a signal peptide of 23 amino acids. The protein sequence and structural features of the beta subunits of mouse, calf, and Torpedo are conserved. A clone coding for the mouse gamma subunit was isolated, and its identity was confirmed by alignment of its sequence to previously published cDNA sequences for the mouse and calf gamma subunits. The clone contained approximately 200 nucleotides more at its 3' end untranslated region than a mouse gamma clone recently described. Northern blot analysis, utilizing as probes these beta and gamma subunit cDNAs and previously characterized alpha and delta subunit cDNAs, shows that the steady-state levels of the four AChR mRNAs increase coordinately during terminal differentiation of cultured C2 and C2i mouse myoblasts. The increase in mRNA levels can account for the rise of cell surface receptors during myogenesis and suggests that the muscle AChR genes may be regulated during development by a common mechanism. Utilization of this oligonucleotide probe should prove useful for screening a variety of libraries made from different species and tissues which are known to express AChRs.

Amino Acid Sequence↗
PubMed1986

Chromosomal localization of muscle nicotinic acetylcholine receptor genes in the mouse.

The chromosomal localization of the genes encoding the four subunits of muscle nicotinic receptor was determined by analyzing restriction fragment length polymorphisms between two mouse species Mus musculus domesticus (DBA/2) and Mus spretus (SPE). Analysis of the progeny of the interspecies mouse backcross (DBA/2 X SPE) X DBA/2 showed that the alpha-subunit gene cosegregates with the alpha-cardiac actin gene on chromosome 17, that the beta-subunit gene is located on chromosome 11, and that the gamma- and delta-subunit genes cosegregate and are located on chromosome 1.

Actins↗
PubMed1986

The effects of inhibiting oligosaccharide trimming by 1-deoxynojirimycin on the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor has a subunit stoichiometry of alpha 2 beta gamma delta; all 5 subunits contain N-linked oligosaccharides. We investigated what role trimming of the oligosaccharides played in the post-translational processing of the subunits and assembly of the receptor by examining the receptor synthesized in the presence of an inhibitor of oligosaccharide trimming, 1-deoxynojirimycin. BC3H-1 cells express one-third fewer receptors when grown in the presence of 1-deoxynojirimycin. The receptor subunits that are expressed have decreased mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating an inhibition of oligosaccharide trimming. In control cells, 40% of the translated alpha subunit acquires the capacity to bind alpha-bungarotoxin with a half-time of 40 min before assembly with the other subunits; the rest is rapidly degraded. In 1-deoxynojirimycin-treated cells approximately the same amount of alpha subunit is translated as in control cells, but that alpha subunit is degraded more rapidly, and only 25% acquires the capacity to bind alpha-bungarotoxin. From these results, we conclude that oligosaccharide processing either may aid in protecting the alpha subunit primary translation product from degradation or may be required for the conformational change or other post-translational modification(s) necessary for formation of the alpha-bungarotoxin binding form of the alpha subunit, which is then protected from proteolytic degradation. The cell surface receptor that is expressed in the presence of 1-deoxynojirimycin, however, is not altered in its affinity for cholinergic ligands. Thus, we conclude that differential N-linked oligosaccharide trimming of the 2 alpha subunits does not appear to play a part in the differences in affinities of the 2 alpha subunits for cholinergic ligands.

1-Deoxynojirimycin↗
PubMed1986

Transcriptional regulation of nicotinic acetylcholine receptor genes during muscle development.

The quantities of nicotinic acetylcholine receptors measured by alpha-bungarotoxin binding on the surface of mouse skeletal muscle C2 cells increase by approximately 10- to 100-fold during their terminal differentiation in culture. By northern blot analysis, we have determined that the steady-state levels of the AChR alpha and delta subunit mRNAs increase by approximately 15-fold during differentiation of C2 cells. To determine if the differences in message levels during myogenesis are due to changes in transcriptional rates of these genes, nuclear run-on experiments were done using nuclei from 3-day-old undifferentiated cells and 7-day-old differentiated cells. The rates of transcription for both the alpha and delta subunit genes changed from levels that could not be detected over background in nuclei from undifferentiated cells to levels that were at least 8-fold over background in differentiated nuclei. As internal controls, we measured the rates of transcription of mouse actin and histone genes. The signals obtained from both undifferentiated and differentiated nuclei were approximately 50-fold over background for both genes, indicating that the absence of detectable transcription of receptor subunit genes in undifferentiated C2 is specific.

Animals↗
PubMed1986

Inhibition of acetylcholine receptor assembly by activity in primary cultures of embryonic rat muscle cells.

Silencing of contractile activity in muscle is known to increase the level of acetylcholine receptor on the cell surface. Both in vivo and in vitro studies indicate that modulation of receptor-specific mRNA levels plays a role in the activity-related regulation, but other mechanisms have not been explored. In this study, we examine the synthesis and post-translational fate of receptor alpha subunit in actively contracting and tetrodotoxin-inhibited rat muscle cultures. Using metabolic labeling and immunoprecipitation with subunit-specific monoclonal antibodies, we find that the increase of alpha subunit synthesis in tetrodotoxin-inactivated cultures is insufficient to account for the increased rate at which new receptors appear on the cell surface. In evaluating stages in the post-translational processing of alpha subunit, we find that in active and inactive cultures, newly synthesized subunit acquires the ability to bind alpha-bungarotoxin with the same kinetics. However, differences were noted at or preceding the stage where alpha subunit becomes assembled with the other subunits to form the 9 S receptor. In inactivated cultures, newly synthesized alpha subunit transits a 5 S precursor pool more rapidly and is assembled more efficiently than in contracting cultures. The possibility that these differences represent a type of post-translational regulation is discussed.

Animals↗