Mouse Acetylcholine Receptor Research Articles

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.

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.

Increased levels of acetylcholine receptor α-subunit mRNA in experimental autoimmune myasthenia gravis

To gain insight into the regulatory mechanisms underlying the blockade and loss of acetylcholine receptor (AChR) in myasthenia, we have followed AChR α-subunit mRNA levels in leg muscles of myasthenic and normal rabbits and rats. Northern blots of RNA preparations from normal and myasthenic animals were hybridized with a mouse AChR α-subunit cDNA probe. Our experiments indicate a specific increase (4–7-fold) in the levels of α-subunit mRNA in animals with experimental autoimmune myasthenia gravis (EAMG), in comparison with control animals. Actin mRNA levels were essentially unchanged. Our results thus suggest that EAMG is accompanied by an increased level of AChR gene transcription.

Transcriptional regulation of nicotinic acetylcholine receptor genes: identification of control elements of a γ-subunit gene

The muscle nicotinic acetylcholine receptor undergoes profound changes in abundance and distribution in response to innervation and denervation. As a start towards understanding the detailed mechanisms of acetylcholine receptor gene regulation, we have identified transcriptional regulatory regions of a receptor subunit gene. A region of genomic DNA at the 5′ end of the mouse acetylcholine receptor γ-subunit gene has been shown to promote transcription of the bacterial chloramphenicol acetyltransferase gene in a cell-type specific manner. In addition, the transcriptional activity of this fragment is developmentally regulated in mouse muscle C2C12 cells. We propose that this fragment contains transcriptional control elements of the mouse muscle acetylcholine receptor γ-subunit gene.

DNase I-hypersensitive sites surround the mouse acetylcholine receptor delta-subunit gene.

The mouse skeletal muscle acetylcholine receptor is regulated, at least in part, by transcriptional mechanisms. As a first step toward defining the cis- and trans-acting factors involved in acetylcholine receptor transcriptional regulation, we examined mouse muscle and fibroblast cells for the presence of DNase I-hypersensitive sites in the chromatin surrounding the delta-subunit gene. We detect six DNase I-hypersensitive sites in muscle DNA blots hybridized with a mouse delta-subunit cDNA. Two hypersensitive sites lie distantly 5' to the delta gene, one lies near the 5' end of the delta gene, one lies near its 3' end, and two hypersensitive sites lie distantly 3'. The pair of distantly 3' hypersensitive sites should by analogy to chicken and human genomes lie near the gamma-subunit gene. Only the distantly 5' pair of hypersensitive sites is present in fibroblasts; the remainder is muscle specific. Two hypersensitive sites, one at the 3' end of the delta gene and one distantly 3', appear only after terminal muscle differentiation and, therefore, are developmentally regulated, similarly to receptor subunit mRNAs. All four muscle-specific hypersensitive sites are excellent candidates for regulatory loci for the delta- and gamma-subunit genes of mouse acetylcholine receptor.

Immunotherapy for myasthenia gravis: a murine model.

In vivo therapy with monoclonal antibody (mAb) GK1.5, which recognizes a glycoprotein antigen designated L3T4 on murine helper T lymphocytes, either prevented or suppressed the development of murine lupus, autoimmune encephalomyelitis, and collagen arthritis. The L3T4 antigen in the mouse is analogous to the human Leu-3/T4 antigen expressed on helper T lymphocytes, because they both participate in the T cell response to class II major histocompatibility complex (MHC) antigens. Class II MHC genes and I-A antigens mediate murine experimental autoimmune myasthenia gravis (EAMG) induced by acetylcholine receptor (AChR) autoimmunity. We studied the efficacy of mAb GK1.5 as an immunotherapeutic agent for murine EAMG. Therapy with mAb GK1.5 not only suppressed established autoimmunity to AChR but also prevented loss of muscle AChR in mice with EAMG. Moreover, permanent remission of clinical muscle weakness was induced if mAb GK1.5 therapy was initiated after the onset of clinical disease. Because the function of the Leu-3/T4 determinant on human helper T lymphocytes is analogous to the murine L3T4 determinant, use of antibody to the Leu-3/T4 determinant as an immunotherapeutic agent may provide a way to control the progression of human MG.

Isolation and sequence of cDNA clones coding for the precursor to the gamma subunit of mouse muscle nicotinic acetylcholine receptor.

cDNA libraries have been constructed in plasmid (pBR322) and bacteriophage lambda gammagt10) vectors with poly (A+) RNA isolated from the nonfusing mouse muscle cell line BC3H-1. The libraries were screened with a restriction fragment derived from a genomic clone coding for a human acetylcholine receptor gamma subunit. Several clones were obtained whose cDNA inserts possessed nucleotide and deduced amino acid sequence homology with acetylcholine receptor gamma subunits from Torpedo californica, chick, calf, and human. One isolate, lambda BMG419, has 88 nucleotides of 5'-untranslated sequence, an open reading frame of 1,557 nucleotides coding for the precursor to the mouse acetylcholine receptor gamma subunit, and 144 nucleotides of 3'-untranslated sequence. Alignment of the lambda BMG419-deduced amino acid sequence with homologs from other species predicts a precursor peptide of 519 amino acids and a mature protein of 497 amino acids, with nonglycosylated molecular weights of 58,744 and 56,424 daltons, respectively. Comparison of the deduced amino acid sequence of the mouse gamma subunit with Torpedo, chick, calf, and human sequences showed overall homologies of 54%, 67%, 90%, and 90%, respectively; however, significantly higher homologies were found in several putative functional domains. Radiolabeled lambda BMG419 has been used to identify homologous RNA species, one of approximately 2 kb and one of about 3.5 kb, in poly (A+) RNA prepared from BC3H-1 cells and denervated mouse limb muscle. gamma Subunit-coding RNA species are considerably more abundant in denervated than in innervated muscle, suggesting that neural regulation of the abundance of the gamma subunit is exerted through regulation of the amount of its mRNA.

Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor

The mouse cell line BC3H-I synthesizes an acetylcholine receptor (AChR) with the pharmacological properties of a muscle nicotinic cholinergic receptor. We have purified mRNA from this cell line and used the size-fractionated poly(A)+RNA to produce a cDNA library of approximately 50,000 clones. The library was screened with a subclone containing genomic sequences coding for the putative acetylcholine-binding site of the alpha-subunit of chicken AChR. We obtained a plasmid, pMAR alpha 15, with a 1,717-base pair insert. The insert cDNA has 26 nucleotides at the 5'-end which code for a portion of the signal peptide followed by a single open reading frame of 1,311 nucleotides which code for a protein of 49,896 daltons. The insert has 377 bases of 3'-untranslated sequence with 3 polyadenylation sites. Radiolabeled plasmid DNA has been used to identify homologous RNA species of about 2 kilobases in Northern blot analyses of poly(A)+ selected RNA from BC3H-I cells. A similar size mRNA is seen in innervated mouse diaphragm and leg muscle, and both mouse and rat brain. Comparisons of the deduced amino acid sequence of the mouse AChR alpha-subunit with Torpedo marmorata, T. californica, chicken, human, and calf sequences show overall homologies of 80%, 80%, 86%, 96%, and 95%, respectively. More detailed analyses reveal a non-random distribution of amino acid substitutions in several structural domains. Based on the absolute conservation of cysteine residues, a new model for the arrangement of the disulfide bonds in the extracellular portion of the alpha-subunit is proposed.

Correction: Isolation and Characterization of a cDNA Clone for the Complete Protein Coding Region of the delta Subunit of the Mouse Acetylcholine Receptor

Correction: Isolation and Characterization of a cDNA Clone for the Complete Protein Coding Region of the delta Subunit of the Mouse Acetylcholine Receptor

Isolation and characterization of a cDNA clone for the complete protein coding region of the δ subunit of the mouse acetylcholine receptor

Isolation and characterization of a cDNA clone for the complete protein coding region of the δ subunit of the mouse acetylcholine receptor

CDNA clone for the alpha subunit of the acetylcholine receptor from the mouse muscle cell line BC3H-1.

Sequences from a gene coding for mouse acetylcholine receptor alpha subunit have been inserted into a recombinant plasmid and cloned in Escherichia coli. mRNAs for acetylcholine receptors occur in low abundance in vertebrate muscle. To clone the mouse alpha-subunit cDNA, we made use of (i) a cell line, BC3H-1, that overproduces the alpha-subunit mRNA and (ii) a polysome fractionation procedure that results in enrichment of alpha-subunit mRNA. Polyadenylylated RNA was used to construct a cDNA library of 750 recombinant clones. Acetylcholine receptor-specific sequences were identified by hybrid-selected translation, followed by monoclonal antibody precipitation and peptide mapping of the translation product. One clone (pA59) that fit these criteria was found in the first 80 isolates. It had a 700-base-pair insert that was excised with Pst I. Blot-hybridization experiments with nick-translated pA59 DNA showed that BC3H-1 cells contain 100-1,000 times more alpha-subunit mRNA than does newborn or adult mouse muscle. Blot hybridization of restriction digests of mouse liver DNA revealed that pA59 is homologous to a very small number (probably one) of genomic sequences.

Myasthenia gravis: passive transfer to mice of antibody to human and mouse acetylcholine receptor.

Mice were given intraperitoneal injections of serum from three patients with myasthenia gravis who had different titers of antibody against mouse acetylcholine receptor (AChR). Almost all mice treated with a high titer of serum antigens showed generalized paralysis, reduced amplitudes of miniature endplate potential (MEPP), or reduced numbers of AChRs. The effects were less marked in serum with lower titers of antibody to mouse AChR, when there was no relation to the severity of effects to the titer of antibody to human AChR.

Experimental myasthenia gravis. A murine system.

Mice from eight inbred strains were immunized with acetylcholine receptor (AChR) purified from Torpedo californica. All mice developed high concentrations of serum antibodies (10(-6) M) against the immunogen and approximately 80% possessed antibodies reactive with mouse nicotinic AChR. 33% of the mice immunized (n = 236) developed muscular weakness and flaccid paralysis. Behavioral, electrophysiological, and pharmacological similarities were found between the experimentally induced muscular weakness and the disease myasthenia gravis. Susceptibility to experimental myasthenia was found to be strain dependent in that the frequency of paralysis was much greater in some strains than others. The occurrence of muscular weakness and flaccid paralysis did not correlate with the concentration of antibodies reactive with T. californica or mouse AChR. Anti-receptor antibodies which increased the rate of AChR degradation on the mouse muscle cell line, BC3H-1, were found in the serum of both myasthenic and nonmyasthenic mice. 40% of the mice tested possessed antibodies reactive with antigenic determinants present on mouse receptor but not T. californica receptor. The occurrence of antibodies unique to mouse receptor did not correlate with myasthenia. Thus, myasthenia in the mouse does not occur simply as a consequence of the presence of antibodies directed against cell surface antigenic determinants of AChR. If anti-AChR antibodies are both necessary and sufficient for the induction of myasthenia, then these studies suggest that populations of a particular structure and/or specificity are required. It is anticipated that the mouse model of myasthenia gravis will permit the regulation of the anti-receptor immune response to be studied in detail.