A 5''-flanking region of the chicken acetylcholine receptor alpha-subunit gene confers tissue specificity and developmental control of expression in transfected cells.

The 5' end and promoter region of the alpha-subunit gene of chicken muscle acetylcholine receptor was mapped and sequenced. It includes a TATA and a CAAT box and a potential Sp1-binding site. When inserted in front of the chloramphenicol acetyltransferase gene, this promoter (including 850 base pairs of upstream sequence) directed high transient chloramphenicol acetyltransferase expression in transfected mouse C2.7 myotubes but not in C2.7 myoblasts or nonmyogenic 3T6 cells.     

Functional activity of the two promoters of the myosin alkali light chain gene in primary muscle cell cultures: comparison with other muscle gene promoters and other culture systems.

Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters.     

鸡乙酰胆碱受体γ亚基基因5′连接区含多元顺式调节元件

本工作利用瞬时表达研究了鸡Ach Rγ亚基基因5′连接区的调控机能。CAT分析表明,γ基因5′连接区-509/-302和-324/+36序列均可独立激活SV40启动子的转录,这种激活作用具有组织特异性,与方向、距离无关,符合增强子样作用。     

Growth and differentiation of permanent and secondary mouse myogenic cell lines on microcarriers

Myogenesis involves the determination of progenitor cells to myoblasts, their fusion to yield multinuclear myotubes, and the maturation of myotubes to muscle fibres. This development is reflected in a time pattern of gene expression, e.g. of genes coding for desmin, the myogenic factors myogenin and myoD, the acetylcholine receptor alpha-subunit and the muscular chloride channel CIC-1. We attempted to improve yields and myogenic differentiation in culture by using three-dimensional microcarrier systems. Out of a variety of carriers tested in stationary cultures, collagen-coated dextran Cytodex3 beads proved optimal for the proliferation and differentiation of the murine myogenic cell line C2C12. With C2C12 myoblasts in stationary and stirred systems (Spinner- and SuperSpinner flasks), surface adherence, differentiation into myotubes and expression of muscle-specific mRNAs on Cytodex3 beads were the same as in conventional cultures. Other carriers tested (DEAE cellulose, glass, plastic, cellulose, polyester) did not support growth and differentiation of C2C12 cells. The secondary mouse myogenic stem cells M12 and M2.7-MDX proliferated and differentiated well in stationary Cytodex3 cultures, but no differentiation occurred in Spinner flasks. As indicated by light and scanning electron microscopy, C2C12 myotubes formed not only on but also in between Cytodex beads. The secondary cell lines may succumb to shear forces under these conditions.     

Interaction of nuclear factors with the upstream region of the alpha-subunit gene of chicken muscle acetylcholine receptor: variations with muscle differentiation and denervation.

The region lying between nucleotides (nt) -110 and -45 of chicken acetylcholine receptor alpha-subunit gene 5' upstream sequence confers developmental control of expression in primary cultures of chicken myotubes. This region interacts with several nuclear factors present in muscle cells as shown by DNase I footprinting and gel-retardation experiments. An Sp1-like factor and a guanine stretch-binding protein were found to bind to overlapping sites immediately upstream of the TATA box. Several factors interacting in the same region with a domain similar to the SV40 enhancer core appeared during in vitro differentiation of myoblasts into myotubes. The concentration of some of these factors increased also after denervation of leg muscle in newborn chickens. The specific interaction of nuclear factors with this domain may thus play a critical role in the regulation of alpha-subunit gene expression by muscle differentiation and electrical activity.     

An acetylcholine receptor alpha-subunit promoter conferring preferential synaptic expression in muscle of transgenic mice.

We have obtained transgenic mice expressing nuclearly targeted beta-galactosidase (nls-beta-gal) under the control of a chicken acetylcholine receptor alpha-subunit promoter. The expression of the transgene was detected in early somites, starting before embryonic day 9.5. In 13-day embryos, the expression pattern of the transgene closely paralleled that of the endogenous mouse alpha-subunit gene, assessed by in situ hybridization. Our results illustrate, with single-cell resolution, the tissue specificity of this alpha-subunit promoter during embryogenesis. After birth, the overall beta-galactosidase activity rapidly decreased with age. However, in diaphragms of newborn animals, beta-galactosidase activity selectively persisted in nuclei underlying the motor endplates. The latter were revealed by an acetylcholinesterase stain. Nls-beta-gal was also visualized by indirect immunofluorescence, while endplates were labelled with fluorescent alpha-bungarotoxin. Confocal microscopy unambiguously identified the more intensely stained nuclei as synaptic 'fundamental nuclei', and allowed estimates of relative staining levels. Thus an 842 bp acetylcholine receptor gene promoter confers preferential synaptic expression to a reporter gene within myofibres in vivo.     

肌肉LIM蛋白增强生肌素对AChRγ启动子的反式激活作用

采用RT PCR技术克隆了大鼠肌肉LIM蛋白 (MLP) 6 40bp的全长cDNA序列 .以此cDNA为探针进行的Northern印迹表明 ,MLP于C2C12细胞在分化的第 3d至第 5d表达 .将MLPcDNA亚克隆至pcDNA3,构建真核表达质粒pcDNA3 MLP ,同时构建AChRγ启动子序列 (96 0bp)调控的荧光素酶报告基因真核表达质粒pGL3 γ .C2C12细胞转染及荧光素酶活性分析表明 ,复合转染pcD NA3 MLP和pGL3 γ的分化肌细胞表达的荧光素酶活性约为对照的 4倍 ;而在 3T3或未分化肌细胞复合转染pcDNA3 MLP和pGL3 γ均未检出报告基因表达 ,说明MLP可促进生肌素对AChRγ亚基基因启动子的反式激活作用 .     

鸡AChRγ亚基基因远上游增强子样作用依赖生肌因子

先前曾报告鸡ＡＣｈＲγ亚基基因５＇连接区－５３８／－２８８片段（含一对Ｅ盒子）与生肌调节因子（ｍｙｏｇｅｎｉｎ）的相互作用,为进一步观察ＤＮＡ－ｍｙｏｇｅｎｉｎ相互作用对γ基因转录激活功能的影响,利用瞬时表达体系研究了鸡ｎＡＣｈＲγ亚基基因５＇端－５３８／－２８８片段调控机能。ＣＡＴ分析表明：γ基因－５３８／－２８８片段可显著激活ｔｋ启动子在分化的Ｃ２Ｃ１２肌细胞中的转录活性,但不能激活在未分化Ｃ２Ｃ１２肌细胞中的转录活性。这种组织特异性转录激活作用与距离无关,因而是一个典型的增强子;－５３８／－２８８片段的增强子样作用既依赖于两毗邻Ｅ盒子的完整性,又依赖于ｍｙｏｇｅｎｉｎ等生肌调节因子的存在。强化表达ｍｙｏｇｅｎｉｎ可激活ｐＢＬＣＡＴ２－γ（－５３８／－４３３）和ｐＢＬＣＡＴ２－γ（－４３２／－２８８）（各含一个Ｅ盒子）在分化肌细胞中的表达,究竟是克隆片段中的独立Ｅ盒子对这种表达起贡献还是出现在克隆位点附近的反向Ｅ盒子（ＧＴＣＧＡＣ）或ｔｋ启动子中的另一个Ｅ盆子起作用？尚不十分清楚。     

Expression of acetylcholine receptor alpha-subunit mRNA during differentiation of the BC3H1 muscle cell line

The accumulation of translatable acetylcholine receptor alpha-subunit mRNA was examined in the BC3H1 muscle cell line in response to serum and cell growth. Relative amounts of alpha-subunit mRNA were quantitated during differentiation by cell-free translation and immunoprecipitation with an alpha-subunit-specific monoclonal antibody. Logarithmically growing cells do not possess cell surface acetylcholine receptors; however, a significant amount of alpha-subunit mRNA is detectable in cells under these conditions. Furthermore, alpha-subunit is synthesized in growing undifferentiated cells at a rate similar to that of differentiated cultures. Following growth arrest of BC3H1 cells, surface receptors are induced to levels greater than 100-fold above that of growing cells. The relative level of translatable alpha-subunit mRNA in differentiated cells, however, is only approximately 4-fold greater than in growing cultures. Induction of alpha-subunit mRNA appears to be reversible since reinitiation of growth in quiescent differentiated BC3H1 cells results in a reduction in relative abundance of this mRNA species to levels comparable to that of undifferentiated cells and the concomitant loss of surface receptors. These results indicate that receptor expression during differentiation is regulated both post-translationally and at the level of receptor subunit mRNA accumulation.     

An altered intron inhibits synthesis of the acetylcholine receptor alpha-subunit in the paralyzed zebrafish mutant nic1.

The acetylcholine receptor (AChR), an oligomeric protein composed of five subunits, is a component of the postsynaptic membrane at the vertebrate neuromuscular junction that plays a central role in synaptic transmission. The zebrafish mutation nic1 blocks the expression of functional and clustered nicotinic muscle AChRs. To understand the mechanisms underlying this lack of AChRs, we characterized the molecular defect in nic1 mutants. Our results suggest that the mutation affects the gene coding for the alpha-subunit of the AChR. Southern blot hybridization and DNA sequence analyses showed that the nic1 AChR alpha-subunit gene lacks part of intron 6 where the splicing branchpoint normally forms. Several lines of evidence suggest that this deletion blocks normal splicing; most nic1 alpha-subunit mRNAs retain intron 6 and are larger and less abundant than wild-type, some nic1 alpha-subunit mRNAs are internally deleted, and wild-type alpha-subunit mRNA rescues nic1 mutant cells. The nic1 mutation reduces the size of an intron, which prevents efficient splicing of the pre-mRNA, thus blocking synthesis of the alpha-subunit and assembly of AChRs. By this route, the nic1 mutation leads to paralysis.     

Heregulin-stimulated acetylcholine receptor gene expression in muscle: requirement for MAP kinase and evidence for a parallel inhibitory pathway independent of electrical activity.

Binding of heregulin (HRG) to its receptor, ErbB3, results in a dimerization with ErbB2/neu and activation of their intrinsic tyrosine kinases, initiating a cascade of events resulting in the stimulation of acetylcholine receptor (AChR) genes in muscle. Here we have examined the signalling downstream of the HRG receptor. We show that phosphatidylinositol 3'-kinase (PI3K) and SHC bind to the HRG-activated ErbB3 in myotubes. Subsequently, p70S6 kinase (p70S6k), and MAP kinase ERK2 and thereby p90rsk are activated. However, inhibition of PI3K and p70S6k by wortmannin and rapamycin, respectively, failed to antagonize AChR alpha-subunit gene expression stimulated by HRG, despite the fact that the activities of the kinases were inhibited. In contrast, these inhibitors elevated AChR alpha-subunit mRNA levels, by themselves, independently of muscle electrical activity. On the other hand, the 17mer antisense oligonucleotide, EAS1, caused a specific depletion of ERK2 and eliminated the ability of HRG to stimulate AChR alpha-subunit gene expression. These results indicate that HRG stimulates expression of AChR genes via ERK2 activation, and provide a physiological example of neurotrophic factor-associated repression of AChR genes by stimulation of p70S6k activity which may contribute to the expression of adult type AChR genes at the neuromuscular junction.     

Expression and characterization of the chick nicotinic acetylcholine receptor alpha-subunit in insect cells using a baculovirus vector

A baculovirus transfer vector was constructed containing an entire cDNA copy of the chick nicotinic acetylcholine receptor (nAChR) alpha-subunit under control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoter. Recombinant baculovirus was obtained by co-transfection of Spodoptera frugiperda cells with infectious, wild-type AcNPV DNA and the transfer vector. Polyhedrin-negative, recombinant viruses were identified which expressed the nAChR alpha-subunit. The insect cell-expressed alpha-subunit protein had a molecular mass of 42 kDa and was shown to be targeted to the plasma membrane by fluorescence microscopy and toxin-binding assays. The levels of expression were low, approximately 1-2% of cell proteins, when compared with the levels of natural polyhedrin protein. The expressed receptor alpha-subunit was recognised by polyclonal antisera raised against purified Torpedo nAChR alpha-subunit and carried the binding site for the snake venom toxin, alpha-bungarotoxin. Bound alpha-bungarotoxin was displaced in competition binding assays by alpha-cobra toxin, carbamylcholine and d-tubocurarine, and thus had a similar pharmacological profile to that obtained with authentic receptors in muscle cells and receptors expressed in other systems i.e. Xenopus oocytes and mammalian cells. We have also shown that when the chick nAChR alpha-subunit is expressed in the absence of other receptor subunits, unexpectedly high concentrations of nicotine (10 mM) were required to displace bound alpha-bungarotoxin.