Delta-subunit Gene Research Articles

Allele-specific expression of the Mgi- phenotype on disruption of the F1-ATPase delta-subunit gene in Kluyveromyces lactis.

Kluyveromyces lactis is a petite-negative yeast that does not form viable mitochondrial genome-deletion mutants (petites) when treated with DNA-targeting drugs. Loss of mtDNA is lethal for this yeast but mutations at three loci termed MGI, for mitochondrial genome integrity, can suppress this lethality. The three loci encode the alpha-, beta- and gamma-subunits of mitochondrial F1-ATPase. In this study we report the isolation and characterization of the KlATPdelta gene encoding the delta-subunit of F1-ATPase. The deduced protein contains 158 amino acids showing 72% identity to the protein from Saccharomyces cerevisiae and a putative mitochondrial targeting sequence of 23 amino acids. Disruption of the gene causes cells to become respiratory deficient while the introduction of ATPdelta from S. cerevisiae restores growth on glycerol. Cells with a disrupted ATPdelta gene, like strains with disruptions of alpha-, beta- and gamma-F1-subunits, do not produce petite mutants when treated with ethidium bromide. However, unlike strains with disruptions in the three largest F1-subunits, disruption of ATPdelta in the presence of some mgi alleles does not abolish the Mgi- phenotype. By contrast, elimination of ATPdelta in other mgi strains removes resistance to ethidium bromide and rho0 mutants are not formed. Hence the ATPdelta subunit of F1-ATPase, while not mandatory for a Mgi- phenotype, aids some mgi alleles in suppressing rho0 lethality.

Cloning and Characterization of a Novel Transcriptional Repressor of the Nicotinic Acetylcholine Receptor δ-Subunit Gene

We have identified a negative cis-acting regulatory element in the nicotinic acetylcholine receptor delta-subunit gene's promoter. This element resides within a previously identified 47-base pair activity-dependent enhancer. Proteins that bind this region of DNA were cloned from a lambdagt11 innervated muscle expression library. Two cDNAs (MY1 and MY1a) were isolated that encode members of the Y-box family of transcription factors. MY1/1a RNAs are expressed at relatively high levels in heart, skeletal muscle, testis, glia, and specific regions of the central nervous system. MY1/1a are nuclear proteins that bind specifically to the coding strand of the 47-base pair enhancer and suppress delta-promoter activity in a sequence-specific manner. These results suggest a novel mechanism of repression by MY1/1a, which may contribute to the low level expression of the delta-subunit gene in innervated muscle. Finally, the gene encoding MY1/1a, Yb2, maps to the mid-distal region of mouse chromosome 6.

Transcription and demethylation of TCR beta gene initiate prior to the gene rearrangement in c-kit+ thymocytes with CD3 expression: evidence of T cell commitment in the thymus.

In order to determine whether the cells immigrating into the thymus have been committed to the T cell lineage, we examined the gene expression of the TCR complex in fetal thymus (FT) and fetal liver (FL) precursors. We previously showed that c-kit bright-positive, Pgp-1 bright-positive and lineage markers negative fetal thymocytes (c-kit+ FT cells) are the most immature cells which do not undergo gene rearrangement of the TCR beta chain. In this study, we demonstrated that the gene rearrangement of TCR gamma as well as beta chains does not occur in c-kit+ FT cells, but that the germline transcript of their TCR beta was found in J-C regions. The TCR beta gene was demethylated in c-kit+ FT cells. CD3 gamma, delta and epsilon subunit genes were also expressed at the mRNA levels in c-kit+ FT cells, but cytoplasmic protein staining divided them into two populations: cytoplasmic CD3 epsilon positive and negative cells. These features were not observed in c-kit+ FL cells. Moreover, Ly-1 expression was found on c-kit+ FT cells but not on c-kit+ FL cells. These results indicate that DNA alteration on the TCR beta gene initiates with other phenotype expression determining the T cell lineage in the thymus prior to TCR gene rearrangement.

Adaptation of nicotinic acetylcholine receptor, myogenin, and MRF4 gene expression to long-term muscle denervation.

Muscle activity alters the expression of functionally distinct nicotinic acetylcholine receptors (nAChR) via regulation of subunit gene expression. Denervation increases the expression of all subunit genes and promotes the expression of embryonic-type (alpha 2 beta delta gamma) nAChRs, while electrical stimulation of denervated muscle prevents this induction. We have discovered that the denervation-induced increases in alpha, beta, gamma, and delta subunit gene expression do not persist in muscles that have been denervated for periods extending beyond a couple of months. However, expression of RNA encoding the epsilon-subunit remains elevated suggesting a return to expression of predominantly adult-type (alpha 2 beta delta epsilon) nAChR in long-term denervated muscles; a finding confirmed by single channel patch-clamp analysis. Since the nAChR subunit genes are regulated by the MyoD family of muscle regulatory factors, and the genes encoding these factors are also induced following short-term muscle denervation, we determined their level of expression in long-term denervated muscle. Although MyoD and myf-5 RNA levels remained elevated, myogenin and MRF4 RNAs were induced only transiently by muscle denervation. Surprisingly, Id-1, a negative regulator of transcription, was gradually induced in denervated muscle with RNA levels peaking about two months after denervation. It is likely that this maintained level of increased Id expression, in conjunction with the returning levels of myogenin and MRF4 expression, account for the reduced level of embryonic receptors in long-term denervated muscle. These changing patterns of gene expression may have important consequences for the ability of muscle to recover function after denervation.

Identification of a DNA element determining synaptic expression of the mouse acetylcholine receptor delta-subunit gene.

mRNAs for acetylcholine receptor genes are highly concentrated in the endplate region of adult skeletal muscle largely as a result of a transcription restricted to the subneural nuclei. To identify the regulatory elements involved, we employed a DNA injection of a plasmid containing a fragment of the acetylcholine receptor delta-subunit gene promoter (positions -839 to +45) linked to the reporter gene lacZ with a nuclear localization signal. Injection of the wild-type construct into mouse leg muscles yielded preferential expression of the reporter gene in the synaptic region. Analysis of various mutant promoters resulted in the identification of a DNA element (positions -60 to -49), referred to as the N box, that plays a critical role in subneural expression. Disruption of this 12-bp element in the context of a mouse delta-subunit promoter from positions -839 to +45 gives widespread expression of the reporter gene throughout the entire muscle fiber, indicating that this element is a silencer that represses delta-subunit gene transcription in extrajunctional areas. On the other hand, this element inserted upstream of a heterologous basal promoter preferentially enhances expression in the endplate region. This element therefore regulates the restricted expression of the delta-subunit gene both as an enhancer at the endplate level and as a silencer in extrajunctional areas. Furthermore, gel-shift experiments with mouse muscle extracts reveal an activity that specifically binds the 6-bp sequence TTCCGG of this element, suggesting that a transcription factor(s) controls the expression of the delta-subunit gene via this element.

Neuregulins are concentrated at nerve-muscle synapses and activate ACh-receptor gene expression.

Two different signalling pathways mediate the localization of acetylcholine receptors (AChRs) to synaptic sites in skeletal muscle. The signal for one pathway is agrin, a protein that triggers a redistribution of previously unlocalized cell surface AChRs to synaptic sites. The signal for the other pathway is not known, but this signal stimulates transcription of AChR genes in myofibre nuclei near the synaptic site. Neuregulins, identified originally as a potential ligand for erbB2 (Neu differentiation factor, NDF), stimulate proliferation of Schwann cells (glial growth factor, GGF), increase the rate of AChR synthesis in cultured muscle cells (AChR-inducing activity) and are expressed in motor neurons. These results raise the possibility that neuregulin is the signal that activates AChR genes in synaptic nuclei. Here we show that neuregulin activates AChR gene expression in C2 muscle cells and that the neuregulin response element in the AChR delta-subunit gene is contained in the same 181 base pairs that confer synapse-specific expression in transgenic mice. We use antibodies to show that neuregulins are concentrated at synaptic sites and that, like the extracellular signal that stimulates synapse-specific expression, neuregulins remain at synaptic sites in the absence of nerve and muscle. We show that C2 muscle cells contain erbB2 and erbB3 messenger RNA but little or no erbB4 mRNA, and that neuregulin stimulates tyrosine phosphorylation of erbB2 and erbB3, indicating that neuregulin signalling in skeletal muscle may be mediated by a complex of erbB2 and erbB3.

BSF1, a novel brain-specific DNA-binding protein recognizing a tandemly repeated purine DNA element in the GABAA receptor delta subunit gene.

The rat GABAA receptor delta subunit gene has been isolated and its promoter mapped to facilitate studies of neuron-specific gene transcription. The gene is transcribed from a single promoter with several clustered transcriptional initiation sites. The major start site maps to a prototype initiator sequence located in the center of a TATA-less CpG-rich island. Analysis of the 5'-flanking region of the GABAA receptor delta subunit gene revealed a novel tandemly repeated purine sequence element. Furthermore, gel retardation assays identified several tissue-specific and ubiquitous proteins that bind to this sequence. One of these factors, brain-specific factor (BSF) 1, was only observed in extracts from brain and was distinct from BETA and PAL, two DNA-binding proteins with similar purine-rich recognition sequences. The distribution of BSF1 in brain closely matched the expression pattern previously determined for the delta subunit mRNA, and its binding activity appeared simultaneously with GABAA receptor delta subunit mRNA during differentiation of cerebellar granule cells in vitro. Our data therefore suggest that BSF1 is a novel brain-specific and neuronal cell type-enriched factor possibly involved in differential transcription of the GABAA receptor delta subunit gene.

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.

An E Box Mediates Activation and Repression of the Acetylcholine Receptor δ-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.

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.

Analysis of binding and activating functions of the chick muscle acetylcholine receptor gamma-subunit upstream sequence.

1. The skeletal muscle acetylcholine receptor comprises several subunits whose coordinated expression during myogenesis is probably controlled by cis elements in the individual subunit genes. We have previously analyzed promoter regions of the alpha and delta genes (Wang et al., 1988, 1990); to gain further insight into receptor regulation, we have now studied the promoter of the chick muscle gamma-subunit gene. 2. This analysis was faciliated by the close upstream proximity of the coding region of the delta-subunit gene and the consequent brevity (740 bp) of the untranslated linker connecting the two genes (Nef et al., 1984). Nuclease protection and primer extension analysis revealed that transcription of the gamma-subunit gene starts at position 56 upstream of the translational initiation site. 3. Nested deletions of the promoter region were employed to identify functionally important elements. A 360-bp sequence (-324 to +36) was found to activate transcription, in a position- and orientation-independent manner, during myotube formation. This sequence comprises 5 M-CAT (Nikovits et al., 1986) similarities and contains, at positions -52/-47 and -33/-28, two CANNTG (Lassar et al., 1989) motifs. 4. Binding experiments were performed by means of gel retardation, gel shift competition, and footprint analysis. The CANNTG motifs were found to bind MyoD and myogenin fusion proteins and to interact with proteins in nuclear extracts from cultured myotubes. 5. Point mutations in the CANNTG motifs revealed that these elements are crucial for full promoter activity in myotubes and essential in fibroblasts cotransfected with a myogenin expression vector. 6. We conclude that the activity of the gamma-subunit gene is determined largely by E boxes, which in vivo are likely to be activated by MyoD family proteins; in addition, other transactivators such as the M-CAT binding protein presumably play a role. Both CANNTG elements and M-CAT motifs are also present in the alpha- and delta-subunit enhancer and may therefore account for the coordinate expression of the three subunits during muscle differentiation.

A 102 base pair sequence of the nicotinic acetylcholine receptor delta-subunit gene confers regulation by muscle electrical activity.

Muscle electrical activity suppresses expression of the embryonic-type (alpha, beta, gamma, and delta) nicotinic acetylcholine receptor (nAChR) genes. The molecular mechanism by which electrical activity regulates these genes is not known. One approach to this problem is to identify regions of the nAChR genes that mediate electrical regulation. Here we report results from such a study of the nAChR delta-subunit gene. We cloned the rat delta-subunit promoter region and created an expression vector in which this DNA controlled the expression of a down-stream luciferase structural gene. The effect that muscle electrical activity had on the expression from this promoter was assayed by introducing this expression vector into electrically stimulated and tetrodotoxin (TTX)-treated rat primary myotubes, and assaying for luciferase activity. These myotubes, when stimulated with extracellular electrodes, suppressed endogenous embryonic-type nAChR gene expression compared to those treated with TTX. Transfection of these cells with delta-promoter-luciferase expression vectors resulted in the delta-promoter conferring electrical regulation on luciferase expression. Additional experiments using deletions from the 5' end of the delta-promoter region have identified an element between -677 and -550 bp that suppressed delta-promoter activity and a minimal 102 bp sequence that promotes and regulates luciferase expression in response to muscle electrical activity. This latter sequence also contains all the necessary elements to confer tissue and developmental stage-specific expression.

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.

The Murine GABAAReceptor δ-Subunit Gene: Structure and Assignment to Human Chromosome 1

The murine chromosomal gene for the GABAA receptor delta subunit was isolated and characterized by high-resolution mapping and DNA sequencing. Spanning 13 kb, it comprises nine exons and displays an intron pattern comparable, but not identical, to that seen in members of the nicotinic acetylcholine receptor family. Notably, the second transmembrane domain thought to line the ion channel and conserved among different GABAA receptor subunits, is interrupted by an intron. The 5'-flanking region of the delta gene displays features characteristic of a CpG island and lacks canonical promoter elements such as TATA and CCAAT consensus sequences in proximity to the transcriptional initiation site. The human delta subunit gene was localized on the short arm of chromosome 1.

Assignment of the human nicotinic acetylcholine receptor genes: the alpha and delta subunit genes to chromosome 2 and the beta subunit gene to chromosome 17

The chromosomal assignments of the genes coding for the alpha, beta and delta subunits of the human nicotinic acetylcholine receptor have been determined from a panel of somatic cell hybrids and by direct in situ hybridization. The results localize CHRNA to 2q24-2q32. CHRNB to 17p11-17p12, and CHRND to chromosome 2q33-2qter.

Two adjacent MyoD1-binding sites regulate expression of the acetylcholine receptor α-subunit gene

Several genes encoding putative myogenic regulatory factors have been isolated on the basis of their ability to convert nonmuscle cells into myoblasts. Four of these genes code for nuclear proteins that belong to a larger family characterized by a conserved helix-loop-helix motif required for DNA-binding and dimerization. At least one protein, MyoD1, can function as a transcription factor and activate muscle-specific genes during differentiation. But the promoter of the delta-subunit gene of the mouse acetylcholine receptor (AChR) was recently reported to be functional in the absence of MyoD1 binding sites and it has been suggested that the genes coding for the AChR could be regulated independently of MyoD1 protein. Here, we identify two functional MyoD1-binding sites in the muscle-specific enhancer of the chicken AChR alpha-subunit gene that are essential for full activity in transfected myotubes.

Primary structure of a precursor for the delta-subunit of sweet potato mitochondrial F1-ATPase deduced from full length cDNA.

A cDNA library constructed from poly(A)-rich RNA of the sweet potato tuberous root using a newly developed plasmid vector carrying tac-SP6 promoters was used to identify full length cDNAs for the nuclear-encoded delta-subunit of mitochondrial F1-ATPase by oligonucleotide-hybridization selection. Selected clones contained cDNA insert which carry the entire coding capacity for the pre-delta-subunit, since the RNA transcribed in vitro from SP6 promoter on the vector directed the synthesis of pre-delta-subunit polypeptide in a wheat germ in vitro translation assay. The nucleotide sequence of one of these cDNAs indicates that it can code for the pre-delta-subunit of 244 amino acids of which 199 amino acids encode the mature subunit. The amino acid sequence of the mature delta-subunit shows similarities of about 18-25% amino acid positional identity with the delta-subunits of bacterial F1-ATPases, about 26% with the delta-subunit of chloroplast CF1-ATPase, and about 32-37% with oligomycin sensitivity conferring proteins of animal and fungal mitochondria. The N-terminal presequence of the precursor composed of maximum of 45 amino acids does not show any obvious sequence homology with either the transit peptide of the nuclear-encoded pre-delta-subunit of chloroplast CF1 or the presequence of the nuclear-encoded pre-oligomycin sensitivity conferring proteins. At least two types of the delta-subunit cDNAs with very similar structures were identified from the library, and the presence of multiple copies of the delta-subunit gene in the hexaploid genome of the sweet potato is also suggested by genomic Southern blot hybridization.

Expression of the acetylcholine receptor delta-subunit gene in differentiating chick muscle cells is activated by an element that contains two 16 bp copies of a segment of the alpha-subunit enhancer.

The acetylcholine receptor is a multimeric membrane protein whose expression is activated during muscle differentiation and upon denervation of adult muscle. To gain insight into the coordinate expression of receptor subunits during myogenesis we have analyzed the chick muscle receptor delta-subunit gene upstream region. The delta-subunit gene lacks canonical promoter elements (CCAAT and TATA boxes). Nuclease protection and primer extension analysis revealed that transcription starts at six major and several minor sites between -110 and -30 upstream of the translational initiation site; two sites, at positions -77 and -66, give rise to approximately 50% of all transcripts. Using nested deletions of the proximal 960 bp of the 5' flanking region of this gene we have identified a 62 bp sequence (-207 to -146) that activates transcription in a position independent manner. This enhancer-like element is activated during myotube formation; it contains two distinct functional moieties, each resembling the same 16 bp portion of the stage and tissue specific alpha-subunit gene enhancer which we have characterized previously [Wang et al. (1988) Neuron, 1, 527-534]. This common element, which also comprises several previously proposed skeletal muscle specific motifs [Buskin, J. N. and Hauschka, S. D. (1989) Mol. Cell Biol., 9, 2627-2640; Mar, J. H. and Ordahl, C. P. (1988) Proc. Natl. Acad. Sci. USA, 85, 6404-6408], may account for the coordinate expression of the two subunits. The cell specificity of the delta-subunit gene 5' flanking region is partly due to the enhancer, partly to an inhibitory element upstream of -207.

Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32----qter.

The nicotinic acetylcholine receptor of skeletal muscle (CHRN in man, Acr in mouse) is a transmembrane protein composed of four different subunits (alpha, beta, gamma, and delta) assembled into the pentamer alpha 2 beta gamma delta. These subunits are encoded by separate genes which derive from a common ancestral gene by duplication. We have used a murine full-length 1,900-bp-long cDNA encoding the gamma subunit subcloned into M 13 (clone gamma 18) to prepare single-stranded probes for hybridization to EcoRI-digested DNA from a panel of human x rodent somatic cell hybrids. Using conditions of low stringency to favor cross-species hybridization, and prehybridization with rodent DNA to prevent rodent background, we detected a single major human band of 30-40 kb. The pattern of segregation of this 30-40 kb band correlated with the segregation of human chromosome 2 within the panel and the presence of a chromosomal translocation in the distal part of the long arm of this t(X;2)(p22;q32.1) chromosome allowing the localization of the gamma subunit gene (CHRNG) to 2q32----qter. The human genes encoding the gamma and delta subunits have been shown to be contained in an EcoRI restriction fragment of approximately 20 kb (Shibahara et al., 1985). Consequently, this study also maps the delta subunit gene (CHRND) to human chromosome 2q32.1----qter. In the mouse, the Acrd and Acrg genes have been shown to be linked to Idh-1, Mylf (IDH1 and MYL1 in humans, respectively) and to the gene encoding villin on chromosome 1. Interestingly, we have recently localized the human MYL1 gene to the same chromosomal fragment of human chromosome 2. These results clearly demonstrate a region of chromosomal homoeology between mouse chromosome 1 and human chromosome 2.

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.