Alpha-subunit cDNA Research Articles

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II. Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the alpha subunit.

The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg much less than Lys much less than Gln much less than Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase alpha subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2(+)-independent kinase activity (5.46 +/- 0.88%) compared to the wild-type Arg283 (1.86 +/- 0.71%) and the Gln283 mutant (2.15 +/- 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2(+)-independent activity, the Arg283 kinase attained maximal Ca2(+)-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2(+)-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2(+)-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.

The leukocyte integrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated.

The functional activity of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes can be regulated by T-cell receptor (TCR) stimulation and pharmacologic agents. It was of interest to determine if functionally active LFA-1 could be reconstituted on a nonhematopoietic, LFA-1-negative cell line. We report the expression of LFA-1 and diethylaminoethyl (DEAE) Mac-1 alpha beta heterodimers on the cell surface of a fibroblastoid cell line, COS, by DEAE dextran cotransfection of the alpha and beta subunit cDNAs. Immunoprecipitation studies demonstrated that the alpha and beta subunit was expressed in heterodimers. The alpha or beta subunit was expressed at lower levels after transfection with the alpha or beta subunit cDNA alone. Cotransfection of the alpha and beta subunit cDNAs, but not transfection of alpha or beta alone, was sufficient to reconstitute intercellular adhesion molecule-1 (ICAM-1) binding activity. Consistent with this observation, LFA-1 on the fibroblastoid cells possesses the activation epitope defined by the L16 monoclonal antibody (mAb). This epitope marks the conversion of LFA-1 from the low to high avidity state on peripheral blood T lymphocytes (PBLs) and is constitutively present on activated cell lines. In contrast to LFA-1 on leukocytes, the functional activity of LFA-1 on fibroblastoid cells was not influenced by phorbol ester treatment. Furthermore, the use of agents that interfere with intracellular signaling, a protein kinase C inhibitor, cAMP analogue, or the combination of a phosphodiesterase inhibitor and adenyl cyclase activator, did not affect the binding of COS cells expressing LFA-1 to purified ICAM-1.

Functional Properties of Rat Brain Sodium Channels Expressed in a Somatic Cell Line

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.

Coexpression of two distinct muscle acetylcholine receptor α-subunits during development

The nicotinic acetylcholine receptor is a ligand-gated channel that mediates signalling at the vertebrate neuromuscular junction. It is a pentameric complex of four different subunits, assembled with a stoichiometry of alpha 2 beta gamma delta. Muscle-like alpha-subunits have been cloned from Torpedo, mouse, calf, rat, chicken, human and Xenopus, and only a single alpha-subunit complementary DNA from each species has been detected. We report here the cloning and characterization of a second muscle alpha-subunit cDNA from Xenopus, and show that this and a previously reported Xenopus alpha-subunit cDNA are encoded by distinct genes. The novel alpha-subunit reported here is expressed uniquely in oocytes; but both types of alpha-subunit are coexpressed throughout muscle development. This latter observation indicates that the expression of these two alpha-subunits is different from a previously reported developmental 'subunit-switch' mechanism used to generate channel diversity.

Expression of NA, K‐ATpase α and β subunit genes during preimplantation development of the mouse

Na,K-ATPase is a plasma membrane enzyme that plays a critical role in eutherian blastocoel formation (cavitation) by pumping Na+ into the extracellular space enclosed by the trophectoderm. Previous experiments with the mouse had shown that the alpha (catalytic) subunit of the enzyme becomes detectable by immunocytochemistry in the late morula, just prior to the onset of cavitation. In the present study we have used cDNAs corresponding to three mRNA isoforms of the alpha subunit and a beta subunit to determine which genes are expressed during preimplantation development and to explore the timing of their expression. Of the three alpha subunit cDNAs tested by Northern blot hybridization with blastocyst RNA, only alpha 1 produced a hybridization signal, recognizing a single mRNA about 4 kb in length. This mRNA is relatively abundant in zygotes but barely detectable by the 2-cell stage and then accumulates steadily thereafter to reach its preimplantation maximum in blastocysts. The beta 1 cDNA detected mRNA of about 2.6-2.8 kb. This mRNA is present in zygotes but could not be detected in 2-, 4-, or 8-cell stages; it is present at a low level in late morulae and is abundant in blastocysts. The temporal profile of accumulation of beta 1 mRNA thus matches more closely than does alpha 1 the timing of appearance of the catalytic subunit. This suggests that the beta subunit may regulate production of the holoenzyme and hence the timing of cavitation.

Molecular characterization of Drosophila Gene Encoding G0 α Subunit Homolog

A Drosophila melanogaster gene (dgo) encoding a G protein alpha subunit has been isolated by screening genomic and adult head cDNA libraries using bovine transducin alpha subunit cDNA as probe. The gene, which maps to 47A on the second chromosome, encodes two proteins which are both 354 amino acids long but differ in seven amino acids in the amino-terminal region. The deduced amino acid sequences of the two proteins are 81% identical to that of a rat Go alpha subunit. Analysis of genomic clones revealed that there are eight coding exons and that the putative transcripts for the two proteins differ in the 5'-noncoding regions and the first coding exons but share the remaining six coding exons. The arrangement of two different 5'-noncoding regions on the gene suggests that two different promoters regulate the expression of the transcripts encoding the two proteins. RNA blot analysis detected three transcripts: a 3.9-kilobase (kb) transcript found at all stages of development; a 5.4-kb transcript present predominantly in adult heads; and a 3.4-kb transcript present only in adult bodies. In situ hybridizations of a cDNA probe to adult tissue sections showed that the gene is expressed abundantly in neuronal cell bodies in the brain, optic lobe, and thoracic ganglia.

Neuronal expression of a newly identified Drosophila melanogaster G protein alpha 0 subunit.

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, which we call DG alpha 0, hybridized to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a trp-E-DG alpha 0 fusion protein recognized a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies revealed expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. DG alpha 0 message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain.

Identification of a Region within the Na,K-ATPase α Subunit That Contributes to Differential Ouabain Sensitivity

To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.

Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.

To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.

Expression and Regulation of Testicular Inhibin α-Subunit Genein Vivoandin Vitro

Inhibin, a gonadal peptide, selectively suppresses FSH release from the pituitary. The cDNAs coding for ovarian inhibin have been isolated and characterized. However, little is known about testicular inhibin. In this study we have isolated inhibin alpha-subunit cDNA from human testicular cDNA libraries and determined inhibin alpha-subunit mRNA levels in testes. The longest cDNA isolated from human testis was 1380 nucleotides long and contained a nucleotide sequence identical to that of human placental inhibin alpha-subunit and isolated human inhibin alpha-subunit gene, but different from human ovarian inhibin alpha-subunit in two amino acids in the signal peptide. A single 1.5-kilobase species of inhibin alpha-subunit mRNA was identified in the testes of several species. This mRNA was the same size as those in human ovary and placenta. The regulation of inhibin alpha-subunit mRNA in rat testis was next examined. The concentration of testicular inhibin alpha-subunit mRNA peaked between 20-25 days of age and gradually declined thereafter. Hypophysectomy decreased testicular inhibin alpha-subunit mRNA levels. Supplementation of hypophysectomized animals with FSH restored inhibin alpha-subunit mRNA levels to those in intact controls. By contrast, treatment with testosterone had no effect. Similarly, in Sertoli cell-enriched cultures, FSH, but not testosterone, increased inhibin alpha-subunit mRNA levels. We conclude that 1) human testicular inhibin alpha-subunit mRNA is similar to that of human ovary and placenta; and 2) inhibin alpha-subunit mRNA in Sertoli cells is regulated by FSH, but not testosterone, both in vivo and in vitro.

A Drosophila melanogaster G protein alpha subunit gene is expressed primarily in embryos and pupae.

A G protein alpha subunit gene has been isolated from a Drosophila melanogaster genomic library using a combination of bovine rod and cone transducin alpha subunit cDNAs as a probe under reduced stringency conditions. The gene, DG alpha 1, encodes a protein with an amino acid sequence 78% identical to bovine Gi alpha 1. However, unlike all reported Gi alpha subunit the DG alpha 1-encoded protein is not expected to be a pertussis toxin substrate, because it lacks a cysteine at the appropriate site. The protein coding region of the gene is split by four introns. The sequence of a head tissue cDNA clone, as well as amino acid similarities to mammalian G proteins, confirms this exon/intron structure. Northern blots of total cellular RNA reveal a major 2.3-kilobase transcript and a less abundant 1.7-kilobase transcript. These transcripts are most abundant in RNA from embryos and pupae. The DG alpha 1 gene is located on band 65C on the left arm of the third chromosome, on the basis of in situ hybridizations to Drosophila salivary gland polytene chromosomes.

Identification of a GTP-binding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin.

Recent molecular cloning of cDNA for the alpha subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called Tr and Tc, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, we have identified a G-protein alpha subunit, which we refer to as Gz alpha, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine Tr alpha-subunit cDNA. The deduced amino acid sequence of Gz alpha is 41-67% identical with those of other known G-protein alpha subunits. However, the 355-residue Gz alpha lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein alpha subunits. We suggest that Gz alpha, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.

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.

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.

Production of human thyroid-stimulating hormone in Chinese hamster ovary cells.

Human thyroid-stimulating hormone (hTSH) has been produced in Chinese hamster ovary (CHO) cells co-transformed with two plasmids: one carrying the alpha subunit cDNA with mouse dihydrofolate reductase gene and the other carrying hTSH beta subunit cDNA. Each cDNA was driven to expression under the control of SV40 early promoter. hTSH and its alpha subunit were secreted into culture media, and their secretion increased with exposure of the cells to increasing concentrations of methotrexate. Gel filtration analysis revealed that the molecular size of the hTSH was the same as that of natural hTSH. Furthermore, the CHO cell-produced hTSH elevated the cyclic AMP level in the rat thyroid cell line FRTL-5 in the same manner as natural hTSH does.

Molecular cloning of a brain-specific calcium/calmodulin-dependent protein kinase.

A calcium/calmodulin-dependent protein kinase type II (CaM-K) alpha-subunit cDNA has been cloned from rat brain. This enzyme is encoded by a 5.1-kilobase mRNA expressed exclusively in the brain. Hybridization histochemistry reveals that the CaM-K mRNA expression corresponds to the distribution of the immunoreactive alpha-subunit protein, suggesting that the high enzyme levels in specific brain areas reflect regional differences in gene expression. The sequence of CaM-K alpha-subunit cDNA indicates a 478-amino acid (54-kDa) protein with three functional domains. The domain organization suggests a structural model for calcium/calmodulin-dependent and independent states that might subserve short- and long-term responses to transient stimuli.

Thyroid Hormone Control of Thyrotropin Gene Expression in Rat Anterior Pituitary Cells*

The relationship between thyroid hormone and TSH subunit mRNA levels and gene transcription in rat pituitaries was investigated. Synthesis of alpha-subunit and TSH-beta mRNAs were measured by incorporation of [32P]UTP into isolated nuclei, followed by hybridization of labeled transcripts to specific cDNAs. Steady state levels of mRNA were measured by Northern blot analysis of mRNA followed by hybridization with labeled TSH beta and alpha-subunit cDNAs. Hybridizing bands were quantitated by densitometry. TSH subunit mRNA synthesis was suppressed by T3 treatment in a manner directly related to T3 receptor occupancy; this effect occurred both in vivo and in cell culture. In the euthyroid rat, alpha-subunit mRNA had a higher level of synthesis [126 parts/million (ppm)] than did TSH beta mRNA (24 ppm). In the hypothyroid animal this situation was reversed, and the synthesis of both alpha-subunit and TSH beta mRNAs was increased to 230 and 331 ppm, respectively. T3 administration to hypothyroid animals suppressed mRNA synthesis maximally to 69 ppm for alpha-subunit and 10 ppm for TSH beta. The alpha-subunit to TSH beta mRNA synthesis ratio can thus vary from 0.7 in the hypothyroid animal to 6.9 in the hyperthyroid animal. The transcription rate for both genes decreased in linear fashion with increased T3 nuclear receptor occupancy. A 50% decrease in transcription occurred at a T3 dose (10(-9) M) similar to the Kd of the thyroid receptor. This agrees with our previous findings in thyrotropic tumors indicating a time- and dose-dependent suppression of the TSH subunit genes with increasing levels of nuclear receptor-bound T3. In primary rat pituitary cultures, T3 directly regulated steady state levels and synthesis of TSH subunit mRNA. With euthyroid pituitary cultures, alpha-subunit mRNA synthesis (50 ppm) was greater than that of TSH beta (12 ppm), but with hypothyroid pituitary cultures both mRNAs were synthesized at nearly equal rates (alpha-subunit, 120 ppm; TSH beta, 105 ppm). T3 (10(-8) M) treatment of hypothyroid cultures for 3 days decreased alpha-subunit (60% of control) and TSH beta (35% of control) mRNAs. Thus, mRNA synthesis is changed coordinately for both TSH subunits, but TSH beta is altered to a greater extent by thyroid status.

Stable expression of transfected Torpedo acetylcholine receptor alpha subunits in mouse fibroblast L cells.

Torpedo californica electric organ cDNA libraries were constructed in lambda gt10 and lambda gt11. Four acetylcholine receptor (AcChoR) subunit cDNA clones were isolated and shown to contain the entire coding region for each of the subunits. When in vitro synthesized AcChoR mRNA was microinjected into Xenopus laevis oocytes, functional cell surface AcChoRs were expressed. A very simple and fast 22Na-uptake experiment was performed on batches of microinjected oocytes to identify oocytes that were expressing large quantities of functional cell surface AcChoRs for use in single-channel recordings. In addition to the transient expression system, DNA-mediated cotransformation is described, which is a method for stably introducing AcChoR cDNAs into the chromosomes of tissue culture cells. Because the AcChoR is composed of four different subunits, it is necessary to integrate four cDNAs into the chromosomes of the same cell before stable expression of a completely functional receptor complex can be established. We show that 80% of the cells that integrated the selectable marker gene into their chromosomes also integrated all four AcChoR cDNAs. When Torpedo alpha-subunit cDNA inserted into an appropriate expression vector was introduced into cells by transfection, alpha-subunit protein was synthesized that migrated on NaDodSO4/polyacrylamide gels with the same molecular mass as native Torpedo alpha subunits and expressed antigenic determinants similar to those of native Torpedo alpha subunits.

Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain.

Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.

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.