Alpha-subunit cDNA Research Articles

Molecular cloning of the rat stomach (H+ + K+)-ATPase.

We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N-terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic.

Mapping of brain areas expressing RNA homologous to two different acetylcholine receptor alpha-subunit cDNAs.

We have used an in situ RNA X RNA hybridization technique to determine, in the central nervous systems of the mouse and rat, the distribution of RNA homologous to cDNA clones encoding the alpha subunit of a putative neural nicotinic acetylcholine receptor and the alpha subunit of the muscle nicotinic acetylcholine receptor. Hybridization of the neural alpha-subunit probe was strongest in the medial habenula but was also detected consistently in the compact part of the substantia nigra and ventral tegmental area, in the neocortex, and in certain parts of the thalamus and hypothalamus. The in situ hybridization technique makes it possible to compile a map of brain regions containing cell bodies expressing RNA coding for a specific receptor type and subsequently to apply the techniques of molecular biology to study these brain receptors.

Expression of Chorionic Gonadotropin-β-Like Messenger Ribonucleic Acid in an α-Subunit-Secreting Pituitary Adenoma*

The mRNA and protein products produced by an alpha-subunit-secreting pituitary tumor were studied to further characterize the early steps in glycoprotein hormone biosynthesis. alpha-Subunit mRNA was readily detected by RNA blot hybridization to alpha-subunit cDNA and was qualitatively normal, with a length of about 800 bases. Although only excess free alpha-subunit was secreted by the tumor, abundant beta-subunit mRNA hybridized to a cDNA which recognized both LH beta and CG beta sequences. Surprisingly, the beta-subunit mRNA in the pituitary tumor was characteristic of CG beta mRNA. The beta-subunit mRNA migrated in parallel with placental CG beta mRNA (approximately 1000 bases) and was easily resolved from LH beta mRNA (approximately 700 bases). Using site-specific hybridization and oligonucleotide-primed extension, the beta-subunit mRNA in pituitary tumor was found to have an extended 5'-untranslated tract (366 bases) characteristic of CG beta gene transcripts rather than LH beta gene transcripts, in which the 5'-untranslated tract is only 9 bases. Activation of the CG beta-like promoter rather than the LH beta promoter indicates that the cellular mechanisms regulating tissue-specific expression of the beta-subunit gene were altered by the neoplastic changes in the pituitary tumor. Protein analysis of tumor homogenate demonstrated large amounts of alpha-subunit in the form of intact FSH and small amounts of intact LH and free alpha-subunit. Despite the presence of abundant CG beta-like mRNA in the tumor, intact hCG or hCG beta was not detected. The uncoupling of beta-subunit gene expression and protein biosynthesis in the setting of ongoing alpha-subunit biosynthesis provides a potential mechanism for unbalanced synthesis and secretion of free alpha-subunit.

Assignment of the gene for the beta subunit of thyroid-stimulating hormone to the short arm of human chromosome 1.

The chromosomal locations of the genes for the beta subunit of human thyroid-stimulating hormone (TSH) and the glycoprotein hormone alpha subunit have been determined by restriction enzyme analysis of DNA extracted from rodent-human somatic cell hybrids. Human chorionic gonadotropin (CG) alpha-subunit cDNA and a cloned 0.9-kilobase (kb) fragment of the human TSH beta-subunit gene were used as hybridization probes in the analysis of Southern blots of DNA extracted from rodent-human hybrid clones. Analysis of the segregation of 5- and 10-kb EcoRI fragments hybridizing to CG alpha-subunit cDNA confirmed the previous assignment of this gene to chromosome 6. Analysis of the patterns of segregation of a 2.3-kb EcoRI fragment containing human TSH beta-subunit sequences permitted the assignment of the TSH beta-subunit gene to human chromosome 1. The subregional assignment of TSH beta subunit to chromosome 1p22 was made possible by the additional analysis of a set of hybrids containing partially overlapping segments of this chromosome. Human TSH beta subunit is not syntenic with genes encoding the beta subunits of CG, luteinizing hormone, or follicle-stimulating hormone and is assigned to a conserved linkage group that also contains the structural genes for the beta subunit of nerve growth factor (NGFB) and the proto-oncogene N-ras (NRAS).

Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA.

We have isolated a cDNA clone for the rat brain Na,K-ATPase alpha subunit. A lambda gt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, lambda rb5, containing a 1200-base-pair cDNA insert expressed a beta-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the alpha subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of lambda rb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney greater than brain greater than liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to lambda rb5 cDNA were amplified approximately 40-fold. Analysis of the lambda rb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase alpha-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the lambda rb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase alpha subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones.

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.

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.

Identification of a cDNA clone coding for the acetylcholine binding subunit of Torpedo marmorata acetylcholine receptor.

A recombinant DNA plasmid has been constructed that contains sequences of the gene coding for the acetylcholine binding subunit (alpha-subunit, 40 000 daltons) of Torpedo marmorata acetylcholine receptor protein (AChR). Polyadenylated RNA purified from Torpedo electric organ was used to construct a cDNA library. The AChR alpha-subunit cDNA clone was then identified by a two-step screening of 700 recombinant clones. As AChR is present in Torpedo electric organ but not in Torpedo liver or spleen, differential screening led to the selection of 12 clones specific for the electric organ. We then tested the ability of cDNA inserts to hybridize alpha-subunit mRNA specifically, as judged by cell-free translation and immunoprecipitation. The insert from one clone, p alpha-1, selectively hybridized with a mRNA species which elicited the synthesis of a 38 000 mol. wt. polypeptide. This polypeptide was precipitated by: (1) a rabbit serum raised against purified denatured alpha-subunit (the pure alpha-subunit displaced the complex); and (2) a rat monoclonal antibody specific for the denatured alpha-subunit. It was thus identified as a precursor of the alpha chain. Blot hybridization analysis of polyadenylated RNA from Torpedo electric organ with the p alpha-1 probe revealed a major species of 2.0 kb, which thus contains approximately 800 non-coding nucleotides.