Molecular characterization and regulation of the human endothelin receptors.

Endothelin receptors (ETRs) are distributed throughout a variety of tissues. Two human cDNAs were identified which encode distinct ETR proteins. One cDNA encoded a 427-amino acid protein that shared 91% identity to rat ETAR. The second cDNA encoded a 442-amino acid protein that was 88% identical to rat ETBR. Ligand binding studies of the cloned receptors expressed in COS cells confirmed that they were pharmacologically ETAR and ETBR subtypes; although the selective antagonist BQ123 showed a potency similar to ET-3 in displacing 125I-ET-1 binding to ETAR. This observation contrasts with rat ETAR pharmacology where BQ123 has a 100-fold higher affinity than ET3. Chinese hamster ovary cells expressing the human ETAR displayed equal potencies in displacing 125I-ET-1 binding, which indicates that rat and human ETAR are pharmacologically distinct. Electrophysiological studies of both ETRs expressed in Xenopus oocytes revealed that they are functional. Northern analysis indicated that the two ETRs are differentially expressed in many tissues. Marmosets maintained on a high fat/high cholesterol diet exhibited 3-fold increase in ETBR mRNA levels with little change in ETAR mRNA levels. Availability of cDNA clones for ETR subtypes can open avenues for future analysis of their role in pathophysiology of various diseases.

Molecular cloning and characterization of the major endothelin receptor subtype in porcine cerebellum.

Endothelin receptors (ETRs) display subtype heterogeneity and are widely distributed throughout the tissues of the periphery and central nervous system. In order to gain further insight into the potential molecular differences of ETRs, we initiated molecular cloning of ETR genes by screening for the appearance of 125I-ET-1 binding activity in COS cells transfected with pools of a porcine cerebellum cDNA expression library. Two independent clones (pPCETR 1.1 and pPCETR 5.6) were identified and isolated by repeated rounds of pool enrichment and COS cell expression. DNA sequence analysis of pPCET 1.1 and pPCET 5.6 indicated that both clones have the same nucleotide sequence; the deduced amino acid sequence indicated that the porcine cerebellum ETR is 443 residues in length and consists of seven potential transmembrane domains, with homology to members of the GTP-binding protein-coupled receptor superfamily. Northern analysis indicated a single mRNA species of about 5 kilobases, which is expressed significantly in cerebellum, lung, kidney, and pituitary. Expression of functional receptor was demonstrated by endothelin-1 (ET-1)-mediated Ca2+ mobilization in COS cells transfected with pPCETR 1.1 (COS/ETR 1.1) and ET-1-mediated electrophysiological responses in Xenopus oocytes injected with RNA derived from pPCETR 1.1. Quantitative comparison of saturation binding of 125I-ET-1 to either porcine cerebellum or COS/ETR 1.1 membranes indicated an identical apparent dissociation constant. The relative efficacy of ET-related peptides to compete for binding of 125I-ET-1 to receptor from porcine cerebellum and COS/ETR 1.1 indicated that both preparations encode a nonselective or ETBR subtype. Chemical cross-linking of 125I-ET-1 to receptor derived from cerebellum or COS/ETR 1 revealed two bands, with apparent molecular masses of 47 and 35 kDa. These data demonstrate that the pPCETR 1.1 encodes the major ETR subtype in the porcine cerebellum.

Cloning, characterization, and expression of two alpha-amylase genes from Aspergillus niger var. awamori.

Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.