Partial amino acid sequence information allowed the isolation of cDNA clones encoding the turkey erythrocyte (3-adrenergic receptor. Antisera raised against synthetic peptides encoded by the cDNA crossreacted with the purified receptor and appropriate tryptic fragments, confirming the identity of the cDNA. The receptor is composed of 483 amino acids and has a molecular mass of 54 kDa. Its sequence suggests that it is arranged predominantly in seven membranespanning sequences and a long cytoplasmic carboxyl-terminal domain. The extracellular amino-terminal domain contains a consensus sequence for N-glycosylation. The /3-adrenergic receptor displays overall structural similarity and weak sequence homology with rhodopsin. Because both proteins act by regulating GTP-binding proteins, a compact structure based on seven membrane-spanning regions may be a general model for receptors that act on G proteins. (1). Functionally, the receptor is phylogenetically conserved. The receptor purified from turkey erythrocytes can efficiently regulate GO from rabbit liver in reconstituted phospholipid vesicles (3, 4), and its selectivity for numerous agonist and antagonist ligands is only slightly discrepant from the mammalian 31-adrenergic receptor (5).Whether the receptor interacts with G. on the hydrophilic cytoplasmic face of the plasma membrane or within the bilayer is unknown; nor is anything known about the structural details of this regulatory interaction. Presumably, all receptors that activate G proteins will share this regulatory domain, and definable differences will exist in the homologous regions of receptors that activate different G proteins.The sites of P-adrenergic ligand binding, regulatory phosphorylation, and stimulatory reduction by thiols (6) are also of great interest. Much of the difficulty in learning about the structure of the f3-adrenergic receptor is due to its low concentration in plasma membranes: P-adrenergic receptor must be purified over 20,000-fold from an already wellpurified plasma membrane fraction. We have therefore undertaken the cloning of the cDNA that encodes the Padrenergic receptor as a first step toward more direct studies of its structure and function. The sequence of the 82-adrenergic receptor from hamster lung has appeared recently (7), and homology between the two sequences and the sequence of rhodopsin suggests functionally important aspects of their structures. METHODSP3-Adrenergic receptor was purified from turkey erythrocyte plasma membrane as described by Brandt and Ross (4). This preparation, which consists mainly of an active 40-kDa proteolysis product and the 53-kDa receptor (1, 4), was separated from digitonin and minor impurities by HPLC on a 300-A pore size, C4 column (Synchrom, Linden, IN) using a linear gradient of0.1% trifluoroacetic acid in water to 0.06% trifluoroacetic acid in 50% 1-propanol (vol/vol). The receptor was cleaved with cyanogen bromide, 60 mg/ml in 70% (vol/vol) formic acid, for 24 hr at room temperature under nitrogen. Sequencing was performed...

We have isolated novel molluskan steroid receptor transcripts orthologous to vertebrate estrogen receptors (ERs) and estrogen receptor-related receptors (ERRs) from the freshwater snail Marisa cornuarietis. Radiolabeled ligand binding analyses showed that neither recombinant receptor protein specifically bound 17beta-estradiol over the range applied (0.3-9.6 nM). These novel receptor transcripts have thus been designated mcER-like and mcERR respectively. Quantitative PCR revealed mcER-like to be expressed ubiquitously throughout a range of male and female structures studied, including neural and reproductive tissues. Highest absolute levels were seen in the male penis-sheath complex. The mcERR mRNA was also expressed ubiquitously throughout all male and female tissues analyzed here, with very low absolute transcript numbers in female accessory sex structures compared to other tissues.

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Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (43/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1-2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy.

The p53 gene is a tumour suppressor gene which has a fundamental role in cell cycle control and division, and in mammals certain genotoxic agents induce specific mutations in p53, leading to tumourigenesis. Fish have been investigated as models for studying carcinogens, but as yet very little data exists that links exposure to specific chemicals with the aetiology of tumours found in wild populations. In this study, p53 was sequenced from five species of fish with a view to the possible use of mutations in the highly conserved domains of p53 to identify genotoxins in the aquatic environment. A 0.8 kb fragment of the cDNA encompassing the conserved DNA-binding domain of p53 was sequenced in three Oncorhynchus salmonid fish: coho (O. kisutch), chum (O. keta), and chinook (O. tshawytscha) and full-length p53 cDNAs were sequenced in the puffer fish (Tetraodon miurus) and the barbel (Barbus barbus). The full-length puffer fish and barbel p53 cDNAs were 1834 bp and 1790 bp in length, encoding a 367 aa protein and a 369 aa protein, respectively. The deduced aa sequences of the p53 cDNA in the Oncorhynchus salmon shared a 100% identity in the five conserved regions (I-V). Comparisons of the deduced aa sequences for puffer fish and barbel p53 with other fish p53s revealed a high homology within the conserved DNA binding domain (68-86% for puffer fish and between 66-88% for barbel). "Conserved" domain I was not highly conserved in fish, as it is in mammals, and, therefore, conserved domains II-V are most likely to provide the valuable sequences in fish p53 for use in mutational studies to fingerprint genotoxins in the aquatic environment.