Molecular Characterization of the Beta-adrenoceptor Gene-family of Rainbow Trout (Oncorhynchus Mykiss).
Molecular Characterization of the Beta-adrenoceptor Gene-family of Rainbow Trout (Oncorhynchus Mykiss).

Author: James G. Nickerson

Publisher:

Published: 2003

Total Pages: 0

ISBN-13:

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Beta-Adrenoceptors (beta-ARs) are seven transmembrane domain, G-protein coupled receptors that transduce the cellular effects of the catecholamines, adrenaline (Adr) and noradrenaline (Nadr). Through their interaction with plasma membrane beta-ARs, catecholamines modulate a number of key physiological parameters aimed at allowing an organism to cope with environmental and physiological stressors. Mammalian species express three distinct beta-AR subtypes (beta 1-, beta2- and beta3-ARs) that exhibit complex modes of regulation and interaction. Relatively few studies have focused on beta-ARs from early branching vertebrates such as fish, particularly at the molecular level. The goal of this study was to characterize the beta-AR gene-family of the rainbow trout (RbT), Oncorhynchus mykiss. Three putative beta-ARs genes were cloned and a phylogenetic analysis predicted one beta2-AR subtype (RbT beta2-AR) and two beta3-AR subtypes (RbT beta3a- and RbT beta 3b-ARs) relative to the established mammalian beta-AR classification. The RbT beta2-, beta3a- and beta3b-AR genes code for proteins of 409, 427 and 477 amino acids, respectively. Hybridization of gene specific probes to trout tissue RNA indicated that RbT beta 2-AR was highly expressed in the liver, red muscle, and white muscle; RbT beta3a-AR was highly expressed in the gill and heart while RbT beta3b-AR was highly expressed in the blood. Pharmacological analysis indicated RbT beta2-AR binding characteristics consistent with mammalian beta2-ARs while the RbT beta3b-AR showed characteristics that were different from all known mammalian beta-AR subtypes. Differences in the potential regulatory phosphorylation profiles between trout beta-ARs suggests subtype specific sensitivities to the classic mechanisms of beta-AR desensitization. This molecular characterization of trout beta-ARs is the first study to demonstrate a beta3-AR homolog in fish, it identifies the trout red blood cell beta-AR as a beta3-subtype and provides support for the presence of a complex and unique beta-AR signaling system in the rainbow trout.

Characterization of a Ras Gene in Rainbow Trout
Characterization of a Ras Gene in Rainbow Trout

Author: Kathy A. Mangold

Publisher:

Published: 1989

Total Pages: 268

ISBN-13:

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The rainbow trout (Oncorhynchus mykiss) model of chemical carcinogenesis is becoming increasingly important as a supplement to rodent studies. However, much of the molecular biology of the carcinogenic response is still unknown in the trout model. The ras gene family has been implicated in the tumorigenesis of both spontaneous and chemically-induced tumors in mammals. This study is the first to characterize a ras proto-oncogene in rainbow trout. To accomplish this, the ras gene sequence was amplified in vitro by using polymerase chain reaction (PCR). Two synthetic and degenerative oligonucleotide sequences based on a consensus mammal/goldfish ras sequence were used as primers in the PCR procedure. An 800 base pair (bp) sequence was amplified from trout genomic DNA and hybridized with a human c-Ha-ras sequence. The initial amplifications of trout liver cDNA using the PCR procedure with the synthetic ras primers resulted in a single product of approximately 216 bps. However, this amplified "trout" 216 by product was subsequently shown to be an artifact of carryover from a human Ki-ras plasmid. Carryover is a common problem found in many laboratories involved with the PCR procedure, and extensive precautions were used to eliminate the problem in our laboratories. The 800 by PCR product was cloned and sequenced using Taq polymerase. RT-8, a clone containing the 800 bp insert, was shown to have 91% homology to the first two exons of mammalian c-Ha-ras gene and lesser homology to other ras genes. Amplification of trout liver cDNA using specific primers based on the RT-8 sequence resulted in the amplification of sequences identical to the sequence of the RT-8 insert without an intron, as well as unique sequences, which may represent additional trout ras genes. The PCR procedure was modified to identify sequence information immediately 3' of the known trout ras sequence. Partial sequences of at least two different trout ras genes are presented. With this new information, analysis of DNA sequence information from chemically initiated tumors may elucidate the role activation of ras genes plays in the trout model of carcinogenesis.