Search

Glutamine synthetase (GSase) is a key enzyme in nitrogen metabolism and encoded by a single gene in mammals. Using PCR cloning techniques, including RTPCR from total RNA and PCR from a cDNA library, we find evidence of four expressed GSase mRNAs for the tetraploid rainbow trout. For two of these mRNAs (OnmyGS01, -GS02) we characterize the full-length coding regions, and for two others (Onmy-GS03, -GS04), we describe partial sequences. Northern analysis of OnmyGS01, -GS02, -GS03 and -GS04 indicates that (1) OnmyGS02 is expressed at higher levels relative to the other transcripts in most adult tissues, with the exception of brain and gill, where Onmy-GS01 is at the highest level, and (2) the tissue with the highest level of expression of all four transcripts is the brain, with decreasing levels in the intestine, liver, red muscle, gill/kidney, white muscle and heart. Clearly, rainbow trout possess multiple GSase genes with differing levels of tissue expression, implying manifold potential routes of regulation for this octameric enzyme. Our data also indicate that caution should be taken when interpreting mRNA expression data of a single gene, unless multiple genes have been ruled out. Consistent with a southern blot, phylogenetic and intron sequence analyses imply that the trout genes are encoded by at least four separate loci, belonging to two distinct evolutionary branches. Our data on rainbow trout, together with those from two full-length zebrafish Danio rerio GSase genes compiled from GenBank ESTs, support the idea that fish GSases are polyphyletic and thai gene duplications have occurred at multiple points and in independent lineages throughout the evolution of bony fishes. [ABSTRACT FROM AUTHOR]

The incorporation of ammonia into glutamine, catalyzed by glutamine synthetase, is thought to be important in the detoxification of ammonia in animals. During early fish development, ammonia is continuously formed as yolk proteins and amino acids are catabolized. We followed the changes in ammonia and urea-nitrogen content, ammonia and urea-nitrogen excretion, glutamine synthetase activity, and mRNA expression of four genes coding for glutamine synthetase (Onmy-GS01-GS04) over 3-80 days post fertilization and in adult liver and skeletal muscle of the rainbow trout (Oncorhynchus mykiss). Both ammonia and urea-nitrogen accumulate before hatching, although the rate of ammonia excretion is considerably higher relative to urea-nitrogen excretion. All four genes were expressed during early development, but only Onmy-GS01 and -GS02 were expressed at appreciable levels in adult liver, and expression was very low in muscle tissue. The high level of expression of Onmy-GS01 and -GS03 prior to hatching corresponded to a linear increase in glutamine synthetase activity. We propose that the induction of glutamine synthetase genes early in development and the subsequent formation of the active protein are preparatory for the increased capacity of the embryo to convert the toxic nitrogen end product, ammonia, into glutamine, which may then be utilized in the ornithine-urea cycle or other pathways. [ABSTRACT FROM AUTHOR]