Characterization and sequencing of glutamine synthetase cDNA from liver of the ureotelic gulf toadfish (Opsanus beta).
The hepatic enzyme, glutamine synthetase (GSase) is a pivotal protein in the regulation of urea synthesis in fish. The sequence of the DNA encoding for GSase from liver of the ureotelic gulf toadfish (Opsanus beta) was analyzed through a suite of molecular techniques (including cDNA cloning, RACE PCR, and genomic PCR). An open reading frame (ORF) was identified in the cDNA sequence which codes for a protein of 394 amino acids with high identity (86%) to dogfish shark GSase. In the course of generating a suitable probe, a partial sequence was also obtained for horned shark GSase which also had high identity with the dogfish shark gene (93%). Like the dogfish shark GSase, the toadfish gene has two methionine translation initiation sites; the downstream site apparently codes for a cytoplasmic isozyme, while the upstream site adds an N-terminal peptide leader sequence of 23 amino acids to the 'cytoplasmic' protein. This leader sequence has characteristics consistent with a mitochondrial targeting peptide, including a cleavage recognition motif (Arg-X-Phe) and the apparent ability to form an amphiphathic helix. Northern analysis revealed that there is a single predominant transcript of approximately 2 kb in size. These results are consistent with the interpretation that in the gulf toadfish GSase cytoplasmic and mitochondrial isozymes are coded for by a single gene and mRNA transcript which is differentially translated at either initiation site. These results are discussed in the context of prior results for enzyme kinetic characteristics and urea synthesis/excretion physiology.
Walsh
P J
PJ
Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, NIEHS Marine and Freshwater Biomedical Sciences Center, Miami, FL 33149-1098, USA. pwalsh@rsmas.miami.edu
Handel-Fernandez
M E
ME
Vincek
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015342:DNA Probes; D018076:DNA, Complementary / Q000235:genetics; D019143:Evolution, Molecular; D005399:Fishes / Q000235:genetics*; D015723:Gene Library; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D015342:DNA Probes; D018076:DNA, Complementary; D005974:Glutamate-Ammonia Ligase