Characterization and sequencing of glutamine synthetase cDNA from liver of the ureotelic gulf toadfish (Opsanus beta). 1999

P J Walsh, and M E Handel-Fernandez, and V Vincek
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

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.

UI MeSH Term Description Entries
D008099 Liver A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances. Livers
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D005399 Fishes A group of cold-blooded, aquatic vertebrates having gills, fins, a cartilaginous or bony endoskeleton, and elongated bodies covered with scales.
D005974 Glutamate-Ammonia Ligase An enzyme that catalyzes the conversion of ATP, L-glutamate, and NH3 to ADP, orthophosphate, and L-glutamine. It also acts more slowly on 4-methylene-L-glutamate. (From Enzyme Nomenclature, 1992) EC 6.3.1.2. Glutamine Synthetase,Glutamate Ammonia Ligase (ADP),Glutamate Ammonia Ligase,Ligase, Glutamate-Ammonia,Synthetase, Glutamine
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D015152 Blotting, Northern Detection of RNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES. Northern Blotting,Blot, Northern,Northern Blot,Blots, Northern,Blottings, Northern,Northern Blots,Northern Blottings
D015342 DNA Probes Species- or subspecies-specific DNA (including COMPLEMENTARY DNA; conserved genes, whole chromosomes, or whole genomes) used in hybridization studies in order to identify microorganisms, to measure DNA-DNA homologies, to group subspecies, etc. The DNA probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the DNA probe include the radioisotope labels 32P and 125I and the chemical label biotin. The use of DNA probes provides a specific, sensitive, rapid, and inexpensive replacement for cell culture techniques for diagnosing infections. Chromosomal Probes,DNA Hybridization Probe,DNA Probe,Gene Probes, DNA,Conserved Gene Probes,DNA Hybridization Probes,Whole Chromosomal Probes,Whole Genomic DNA Probes,Chromosomal Probes, Whole,DNA Gene Probes,Gene Probes, Conserved,Hybridization Probe, DNA,Hybridization Probes, DNA,Probe, DNA,Probe, DNA Hybridization,Probes, Chromosomal,Probes, Conserved Gene,Probes, DNA,Probes, DNA Gene,Probes, DNA Hybridization,Probes, Whole Chromosomal
D015723 Gene Library A large collection of DNA fragments cloned (CLONING, MOLECULAR) from a given organism, tissue, organ, or cell type. It may contain complete genomic sequences (GENOMIC LIBRARY) or complementary DNA sequences, the latter being formed from messenger RNA and lacking intron sequences. DNA Library,cDNA Library,DNA Libraries,Gene Libraries,Libraries, DNA,Libraries, Gene,Libraries, cDNA,Library, DNA,Library, Gene,Library, cDNA,cDNA Libraries

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