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Evolution of glutamine synthetase in vertebrates: multiple glutamine synthetase genes expressed in rainbow trout (Oncorhynchus mykiss)

Brent W. Murray^1,*, Ellen R. Busby², Thomas P. Mommsen² and Patricia A. Wright^1,

¹ Department of Zoology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
² Biochemistry and Microbiology, University of Victoria, PO Box 3055, Victoria, BC V8W 3P6, Canada
^* Present address: Biology Program, College of Science and Management, University of Northern British Columbia, Prince George, BC, Canada V2N 4Z9

View larger version (24K): [in a new window]   Fig. 1. Rainbow trout (Oncorhynchus mykiss, Onmy) glutamine synthetase cDNA sequences. A summary of the sequence information for each of the four contiguous sequences, Onmy-GS01 – Onmy-GS04, is shown in black above the supporting clones. Corresponding GenBank accession numbers are shown in parentheses. Coding sequence (CDS) information is indicated by a black rectangle, while the 5' and 3' untranslated regions (UTR) are indicated by a thick line. Positions of the start and stop codons and the location of the supporting clones are shown in base pairs. Asterisks indicate possible polyadenylation signal. Supporting clones are shown as either grey or hatched rectangles, indicating clones isolated from the primary cDNA library (S. F. Perry, personal communication) or an alevin RNA preparation, respectively.

View larger version (67K): [in a new window]   Fig. 2. Alignment of vertebrate glutamine synthetase genes. Identity to the consensus sequence is shown with a dash, while an insertion/deletion is shown with an asterisk and a stop codon with an exclamation mark. Tentative active site residues of the glutamate binding site based on the residues identified in Salmonella typhimurium (Gill and Eisenberg 2001) are shown in bold, underlined and indicated with a check mark '. The location of intron 4 is indicated by a downward arrow (). Three periods (...) indicate incomplete or missing sequence information. The sequences are identified with a four-letter code based on their species name and followed by either their unique GenBank accession number, Joint Genome Institute genescan model number or, for sequences reported here, a unique gene indicator, i.e. GS01. Species included are Oncorhynchus mykiss, Onmy; Danio rerio, Dare; Opsanus beta, Opbe; Bostrichthys sinensis, Bosi; Takifugu rubripes, Fugu; Xenopus laevis, Xela; Cricetulus griseus, Crgr; Acomys cahirinus, Acca; Rattus norvegicus, Rano; Mus musculus, Mumu; Sus scrofa, Susc; Homo sapiens, Hosa; Gallus gallus, Gaga; Heterodontus francisci, Hefr and Squalus acanthias, Sqac.

View larger version (31K): [in a new window]   Fig. 3. Maximum-likelihood phenogram based on a DNA alignment of the coding sequence of glutamine synthetase genes in vertebrates. The nucleotide alignment used (available upon request) is identical in form to the amino acid alignment (Fig. 2). Nomenclature of genes is similar to that used in Fig. 2, except that unique GenBank accession numbers have been added to the sequences reported to assist comparison with other papers that may use slightly different nomenclature schemes (i.e. Walsh et al., 2003). Genes coding for mitochondrial isozymes are underlined. Bootstrap values above 50%, based on 200 bootstraps, are placed at the appropriate nodes.

View larger version (12K): [in a new window]   Fig. 4. Dot-plot comparisons of rainbow trout (Oncorhynchus mykiss) glutamine synthetase intron 4 sequences. The last and first 10 base pairs of exons 4 and 5 respectively, flanking intron 4, are included in the comparison.

View larger version (93K): [in a new window]   Fig. 5. Tissue distribution of rainbow trout glutamine synthetase (GS) using high-stringency northern analysis. Membranes were probed using 32P-labelled random-primed cDNA probes for four GSase genes (Onmy-GS01, Onmy-GS02, Onmy-GS03 and Onmy-GS04) and ß-actin, and hybridized at 65°C. Each lane contained 10 µg of total RNA. K, kidney; H, heart; L, liver; G, gill; Int, intestine; W, white muscle; R, red muscle; B, brain. Arrows indicate the location of the respective band.