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First published online May 13, 2004
Journal of Experimental Biology 207, 2033-2042 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00958

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Dogmas and controversies in the handling of nitrogenous wastes: Expression of arginase Type I and II genes in rainbow trout: influence of fasting on liver enzyme activity and mRNA levels in juveniles

Patricia A. Wright^*, Alisha Campbell, Robyn L. Morgan, Andrew G. Rosenberger and Brent W. Murray

Department of Zoology, University of Guelph, Guelph, ON, Canada N1G 2W1

View larger version (19K): [in a new window]   Fig. 1. Rainbow trout (Oncorhynchus mykiss; Onmy) arginase cDNA and expressed sequence tag (EST) sequences. A summary of the sequence information for each of the four contiguous sequences, Onmy-ARG01–Onmy-ARG02b, is shown above the supporting clones and EST contigs. Corresponding Third Party Annotation (TPA) and GenBank (GB) 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 (in base pairs) of the start and stop codons are shown above the black line while the location of the supporting clones is shown below the black line. Supporting clones are shown as either grey or hatched rectangles, indicating clones isolated from the primary cDNA library (S. F. Perry, personal communication) or database-derived EST sequence contigs, respectively.

View larger version (119K): [in a new window]   Fig. 2. Amino acid alignment of vertebrate arginase genes. Strictly conserved residues are boxed, bolded and darkly shaded while residues that maintain the physio-chemical properties of a position are boxed and lightly shaded. A previous comparison of 21 arginase enzymes in both eukaryotes and prokaryotes (Perozich et al., 1998) identified residues that are strictly conserved, highly conserved (at least 80% conserved) and display invariant similarity (i.e. D/E, S/T or V/I/L/M). These positions are indicated beneath the alignment by *, # and ^, respectively. 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 or a unique gene indicator, i.e. ARG01. Species included are Oncorhynchus mykiss (Onmy), Danio rerio (Dare), Takifugu rubripes (Taru), Rana catesbeiana (Raca), Xenopus laevis (Xela), Rattus norvegicus (Rano), Mus musculus (Mumu), Sus scrofa (Susc) and Homo sapiens (Hosa).

View larger version (24K): [in a new window]   Fig. 3. Maximum likelihood phenogram (mid-point rooted) based on a DNA alignment of the coding sequence of arginase genes in vertebrates and three nonvertebrate eukaryotes. Nomenclature of genes is identical to that used in Fig. 2, with the inclusion of three nonvertebrate eukaryote arginase sequences: Neurospora crassa (Necr), Schizosaccharomyces pombe (Scpo) and Schistosoma japonicum (Scja). The location of the common ancestor of all vertebrate cytosolic arginase genes is shown by the arrow. Bootstrap values, based on 300 bootstraps replicates, are placed to the left of the appropriate nodes.

View larger version (14K): [in a new window]   Fig. 4. The tissue distribution of arginase genes in adult rainbow trout (Onmy-ARG01 and Onmy-ARG02) is shown relative to ß-actin. Total RNA (10 µg) was loaded into each lane. Tissues include liver, kidney, gill, intestine, red muscle and heart. Intensity of signal was quantified by denisitometry (see Materials and methods). Means ± S.E.M. (N=3). Asterisks denote significant difference from the ratio of Onmy-ARG01:ß-actin levels (P<0.05).

View larger version (15K): [in a new window]   Fig. 5. The ratio of liver arginase genes, Onmy-ARG01 and Onmy-ARG02, mRNA levels relative to ß-actin mRNA levels from northern blot analysis after 6 weeks of feeding (control) or fasting in juvenile O. mykiss. Total RNA (10 µg) was loaded into each lane. Intensity of the signal was quantified by denisitometry (see Materials and methods). Means ± S.E.M. (N=4). (A) Onmy-ARG01:ß-actin levels, (B) Onmy-ARG02:ß-actin levels. Asterisk denotes significant difference from fed group (P<0.05).