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A second glutamine synthetase gene with expression in the gills of the gulf toadfish (Opsanus beta)

Patrick J. Walsh1,*, Gregory D. Mayer1, Mónica Medina2, Matthew L. Bernstein1, John F. Barimo1 and Thomas P. Mommsen3

1 NIEHS Marine and Freshwater Biomedical Sciences Center, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149, USA
2 Joint Genome Institute, Genomic Diversity Laboratory, Walnut Creek, CA 94598, USA
3 Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada



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  		Fig. 3. Amino acid sequence comparison of `liver' and `gill' forms of GSase from gulf toadfish Opsanus beta. Residues contributing to the active site are noted by an asterisk. Solid shading indicates identical residues; grey shading, similar residues.

 


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  		Fig. 1. Western blots of several tissues from unconfined and confined representative individuals of the gulf toadfish Opsanus beta. The antibody is to a conserved oligopeptide sequence of GSases (homologous to amino acids 326-343 of the liver Gsase; Fig. 3). (A) Lanes 1 and 10 are molecular mass marker lanes. Even-numbered lanes are samples from unconfined toadfish and odd-numbered lanes from confined toadfish. Samples in lanes 2–3 are derived from brain; lanes 4–5, liver; lanes 6–7, kidney; lanes 8–9, stomach. (B) Lanes 1 and 9 are molecular mass marker lanes, and lane 10 is from unconfined brain (the same sample as in A, lane 2) for cross reference between gels. Even-numbered lanes are samples from unconfined toadfish and odd-numbered lanes from confined toadfish. Samples in lanes 2–3 are derived from intestine; lanes 4–5, gill; lanes 6–7, white muscle. Lane 8 is an empty well.

 


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  		Fig. 2. Nucleotide sequence for novel GSase isolated from gill tissue cDNA of the gulf toadfish Opsanus beta (GenBank AF532312). The location of various primers used and other features are noted in boldface type (see text for details).

 


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  		Fig. 4. Minimum evolution phylogenetic tree of several vertebrate GS proteins. Drosophila mitochondrial and cytosolic GSase sequences were used as outgroups. Support for nodes (50% majority rule) is depicted on corresponding branches (ME bootstrap support/quartet-puzzling reliability values/Bayesian posterior probabilities are represented from left to right). Dashes indicate that there is no 50% majority rule support for the node under that analysis. Species are generally coded as in Murray et al. (2003Go), where the four-letter code refers to the first two letters of genus and species, followed by an accession number in either Genbank or SwissProt databases. Species are: Bosi, Bostrichthyes sinensis (sleeper); Onmy, Oncorhynchus mykiss (rainbow trout); Opbe, Opsanus beta (L,G, liver and gill) (gulf toadfish); Orni, Oreochromis niloticus (tilapia); SINFRUP, Takifugu rubripes (pufferfish); Dare, Danio rerio (zebrafish); Hefr, Heterodontus francisci (horned shark); Sqac, Squalus acanthias (spiny dogfish shark); Gaga, Gallus gallus (chicken); Hosa, Homo sapiens (human); Susc, Sus scrofa (pig); Acca, Acomys cahirinus (Egyptian spiny mouse); Crgr, Cricetulus griseus (Chinese hamster); Mumu, Mus musculus (mouse); Rano, Rattus norvegicus (rat); Xela, Xenopus laevis (clawed toad); Pali, Panulirus argus (spiny lobster); Paar, Paracentrotus lividus (sea urchin); Drme, Drosophila melanogaster (fruit fly). Scale bar represents proportion of amino acids per site.

 


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  		Fig. 5. Relative expression of `liver' and `gill' mRNA/cDNA in tissues of gulf toadfish Opsanus beta. Representative agarose gel of a restriction digest experiment of mRNAs obtained after RT-PCR with primer pair GS 501 and 301. Lanes 1 and 14 are molecular mass markers, lanes 2–5, gill; lanes 6–9, liver; lanes 10–13, stomach. From left to right, for each tissue: uncut PCR products, PCR products digested with StuI, PCR products digested with PvuII, and PCR products digested with StuI plus PvuII.

 

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© The Company of Biologists Ltd 2003