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Glutamine synthetase expression in liver, muscle, stomach and intestine of Bostrichthys sinensis in response to exposure to a high exogenous ammonia concentration

P. M. Anderson¹, M. A. Broderius¹, K. C. Fong², K. N. T. Tsui², S. F. Chew³ and Y. K. Ip²

¹ Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth, Duluth, MN 55812, USA
² Department of Biological Sciences, National University of Singapore, Singapore 117543
³ Natural Sciences Academic Group, Nanyang Technological University, National, Institute of Education, 1 Nanyang Walk, Singapore 637616

View larger version (80K): [in a new window]   Fig. 1. Alignment of the deduced amino acid sequences of glutamine synthetase (Gsase) from liver and stomach of B. sinensis and two other GSases. (A) The amino acid sequence that corresponds to the oligopeptide used for preparing antibody to the GSase is underlined with a wavy line (B. sinensis stomach GSase). The amino acid sequences with codons that correspond to the two consensus primers, LcGS1 and RcGS1, used for obtaining the two complete fragments of the cDNA from stomach and liver by PCR with each of the consensus sequences and AP1, respectively, are indicated by lines; arrows indicate the direction copied above the corresponding sequences for B. sinensis stomach GSase. Amino acid sequences with codons that correspond to the two specific primers used for preparing the probe for RPAs are indicated by solid lines below the corresponding sequences for B. sinensis liver GSase; they are connected by a dotted line corresponding to the entire probe sequence (110 amino acids, 330 base pairs). Mitochondrial targeting sequences are identified by a dashed underline. (B) Base sequences of the 5' end of the B. sinensis liver and stomach mRNA GSase transcripts. The beginning of the open reading frame is underlined with the corresponding N-terminal amino acid sequence indicated above the sequence. Bost., B. sinensis; Toad, Opsanus beta; Dogfish shark, Squalus acanthias. *Identical residues in the alignment; colons, conservative amino acid replacements in the alignment (i.e. similar amino acids); stops, dissimilar amino acids in the alignment.

View larger version (44K): [in a new window]   Fig. 2. Western blots showing changes in level of expression of GSase protein following exposure of B. sinensis to NH4Cl. Total protein (0.5 µg) loaded onto the gels is equivalent in all lanes. The bands correspond to the expected molecular size of GSase as determined by the migration of standards and the predicted molecular mass of B. sinensis Gsase, based on its amino acid sequence.

View larger version (45K): [in a new window]   Fig. 3. Expression of GSase mRNA in control- and NH4Cl-exposed B. sinensis in different tissues. mRNA was detected using ribonuclease protection assays as described in the text. Each lane was loaded with sample that originally contained 10µg of total RNA; when yeast RNA was substituted for mRNA from B. sinensis as a control, no visible band was obtained (data not shown). Lanes P correspond to either the GSase or ß-actin probe loaded with yeast RNA without the RNase step.