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PubMed2001

Glutamine transport in C6 glioma cells: substrate specificity and modulation in a glutamine deprived culture medium.

A previous study has shown that glutamine (Gln) uptake in C6 cells grown in a standard medium containing 2 mM Gln, is predominantly mediated by a sodium-dependent system that is inhibited by ASC system substrates alanine (Ala), serine (Ser), cysteine (Cys) and threonine (Thr), shows pH sensitivity and partial tolerance to substitution of Na+ by Li+, features compatible with system ASCT2 that is strongly expressed in cultured astrocytes. The uptake was not inhibited by the model system A substrate alpha-(methylamino)isobutyric acid (MeAiB), and glycine (Gly) or proline (Pro), indicating that the substrate-regulated system A as defined by routine criteria is relatively inactive in these cells (Dolinska et al., 2000). In this study we compared the uptake of radiolabeled Gln and a model ASC substrate -Thr in cells grown to the same density in Gln-containing and Gln-deprived media. Cells grown in the absence of Gln showed a reduced activity of system ASC-mediated Gln uptake, and the system lost tolerance for Li+ and became somewhat more resistant to lowering pH of the medium. In contrast to cultured astrocytes deprived of Gln, the overall Gln uptake activity in C6 cells adapted to grow in a medium without Gln was lower than in cells grown in a Gln containing medium, and the uptake by system A remained inactive. C6 cells cultured both in the presence and absence of Gln expressed ASCT2 mRNA, indicating that system ASCT2-mediated Gln uptake is modulated at a posttranscriptional level. In contrast to Gln uptake, Thr uptake was more active in cells cultured in the absence of Gln and showed neither pH dependence nor lithium tolerance in either medium, which is typical of an uptake mediated by the widespread ASCT1 isoform of system ASC. In C6 cells grown in the presence or absence of Gln alike, approximately 20% of the sodium-dependent Gln uptake was resistant to MeAiB+Thr, indicating contribution of system N. The N system-mediated uptake in C6 cells grown in the absence, but not in the presence of Gln was not inhibited by glutamate (Glu) that conforms to the characteristics of the glial N system variant, SN1.

Amino Acid Transport System ASC↗
PubMed2003

Low plasma glutamine after multiple trauma: relationship with intracellular glutamine in polymorphonuclear neutrophils during prolonged ICU stay.

BACKGROUND: Aim of the study was to evaluate whether low plasma glutamine (GLN) is related to low intracellular GLN in stress-affected cells such as polymorphonuclear neutrophil (PMN). We hypothesized, that because low plasma GLN is assumed to have an impact on clinical outcome, stress-affected cells may also show low GLN contents. METHODS: Thirty-nine consecutive severely injured trauma patients staying at least 10 days at a surgical intensive care unit (ICU) of a university hospital were separated into two groups: group one (n = 16) with low plasma GLN (< 420 micromol/l in average during ICU stay), and group two (n = 23) with normal plasma GLN. Initial blood samples for GLN analyses were collected within 24 h of admission at ICU. Further blood samples were taken on days 5 and 10 at 08:00 hours. RESULTS: Patients in both groups showed no differences regarding demographic data, surgical interventions or infections. Acute physiology and chronic health evaluation (APACHE) II and the sequential organ failure assessment (SOFA) score and mortality rate were also comparable. During the study period, intracellular PMN GLN contents and concentrations did not differ between both groups. On the first day, intracellular PMN GLN content in the low plasma GLN group peaked at 5.01 +/- 3.06 x 10(-16) mol and in normal plasma GLN group at 4.73 +/- 2.57 x 10(-16) mol above the level of healthy individuals. In both groups, content decreased significantly towards the end of the observation period (group one: 2.79 +/- 1.59 x 10(-16) mol and group two: 2.63 +/- 1.71 x 10(-16) mol). A correspondent course could be observed for cell volumes. In contrast, variation of intracellular GLN concentrations remained within the reference range throughout the observation period: group one 836 +/- 510 micromol/l on day 1 and 582 +/- 331 micromol/l on day 10, and group two 788 +/- 428 micromol/l on day 1 and 548 +/- 356 micromol/l on day 10. No correlation between plasma GLN and intracellular GLN was found in either group. CONCLUSION: No association between low plasma GLN and low intracellular GLN in PMN was found in a cohort of severely injured trauma patients with a minimum stay of 10 days at ICU.

Adult↗
PubMed2001

Growth hormone together with glutamine-containing total parenteral nutrition maintains muscle glutamine levels and results in a less negative nitrogen balance after surgical trauma.

BACKGROUND: Muscle protein catabolism, reflected by a decrease in glutamine (GLN), a decrease in muscle protein synthesis, and a negative nitrogen balance can be reduced by either administration of GLN or growth hormone (GH). In this study, the effects of a combination of GH and GLH were studied. METHODS: Patients (n = 16) undergoing abdominal operation were given total parenteral nutrition (TPN) containing either GLN alone or GLN together with GH (GH/GLN) during 3 postoperative days. The amino acid concentration and protein synthesis in muscle tissue and the nitrogen balance were measured. RESULTS: GH/GLN reduced nitrogen losses compared with GLN alone (-5.8 +/- 1.4 g nitrogen versus -10.6 +/- 1.1 g nitrogen, P <.05). GH/GLN maintained muscle GLN at preoperative levels compared with a 47.5% +/- 6.3% decline in the GLN group. A similar decrease was seen in the fractional synthesis rate of muscle protein postoperatively in both groups. CONCLUSIONS: GH has an additive effect given together with GLN on muscle amino acid metabolism, preventing the decrease in the GLN concentration in skeletal muscle and diminishing the loss of whole body nitrogen. However, the improvements in muscle amino acid concentrations and nitrogen loss were not associated with differences between the groups in muscle protein synthesis postoperatively.

Abdomen↗
PubMed1996

Butachlor impact on protein, free amino acid and glutamine contents, and on activity levels of aminotransferases, glutamate dehydrogenase and glutamine synthetase in the fresh water snail, Pila globosa (Swainson).

Biochemical changes followed in the freshwater snail Pila globosa (Swainson) during exposure to sublethal concentrations of the herbicide butachlor (26.6 ppm) in the ambient medium, at 3,6,12,24 and 48 h intervals, were marked by a significant decrease in total and soluble proteins, and an increase in free amino acids in foot and hepatopancreas up to 12 h before gradually recovering. Aminotransferase activities and glutamine content decreased during the early periods of exposure, while glutamate dehydrogenase activity increased. After an initial elevation, glutamate synthetase activity decreased at later intervals. Maximum effect of butachlor on the enzymes was seen after 12 h exposure. The extent of increase or decrease in different parameters examined varied between the two tissues studied. These changes are discussed in relation to the toxic stress of butachlor.

Acetanilides↗
PubMed1997

Both growth hormone and exogenous glutamine increase gastrointestinal glutamine uptake in trauma.

OBJECTIVE: The authors studied the effect of exogenous glutamine (GLN), with and without growth hormone (GH), pretreatment, on gastrointestinal, hepatic, femoral, and renal GLN fluxes. SUMMARY BACKGROUND DATA: Growth hormone treatment increases gastrointestinal uptake of GLN despite a reduced skeletal muscle and whole body release. METHODS: Piglets were randomized to a GH + GLN group (n = 8), a GLN group (n = 8), a GH group (n = 8), and a control group (CON; n = 8). Genotropin (Pharmacia, Stockholm, Sweden; 24 international units; correspondingly saline in the GLN and the CON group) was given daily 3 days before and at the onset of trauma (surgery). Organ fluxes and whole body release of GLN were determined 1 and 5 hours after surgery. An infusion of GLN 36 micrograms/kg per minute was started after the first measurement in the GH + GLN and the GLN groups. RESULTS: Both GH treatment and exogenous GLN increased gastrointestinal GLN uptake (p = 0.001 and p = 0.02, respectively). Growth hormone treatment reduced hepatic GLN uptake (p = 0.001). Hepatic GLN uptake was lower in the GH + GLN group versus the GH group (p = 0.02), but not in the GLN group versus the CON group (p = 0.98). Growth hormone treatment reduced femoral and whole-body GLN release (p = 0.0001 and p = 0.02, respectively). Renal GLN uptake was higher in the two GH-treated groups (p = 0.003). CONCLUSION: Both exogenous GLN and GH increased gastrointestinal GLN uptake, and the combination was additive. In contrast to exogenous GLN, GH reduced hepatic uptake and consequently facilitated the increased gastrointestinal GLN uptake that occurred despite reduced femoral and whole-body release.

Animals↗
PubMed1967

Regulation of glutamine synthetase. X. Effect of growth conditions on the susceptibility of Escherichia coli glutamine synthetase to feedback inhibition.

The kinetic properties of Escherichia coli glutamine synthetase are markedly influenced by the manner in which the organism is grown. Enzyme obtained from stationary-phase cells grown on glycerol and glutamate is strongely inhibited by each of the eight feedback effectors known to influence this enzyme; however, the enzyme from log-phase cells grown on glucose and growth-limiting concentrations of NH(4)Cl is stimulated by some of these effectors. Of the growth variables examined, nitrogen source and time of harvest were the most important; carbon source and aeration seemed to have no effect. Two purified enzyme preparations have been obtained from cells grown under two different conditions, designated enzymes I and II for convenience. Enzyme I is stimulated by adenosine 5'-monophosphate, histidine, and tryptophan in the transfer assay, whereas enzyme II is strongly inhibited by all effectors tested. Enzyme I has a higher specific activity in the forward assay in the presence of Mg(++) or Co(++), whereas enzyme II is more active in the presence of Mn(++).

Adenine Nucleotides↗
PubMed2005

Intestinal renal metabolism of L-citrulline and L-arginine following enteral or parenteral infusion of L-alanyl-L-[2,15N]glutamine or L-[2,15N]glutamine in mice.

Previously, we observed increased plasma arginine (ARG) concentrations after glutamine (GLN)-enriched diets, in combination with clinical benefits. GLN delivers nitrogen for ARG synthesis, and the present study was designed to quantify the interorgan relationship of exogenous L-GLN or GLN dipeptide, by enteral or parenteral route, contributing to intestinal citrulline (CIT) and renal de novo ARG synthesis in mice. To study this, we used a multicatheterized mouse model with Swiss mice (n = 43) in the postabsorptive state. Stable isotopes were infused into the jugular vein or into the duodenum {per group either free L-[2,(15)N]GLN or dipeptide L-ALA-L-[2,(15)N]GLN, all with L-[ureido-(13)C-(2)H(2)]CIT and L-[guanidino-(15)N(2)-(2)H(2)]ARG} to establish renal and intestinal ARG and CIT metabolism. Blood flow was measured using (14)C-para-aminohippuric acid. Net intestinal CIT release, renal uptake of CIT, and net renal ARG efflux was found, as assessed by arteriovenous flux measurements. Quantitatively, more de novo L-[2,(15)N]CIT was produced when free L-[2,(15)N]GLN was given than when L-ALA-L-[2,(15)N]GLN was given, whereas renal de novo L-[2,(15)N]ARG was similar in all groups. In conclusion, the intestinal-renal axis is hereby proven in mice in that L-[2,(15)N]GLN or dipeptide were both converted into de novo renal L-[2,(15)N]ARG; however, not all was derived from intestinal L-[2,(15)N]CIT production. In this model, the feeding route and form of GLN did not influence de novo renal ARG production derived from GLN.

Algorithms↗
PubMedDate not supplied

Enteral glutamine spares endogenous glutamine in chronic acidosis.

Metabolic acidosis associated with the catabolic state mobilizes muscle nitrogen and releases it into blood as glutamine (GLN) targeted for renal consumption and base generation. Because GLN removed by the kidneys during acidosis is a major drain on the GLN available to other sites, subsequent deprivation may lead to impaired organ function. Conversely, GLN supplementation may spare endogenous supplies and restore organ function. To test this, Sprague-Dawley rats weighing between 250 and 350 g were pair-fed elemental diets supplemented with GLN 4.9 g/L (GLN-ED) or an equivalent mixture of neutral amino acids substituted for GLN (ED). Acid loading was effected by adding hydrochloric acid to the liquid diet (110 mmol/L). Animals were studied in metabolic cages for five consecutive 24-hour urine collection periods and then anesthetized for short-term studies of interorgan fluxes and tissue GLN content. Acidosis effected an increase in ammonium nitrogen excretion (fivefold) and a reciprocal decrease (24%) in urea nitrogen excretion. Enteral GLN had no effect on the acidosis-effected ammonium (2170 +/- 71 vs 2059 +/- 361 mumol/100 g, ED vs GLN-ED, respectively) or urea excretion (5522 +/- 95 vs 5915 +/- 984 mumol/100 g, ED vs GLN-ED, respectively). Although arterial blood GLN was not increased in the GLN-ED group (531 +/- 58 vs 438 +/- 51 nmol/mL, p = .10), both liver and muscle GLN were elevated (11,650 +/- 1137 nmol/g vs 7063 +/- 578 and 5503 +/- 489 and 4742 +/- 333 nmol/g, respectively, each p < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMedDate not supplied

Glutamine-enriched enteral diet enhances bacterial clearance in protected bacterial peritonitis, regardless of glutamine form.

BACKGROUND: The effects of glutamine (Gln)-enriched enteral diets on bacterial clearance were investigated in a rat protracted peritonitis model. The effects of the Gln form, peptide-based vs free amino acid-based, were also compared. METHODS: Twenty-three rats underwent gastrostomy. An osmotic pump was implanted in the peritoneal cavity. The rats received a continuous intragastric infusion of one of three diets: Gln-depleted (Gln 0), Gln-enriched with the Gln in free amino acid form (Gln F), or Gln-enriched with the Gln in oligopeptide form (Gln P). The three formulas were isocaloric and isonitrogenous. The pumps delivered a continuous infusion of Escherichia coli, starting at 48 hours after implantation, for 24 hours. Then, the animals were killed. RESULTS: Bacterial numbers in peritoneal lavaged fluid (PLF) and the liver were significantly lower in the Gln P and Gln F groups than in the Gln 0 group. The bacterial number in PLF correlated with that in the liver. Neither the number nor the population of peritoneal exudative cells differed among groups. Plasma levels of proline, alanine and citrulline were significantly higher in the Gln P and Gln F groups than in the Gln 0 group. Both Gln supplemented groups showed significantly greater villous height, crypt depth, and numbers of mitoses per crypt in the small intestine than the Gln 0 group. CONCLUSIONS: Supplemental Gln enhances peritoneal and hepatic bacterial clearance, regardless of Gln form. Gln-enriched may be more beneficial than Gln-depleted enteral diets in peritonitis.

Animals↗
PubMed1993

Modulation of glutamine synthetase gene expression in tobacco by the introduction of an alfalfa glutamine synthetase gene in sense and antisense orientation: molecular and biochemical analysis.

A glutamine synthetase (GS) cDNA isolated from an alfalfa cell culture cDNA library was found to represent a cytoplasmic GS. The full-length alfalfa GS1 coding sequence, in both sense and antisense orientation and under the transcriptional control of the cauliflower mosaic virus 35S promoter, was introduced into tobacco. Leaves of tobacco plants transformed with the sense construct contained greatly elevated levels of GS transcript and GS polypeptide which assembled into active enzyme. Leaves of the plants transformed with the antisense GS1 construct showed a significant decrease in the level of both GS1 and GS2 polypeptides and GS activity, but did not show any significant decrease in the level of endogenous GS mRNA. We have proposed that antisense inhibition using a heterologous antisense GS RNA occurs at the level of translation. Our results also suggest that the post-translational assembly of GS subunits into a holoenzyme requires an additional factor(s) and is under regulatory control.

Alcohol Oxidoreductases↗
PubMed1988

Genetic determinants of glutamine synthetase in Drosophila melanogaster: a gene for glutamine synthetase I resides in the 21B3-6 region.

Recombinational and deletion mapping of electrophoretic variants of the glutamine synthetase I isozyme (GSI) in Drosophila melanogaster locates the gene in the 21B region on the second chromosome. We have conducted a genetic analysis of the region extending cytologically from 21A to 21B4-6. Recessive lethal mutations were generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU) mutagenesis and by hybrid dysgenesis (HD). These lethals fall into seven functional groups, which were partially ordered by complementation with cytologically defined deficiencies of this region generated by hybrid dysgenesis. Two of the EMS- and two of the ENU-induced lethals fulfill biochemical criteria expected for null alleles of the GSI gene.

Animals↗
PubMed1985

Distribution of glutamine synthetase and glial fibrillary acidic protein and correlation of glutamine synthetase with glutamate decarboxylase in different regions of the rat central nervous system.

The concentration of soluble glial fibrillary acidic (GFA) protein and the specific activity of glutamine synthetase (GS) were estimated in 11 central nervous system (CNS) regions of the 90-day-old rat. Marked differences were observed in the regional distribution of these astrocyte marker proteins. The striatum and spinal cord contained the lowest concentration (per g wet weight) of GFA protein and GS activity, respectively, while the olfactory bulbs had the highest level of both astrocytic proteins. Differences between the lowest and the highest values were 3-fold for GS and 4-fold for GFA protein. More significant was the marked variation in the ratio of GS to GFA protein in different CNS regions; the highest and lowest values were in the striatum and the spinal cord respectively, and the difference between the highest and the lowest value was about 5-fold. The spinal cord contained low GS and high GFA protein; on the other hand, the colliculi had high GS and relatively low GFA protein. Immunochemical detection of GS and GFA proteins in whole homogenates of different regions showed that the variation of the specific activities of GS and the concentration of soluble GFA protein were due to the differences in their absolute protein concentrations. In different regions of the brain the activity of GS was significantly correlated with that of glutamate decarboxylase, but not with that of choline acetyltransferase. These observations provide further evidence for differing biochemical properties of astrocytes from various CNS regions and for the involvement of GS in processes associated with amino acid neurotransmission.

Animals↗
PubMed2002

Glutamine synthetase isolated from human brain: octameric structure and homology of partial primary structure with human liver glutamine synthetase.

Glutamine synthetase (GS) has been purified from the cytosolic fraction of non-frozen human brain tissue. The purified GS migrated as a main band around 44 kD on reducing SDS-PAGE. Two-dimensional electrophoresis revealed heterogeneity within subunits of GS. The masses of eight different peptides from a tryptic digest of GS as measured by high resolution MALDI-MS matched with the respective masses from an in silico tryptic fingerprint of the Swiss-Prot database entry of human liver GS, proving that at least 24% of the primary sequences of GS from brain and liver are identical. Sedimentation equilibrium profiles obtained from analytical ultracentrifugation experiments at 10 degrees C showed that human brain GS is mainly octameric. The quaternary structure of human brain GS at 10 microM (subunit concentration) was not significantly affected by cations, such as magnesium (5 and 20 mM) or manganese (0.2 and 1 mM) within the range of pH 7.1-7.8.

Amino Acid Sequence↗
PubMed1992

Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase.

The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs. Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins. To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA. Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals. A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal. Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity. Both could have resulted from third-base-codon substitutions. A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme. The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme. The complete sequence of chicken retinal GS is also identical to that of the liver enzyme. GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s).

Amino Acid Sequence↗
PubMed2004

Overexpression of GLUTAMINE DUMPER1 leads to hypersecretion of glutamine from Hydathodes of Arabidopsis leaves.

Secretion is a fundamental process providing plants with the means for disposal of solutes, improvement of nutrient acquisition, and attraction of other organisms. Specific secretory organs, such as nectaries, hydathodes, and trichomes, use a combination of secretory and retrieval mechanisms, which are poorly understood at present. To study the mechanisms involved, an Arabidopsis thaliana activation tagged mutant, glutamine dumper1 (gdu1), was identified that accumulates salt crystals at the hydathodes. Chemical analysis demonstrated that, in contrast with the amino acid mixture normally present in guttation droplets, the crystals mainly contain Gln. GDU1 was cloned and found to encode a novel 17-kD protein containing a single putative transmembrane span. GDU1 is expressed in the vascular tissues and in hydathodes. Gln content is specifically increased in xylem sap and leaf apoplasm, whereas the content of several amino acids is increased in leaves and phloem sap. Selective secretion of Gln by the leaves may be explained by an enhanced release of this amino acid from cells. GDU1 study may help to shed light on the secretory mechanisms for amino acids in plants.

Amino Acid Sequence↗
PubMed1989

Glutamine auxotrophs of Bacillus subtilis that overproduce glutamine synthetase antigen have altered conserved amino acids in or near the active site.

A number of mutations within the Bacillus subtilis glutamine synthetase (GS) gene result in altered catalytic properties and overproduction of the GS antigen. The restriction fragments containing mutations from three such mutants were sequenced, and they all had amino acid changes in conserved residues found either within or near sequences contributing to the active site of the Salmonella typhimurium GS.

Amino Acid Sequence↗
PubMed1993

Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences.

The gene glnA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sulfolobus solfataricus glnA gene as the probe. An operon reading frame of 448 amino acids was identified within a DNA segment of 1,528 bp. The encoded protein was 49% identical with the GSI of Methanococcus voltae and exhibited conserved regions characteristic of the GSI family. The P. woesei GSI was aligned with available homologs from other archaea (S. solfataricus, M. voltae) and with representative sequences from cyanobacteria, proteobacteria, and gram-positive bacteria. Phylogenetic trees were constructed from both the amino acid and the nucleotide sequence alignments. In accordance with the sequence similarities, archaeal and bacterial sequences did not segregate on a phylogeny. On the basis of sequence signatures, the GSI trees could be subdivided into two ensembles. One encompassed the GSI of cyanobacteria and proteobacteria, but also that of the high-G + C gram-positive bacterium Streptomyces coelicolor (all of which are regulated by the reversible adenylylation of the enzyme subunits); the other embraced the GSI of the three archaea as well as that of the low-G + C gram-positive bacteria (Clostridium acetobutilycum, Bacillus subtilis) and Thermotoga maritima (none of which are regulated by subunit adenylylation). The GSIs of the Thermotoga and the Bacillus-Clostridium lineages shared a direct common ancestor with that of P. woesei and the methanogens and were unrelated to their homologs from cyanobacteria, proteobacteria, and S. coelicolor. The possibility is presented that the GSI gene arose among the archaea and was then laterally transferred from some early methanogen to a Thermotoga-like organism. However, the relationship of the cyanobacterial-proteobacterial GSIs to the Thermotoga GSI and the GSI of low-G+C gram-positive bacteria remains unexplained.

Amino Acid Sequence↗
PubMed2003

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

Glutamine synthetase (GSase) is a key enzyme in nitrogen metabolism and encoded by a single gene in mammals. Using PCR cloning techniques, including RT-PCR from total RNA and PCR from a cDNA library, we find evidence of four expressed GSase mRNAs for the tetraploid rainbow trout. For two of these mRNAs (Onmy-GS01, -GS02) we characterize the full-length coding regions, and for two others (Onmy-GS03, -GS04), we describe partial sequences. Northern analysis of Onmy-GS01, -GS02, -GS03 and -GS04 indicates that (1) Onmy-GS02 is expressed at higher levels relative to the other transcripts in most adult tissues, with the exception of brain and gill, where Onmy-GS01 is at the highest level, and (2) the tissue with the highest level of expression of all four transcripts is the brain, with decreasing levels in the intestine, liver, red muscle, gill/kidney, white muscle and heart. Clearly, rainbow trout possess multiple GSase genes with differing levels of tissue expression, implying manifold potential routes of regulation for this octameric enzyme. Our data also indicate that caution should be taken when interpreting mRNA expression data of a single gene, unless multiple genes have been ruled out. Consistent with a southern blot, phylogenetic and intron sequence analyses imply that the trout genes are encoded by at least four separate loci, belonging to two distinct evolutionary branches. Our data on rainbow trout, together with those from two full-length zebrafish Danio rerio GSase genes compiled from GenBank ESTs, support the idea that fish GSases are polyphyletic and that gene duplications have occurred at multiple points and in independent lineages throughout the evolution of bony fishes.

Amino Acid Sequence↗