Cloning and expression profile of human inorganic pyrophosphatase.

We have cloned a 1.23 kb cDNA from a human heart library which encodes a 32 kDa protein that is 94% identical to bovine inorganic pyrophosphatase. The protein contains an aspartate-rich signature sequence that was previously identified in yeast and prokaryotic pyrophosphatases. Our clone detects a single band on Northern blots and is expressed at modest levels in all tissues examined. The cDNA shows linkage to markers on the long arm of chromosome 10.

Evaluation of gene therapy for citrullinaemia using murine and bovine models.

Citrullinaemia is an autosomal recessive disorder caused by the deficiency of argininosuccinate synthase. The deficiency of this enzyme results in an interruption in the urea cycle and the inability to dispose of excess ammonia derived from the metabolism of protein. The only treatment for this disorder has been dietary restriction of protein and supplementation with medications allowing for alternative excretion of excess nitrogen. Gene therapy offers the possibility of a long-term cure for disorders like citrullinaemia by expressing the deficient gene in the target organ. We have explored the use of adenoviral vectors as a treatment modality for citrullinaemia in two animal models, a naturally occurring bovine model and a murine model created by molecular mutagenesis. Mice treated with adenoviral vectors expressing argininosuccinate synthase lived significantly longer than untreated animals (11 days vs 1 day; however, the animals did not exhibit normal weight gain during the experiment, indicating that the therapeutic effectiveness of the transducing virus was suboptimal. It is speculated that part of the failure to observe better clinical outcome might be due to the deficiency of arginine. In the bovine model, the use of adenoviral vectors did not result in any change in the clinical condition of the animals or in the level of plasma ammonia. However, the use of 15N isotopic ammonia allowed us to assess the flux of nitrogen through the urea cycle during the experiment. These studies revealed a significant increase in the flux through the urea cycle following administration of adenoviral vectors expressing argininosuccinate synthase. We conclude that the use of adenoviral vectors in the treatment of citrullinaemia is a viable approach to therapy but that it will be necessary to increase the level of transduction and to increase the level of enzyme produced from the recombinant viral vector. Future experiments will be designed to address these issues.

Exploring the role of histidines in the catalytic activity of duck delta-crystallins using site-directed mutagenesis.

The duck delta 2-crystallin gene encodes an enzymatically-active argininosuccinate lyase while the delta 1-crystallin gene product, although 94% identical, is enzymatically inactive. Four histidine residues in the duck delta 2-crystallin. His91, His110, His162 and His178, were converted to asparagine residues in an effort to define the role of histidines in the catalytic process of this enzyme-crystallin and to explain the lack of enzyme activity in the delta 1-crystallin protein. The recombinant mutant proteins were expressed in E. coli and purified to homogeneity for analysis. These four residues were chosen because they fall within highly conserved regions of argininosuccinate lyases from several species. This analysis revealed that change of His91 or His162 for asparagine resulted in complete loss of activity. The His110 enzyme had a reduced Vmax and the His178 enzyme was near normal in its kinetic properties. These data confirm the roles of histidine in the catalytic process of this enzyme-crystallin and suggest that the change of His91 to Gln91 observed in the duck and chicken delta 1-crystallin molecules may be sufficient to account for the lack of enzymatic activity of those proteins.

Generation of a mouse model for citrullinemia by targeted disruption of the argininosuccinate synthetase gene.

Argininosuccinate synthetase (ASS) is a urea cycle enzyme that forms argininosuccinate from citrulline and aspartate. Mutations at the ASS locus in man cause the inherited disease, citrullinemia. Citrullinemia is inherited as an autosomal recessive trait and is characterized, biochemically, by elevated levels of blood citrulline and ammonia and often results in early neonatal death if untreated. We have used homologous recombination in embryonic stem cells to generate a line of mice having a targeted disruption of the Ass gene. Homozygous mutant animals develop high levels of blood citrulline, become hyperammonemic, and die within one or two days after birth. Because the phenotype of the mutant mice closely resembles that of humans who lack the ASS enzyme, we expect that these mice will serve as a useful model for exploring new treatments for citrullinemia including somatic gene therapy.

Constitutive and glucocorticoid-mediated activation of glutamine synthetase gene expression in the developing chicken retina.

This report correlates constitutive and glucocorticoid-mediated alterations in glutamine synthetase gene expression observed in chicken retinal organ cultures with activation of the gene during terminal differentiation. Because glucocorticoid hormones are potent inducers of glutamine synthetase in embryonic retinae, they have been considered as primary mediators of the developmental rise. However, unlike what might be expected of a developmental inducer, our data demonstrate that glucocorticoids act as reversible regulators of glutamine synthetase transcription. Moreover, long-term organ culture of embryonic retina in the absence of glucocorticoids leads to a dramatic increase in expression of glutamine synthetase mRNA. These studies argue that although glucocorticoids may potentiate transcription of glutamine synthetase, the retina has been programmed as early as embryonic day 6 to express this enzyme at a later time via a glucocorticoid-independent process. Interestingly, transcription of retinal glutamine synthetase increases by only approximately 10-fold during development, based on nuclear run-on measurements. Therefore, post-transcriptional control mechanisms might also contribute to the 130-fold increase in glutamine synthetase mRNA observed during terminal differentiation.

Multiple mechanisms by which glutamine synthetase levels are controlled in murine tissue culture cells.

We report the isolation of a complimentary DNA (cDNA) clone encoding glutamine synthetase, derived from a population of methionine sulfoxime-resistant mouse GF1 fibroblasts. When GF1 cells are incubated for 48 h in the presence of the glucocorticoid hormone dexamethasone, the specific activity of glutamine synthetase (GS), assayed as glutamyltransferase activity, increases by threefold. Based on dot hybridization analysis, hormonal treatment also produces a similar increase in the level of GS mRNA. When GF1 cells or mouse Neuro 2A neuroblastoma cells are transferred from medium containing 4 mM glutamine to glutamine-free medium, glutamyltransferase activity increases by at least fivefold. However, the presence or absence or glutamine in the medium does not affect the relative level of glutamine synthetase mRNA in either cell line. With both GF1 and Neuro 2A cells, the half-time for the decline in glutamine synthetase enzyme activity on addition of glutamine to the medium is approximately 1.5 h. This rapid decline, coupled with the lack of effect of glutamine on the level of GS messenger RNA in Neuro 2A cells, renders it unlikely that neural cells alter glutamine synthetase levels in response to glutamine by a biosynthetic mechanism, as suggested by previous authors [L. Lacoste, K.D. Chaudhary, and J. Lapointe (1982) J. Neurochem. 39, 78-85].

Developmentally regulated primary glucocorticoid hormone induction of chick retinal glutamine synthetase mRNA.

We have characterized the glucocorticoid hormone induction of glutamine synthetase mRNA in embryonic chick retinal organ cultures by quantitative dot hybridization using a cDNA clone derived from chick retinal RNA. Hydrocortisone (Kapp = 3-4 nM) and dexamethasone (Kapp = 1-2 nM) produce an approximate 30-fold increase in glutamine synthetase mRNA after incubation of organ cultures derived from embryonic day 12 retinae with either hormone for 3 hr. Progesterone is a poor inducer. The glucocorticoid-mediated rise is rapid (t1/2 = 2-3 hr) and occurs in the presence of either of the protein synthesis inhibitors cycloheximide or puromycin, indicating that the induction is a primary or direct response to the hormone. However, the magnitude of the hormonal response observed in culture increases markedly during retinal development. These observations, coupled with the previously reported absence of a hormonal induction in embryonic liver, raise the possibility of a synergistic mechanism, involving tissue-specific regulatory molecules in addition to the glucocorticoid hormone receptor, to explain the retinal-specific primary glucocorticoid hormone induction of glutamine synthetase mRNA.

Tissue-specific regulation of avian glutamine synthetase expression during development and in response to glucocorticoid hormones.

We have isolated a glutamine synthetase cDNA clone derived from chicken retinal RNA. The clone detects a 3.2-kilobase RNA in chicken retina, liver, and brain, based on Northern blotting analysis. The dramatic developmental rise observed for the retinal enzyme, assayed as glutamyl transferase activity, is accompanied by a corresponding rise in this RNA. Injection of hydrocortisone 21-phosphate into the yolk sac of day 10 embryos produces an increase in retinal glutamine synthetase mRNA and glutamyl transferase activity, assayed 4 days after injection. An increase in glutamine synthetase mRNA is also observed within 2 h of incubation of retinal organ cultures with hydrocortisone. Moreover, incubation of these cultures with cycloheximide at a concentration that inhibits protein synthesis by 93% affects neither the basal level nor the hydrocortisone-mediated induction of glutamine synthetase mRNA. Although expression of this RNA is developmentally regulated in the brain, steroid hormone injection does not result in a substantial induction. Hepatic glutamine synthetase mRNA is expressed constitutively between embryonic day 10 and 6 days after hatching and is also not hormone inducible. Southern blotting data with chicken DNA digested with EcoRI, HindIII, and BamHI are best interpreted in terms of the cDNA clone detecting only one gene. If so, several cell-type-specific regulatory mechanisms must function to modulate expression of this gene during development.