Expression of the human argininosuccinate synthetase gene in hamster transferents.

Abstract

The structural gene for human argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5] was transferred to argininosuccinate synthetasedeficient Chinese hamster cells via metaphase chromosomes isolated-from human lymphoblast line MGL8D1, a constitutive overproducer of argininosuccinate synthetase, and from its repressible parent, MGL8B2. Argininosuccinate synthetase expression was selected for ih citrulline-containing medium, and the human origin of the argininosuccinate synthetase expressed by seven transferents was identified by isoelectric focusing. Stable transferents expressing MGL8D1 argininosuccinate synthetase fell into two classes: (i) those whose argininosuccinate synthetase activity was reduced to 10-50% by arginine, similar to the repression of argininosuccinate synthetase synthesis observed in normal human lymphoblasts, and (ii) those that constitutively expressed argininosuccinate synthetase when grown in the presence of arginine or citrulline. Two transferents from the MGL8B2 donor constitutively expressed human argininosuccinate synthetase. Three hamster revertants were isolated that constitutively expressed hamster argininosuccinate synthetase. Transferents and revertants exhibited growth-dependent changes in argininosuccinate synthetase activity, in contrast to the constant synthetase activity levels in donor lymphoblasts during growth. The isolation of stable transferents that constitutively or repressibly express argininosuccinate synthetase makes possible the analysis of regulatory signals influencing expression of the argininosuccinate synthetase gene.Functional genes can be transferred to mammalian cells and stably transmitted to progeny cells when either metaphase chromosomes (1-11) or DNA (12-16) is incubated with recipient cells. Stable transferents appear to have integrated the donor gene into their genomes with no apparent site specificity (7)(8)(9)13). The integrated DNA, estimated to represent less than 0.25% of the haploid donor genome in chromosome transfer experiments (4-6) and approximately 3.4 kilobases for the viral thymidine kinase gene (12,13), is large enough to include regulatory sequences adjacent to the structural gene sequence(s). These features of gene transfer make it attractive for analyzing regulatory signals influencing gene expression, as well as for mapping these regulatory sequences.We have transferred the human gene for the urea cycle enzyme argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5], which has been localized to the distal portion of the long arm of chromosome 9 (17), to enzyme-deficient hamster fibroblasts and examined the regulation of the argininosuccinate synthetase expressed in transferent cells. The synthesis of argininosuccinate synthetase in human lymphoblast line MGL8B2 is normally repressed when arginine is present in the medium (18). Constitutive overproducers of argininosuccinate synthetase, however, have been isolated by selecting human lymphoblasts resistant to c...

Sequence for human argininosuccinate synthetase cDNA

“…The assigned coding region predicted an amino acid content in close agreement with that published for bovine liver enzyme (10). The content of basic amino acids (21 arginines and 33 lysines in 412 amino acids) was unusually high and was consistent with the pI of 9.0 reported for the human enzyme (13).…”

Increased translatable messenger ribonucleic acid for argininosuccinate synthetase in canavanine-resistant human cells

“…The difference in the activity in the CanT cell line was not consistently observed and was not thought to be significant. The data from multiple experiments showed similar relative activities to those shown in Table I although the absolute activities varied slightly between experiments, probably related to culture conditions and cell density (Hudson et al, 1980). The CanT cell line consistently yielded argininosuccinate synthetase activity in the range of 20-30 nmol min'1 (mg of protein)-1.…”

Argininosuccinate synthetase from the urea cycle to the citrulline–NO cycle

“…ASS deficiency in human causes citrullinemia (see Introduction) and the classic neonatal CTLN1‐form of the disease frequently leads to neonatal death [3]. This stimulated the development of gene‐transfer strategies ≈ 20 years ago [173,174]. Using retroviral vectors, long‐term expression of the human enzyme was obtained in mice receiving bone marrow[175], and by administration of an adenoviral vector expressing human ASS, partial correction of the enzyme defect was observed in a neonatal bovine model of citrullinemia [176].…”