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Expanded newborn screening detects patients with modest elevations in citrulline; however it is currently unclear how to treat these patients and how to counsel their parents. In order to begin to address these issues, we compared the clinical, biochemical, and molecular features of 10 patients with mildly elevated citrulline levels. Three patients presented with clinical illness whereas seven came to attention as a result of expanded newborn screening. One patient presented during pregnancy and responded promptly to IV sodium phenylacetate/sodium benzoate and arginine therapy with no long-term adverse effects on mother or fetus. Two children presented with neurocognitive dysfunction, one of these responded dramatically to dietary protein reduction. ASS enzyme activity was not deficient in all patients with biallelic mutations suggesting this test cannot exclude the ASS1 locus in patients with mildly elevated plasma citrulline. Conversely, all symptomatic patients who were tested had deficient activity. We describe four unreported mutations (p.Y291S, p.R272H, p.F72L, and p.L88I), as well as the common p.W179R mutation. In silico algorithms were inconsistent in predicting the pathogenicity of mutations. The cognitive benefit in one patient of protein restriction and the lack of adverse outcome in seven others restricted from birth, suggest a role for protein restriction and continued monitoring to prevent neurocognitive dysfunction., ((c) 2008 Wiley-Liss, Inc.)

Background: Urea cycle disorders are relatively rare but well-established causes of postpartum coma and death. Such clinical presentations have been reported previously in ornithine transcarbamylase and carbamyl phosphate synthetase deficiencies., Case: We describe a woman, without prior symptoms of metabolic disease, who presented with hyperammonemia and psychiatric symptoms in the postpartum period. Initial diagnoses included acute fatty liver of pregnancy and postpartum psychosis. She was later found to have argininosuccinate synthetase deficiency after further metabolic investigations. Rare heterozygous mutations in the argininosuccinate synthetase gene were identified., Conclusion: Urea cycle disorders may present initially with postpartum psychiatric symptoms and may represent an underrecognized cause of "postpartum psychosis." We recommend obtaining metabolic studies in women with neurologic or severe psychiatric symptoms in the postpartum period.

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.

Results from biochemical and pharmacologic studies suggest that Lcitrulline is taken up by cerebral perivascular nerves and is converted to Larginine for synthesizing nitric oxide (NO). The current study was designed using morphologic techniques to determine whether Lcitrulline is taken up into axoplasm of perivascular nerves and to explore the possibility that conversion of Lcitrulline to Larginine in these nerves is through the argininosuccinate pathway in porcine cerebral arteries. Results from light and electron microscopic autoradiographic studies indicated that dense silver grains representing L-[3H] citrulline uptake were found in cytoplasm of perivascular nerves, smooth muscle cells, and endothelial cells. The neuronal silver grains were significantly decreased in arteries pretreated with glutamine, which has been shown biochemically to block neuronal uptake of Lcitrulline. Results from light and electron microscopic immunohistochemical and histochemical studies indicate that dense nitric oxide synthase-immunoreactive (NOS-I), argininosuccinate synthetase-immunoreactive (ASS-I), and argininosuccinate lyase-immunoreactive (ASL-I) fibers were found in the adventitia of cerebral arteries. NOS-, ASS-, and ASL-immunoreactivities fibers were found in the axoplasm and in the endothelium. In whole-mount preparations, the NOS-I, ASS-I, and ASL-I fibers were completely coincident with NADPH diaphorase fibers, suggesting that axoplasmic ASS, ASL, and NOS were co-localized in the same neurons. These studies provide the first morphologic evidence indicating that Lcitrulline is taken up into cytoplasm of cerebral perivascular nerves and that the axoplasmic enzymes catalyzing the conversion of Lcitrulline to Larginine (for synthesizing NO) by argininosuccinate pathway always are co-localized in same neurons. These results support the hypothesis that Lcitrulline, the by-product of NO synthesis, is recycled to form Larginine for synthesizing NO in perivascular nerves to mediate cerebral neurogenic vasodilation. Results of the current morphologic studies also support the presence of Lcitrulline-Larginine cycle in cerebral vascular endothelium.

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.

Deficiency of argininosuccinate synthetase (ASS) causes citrullinemia in human beings. Type II citrullinemia is found in most patients with adult-onset citrullinemia in Japan, and ASS deficiency is found specifically in the liver. Previous studies have shown that the decrease of hepatic ASS activity is caused by a decrease in enzyme protein with normal kinetic properties and that there were no apparent abnormalities in the amount, translational activity, and gross structure of hepatic ASS mRNA. In the present work, we show by sequencing analysis that there was no mutation in the ASS mRNA from two patients with type II citrullinemia. We also report RFLP analysis of a consanguineous family with type II citrullinemia, by using three DNA polymorphisms located within the ASS gene locus. In spite of having consanguineous parents, the patient was not a homozygous haplotype for the ASS gene. The RFLP analysis of 16 affected patients from consanguineous parents showed that 5 of 16 patients had the heterozygous pattern for one of the three DNA probes and that the frequency of the heterozygous haplotype was not different from the control frequency. These results suggest that the primary defect of type II citrullinemia is not within the ASS gene locus.

The distribution of the urea cycle enzyme, argininosuccinate synthetase, in the rat brain was determined using immunohistochemistry. This enzyme participates in the only known metabolic pathway for citrulline, its condensation with aspartate to form argininosuccinate, which can then be cleaved to fumarate and arginine. It may thus provide a mechanism to recycle citrulline, formed in the nervous system via nitric oxide synthase activity, back to the nitric oxide precursor, L-arginine. Argininosuccinate synthetase immunoreactivity was detected in discrete populations of neurons throughout the brain. Double-staining with nicotinamide adenine dinucleotide phosphate (reduced form)-diaphorase histochemistry for the localization of nitric oxide synthase demonstrated that argininosuccinate synthetase coexists with nitric oxide synthase in some brain regions. However, many neurons were found that contained one of these two enzymes, but not the other. Thus some nitric oxide synthase-containing neurons appear able to recycle citrulline via argininosuccinate, while others do not. Additional roles for argininosuccinate synthetase in the brain are discussed.

Amphotropic and ecotropic packaging cell lines were used to obtain high titers (greater than 10(6) colony forming units/ml) of retroviruses encoding human argininosuccinate synthetase, and these viruses were used to transduce murine bone marrow cells using cocultivation in vitro. The bone marrow cells were transplanted into lethally irradiated recipient mice, and argininosuccinate synthetase activity was measured in peripheral blood. Transduction with amphotropic retrovirus resulted in short-term expression for a period of 1-8 weeks, and no animals expressed the human gene after 25 weeks. Over 60% of the animals transplanted with cells transduced with ecotropic retrovirus expressed the human gene 44 weeks post-transplant, although the level of expression varied over a wide range. Analysis of the DNA from transplanted animals demonstrated the presence of the human sequence in expressing animals, and S1 nuclease analysis of RNA confirmed the presence of the human RNA transcripts. Analysis of granulocyte/macrophage (GM) colonies derived from the bone marrow of transplanted, expressing animals revealed a correlation between the level of expression of the transduced gene with the percentage of GM colonies carrying the human gene sequence. These data demonstrate the feasibility of obtaining long-term expression of genes introduced into bone marrow cells using retroviral vectors and the feasibility of obtaining expression of a gene not normally expressed in bone marrow.

The cDNA and gene encoding murine argininosuccinate synthetase were cloned and characterized. The cDNA sequence predicts a peptide of 412 amino acids (aa) including the initiator methionine. There is 98% identity with the aa sequence of the human enzyme. The 3'-untranslated region of the cDNA includes two regions of sequence which are conserved between mouse, rat, human and cow. The murine gene contains 16 exons with the start codon occurring in exon 3. Although alternative splicing occurs in primates to include or exclude exon 2, exon 2 sequences were included in the murine mRNA in all tissues and developmental stages examined. The inclusion of exon 2 in murine mRNA, compared to the usual exclusion in human mRNA, may be explained by differences in the donor splice sequences for exon 2.

Deficiency of argininosuccinate synthetase causes arginine auxotrophy in lower organisms and causes citrullinemia in humans and cattle. Previously, seven missense mutations, four mutations associated with an absence of an exon in mRNA, and one splicing mutation have been identified in human neonatal citrullinemia. Reverse transcription of mRNA, amplification of cDNA and sequencing of cDNA clones were used to identify two additional missense mutations causing citrullinemia. One mutation involves substitution of leucine for serine at position 18 (S18L) and the other a substitution of cysteine for arginine at position 86 (R86C). Both of these mutations represent C----T transitions in CpG dinucleotides, and eight of nine missense mutations causing human citrullinemia involve similar transitions in CpG dinucleotides. The nucleotide coding sequence and deduced amino acid analysis are available for four mammalian species, yeast and three bacterial species. Six of nine missense mutations in humans occur in amino acid positions that are completely conserved in these organisms. Mutations causing human citrullinemia are extremely heterogeneous, and all non-consanguineous individuals studied to date are compound heterozygotes.

Citrullinemia is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase. In order to characterize mutations, RNA was isolated from cultured fibroblasts from 13 unrelated patients with neonatal citrullinemia. Ten mutations were identified by sequencing of amplified cDNA. Seven single base missense mutations were identified: Gly14----Ser, Ser180----Asn, Arg157----His, Arg304----Trp, Gly324----Ser, Arg363----Trp, and Gly390----Arg. Six of these missense mutations involved conversion of a CpG dinucleotide in the sense strand to TpG or CpA, and six of the seven mutations alter a restriction enzyme site in the cDNA. Two mutations were observed in which the sequences encoded by a single exon (exon 7 or 13) were absent from the cDNA. One mutation is a G----C substitution in the last position of intron 15 resulting in splicing to a cryptic splice site within exon 16. There is extreme heterogeneity of mutations causing citrulinemia. This heterogeneity may prove typical for less common autosomal recessive human genetic diseases.

In order to develop linkage markers for the murine argininosuccinate synthetase locus (Ass-1), we have searched for restriction fragment length polymorphisms in the mouse genome using cloned sequences from the mouse arginosuccinate synthetase structural gene. Five restriction fragment length polymorphisms were found among the recombinant inbred progenitor strains AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J. Of these, four polymorphisms were found to distinguish the SWR/J strain from the other six strains, which all had the same fragment. The fifth polymorphism revealed differences among the progenitor strains for recombinant inbred strain sets AKXL, BXD, and SWXL. The strain distribution pattern for this polymorphism indicated close linkage of Ass-1 to Hc (the fifth component of complement) on proximal mouse chromosome 2 with a recombination fraction of 0.016 and a 95% confidence interval of 0.003 to 0.054. These data place Ass-1 in a syntenic group with the genes Hc, Abl, Fpgs, and Ak-1 whose linkage has been conserved between human chromosome 9q and mouse chromosome 2.

Regulation of argininosuccinate synthetase (AS) was studied by using minigenes containing 3 kilobases of DNA upstream from the TATAA box and 9 kilobases downstream (including the first four exons of the AS gene) ligated to either the cDNA for AS or to the chloramphenicol acetyltransferase (CAT) gene. Unlike the endogenous AS gene, expression of the CAT minigene was not elevated in Canr1 cells, which overproduce AS compared with parental RPMI-2650 cells. Expression of the CAT minigene in both stable and transient analyses was four- to five-fold higher in RPMI-2650 cells grown in citrulline medium than in cells grown in arginine medium. Although endogenous AS activity is not subject to metabolite regulation in Canr1 cells and expression of the CAT minigene in Canr1 cells was not increased when cells were grown in citrulline medium, expression of the CAT minigene was 10- to 22-fold greater when intracellular arginine pools were depleted by transient starvation for arginine and citrulline.

Citrullinaemia is a human inborn error of metabolism resulting from the deficiency of argininosuccinate synthetase. In a previous study of cultured skin fibroblasts from citrullinaemia patients, we showed that the presumed defects in DNA were not detectable by Southern blotting analysis, and that only 2 of 11 cell lines contained detectable enzyme antigen. All citrullinaemia cell lines contained hybridizable mRNA but slight size heterogeneity was noted. Here we report the extension of the analysis of the RNA using S1 nuclease mapping techniques. Among six cell lines examined, five showed an abnormality of mRNA detectable by S1 nuclease analysis. The data indicate that a minimum of three out of five non-consanguineous patients represent compound heterozygotes. The S1 nuclease detectable defects may represent deletions or rearrangements in the genomic DNA, or more probably represent examples of abnormal RNA splicing. The approach used here is useful for molecular analysis of genetic defects, for prenatal diagnosis, and for study of genetic variation.

We have studied the transcription of the argininosuccinate synthetase gene in cultured RPMI 2650 cells under conditions where the enzyme is subject to metabolite regulation and in canavanine-resistant variants (Canr1 cells) which overproduce the enzyme greater than 200-fold. When grown continuously in medium with citrulline substituted for arginine, the argininosuccinate synthetase activity of RPMI 2650 cells increases 5- to 10-fold. In these cells, expression of a transfected minigene containing the 5'-flanking region of the argininosuccinate synthetase gene was increased 20-fold by short term starvation for arginine and 10-fold by short term starvation for leucine. Levels of nuclear RNA from the first intron of the gene correlated with enzyme activity; i.e. RPMI 2650 cells cultured in arginine medium less than RPMI 2650 cells cultured in citrulline medium less than Canr1 cells. Run-off transcription experiments showed that the transcription of argininosuccinate synthetase increased in RPMI 2650 cells starved for either arginine or leucine. While expression of the minigene and the transcription rate for argininosuccinate synthetase were increased during 48 to 72 h of starvation, the endogenous enzyme activity did not increase in RPMI 2650 cells. Amino acid starvation did not affect the rate of transcription of argininosuccinate synthetase in Canr1 cells. The results indicate that the steady state levels of argininosuccinate synthetase expression in Canr1 cells and in citrulline-adapted RPMI 2650 cells are largely determined by the rate of transcription. The failure of increased transcription rate to correlate with increased enzyme activity during acute starvation for arginine or leucine may suggest the involvement of post-transcriptional regulatory mechanisms for argininosuccinate synthetase or may merely be due to amino acid deprivation. The finding that leucine starvation has effects similar to arginine starvation raises the question of whether mammalian cells have general control mechanisms which are similar to the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

There are multiple, processed, dispersed pseudogenes for human argininosuccinate synthetase. Chinese hamster X human somatic cell hybrids were used to map DNA fragment groups corresponding to the single expressed gene and 14 pseudogene loci. Each chromosomal assignment was confirmed using hybrids containing very few human chromosomes and/or by demonstrating monosomic or trisomic dosage in human cell lines with chromosomal abnormalities. Pseudogenes were mapped to chromosomes 2cen-p25, 3q12-qter, 4q21-qter, 5 (two loci), 6, 7, 9p13-q11, 9q11-q22, 11q, 12, Xp22-pter, Xq22-q26, and Ycen-q11. DNA fragments from the expressed gene were mapped to 9q34-qter in agreement with the previous assignment for enzyme activity. A high-frequency restriction fragment length polymorphism mapped to 9q11-q22. The analyses emphasized the feasibility of using chromosomally abnormal human cell lines for confirmation and regionalization of gene-mapping assignments made using somatic-cell hybrids. Conversely, cloned DNA probes, once mapped and characterized, can be very valuable for determining the chromosomal composition of interspecies hybrids and the dosage of loci in human cells. The argininosuccinate synthetase cDNA is a convenient reagent for dosage analysis of 15 human loci on 11 different chromosomes. Improved reagents could be designed that would simplify Southern blot patterns by eliminating overlapping DNA fragments and providing a single DNA fragment for each locus.

The cDNA sequence for human argininosuccinate synthetase (AS) was introduced into plasmid expression vectors with an SV40 promoter or Rous sarcoma virus promoter to construct pSV2-AS and pRSV-AS, respectively, and human enzyme was synthesized after gene transfer into Chinese hamster cells. The functional cDNA was inserted into the retroviral vectors pZIP-NeoSV(X) and pZIP-NeoSV(B). Ecotropic AS retrovirus was produced after calcium-phosphate-mediated gene transfer of these constructions into the packaging cell line psi-2, and viral titers up to 10(5) CFU/ml were obtained. Recombinant AS retrovirus was evaluated by detecting G-418-resistant colonies after infection of the rodent cells, XC, NRK, and 3T3. Colonies were also obtained when infected XC cells were selected in citrulline medium for expression of AS activity. Southern blot analysis of infected cells demonstrated that the recombinant retroviral genome was not altered grossly after infecting some rodent cells, while other cells showed evidence of rearrangement. A rapid assay for detecting AS retrovirus was developed based on the incorporation of [14C]citrulline into protein by intact 3T3 cells or XC cells.

We have analyzed cultured skin fibroblasts derived from patients with argininosuccinate synthetase deficiency for alterations in gene structure, mRNA content, and protein structure. Genomic DNA was digested with the endonucleases EcoRI or HindIII, and the fragments were analyzed by Southern blotting and hybridization with a cDNA probe for argininosuccinate synthetase. The blot pattern is complex because there are at least 10 copies of argininosuccinate synthetase-like genes scattered over multiple human chromosomes. All nine patients studied showed patterns of DNA fragments that were indistinguishable from the normal control cell lines, and despite the possibility that the complexity could mask some changes, major deletions of the active gene(s) were not present. Blot hybridization of RNA indicated the presence of hybridizable mRNA of approximately normal size in seven of seven individuals examined with a suggestion of some heterogeneity. Analysis of enzyme antigen by protein transfer from NaDodSO4 containing polyacrylamide gels revealed considerable heterogeneity. This analysis revealed no cross-reacting material (CRM) in nine cell lines, CRM of normal molecular weight in one cell line, and CRM of reduced molecular weight in one cell line. These findings suggest that the genes for argininosuccinate synthetase in most citrullinemia patients are transcribed and produce stable mRNA. These mRNA either are not translated, or the translation product (enzyme) is rapidly degraded or is immunologically nonreactive. Defective gene expression in this disorder appears to involve abnormal mRNA, which may be altered by point mutations, frame shift mutations, deletions, insertions or particularly by abnormal RNA processing.

Canavanine-resistant (Canr) human cells overproduce argininosuccinate synthetase without the occurrence of gene amplification. Using calcium phosphate precipitation, genomic DNA from Canr human cells was used to carry out gene transfer into Chinese hamster cells, which do not express argininosuccinate synthetase activity. Growth in tissue culture medium with citrulline substituted for arginine was adequate to select enzyme-positive colonies. Six independent isolates were selected for detailed analysis by enzyme assay, Southern blotting, Northern blotting, and S1 nuclease analysis, the last of which distinguishes human and hamster mRNA. Five isolates were transferrants containing the human structural gene and synthesizing human enzyme. One isolate represented a cell line synthesizing Chinese hamster enzyme. The data document gene transfer of DNA fragments at least 80 kb in length, the low level of spontaneous activation of the argininosuccinate synthetase locus in Chinese hamster cells, the feasibility of this expression and selection system for DNA-mediated gene transfer, and a method for distinguishing the human and hamster gene products at an RNA level.

The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.

Citrullinemia is an inborn error of metabolism due to deficiency of the urea cycle enzyme, argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5]. The disease was first described in humans but was recently reported in dairy cattle in Australia. Here we report the nucleotide sequence of the normal bovine cDNA for argininosuccinate synthetase and the mutation present in animals with citrullinemia. Analysis of DNA from affected animals by Southern blotting did not readily identify the mutation in the bovine gene. RNA (Northern) blotting revealed a major reduction in the steady-state amount of mRNA in the liver of affected animals to less than 5% of controls. The bovine cDNA was cloned and sequenced and revealed 96% identity with the deduced human sequence at the amino acid level. Starting with mutant bovine liver, the mRNA was reverse-transcribed; the cDNA product was amplified with the polymerase chain reaction, cloned, and sequenced. The sequence revealed a C----T transition converting arginine-86 (CGA) to a nonsense codon (TGA). A second C----T transition represented a polymorphism in proline-175 (CCC----CCT). The mutation and the polymorphism were confirmed by amplification of genomic DNA and demonstration with restriction endonuclease enzymes of both the loss of an Ava II site in DNA from mutant animals at codon 86 and the presence or absence of a Dde I site at codon 175. The loss of the Ava II site can be used for rapid, economical, nonradioactive detection of heterozygotes for bovine citrullinemia.

The level of argininosuccinate synthetase activity in the human tissue culture cell line RPMI 2650 was 6-fold higher when citrulline was substituted for arginine in the culture medium. Canavanine-resistant (Canr) variants were isolated and had enzyme activity up to 25 nmol min-1 (mg of protein)-1 or 180-fold higher than that of the wild-type cells grown in arginine. The differences in enzyme activity were paralleled by differences in the amount of enzyme determined immunologically. The micrograms of enzyme per milligrams of protein, determined by complement fixation, were 0.03 for wild-type cells grown in arginine, 0.29 for wild-type cells grown in citrulline, and 6.73 for a Canr variant. In vivo labeling studies suggested increased synthesis of argininosuccinate synthetase in Canr cells, and in vitro translation of poly(adenylic acid) [poly(A)] messenger ribonucleic acid (mRNA) from wild-type and Canr cells confirmed a quantitatively compatible increase in translatable poly(A) mRNA for the enzyme in Canr cells. No precursor for the enzyme was recognized by using in vitro translation, and the poly(A) mRNA for the enzyme had a sedimentation value of 16 S by sucrose-gradient analysis. The levels of argininosuccinate synthetase activity in the Canr cells were similar to those found in normal liver.

Heritable deficiency of the enzyme argininosuccinate synthetase results in the autosomal recessive disease citrullinemia. RNA was isolated from cultured fibroblasts from citrullinemic patients for synthesis of complementary DNA (cDNA). The mutant mRNAs were reverse transcribed, amplified and cloned for sequencing. The cDNA sequence from one mutant allele revealed the absence of exon 5 sequences, while the sequence from a second mutant allele revealed the absence of exon 6. Genomic DNA from 25 citrullinemic patients was analyzed using intron probes derived from the argininosuccinate synthetase gene. One polymorphic probe located between exons 5 and 6 identified two novel alleles amongst 48 citrullinemia chromosomes examined. Each of these alleles appears to involve deletion of genomic DNA from this region. These two mutations, which were detected as alterations of the gene structure, are the same two mutations that result in deletion of exon 5 or 6, respectively, from the mRNA. These two mutations account for approximately 10% of the chromosomes carrying mutations that cause citrullinemia.

In the human genome there is one expressed gene for argininosuccinate synthetase and 14 pseudogenes. A cDNA coding for human argininosuccinate synthetase was used to screen a human genomic library. Twenty-five unique genomic clones were isolated and extensively characterized. At least seven clones represented processed argininosuccinate synthetase pseudogenes that lost the introns in the expressed gene. Restriction mapping demonstrated that these processed pseudogenes were located in distinct regions of the human genome. Complete nucleotide sequences of two processed pseudogenes, psi AS-1 and psi AS-3, and a partial sequence of psi AS-7 were determined. Both psi AS-1 and psi AS-3 had an adenine-rich region at their 3' end and were flanked by distinct imperfect direct repeats. A comparison of these pseudogene sequences to that of the cDNA demonstrated that psi AS-1 and psi AS-3 were 93% homologous to the cDNA, whereas psi AS-7 was 89% homologous to the cDNA. Therefore, it is estimated that psi AS-1 and psi AS-3 were created 10-11 million years ago, whereas psi AS-7 arose approximately 21 million years ago. We have estimated the evolutionary rate for the expressed argininosuccinate synthetase gene based on the sequences of psi AS-1 and psi AS-3. These data indicate that the expressed argininosuccinate synthetase gene is evolving at a rate similar to that of the beta-globin gene and much faster than the alpha-tubulin gene. Furthermore, a comparison of the sequences of psi AS-1 and psi AS-3 suggests the possibility that these pseudogenes arose from a common intermediate.

We have identified three restriction fragment length polymorphisms (RFLPs) from within the argininosuccinate synthetase (ASS) gene which maps to human chromosome 9q34-qter. Although RFLPs at pseudogene loci are detected by the cDNA, these are the first polymorphisms reported at the ASS locus. The three RFLPs are in linkage equilibrium with each other, and haplotypes for the ASS locus are highly informative. Two-locus recombination estimates between ASS and seven other 9q markers indicated that ASS is closest to the ABO blood group with a recombination fraction of 0.04 (0.005-0.11). A multilocus lod score analysis with these seven 9q markers indicated that ASS maps between ABL and MCT136 close to ABO, but it is uncertain if ASS is centromeric or telomeric to ABO.

The nucleotide sequence for human argininosuccinate synthetase cDNA was determined by analysis of six clones isolated from a single experiment. The sequence covered 1623 nucleotides including 76 bases of poly(A) and contained a 1236 nucleotide open reading frame encoding a protein of 46,434 daltons. In one cDNA isolate, a cloning artifact or perhaps RNA polymerase error involving addition of an A in a region of six A's within the coding sequence was documented. Single base variations in the 3' untranslated region were examined in detail since detection of DNA polymorphisms in the cDNAs could imply over-expression of both alleles at the active locus in canavanine-resistant cells, i.e. a trans-acting mechanism for enzyme overproduction. However, the sequence from five cDNAs suggested some single base artifacts, and DNA polymorphism remains uncertain. The occurrence of three tandem arginine codons in the 5' untranslated region of the cDNA suggested the possibility of an interaction of arginyl-tRNA with mRNA to regulate RNA processing or half-life as a mechanism for arginine-mediated repression.

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.

Previous studies of the human cell line RPMI-2650 (wild type) and its canavanine-resistant variants have demonstrated differences in argininosuccinate synthetase activity as follows: canavanine-resistant much greater than wild type grown in citrulline greater than wild type grown in arginine (Su, T.-S., Beaudet, A. L., and O'Brien, W. E. (1981) Biochemistry 20, 2956-2960). A recombinant plasmid containing a 1.55-kilobase insert complementary to the mRNA for human argininosuccinate synthetase was isolated by the combined use of differential colony hybridization and immunoprecipitation of the products of plasmid-selected mRNA translation. Both blot and dot hybridization analysis of polyadenylated RNA indicated a major mRNA species of 1.67 kilobase in all cells, and the levels of mRNA correlated well with the levels of enzyme activity: canavanine-resistant, 180; wild type grown in citrulline, 7; and wild type grown in arginine, 1. One major mRNA species of 1.67 kilobase and one minor species of 2.68 kilobase were observed in wild type and canavanine-resistant cell lines. Reassociation kinetics of pAS1 with genomic DNA from human liver, canavanine-resistant cells, and wild type cells were not significantly different. Blot hybridization of genomic DNA revealed no detectable differences between wild type cells, canavanine-resistant cells, and human leukocytes. The data demonstrated that there were multiple copies, perhaps 10 or more, of argininosuccinate synthetase-like sequences in human DNA and that the canavanine-resistant phenotype was not due to gene amplification.

As part of an effort to develop a model system for somatic gene therapy, a human argininosuccinate synthetase (AS) cDNA has been introduced into two gag+ retroviral vectors, and high titer clones were obtained for both constructs. The presence of proviral DNA sequences was detected in individual spleen colonies after infection of primary murine marrow cells with each virus. Mice were reconstituted for long-term survival with marrow infected with one virus, and they demonstrated elevated levels of AS expression in peripheral blood for up to eight weeks posttransplantation.

DNA sequences closely homologous to argininosuccinate synthetase are present at ten or more distinct locations in the human genome, including sites on chromosomes 6, 9 and X. Argininosuccinate synthetase thus represents one of the most widely dispersed multigene families described to date, the first instance of a multigene family associated with an enzyme of intermediary metabolism and, perhaps most striking, the first instance of a multigene family with members on both autosomes and sex chromosomes.