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Biomedical subjects

A L Beaudet

Publications and source records attributed to A L Beaudet.

At least 181 records · Page 10Linked to original sources
PubMed1986

Expression of human argininosuccinate synthetase after retroviral-mediated gene transfer.

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.

Animals↗
PubMed1986

Risk of serious illness in heterozygotes for ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder associated with hyperammonemia. Heterozygous females have variable clinical expression, ranging from asymptomatic illness to recurrent episodes of hyperammonemic coma. We studied 17 OTC-deficient kindreds containing 114 women at risk for heterozygosity. Sixty-one of these women were designated heterozygotes by pedigree analysis, history of protein intolerance, protein tolerance tests, or DNA probe studies. Eleven (18%) of the 61 heterozygotes had experienced encephalopathic episodes; nine (82%) girls died during these episodes. Our findings indicate that there is a significant risk of symptomatic hyperammonemia in females heterozygous for OTC deficiency. We suggest that, within OTC-deficient kindreds, females at risk should be identified early, by means of protein tolerance tests and DNA probe studies. Those who develop significant hyperammonemia after a protein load should be considered for long-term alternate pathway therapy and should receive aggressive therapy during hyperammonemic episodes.

Amino Acid Metabolism, Inborn Errors↗
PubMed1986

Arginine-mediated regulation of an argininosuccinate synthetase minigene in normal and canavanine-resistant human cells.

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.

Arginine↗
PubMed1986

A fine structure physical map of the short arm of chromosome 5.

A series of somatic cell hybrids that retain abnormal chromosomes 5 from 11 different persons with deletions or translocations involving 5p have been isolated. One hundred twenty DNA fragments isolated from a genomic library enriched for sequences from 5p were regionally localized by Southern blot analysis of the hybrid cell deletion mapping panel, including five DNA fragments that reveal restriction fragment length polymorphisms. The fine structure physical map of 5p together with the identification of additional polymorphic loci will facilitate the construction of a complete linkage map of this region. In addition, DNA fragments localized to a region near the 5p15.2-5p15.3 border, which appears to be the segment of 5p that is critical in producing the phenotype associated with the cri du chat syndrome when it is rendered hemizygous by deletion, will be useful in a molecular and DNA level analysis of this deletion syndrome.

Animals↗
PubMed1986

Linkage of DNA markers to cystic fibrosis in 26 families.

Two DNA markers, the met oncogene and the anonymous probe, pJ3.11, previously reported to be tightly linked to cystic fibrosis (CF), were used for linkage analysis in 26 families with two or more individuals affected with CF. A new high frequency polymorphism was identified using BanI and the pmetD probe. The results of linkage analysis were as follows: between met and CF, lod score of 18.2 at theta of .009; between pJ3.11 and CF, lod score of 12.1 at theta of 0; and between met and pJ3.11, lod score of 16.7 at theta of 0. These data indicate that most or all of CF is due to an abnormality at a single locus and that the DNA markers are useful for prenatal diagnosis and heterozygote detection within affected families.

Cystic Fibrosis↗
PubMed1986

New mutation and prenatal diagnosis in ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) (E.C.2.1.3.3) is an X-linked hepatic enzyme in the urea cycle necessary for ammonia detoxification. Deficiency of OTC results in neonatal hyperammonemia, coma, and death in childhood. Because fibroblasts do not express OTC, prenatal diagnosis in the past has required fetal liver biopsy. Using a complementary DNA (cDNA) for OTC for Southern blot analysis of genomic DNA, we have found probands with complete OTC deficiency from two unrelated families in whom the same TaqI restriction endonuclease site has been altered because of independent, but not necessarily identical, mutations in the OTC gene, suggesting that this site may be a relative hotspot for mutation at a location that is critical for normal gene function. This TaqI alteration has allowed the identification of the individual in each family in whom the mutation originated as well as the exclusion of a recurrence of OTC deficiency in a male fetus at risk for the disease. OTC deficiency joins the growing list of genetic disorders for which Southern blot analysis allows accurate heterozygote detection and prenatal diagnosis in conditions for which they were not previously available.

Child, Preschool↗
PubMed1985

A study of restriction fragment length polymorphisms at the human alpha-1-antitrypsin locus.

A cloned cDNA for alpha-1-antitrypsin (alpha-1-AT) was selected from a human liver cDNA library. The identity of the clone was established by hybrid-selected translation and partial DNA sequencing. The cDNA was used as a probe to search for restriction site polymorphisms (RSPs) near the alpha-1-AT gene. Only two RSPs were found using 29 different restriction enzymes. Each of these polymorphisms resulted from the loss of a restriction site, one for EcoRI and the other for Taq I. The frequency of polymorphic restriction was calculated to be 1.1% to 2.6% of all sites tested, a figure lower than the 9.3% value observed for 12 RSPs in the human beta-globin gene cluster. Since the corresponding figure for detectable polymorphisms at the alpha-1-AT locus at the protein level is 12%, restriction enzymes are comparatively inefficient in detecting genetic variability. The basis of this inefficiency was studied by computing the nucleotide diversity from the RSP data. On the average, one in 500 to 1000 bases is polymorphic around the alpha-1-At locus. This value is comparable to that which we have calculated for the human beta-globin gene cluster and the human growth hormone gene cluster (both one in 500). These data demonstrate the limited usefulness of linked RSPs for genetic linkage studies at the alpha-1-AT locus.

Base Sequence↗
PubMed1984

Molecular structures of human argininosuccinate synthetase pseudogenes. Evolutionary and mechanistic implications.

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.

Argininosuccinate Synthase↗
PubMed1984

Genomic DNA-mediated gene transfer for argininosuccinate synthetase.

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.

Animals↗
PubMed1984

A method for analyzing transcription using permeabilized cells.

Brief exposure of Friend cells to a buffered hypotonic solution containing 1% Tween 80 caused permeabilization and allowed incorporation of [3H]UTP into RNA. The incorporation was inhibited 85-97% by 20 micrograms/ml actinomycin D and the reaction product was completely hydrolyzed by 0.1 M KOH. UMP incorporation was nearly linear for 60 min at 23 degrees C; however, at 37 degrees C it ceased after 15-20 min of rapid incorporation. The inhibition of UMP incorporation by 2 micrograms/ml alpha-amanitin was much greater at 23 degrees C than at 37 degrees C. The molecular weight of the RNA synthesized in permeabilized cells is broadly distributed with about 83% larger than 18 S. In vitro transcription of the mouse beta-major globin gene was studied by hybridizing 32P-labeled nascent RNA to filter-bound DNA sequences representing this gene and its flanking regions. After induction by hexamethylene-bisacetamide, Friend cells exhibited more than fivefold increases in the rate of transcription for the beta-major globin gene as compared to the uninduced control cells. Induction also caused an increase in the transcription rate of the 3'-flanking region located downstream from the poly(A) addition site. Thus, the primary transcription unit of beta-major globin gene is essentially the same in permeabilized cells as that previously reported for nuclei isolated from the same cell line. In addition, permeabilized cells actively initiate RNA synthesis as determined by the incorporation of a thiol group at the 5' initiating nucleotide, when synthesis was in the presence of [gamma-S]-labeled nucleoside triphosphates. Permeabilized cells are about 7-11 times more active than isolated nuclei in the synthesis of both in vitro-initiated and total RNA.

Animals↗
PubMed1984

Molecular structure of the human argininosuccinate synthetase gene: occurrence of alternative mRNA splicing.

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.

Animals↗
PubMed1984

Human chromosomal assignments for 14 argininosuccinate synthetase pseudogenes: cloned DNAs as reagents for cytogenetic analysis.

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.

Animals↗