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W E O'Brien

Publications and source records attributed to W E O'Brien.

At least 91 records · Page 5Linked to original sources
PubMed1988

Gene sharing by delta-crystallin and argininosuccinate lyase.

The lens structural protein delta-crystallin and the metabolic enzyme argininosuccinate lyase (ASL; L-argininosuccinate arginine-lyase, EC 4.3.2.1) have striking sequence similarity. We have demonstrated that duck delta-crystallin has enormously high ASL activity, while chicken delta-crystallin has lower but significant activity. The lenses of these birds had much greater ASL activity than other tissues, suggesting that ASL is being expressed at unusually high levels as a structural component. In Southern blots of human genomic DNA, chicken delta 1-crystallin cDNA hybridized only to the human ASL gene; moreover, the two chicken delta-crystallin genes accounted for all the sequences in the chicken genome able to cross-hybridize with a human ASL cDNA, with preferential hybridization to the delta 2 gene. Correlations of enzymatic activity and recent data on mRNA levels in the chicken lens suggest that ASL activity depends on expression of the delta 2-crystallin gene. The data indicate that the same gene, at least in ducks, encodes two different functions, an enzyme (ASL) and a structural protein (delta-crystallin), although in chickens specialization and separation of functions may have occurred.

Amino Acid Sequence↗
PubMed1988

Characterization of point mutations in the same arginine codon in three unrelated patients with ornithine transcarbamylase deficiency.

Point mutations in the X-linked ornithine transcarbamylase (OTC) gene have been detected at the same Taq I restriction site in 3 of 24 unrelated probands with OTC deficiency. A de novo mutation could be traced in all three families to an individual in a prior generation, confirming independent recurrence. The DNA sequence in the region of the altered Taq I site was determined in the three probands. In two unrelated male probands with neonatal onset of severe OTC deficiency, a guanine (G) to adenine (A) mutation on the sense strand (antisense cytosine [C] to thymine [T]) was found, resulting in glutamine for arginine at amino acid 109 of the mature polypeptide. In the third case, where the proband was a symptomatic female, C to T (sense strand) transition converted residue 109 to a premature stop. These results support the observation that Taq I restriction sites, which contain an internal CG, are particularly susceptible to C to T transition mutation due to deamination of a methylated C in either the sense or antisense strand. The OTC gene seems especially sensitive to C to T transition mutation at arginine codon 109 because either a nonsense mutation or an extremely deleterious missense mutation will result.

Adult↗
PubMed1988

Uniparental disomy as a mechanism for human genetic disease.

A female with cystic fibrosis and short stature was investigated for molecular or cytogenetic abnormalities that might explain the combined phenotype. Analysis with polymorphic DNA markers indicated that the father did not contribute alleles to the propositus for markers near the CF locus or for centromeric markers on chromosome 7. High-resolution cytogenetic analysis was normal, and the result could not be explained on the basis of nonpaternity or a submicroscopic deletion. All of the data indicate that the propositus inherited two identical copies of maternal sequences for much or all of chromosome 7. The occurrence of uniparental disomy could be explained by models postulating postfertilization error, gamete complementation, monosomic conception with subsequent chromosome gain, or trisomic conception followed by chromosome loss. Uniparental disomy in an individual with a normal chromosome analysis is a novel mechanism for the occurrence of human genetic disease.

Adolescent↗
PubMed1988

Extensive DNA polymorphism at the factor XIIIa (F13A) locus and linkage to HLA.

A 1,161-bp EcoRI fragment from the 5' end of the cDNA coding for human factor XIIIa (gene symbol F13A) was used to identify RFLPs in human DNAs. Several different RFLPs were identified with 15 different restriction enzymes. Two RFLPs detected with the restriction enzyme BamHI and one multiallelic RFLP detected with BclI were used for further studies. Linkage relationships between these three polymorphisms and the HLA complex were studied in DNA samples from the 40 Centre d'Etude du Polymorphisme Humain families. Combining all of the data to form highly informative haplotypes, we found linkage to HLA with a maximum lod score of 11.44 at a recombination fraction of .25 for males and .35 for females. These three RFLPs at the FXIIIa locus provide a highly informative marker for the short arm of chromosome 6 with an observed heterozygosity of 91%. Using this marker and the HLA locus, one can confirm or exclude the assignment of gene loci to most of chromosome 6p.

Chromosome Mapping↗
PubMed1988

Prenatal diagnosis of cystic fibrosis: microvillar enzymes and DNA analysis compared.

We have established reference ranges for three microvillar intestinal enzymes--alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.1), and leucine aminopeptidase (EC 3.4.1.1)--measured in amniotic fluid in a reference population of 1875 women presenting for routine amniocentesis. These data were derived for use in prenatal diagnostic studies in a population at risk (1:4) for cystic fibrosis. False-positive or indeterminate results were noted for fewer than 3.5% of all low-risk cases for each enzyme evaluated. Total alkaline phosphatase and its isoenzymes and leucine amino-peptidase and gamma-glutamyltransferase were measured in amniotic fluid sampled between the 15th and 19th weeks of gestation. Restriction fragment length polymorphism analysis of DNA was also performed when possible. In 52 cases examined for cystic fibrosis thus far, 46 were diagnosed on the basis of DNA analysis and (or) by sweat testing; for the other six cases, only abnormal enzyme results were obtained before termination of pregnancy. Predictions based on microvillar enzyme results were falsely negative in three cases. In only one case was there a discrepancy between enzyme results and DNA analysis. Diagnostic accuracy was highest during the 17th and 18th week of gestation. Preliminary results suggest the false-negative rate of this diagnostic strategy may be greater than or equal to 10%.

Alkaline Phosphatase↗
PubMed1987

Prenatal diagnosis of cystic fibrosis using linked DNA markers and microvillar intestinal enzyme analysis.

Prenatal diagnosis was performed for 47 pregnancies with 1 in 4 risk of cystic fibrosis, including 7 cases analyzed with linked DNA markers, 16 cases analyzed by microvillar intestinal enzyme testing, and 24 cases where both methods of testing were attempted. DNA was obtained by chorionic villus sampling in 10 cases and by amniocentesis in 21 cases, and diagnosis was based on analysis with the tightly linked DNA markers D7S8 and met. DNA analysis using these probes was fully informative in 74.4% of 90 couples with 1 in 4 risk. In 18 cases where both DNA results and microvillar intestinal enzyme data were diagnostic, there was agreement regarding the predicted status of the fetus. No adequate diagnosis was achieved in two cases where both diagnostic tests were attempted. Outcome is known for 24 pregnancies including 10 where DNA analysis was diagnostic, and no errors in diagnosis were detected. Prenatal diagnosis of cystic fibrosis using DNA markers is highly informative and accurate, but microvillar intestinal enzyme analysis remains a valuable part of a complete diagnostic program.

Alkaline Phosphatase↗
PubMed1987

Regulation of mRNA levels for five urea cycle enzymes in rat liver by diet, cyclic AMP, and glucocorticoids.

Adaptive changes in levels of urea cycle enzymes are largely coordinate in both direction and magnitude. In order to determine the extent to which these adaptive responses reflect coordinate regulatory events at the pretranslational level, measurements of hybridizable mRNA levels for all five urea cycle enzymes were carried out for rats subjected to various dietary regimens and hormone treatments. Changes in relative abundance of the mRNAs in rats with varying dietary protein intakes are comparable to reported changes in enzyme activities, indicating that the major response to diet occurs at the pretranslational level for all five enzymes and that this response is largely coordinate. In contrast to the dietary changes, variable responses of mRNA levels were observed following intraperitoneal injections of dibutyryl cAMP and dexamethasone. mRNAs for only three urea cycle enzymes increased in response to dexamethasone. Levels of all five mRNAs increased severalfold in response to dibutyryl cAMP at both 1 and 5 h after injection, except for ornithine transcarbamylase mRNA which showed a response at 1 h but no response at 5 h. Combined effects of dexamethasone and dibutyryl cAMP were additive for only two urea cycle enzyme mRNAs, suggesting independent regulatory pathways for these two hormones. Transcription run-on assays revealed that transcription of at least two of the urea cycle enzyme genes--carbamylphosphate synthetase I and argininosuccinate synthetase--is stimulated approximately four- to fivefold by dibutyryl cAMP within 30 min. The varied hormonal responses indicate that regulatory mechanisms for modulating enzyme concentration are not identical for each of the enzymes in the pathway.

Animals↗
PubMed1986

Atypical presentation and neuropathological studies in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

A 6 1/2-month-old male offspring of consanguineous Egyptian parents was first seen because of fever, somnolence, vomiting, right focal motor seizures, right hemiparesis, elevated transaminase levels, hyperammonemia, and acidosis. A computed tomographic scan of the head suggested swelling of the left cerebral hemisphere, and an electroencephalogram indicated left frontotemporal abnormalities, but brain biopsy demonstrated diffuse white matter spongiosis and gliosis. Subsequently, urine organic acid analysis and enzyme assays were diagnostic of 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency.

Brain↗
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

Cloning and sequence analysis of cDNA for human argininosuccinate lyase.

Using antibodies specific for argininosuccinate lyase (EC 4.3.2.1), we isolated two cDNA clones by screening a human liver cDNA library constructed in the lambda gt11 expression vector. The identity of these isolates was confirmed by in vitro translation of plasmid-selected mRNA. One of these isolates was used to rescreen the cDNA library and a 1565-base-pair (bp) clone was identified. The entire nucleotide sequence of this clone was determined. An open reading frame was identified which encoded a protein of 463 amino acids with a predicted molecular weight of 51,663. The clone included 115 bp of 5' untranslated sequence and 46 bp of 3' untranslated sequence. A canonical poly(A) addition site was present in the 3' end, 16 bp from the beginning of the poly(A) tract. Comparison of the deduced amino acid sequence of the human enzyme with that of the yeast enzyme revealed a 56% homology, when conservative amino acid changes were taken into consideration. The yeast protein is also 463 amino acids long, with a molecular weight of 51,944. By use of a genomic DNA panel from human-Chinese hamster somatic cell hybrids, the human gene was mapped to chromosome 7. Another hybridizing region, corresponding to a portion of the 5' end of the cDNA, was found on chromosome 22.

Amino Acid Sequence↗
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

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↗