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W E O'Brien

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

At least 55 records · Page 3Linked to original sources
PubMed1992

Long-term expression of human argininosuccinate synthetase in mice following bone marrow transplantation with retrovirus-transduced hematopoietic stem cells.

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.

Animals↗
PubMed1991

Expression of duck lens delta-crystallin cDNAs in yeast and bacterial hosts. Delta 2-crystallin is an active argininosuccinate lyase.

The major soluble protein in the lenses of most birds and reptiles is delta-crystallin. In chickens and ducks the delta-crystallin gene has duplicated, and in the duck both genes contribute to the protein in the lens, while in the chicken lens there is a great preponderance of the delta 1 gene product. Purified delta-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. In order to determine the enzymatic properties of the two duck delta-crystallins their corresponding cDNA molecules were placed in yeast and bacterial expression plasmids. In Saccharomyces cerevisiae, the activity of each crystallin was assessed by transformation of the expression plasmids into a strain deficient for argininosuccinate lyase activity. The ability of the resulting yeast to grow on arginine deficient medium was used as a measure of enzymatic activity. Yeast expressing the duck delta 2-crystallin protein grew rapidly, while those expressing delta 1-crystallin failed to grow. Enzyme activity measurements confirmed the presence of activity in the delta 2-crystallin-expressing yeast, and no detectable activity could be demonstrated in the delta 1-crystallin-expressing yeast. Northern blotting of RNA from the transformed yeast revealed equal levels of mRNA species from the two constructs. For further analysis, the delta 2-crystallin cDNA was placed in the bacterial expression plasmid, pET-3d. The delta 2-crystallin protein produced in Escherichia coli was purified to homogeneity and analyzed to determine the kinetic properties. A Km of 0.35 mM was determined for argininosuccinate and a Vm of 3.5 mumols/min/mg was determined. These data demonstrate that, following duplication of the primordial argininosuccinate lyase gene, one of the genes maintained its role as an enzyme (delta 2-crystallin) while also serving as a crystallin and the other has evolved to specialize as a structural protein in the lens (delta 1-crystallin), presumably losing most or all of its catalytic capacity.

Animals↗
PubMed1991

Molecular characterization of the murine argininosuccinate synthetase locus.

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.

Amino Acid Sequence↗
PubMed1991

Analysis of naturally occurring and site-directed mutations in the argininosuccinate lyase gene.

Argininosuccinic aciduria is an inborn error of metabolism due to the genetic deficiency of argininosuccinate lyase. In order to determine the molecular basis for the disease, RNA isolated from cultured skin fibroblasts derived from four unrelated patients was reverse-transcribed and amplified using the polymerase chain reaction and the products were cloned and sequenced. Three single base missense mutations were identified: Arg111----Trp, Gln286----Arg, and Arg193----Gln. One single base amber mutation was identified at Gln454. One mutation involved a 13-base pair deletion within exon 13, and it was noted that the majority of the mature RNA derived from this allele was deleted for the entire exon rather than containing the exon with the 13 bases deleted. A final mutation was observed in which exon 2 was deleted from the mature RNA. The molecular basis for this deletion was not determined. Of the eight potential mutations present in the four cell lines studied, six mutations were identified and further data indicate that the remaining two unidentified mutations were different from those identified. Two site-directed mutations were created in the cDNA, Lys51----Asn and His89----Gln, and these were expressed in yeast. The Lys51 mutation caused an approximate 2-fold reduction in activity and the His89 mutation resulted in an approximate 10-fold reduction in activity. The combination of determination of naturally occurring mutations and the study of the effect of site-directed mutations on the activity of argininosuccinate lyase provide insight into the amino acid residues critical to the function of the enzyme.

Amino Acid Sequence↗
PubMed1991

Assignment of the gene for intercellular adhesion molecule-1 (Icam-1) to proximal mouse chromosome 9.

Intercellular adhesion molecule-1 (ICAM-1) is an integral membrane protein, a member of the immunoglobulin superfamily, and a ligand for LFA-1, a beta 2 leukocyte integrin. ICAM-1 has a tissue distribution similar to that of the major histocompatibility complex class II antigens and is likely to play a role in inflammatory responses. We have mapped this gene to proximal mouse chromosome 9 by using mouse-hamster somatic cell hybrids and an interspecies backcross. Since human ICAM-1 maps to chromosome 19, it joins the LDL receptor to establish a new conserved syntenic segment between human chromosome 19 and proximal mouse chromosome 9. Murine Icam-1 maps between Cbl-2 and the centromere in the same region as one of the susceptibility genes for insulin-dependent diabetes mellitus (Idd-2) that is postulated to play a role in immune function and inflammation leading to insulitis. The mapping of Icam-1 to the region known to contain the Idd-2 gene raises the question of whether the phenotypic differences attributed to the Idd-2 locus might be due to genetic variation in Icam-1.

Animals↗
PubMed1991

Characterization of the human argininosuccinate lyase gene and analysis of exon skipping.

A cosmid clone containing the entire human argininosuccinate lyase gene was isolated and characterized. The gene is approximately 35 kb in length and is divided into 16 exons. The sequence surrounding all splice sites was determined, and one unusual 5' splice site sequence in which a GC dinucleotide occurred instead of the consensus GT dinucleotide was observed. The exon structure of the gene is identical to that of the rat and similar to that of the delta-crystallin genes in the chicken. The transcription initiation site was identified, and 252 bases 5' of that site were sequenced. Within that region, the GC content is 75%, no TATA element was observed, and multiple potential Spl and CACCC binding sites were noted. Also, a putative consensus sequence that may play a role in the regulation of urea cycle genes was identified within this region. Analysis of the structure of the mature mRNA derived from this gene revealed that the sequences encoded by exon seven were deleted in approximately 5-10% of the mature mRNA in all tissue sources examined.

Argininosuccinate Lyase↗
PubMed1991

Localization of arginine biosynthetic enzymes in renal proximal tubules and abundance of mRNA during development.

Argininosuccinate synthetase and argininosuccinate lyase catalyze the conversion of citrulline to arginine in kidney. Immunohistochemical staining of mouse kidney sections with antibodies to these two enzymes, compared with the staining patterns of known markers for proximal tubules, demonstrated that these enzymes are localized within the proximal tubules. The relative abundance of mRNA encoding argininosuccinate synthetase and argininosuccinate lyase during fetal and postnatal development of mouse kidney was also determined. Changes in relative abundance of these mRNA in kidney are coordinate during development, paralleling the developmental profile of phosphoenolpyruvate carboxykinase mRNA, which is also expressed in proximal tubules. Although relative abundances of the mRNA are comparable in liver and kidney of adult mice, the profiles of mRNA abundance during development of these two organs are distinct. The results indicate that these enzymes and their corresponding mRNA can serve as useful markers for examining the differentiation and development of renal proximal tubules in vivo and in cultured explants.

Animals↗
PubMed1991

Additional mutations in argininosuccinate synthetase causing citrullinemia.

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.

Amino Acid Metabolism, Inborn Errors↗
PubMed1990

Heterogeneity of mutations in argininosuccinate synthetase causing human citrullinemia.

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.

Amino Acid Sequence↗
PubMed1990

Mutation analysis for heterozygote detection and the prenatal diagnosis of cystic fibrosis.

The cystic fibrosis gene was recently cloned, and a three-base deletion removing phenylalanine 508 from the coding region was identified as the mutation on the majority of cystic fibrosis chromosomes. We used the polymerase chain reaction and hybridization with allele-specific oligonucleotides to analyze the presence or absence of this mutation on 439 cystic fibrosis chromosomes and 433 normal chromosomes from non-Ashkenazic white families. This mutation was present on 75.8 percent of the cystic fibrosis chromosomes. Using the DNA markers XV-2c and KM-19, we found that 96 percent of cystic fibrosis chromosomes with the mutation had a single DNA haplotype that occurs frequently with cystic fibrosis chromosomes. This haplotype was also found on 54 percent of the cystic fibrosis chromosomes without the three-base deletion. The three-base deletion was found on only 30.3 percent of cystic fibrosis chromosomes from Ashkenazic families, although the common cystic fibrosis haplotype was present on 97 percent of cystic fibrosis chromosomes from Ashkenazic families. The ability to detect the common mutation causing cystic fibrosis represents a major improvement in prenatal diagnosis and heterozygote detection, particularly in families in which no DNA sample is available from the affected child, and provides an improved method of testing for spouses of carriers of cystic fibrosis. Mutation analysis introduces the possibility of population-based screening programs for carriers, which on the basis of the sample in this study, would currently identify about 57 percent of the non-Ashkenazic white couples at risk.

Base Sequence↗
PubMed1990

Prenatal diagnosis of citrullinaemia: review of a 10-year experience including recent use of DNA analysis.

Prenatal diagnosis of citrullinaemia has been accomplished by three different methods to date: (1) enzyme assay of cultured fetal cells; (2) quantification of citrulline in amniotic fluid supernatant; and (3) incorporation of [14C]citrulline into protein by cultured fetal cells. Our laboratory has used these methods to perform prenatal diagnosis for 28 fetuses over a 10-year period. More recently, DNA polymorphisms were used for prenatal diagnosis by linkage analysis. Of the 28 fetuses studied, 23 were predicted to be unaffected, four were predicted to be affected, and results were conflicting in one case where [14C]citrulline incorporation erroneously indicated an affected fetus but linkage analysis correctly predicted an unaffected fetus. Because of low levels of enzyme activity in heterozygotes and in certain amniotic fluid cell types, biochemical diagnosis of citrullinaemia is complicated by the risk of false affected results, although [14C]citrulline incorporation is relatively reliable. When informative, linkage analysis is the preferable method for cases with a 25 per cent risk. The risk of false affected results makes prenatal diagnosis for cases with less than 25 per cent risk of questionable value.

Amino Acid Metabolism, Inborn Errors↗
PubMed1990

Assignment of the structural gene for argininosuccinate synthetase to proximal mouse chromosome 2.

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.

Animals↗
PubMed1990

Deletion and linkage mapping of eight markers from the proximal short arm of chromosome 6.

Eight chromosome 6p markers (MUT, D6S4, D6S5, D6S19, D6S29, PIM, HLA, and F13A) were regionally mapped using somatic cell hybrid deletion cell lines that retained different regions of chromosome 6p. New restriction fragment length polymorphisms were identified at the D6S5 and PIM loci using newly isolated genomic clones at these loci. Genetic linkage among the eight loci was determined using the 40 CEPH reference families. Linkage analyses showed that these loci are in one linkage group spanning 48 cM in males and 128 cM in females. Using both the deletion mapping data and multipoint linkage analyses, chromosomal order for these loci was determined as centromere-(MUT, D6S4)-(D6S5, D6S19)-(D6S29, PIM)-HLA-F13-A-telomere. Analyses of sex-specific recombination frequencies revealed a higher rate of recombination in females in the region between D6S4 and D6S29, while the recombination rate in males was higher for the interval between D6S29 and the HLA loci.

Chromosome Deletion↗
PubMed1990

Benign missense variations in the cystic fibrosis gene.

The common mutation causing cystic fibrosis is a deletion of phenylalanine 508 (delta F508), which occurs in a putative nucleotide-binding fold of the gene product. We report two additional mutations, substitution of cysteine for phenylalanine 508 (F508C) and substitution of valine for isoleucine 506 (I506V). Three compound heterozygous persons, two delta F508/F508C and one delta F508/I506V, had normal clinical and epithelial physiological studies indicating that the F508C and I506V mutations are benign. This opportunity to study the in vivo function of these mutations suggests that amino acid substitutions are more benign than changes in the length of this portion of the putative nucleotide-binding fold. These mutations must be taken into account when performing molecular diagnosis and carrier detection for cystic fibrosis.

Adult↗