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A L Beaudet

Publications and source records attributed to A L Beaudet.

At least 127 records · Page 7Linked to original sources
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

Methods for analysis of multiple cystic fibrosis mutations.

A large number of mutations causing cystic fibrosis (CF) have been reported. In an attempt to improve methods for genetic diagnosis and for heterozygote screening, we evaluated methods for efficient analysis of the delta F508, G542X, G551D, R553X, and N1303K mutations. We found that multiple mutations can be analyzed simultaneously using hybridization with allele-specific oligonucleotides. Alternatively all of these mutations can be detected by amplification of DNA followed by restriction enzyme digestion and analysis on polyacrylamide gels. A previously reported method for use of modified primers for DNA amplification to allow detection of virtually any single-base change by restriction enzyme analysis proved particularly useful. The common delta F508 mutation and three mutations in exon 11 were analyzed using a multiplex amplification reaction followed by double digestion with restriction enzymes and electrophoresis in a single lane on a polyacrylamide gel. In a sample of 439 CF chromosomes from North American Caucasians, the frequencies of various mutations were as follows: delta F508 = 75.8%, G542X = 2.7%, G551D = 3.2%, R553X = 1.4%, and N1303K = 1.4% for a total of 84.5% detection of CF chromosomes by analysis for these five mutations.

Base Sequence↗
PubMed1991

Linkage mapping and fluorescence in situ hybridization of TCTE1 on human chromosome 6p: analysis of dinucleotide polymorphisms on native gels.

Highly informative dinucleotide repeat polymorphisms were identified at the T-complex-associated-testes-expressed-1 (TCTE1) locus on human chromosome 6p. Electrophoresis of single-stranded DNA on native gels facilitated the analysis of the dinucleotide polymorphisms. Linkage mapping positions this marker midway between the centromere and HLA with recombination fractions as follows: D6Z1-0.21-TCTE1-0.24-HLA. Two-color fluorescence in situ hybridization places TCTE1 proximal to CRIL171 (D6S19). Together, linkage and in situ hybridization indicate that the order of the loci is D6Z1-D6S4-D6S90-TCTE1-D6S19-D6S29-HL A-telomere. A sequence tagged site (STS) was established, and three yeast artificial chromosome (YAC) clones were identified for the TCTE1 locus.

Alleles↗
PubMed1991

Mapping of multiple subunits of the neuronal nicotinic acetylcholine receptor to chromosome 15 in man and chromosome 9 in mouse.

The alpha 3, alpha 5, and beta 4 genes (human gene symbols CHRNA3, CHRNA5, and CHRNB4 respectively; mouse gene symbols Acra-3, Acra-5, and Acrb-4, respectively) are members of the nicotinic acetylcholine receptor gene family and are clustered within a 68-kb segment of the rat genome (Boulter et al., 1990, J. Biol. Chem. 265:4472). By somatic cell hybrid analysis, three cDNAs corresponding to these genes were used to map the homologous loci to human chromosome 15 and to mouse chromosome 9. Linkage analysis using CEPH pedigrees showed that the CHRNA5 gene was closely linked to the following chromosome 15 loci: D15S46, D15S52, D15S28, D15S34, and D15S35. Using interspecies crosses in mice, the Acra-5 gene was found closely linked to the Mpi-1 locus. The mapping of these members of a neurotransmitter receptor gene family may facilitate the identification of relationships between the neurotransmitter receptors and murine or human phenotypes.

Animals↗
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

Molecular cloning and sequence analysis of the murine cDNA for the cystic fibrosis transmembrane conductance regulator.

We have cloned the mouse homolog of the human cystic fibrosis transmembrane conductance regulator (CFTR) using clones isolated from a mouse lung cDNA library and using amplification of cDNA to isolate specific regions. The cDNA was 6304 bp in length and encoded a polypeptide of 1476 amino acids. Comparison of the deduced amino acid sequence showed that the mouse protein has high homology to the human protein; overall identity was 78.3%. The amino acid identity was high for both transmembrane domains (first transmembrane domain, 86.7%; second transmembrane domain, 81.1%) and for both ATP-binding folds (first ATP-binding fold, 80.5%; second ATP-binding fold, 83.9%), suggesting the functional importance of these regions. On the other hand, the R domain was less well conserved (68.9% identity). All of the published missense mutation sites and the site of the common delta F508 mutation were conserved between human and mouse.

Amino Acid Sequence↗
PubMed1991

Adherence of neutrophils to canine cardiac myocytes in vitro is dependent on intercellular adhesion molecule-1.

The adhesiveness of isolated canine cardiac myocytes for neutrophils is greatly increased by stimulation with cytokines such as tumor necrosis factor alpha (TNF alpha). Since this adhesion is significantly inhibited by an anti-CD18 MAb, experiments were performed to test the hypothesis that the newly expressed adhesion molecule on the cardiac myocytes was intercellular adhesion molecule-1 (ICAM-1). A newly developed MAb, CL18/6, was found to exhibit the functional and binding characteristics with canine neutrophils and canine jugular vein endothelial cells expected of an antibody recognizing ICAM-1. MAb CL18/6 also bound to isolated cardiac myocytes after stimulation of the myocytes with cytokines, and it blocked by greater than 90% the adhesion of neutrophils to stimulated myocytes. A partial cDNA clone for canine ICAM-1 was isolated, and ICAM-1 mRNA was found to be increased in both endothelial cells and cardiac myocytes after cytokine stimulation. Cytokines that both increased the CL18/6-inhibitable adhesion of neutrophils to myocytes and induced expression of ICAM-1 were IL-1 beta, TNF alpha, and LPS. These results are consistent with the conclusion that canine endothelial cells and cardiac myocytes express ICAM-1 in response to cytokine stimulation, and that ICAM-1 functions as an adhesive molecule for neutrophils on both cell types.

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↗