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Biomedical subjects

R G Gregg

Publications and source records attributed to R G Gregg.

At least 55 records · Page 3Linked to original sources
PubMed1991

Current issues in neonatal screening for cystic fibrosis and implications of the CF gene discovery.

Many questions remain regarding the efficacy, toxicity, and costs of CF neonatal screening. It would be premature, in our opinion, to implement mass population screening of newborns for CF until the benefits and risks have been fully defined, and an adequate and logistically feasible testing system developed and/or highly effective therapy for CF lung disease becomes available. In addition, the ethical issues described herein need to be resolved. This pertains not only to the CF patient but also the heterozygote carrier. These reservations notwithstanding, the discovery of the CF gene should have a favorable impact both directly and indirectly on neonatal screening for the disease. Mutation analysis coupled to IRT testing seems most attractive at this time, at least on a research basis, but primary molecular diagnostic procedures might supervene in the future, particularly if they are financially feasible.

Chromosome Mapping↗
PubMed1991

Waisman syndrome, a human X-linked recessive basal ganglia disorder with mental retardation: localization to Xq27.3-qter.

Linkage of the gene responsible for an X-linked early onset parkinsonism disorder with mental retardation (McKusick 311510) to DNA probes that detect restriction fragment length polymorphisms is described. The disease gene is linked to the F8C gene, and to DNA probes detecting polymorphic loci DXS52, DXS15, DXS134, and DXS374 with maximum lod scores at theta = 0 of 5.08, 5.19, 5.00, 5.03, and 4.46, respectively. Multipoint linkage analysis gives a maximum multipoint lod score of 6.75 at the F8C gene. This places the disease gene in chromosomal region Xq27.3-qter.

Basal Ganglia Diseases↗
PubMed1991

Assignment of the human gene for the alpha 1 subunit of the cardiac DHP-sensitive Ca2+ channel (CCHL1A1) to chromosome 12p12-pter.

A human clone corresponding to the gene encoding the alpha 1 subunit of the cardiac dihydropyridine-sensitive calcium channel (CCHL1A1) has been isolated and partially sequenced. Oligonucleotides based on the human sequence were constructed and used in the polymerase chain reaction to amplify specifically this human gene in human-rodent somatic cell hybrids. Using somatic cell hybrids that contained defined regions of human chromosome 12, the human alpha 1 subunit of the cardiac dihydropyridine-sensitive calcium channel has been assigned to the short arm of chromosome 12 in the interval 12p12-pter.

Amino Acid Sequence↗
PubMed1990

Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.

Animals↗
PubMed1988

Regulated expression of genes inserted at the human chromosomal beta-globin locus by homologous recombination.

We have examined the effect of the site of integration on the expression of cloned genes introduced into cultured erythroid cells. Smithies et al. [Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. & Kucherlapati, R.S. (1985) Nature (London) 317, 230-234] reported the targeted integration of DNA into the human beta-globin locus on chromosome 11 in a mouse erythroleukemia-human cell hybrid. These hybrid cells can undergo erythroid differentiation leading to greatly increased mouse and human beta-globin synthesis. By transfection of these hybrid cells with a plasmid carrying a modified human beta-globin gene and a foreign gene composed of the coding sequence of the bacterial neomycin-resistance gene linked to simian virus 40 transcription signals (SVneo), cells were obtained in which the two genes are integrated at the beta-globin locus on human chromosome 11 or at random sites. When we examined the response of the integrated genes to cell differentiation, we found that the genes inserted at the beta-globin locus were induced during differentiation, whereas randomly positioned copies were not induced. Even the foreign SVneo gene was inducible when it had been integrated at the beta-globin locus. The results show that genes introduced at the beta-globin locus acquire some of the regulatory properties of globin genes during erythroid differentiation.

Animals↗
PubMed1987

Hepatic cholesterol synthesis and hydroxymethylglutaryl CoA reductase activity after injection of methylazoxymethanol acetate.

Cholesterol synthesis and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) in the liver of rats at various times (7, 22, 45 and 314 days) after injection with the carcinogen, methylazoxymethanol acetate (MAMA) is reported. Seven days after treatment, an increase in both cholesterol synthesis and HMG-CoA reductase activity was observed. Elevated HMG-CoA reductase activity and reduced dietary feedback was present 22 days after carcinogen. Cholesterol synthesis was normal at this time but dietary cholesterol failed to significantly reduce synthesis. Forty-five days after carcinogen both cholesterol synthesis and HMG-CoA reductase activity had returned to normal. Both parameters were normal 314 days after carcinogen. The enzyme gamma-glutamyl transferase was also elevated at 7, 22 and 314 days. These results indicate that HMG-CoA reductase activity and cholesterol synthesis exhibit different regulatory characteristics during the early stages of hepatocarcinogenesis initiated by MAMA injection.

Animals↗
PubMed1986

Cholesterol synthesis and HMG CoA reductase activity during hepatocarcinogenesis in rats.

Two treatment regimes were used to produce preneoplastic foci (as determined by the presence of gamma-glutamyl transferase) in rat liver. Increased [14C]acetate incorporation into cholesterol and 3-hydroxy-3-methyl glutaryl CoA reductase activity were associated with high levels of gamma-glutamyl transpeptidase and foci formation. Dietary feedback inhibition of both [14C]acetate incorporation and 3-hydroxy-3-methyl glutaryl CoA reductase activity was reduced at a selected time when gamma-glutamyl transpeptidase activity was high. These changes could not be accounted for by a regenerative response in the liver.

2-Acetylaminofluorene↗
PubMed1986

Efficient transformation and frequent single-site, single-copy insertion of DNA can be obtained in mouse erythroleukemia cells transformed by electroporation.

Electroporation has recently been shown to have advantages over the commonly used method of calcium phosphate precipitation for obtaining DNA-mediated transformation of certain types of cells. Although mouse erythroleukemia cells and other cells of hematopoietic origin are not transformed at useful frequencies by calcium phosphate-DNA precipitation methods, we obtained high frequencies of transformation (approximately 10(-5)) of these cells with electroporation. Even higher transformation frequencies (approximately 10(-3)) were obtained with human fibroblasts. Another advantage of electroporation was found when analysis of Southern blots of DNA from 243 transformed erythroleukemia cell colonies indicated that, under appropriate conditions, about 79% of the transformed cells had the exogenous DNA integrated in single copies at single sites. Under conditions of higher DNA and lower cell concentrations using fibroblasts, cotransformation was obtained with two plasmids that confer HAT or G418 resistance when integrated into cellular DNA. About 23% of the transformed cells developed both types of resistance. We describe a simple, inexpensive apparatus for carrying out electroporation.

Animals↗
PubMed1985

Effect of assay temperature on the kinetics of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in rat liver and Morris hepatoma 5123C.

A procedure is described for the assay of 3-hydroxy-3-methylglutaryl CoA-reductase (HMG-CoA reductase) in a large number of samples with minimal benchwork and within a 24-hr period. The Michaelis constants for HMG-CoA reductase were determined for microsomal enzyme from the liver of normal and cholesterol-fed rats and Morris hepatoma 5123C. The apparent Km D-HMG-CoA was ca. 3.5 microM and was not affected by assay temperature or cholesterol feeding. The apparent Km NADPH for microsomal HMG-CoA reductase was 10-15 microM and similarly was not affected by assay temperature. The Arrhenius plot parameters (activation energy and transition temperatures) were the same whether determined using the reaction velocity from fixed substrate concentrations or V from subtraction curves. This confirmed that values obtained using fixed saturating substrate concentrations are valid and not affected by a temperature-dependent alteration in the affinity of the enzyme for its substrates.

Animals↗
PubMed1983

Reversible phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase in Morris hepatomas.

The reversible phosphorylation of microsomal 3-hydroxy-3-methylglutaryl CoA reductase in host liver and hepatoma 5123C has been investigated. The percentage of the total enzyme activity in vivo was similar in the normal liver, host liver and hepatoma 5123C. The inclusion of 30 mM EDTA and 10 mM mevalonic acid in assays of 3-hydroxy-3-methylglutaryl CoA reductase inactivation in vitro eliminated artifacts generated by the presence of mevalonate kinase. Inactivation of 3-hydroxy-3-methylglutaryl CoA reductase from normal liver, host liver and hepatoma occurred at a similar rate with similar half-times. We conclude that phosphorylation/dephosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase occurs in hepatomas and that the lack of dietary cholesterol feedback inhibition in the hepatomas is not a result of a defect in this particular aspect of the reversible phosphorylation system.

Adenosine Triphosphate↗
PubMed1982

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in rat liver and Morris hepatomas 5123C, 9618A and 5123t.c.

Characteristics of 3-hydroxy-3-methylglutaryl-CoA reductase from normal liver, Morris hepatomas 5123C, 5123t.c. and 9618A, and host liver were studied. Animals were fed on control and 5%-cholesterol diets. Microsomal membranes from all tissues were found to accumulate cholesterol after 3 days on the 5%-cholesterol diet. The enzyme of the tumours showed no feedback inhibition by dietary cholesterol, and that of host liver gave a variable response, whereas that of control liver was constantly inhibited by 90% or more. Arrhenius-plot analysis was conducted on the microsomal enzyme isolated from the various tissues. Control animals showed that the phase transition present at 27 degrees C was removed when animals were fed on 5%-cholesterol diet for 12 h. The hepatomas failed to show this change even after 3 days of 5%-cholesterol diet and a significant increase in microsomal cholesterol. This failure to remove the break in Arrhenius plots also occurred in host liver, even though enzyme inhibition occurred. The reason why hepatomas fail to regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in response to dietary cholesterol may be a decreased membrane-enzyme interaction.

Animals↗
PubMedDate not supplied

Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination.

A 'rescuable' plasmid containing globin gene sequences allowing recombination with homologous chromosomal sequences has enabled us to produce, score and clone mammalian cells with the plasmid integrated into the human beta-globin locus. The planned modification was achieved in about one per thousand transformed cells whether or not the target gene was expressed.

Base Sequence↗