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R DeMars

Publications and source records attributed to R DeMars.

At least 91 records · Page 5Linked to original sources
PubMed1980

Autonomous gene expression on the human inactive X chromosome.

Local derepression of the hpt locus on the human inactive X chromosome obtained in human female fibroblast x mouse L cell somatic cell hybrids was not correlated with the presence or absence of any specific human chromosome in the hybrids. Loss of the human active X, in particular, did not result in observable derepression of genes on the inactive X. Introduction of an active X, via a second hybridization of human cells having an active X with hybrid cells containing a locally derepressed X chromosome, did not restore repression of the derepressed hpt allele. The rate of hpt locus derepression in hybrid cells was estimated to be 10(-6) per inactive X chromosome per cell generation.

Animals↗
PubMed1980

Hemizygous HLA mutants of lymphoblastoid cells: a new cell type for histocompatibility testing.

HLA variants that have lost expression of multiple cis-linked alleles as determined serologically and enzymatically were analyzed for expression of HLA-D PLT-stimulating (PL) determinants using the primed lymphocyte test. All 19 variants that had lost HLA-DRw3 expression simultaneously had lost expression of the HLA-D region-associated PL specificity. PLT cells made by priming to a variant that was hemizygous for HLA genes resulted in priming to HLA-D PL determinants encoded for by just one haplotype, analogous to priming to an HLA homozygous cell.

Alleles↗
PubMed1980

Gamma ray-induced loss of expression of HLA and glyoxalase I alleles in lymphoblastoid cells.

Gamma rays from a cesium source were used to generate human lymphoblastoid cell line variants that had lost expression of all major histocompatibility complex antigens coded for by a single haplotype. The cell line was heterozygous at the glyoxalase I locus and had the HLA haplotypes HLA-A1, B8, DRw3, and HLA-A2, B5, DRw1. We selected with anti-HLA-B8 antiserum in a population of cells that had been irradiated with 300 R. The incidence of B8-loss variants was 4.1 X 10(-5) on day 5 after irradiation. Analysis of variants showed that expressions of HLA and GLO alleles trans to B8 were retained. However, expression of additional cis-linked HLA and GLO gene products was lost in 12 of 17 variants. Variants that had lost expression of (i) HLA-B8, (ii) HLA-B8, A1, (iii) HLA-B8, A1, DRw3, or (iv) HLA-B8, A1, DRw3 and the cis-linked glyoxalase I allele were obtained. Karyotype analysis was performed on eight variants that had lost expression of two or more cis-linked alleles. Three variants had two normal appearing no. 6 chromosomes, four variants had a deletion that included the region coding for HLA genes on the short arm of one no. 6 chromosome, and one variant had an inversion or translocation involving the short arm of one no. 6.

Cell Line↗
PubMed1976

Abnormal ornithine carbamoyltransferase in mice having the sparse-fur mutation.

Mice with the X-chromosomal sparse-fur (spf) mutation frequently have urinary bladder stones composed mostly of orotic acid, which was identified by the following criteria: ultraviolet and infrared absorption, spectra, chromatographic behavior, melting point, and reactivity in a specific color test. This clue led to the discovery that spf-bearing mice have an abnormal form of liver ornithine carbamoyltransferase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3). Normal ornithine carbamoyltransferase has maximum activity at pH 7.6-8.0 and 80% of maximum activity at pH 10.0.

Alleles↗
PubMed1975

Adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase immunoprecipitation reactions in human-mouse and human-hamster cell hybrids.

Male New Zealand White rabbits were immunized with human adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which were purified about 2000-fold and 800-fold, respectively, from erythrocytes by DEAE-cellulose chromatography, ammonium sulfate precipitation and preparative polyacrylamide gel electrophoresis. Specific immunoprecipitations of APRT and HGPRT were achieved with the antisera that were obtained and by using polyethylene glycol as a substitute for goat anti-(rabbit) gamma globulin. The activities of the human forms of these enzymes, whether from red blood cells or from cultured cells, were almost completely eliminated under the conditions of immunoprecipitation used. Little or no reduction of APRT and HGPRT activities from mouse and Chinese hamster cells was observed. This discriminatory capacity of the antisera was successfully used for the identification of human APRT and HGPRT in human-mouse and human-hamster cell hybrids using the immunoprecipitation reaction.

Adenine Phosphoribosyltransferase↗
PubMed1975

Localized Derepression on the Human Inactive X Chromosone in Mouse-Human Cell Hybrids.

Evidence for derepression of the gene for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) on the human inactive X chromosome was obtained in hybrids of mouse and human cells. The mouse cells lacked HPRT and were also deficient in adenine phosphoribosyltransferase (APRT; AMP: pyrophosphate phosphoribosyltransferase; EC2.4.2.7). The human female fibroblasts were HPRT-deficient as a consequence of a mutation on the active X but contained a normal HPRT gene on the inactive X. The two human X chromosomes were further distinguished by differences in morphology: the inactive X was morphologically normal while the active X included most of the long arm of autosome no. 1 translocated to the distal end of the X long arm. Forty-one hybrid clones were first isolated by selection for the presence of APRT; when these clones were selected for HPRT, six of them yielded derivatives having human HPRT with incidences of about 1 in 10-6 APRT-selected hybrid cells. The HPRT-positive derivatives contained a normal-appearing X chromosome indistinguishable from the inactive X of the parental human fibroblasts. The active X with the translocation was not found in any of the HPRT-positive hybrid cells. Human phosphoglycerokinase (ATP:3-phospho-D-glycerate 1-phosphotransferase. EC 2.7.2.3) and glucose-6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP 1-oxidoreductase, EC 1.1.1.49), which are specified by X-chromosomal loci, were not detected in the hybrids expressing HPRT even though they contained an apparently intact X chromosome. The observations are most simply explained by the infrequent, stable derepression of inactive X chromosome segments that include the HPRT locus but not the phosphoglycerokinase and glucose-6-phosphate dehydrogenase loci.

Adenine Phosphoribosyltransferase↗
PubMed1974

The locus for human adenine phosphoribosyltransferase on chromosome no. 16.

Evidence for assigning the locus determining the structure of adenine phosphoribosyltransferase (APRT) to human chromosome No. 16 is presented. Hybrids of APRT-deficient mouse cells and of human fibroblasts having normal APRT were isolated by fusing the parental cells with Sendai virus, blocking de novo purine nucleotide synthesis with azaserine and selecting for hybrids that could use exogenous adenine. The hybrid clones that were studied had only APRT activity that was indistinguishable from human APRT with regard to electrophoretic migration and reaction with antibodies against the partially purified human enzyme. No. 16 was the only human chromosome consistently present in all of the clones, and in one clone, it was the only human chromosome detected. Selection against hybrid cells with 2,6-diaminopurine (DAP) yielded DAP-resistant survivors that lacked chromosome No. 16. One hybrid that originally had an intact No. 16 yielded adenine-utilizing subclones that lacked No. 16 but had a new submetacentric chromosome. The distribution of centromere-associated heterochromatin and the fluorescence pattern indicated that this chromosome consisted of a mouse telocentric chromosome and the long arm of No. 16. Cells having the submetacentric chromosome had human APRT. Both the enzyme and the chromosome were absent in DAP-resistant derivatives. These results suggest that the structure of APRT is defined by a locus on the long arm of human chromosome No. 16.

Adenine↗
PubMed1973

Diaminopurine-resistant mutants of cultured, diploid human fibroblasts.

Clones of cells resistant to 2,6-diaminopurine were detected in skin fibroblast cultures derived from 13 of 21 normal humans of both sexes from 17 unrelated families. Almost all of the cultures that yielded mutants were chosen for further study from among a total of 83 surveyed because they displayed a slight resistance to low concentrations of diaminopurine. The incidences of mutant colonies ranged between about 10(-5) and 10(-4) per cell surviving prior mutagenic treatment with MNNG. The incidences of spontaneous mutants were about 10(-7) to 10(-5) in three unrelated cultures. Most independent mutants had distinctly reduced activity of adenine phosphoribosyltransferase but some had apparently normal amounts of activity. Two mutants from unrelated boys had little or no detectable enzyme activity and were unable to effectively use exogenous adenine for growth when purine biosynthesis was blocked with azaserine. Most mutants could utilize exogenous adenine, just as most azaguanine-resistant fibroblast mutants can utilize exogenous hypoxanthine, even when their hypoxanthine-guanine phosphoribosyltransferase activity is reduced. Diverse genetic changes conferred diaminopurine resistance but their specific natures are still undefined. Gross numerical or structural chromosome abnormalities were not observed in the mutants examined so far. Since at least one gene responsible for adenine phosphoribosyltransferase activity is on autosome No. 16 our results suggest that at least some of the cultures yielding mutants were heterozygous and that alleles conferring diaminopurine resistance may be frequent enough to comprise a polymorphism.

Adenine↗
PubMed1969

Lesch-Nyhan mutation: prenatal detection with amniotic fluid cells.

Cells cultured from the amniotic fluid of a 22-week fetus in a heterozygote for the X-linked Lesch-Nyhan mutation, which results in neurological and developmental disorders, lacked sex chromatin and were unable to incorporate hypoxanthine. The diagnosis of a mutant male was confirmed upon birth of enzyme-deficient, hyperuricemic twin boys whose amniotic membrane cells failed to incorporate hypoxanthine.

Amniotic Fluid↗
PubMed1969

Purine requirement of cells cultured from humans affected with Lesch-Nyhan syndrome (hypoxanthine-guanine phosphoribosyltransferase deficiency).

Humans with the Lesch-Nyhan syndrome have an X-chromosomal mutant gene that causes severe neurological and developmental abnormalities. The patients are deficient in hypoxanthine-guanine phosphoribosyltransferase, which converts hypoxanthine to inosinic acid, a major precursor of adenine and guanine nucleotides. Paradoxically, the enzyme defect causes hypernormal de novo synthesis of inosinic acid, which manifests itself as excesses of hypoxanthine, xanthine, and uric acid. The first step in the de novo pathway is thought to be rate-limiting, due to feedback repression by adenine and guanine nucleotides. The derepressed rate of purine production in mutants and their failure to thrive could result from reduction in the amounts of nucleotides derived from inosinic acid to levels that are inadequate for normal feedback control and for nucleic acid synthesis needed in growth. Studies with cultured cells, reported here, support the interpretation that mutants are, in effect, nucleotide-deficient. Skin fibroblasts from patients fail to proliferate in media that do not contain supplementary adenine or folic acid, a participant in two stages of purine biosynthesis. The folic acid requirement of mutant cells is at least 50-fold greater than that of normal cells, which can synthesize all the nucleotides needed for growth without exogenous adenine. Both folic acid and adenine supplements are thought to provide mutant cells with the means of making more inosinic acid available for conversion to adenine and guanine nucleotides. It is not clear why the availability of inosinate or its conversion to other nucleotides is impaired. Therapy with adenine or folic acid begun at the time of birth may avert development of the disease in mutant males.The relevant gene is X-linked and shows clonal, single-allele-expression: phenotypically normal and phenotypically mutant clones have been derived from females heterozygous for the mutant gene. The phenotypically mutant heterozygous clones have the same requirement for adenine or folic acid as cells from hemizygous mutant males, an indication that the normal allele is repressed in these clones. The adenine-folic acid requirement of mutant cells provides a method of direct, clonal selection for rare, phenotypically normal cells in mutant populations, which is applicable to the single-active-X problem and other in vitro genetic studies.

Adenine↗