Reexpression of the Rat Hypoxanthine Phosphoribosyltransferase Gene in Rat-Human Hybrids

Croce, Carlo M.; Bakay, Bohdan; Nyhan, William L.; Koprowski, Hilary

doi:10.1073/pnas.70.9.2590

Cited by 39 publications

(9 citation statements)

References 22 publications

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“…HeLa cells are grown at 370 in an atmosphere of 5% C02-95% air on plastic petri plates (Falcon, 100 mm style dishes) in DME medium consisting of Dulbecco's modified Eagle's medium (Gibco, 13.5 g of powdered media per liter) containing 10% calf serum (Irvine Scientific), 22 mM sodium bicarbonate, andl5 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic buffer) adjusted to pH 7.2. All cell lines discussed in this paper have a generation time of [15][16][17][18][19][20] hr and a plating efficiency of 50-70%.…”

Section: Methodsmentioning

confidence: 99%

Analysis of HeLa cell hypoxanthine phosphoribosyltransferase mutants and revertants by two-dimensional polyacrylamide gel electrophoresis: evidence for silent gene activation.

Milman¹,

Lee²,

Ghangas³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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The spot corresponding to hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) has been identified in twodimensional polyacrylamide gels of HeLa cell extracts. This spot is absent in gels of 24 HPRT deficient mutants. A missense mutant displays a new HPRT spot at the same molecular weight but different isoelectric focusing position. Five (3)(4)(5)(6). In the present work, we demonstrate that HPRT mutants and revertants can be analyzed by two-dimensional polyacrylamide gel electrophoresis of crude cell extracts. In this procedure, proteins are separated in one dimension by isoelectric focusing, and then in a second dimension by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (7).We have identified the spot corresponding to HPRT on two dimensional gels of HeLa cell extracts even though the enzyme represents only 0.02% of the soluble protein. We have analyzed 24 HPRT deficient mutants, and for every one, the spot corresponding to the wild-type enzyme disappears. A missense mutant labeled H23 displays a new spot at the same molecular-weight location, but at a different isoelectric focusing position. Unexpectedly, five independently isolated revertants of H23 display spots corresponding to both the wild-type and Abbreviations: HPRT, hypoxanthine phosphoribosyltransferase; DME medium, Dulbecco's modified Eagle's medium; TG medium, DME medium containing 6-thioguanine; MTH medium, DME medium containing methotrexate, thymidine, and hypoxanthine; TH medium, DME medium containing thymidine and hypoxanthine. * Address reprint requests to this author. Mutagen Treatment. One million wild-type HeLa cells growing exponentially are placed in plates containing 10 ml of fresh DME medium. The medium is removed after 12-24 hr and replaced with DME medium containing mutagen. The mutagens used are ethyl methanesulfonate at 400, 500, and 600 ,ug/ml and N-methyl, N'-nitro, N-nitrosoguanidine at 4, 5, and 6 ,ug/ml. After 24 hr, the medium containing mutagen is removed, the cells are rinsed with 5 ml of phosphate-buffered saline, and 10 ml of fresh DME medium is added to the plates. The cells are grown in DME medium for 5-10 days prior to the addition of selective medium to allow time for the residual HPRT concentration in mutants to decrease. During this time, the cells are transferred so that they do not exhaust the medium. The three levels of ethyl methanesulfonate and nitrosoguanidine kill approximately 60%, 90%, and 98% of the cells, respectively.Mutant Selections. Ten milliliters of selective "TG medium" containing 0.1 mM 6-thioguanine in DME-medium is added to plates containing approximately 4 to 8 X 106 mutagentreated cells. After 3-7 days, the medium and cell debris are removed and fresh TG medium is added. Colonies are observed 14-21 days after addition of TG medium. Colonies are removed with a pasteur pipet and grown in TG medium. Each mutant cell line is recloned by plating the cells at a low density and isolating individual colonies. To avoid duplication o...

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Section: Methodsmentioning

confidence: 99%

Analysis of HeLa cell hypoxanthine phosphoribosyltransferase mutants and revertants by two-dimensional polyacrylamide gel electrophoresis: evidence for silent gene activation.

Milman¹,

Lee²,

Ghangas³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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“…The early increases in saturation density of cells transferred after prolonged confluence also occur in a large fraction of the population in a uniform, graded manner before dense foci appear (2,20). Evidence of population-wide damage to cells is also seen after x-irradiation (6) and is followed by elevated frequencies of certain complex types of mutation associated with the hemizygous HPRT locus (29) but no increase in specific point mutations (30). The evidence for epigenetic change is not restricted to cells adapted for culture.…”

Section: Discussionmentioning

confidence: 99%

Neoplastic development: paradoxical relation between impaired cell growth at low population density and excessive growth at high density.

Rubin

Yao

Chow

1995

Proc. Natl. Acad. Sci. U.S.A.

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The role of heritable, population-wide cell damage in neoplastic development was studied in the 28 L subline of NIH 3T3 cells. These cells differ from the 173c subline used previously for such studies in their lower frequency of "spontaneous" transformation at high population density and their greater capacity to produce large, dense transformed foci. Three cultures of the 28 L subline of NIH 3T3 cells were held under the constraint ofconfluence for 5 wk (5 wk 10 assay) and then assayed twice in succession (20 and 3°assays) for transformed foci and saturation density. After the 20 assay, the cells were also passaged at low density to determine their exponential growth rates and cloned to determine the size and morphological features of the colonies. Concurrent measurements were made in each case with control cells that had been kept only in frequent low-density passages and cells that had been kept at confluence for only 2 wk (2 wk 10). Two of the three cultures transferred from the 20 assay of the 5 wk 1°cultures produced light transformed foci, and the third produced dense foci. The light focusforming cultures grew to twice the control saturation density in their 20 assay and 6-8 times the control density in the 30 assay; saturation densities for the dense focis formers were about 10 times the control values in both assays. All three of the cultures transferred from the 20 assay ofi the 5 wk 10 cultures multiplied at lower rates than controls at low densities, but the dense focus formers multiplied faster than the light focus formers. The reduced rates of multiplication of the light focus formers persisted for >50 generations of exponential multiplication at low densities. Isolated colonies formed from single cells of the light focus formers were of a lower population density thaR, controls; colonies formed by the dense focus formers were slightly denser than the controls but occupied only half the area. A much higher proportion of the colonies from the 5 wk 10 cultures than the controls consisted of giant cells or mixtures of giant and normal-appearing cells, The'results reinforce the previous conclusion that the early increases in saturation density and light fo&us formation are associated with, and perhaps caused by, heritable, populationwide damage to cells that is essentially epigenetic in nature.

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“…Initial strategies were aimed at establishing a panel of somatic cell hybrids by somatic cell hybridization and random elimination of a majority of human chromosomes from fused nuclei of human and rodent hybrids (33)(34)(35). This hybridization technique was subsequently improved for establishing monochromosomal hybrids by employing fusion of micronuclei (36)(37)(38).…”

Section: Monochromosomal Hybridsmentioning

confidence: 99%

Monochromosomal Hybrids and Chromosome Transfer: A Functional Approach for Gene Identification

Kandpal

Sandhu

Kaur

et al. 2017

CGP

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Reexpression of the Rat Hypoxanthine Phosphoribosyltransferase Gene in Rat-Human Hybrids

Cited by 39 publications

References 22 publications

Analysis of HeLa cell hypoxanthine phosphoribosyltransferase mutants and revertants by two-dimensional polyacrylamide gel electrophoresis: evidence for silent gene activation.

Analysis of HeLa cell hypoxanthine phosphoribosyltransferase mutants and revertants by two-dimensional polyacrylamide gel electrophoresis: evidence for silent gene activation.

Neoplastic development: paradoxical relation between impaired cell growth at low population density and excessive growth at high density.

Monochromosomal Hybrids and Chromosome Transfer: A Functional Approach for Gene Identification

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