Incorporation of isolated chromosomes and induction of hypoxanthine phosphoribosyltransferase in chinese hamster cells

Evidence is presented for the uptake of radioactive-labeled isolated Chinese hamster chromosomes following incubation with Chinese hamster cells. Metaphases were found which contained radioactive labeled chromosomes in a very low frequency, and in some of the labeled chromosomes only one chromatid was labeled. Incubation of hypoxanthine phosphoribosyltransferase (HPRT)-deficient Chinese hamster cells with chromosomes isolated from HPRT ⁺ Chinese hamster or human cells resulted in the appearance of HPRT⁺ cells. Clones derived from these cells were isolated in HA T medium. Cells in mitosis during incubation with the chromosomes yielded three times more HPRT⁺ clones than did cells in interphase. The intraspecies combination involving recipient cells and chromosomes from Chinese hamster origin yielded significantly higher numbers of HPRT⁺ clones than did the interspecies system using human chromosomes and Chinese hamster recipient cells (5×10^–5 and 6×10^–6 respectively). Electrophoresis of HPRT from Chinese hamster cells treated with human chromosomes revealed the pattern of the human enzyme.     

Derepression of genes on the human inactive X chromosome: Evidence for differences in locus-specific rates of derepression and rates of transfer of active and inactive genes after DNA-mediated transformation

Mouse-human hybrid cells that contained an inactive human X chromosome were treated with agents known to alter gene expression and to perturb DNA methylation. 5-Azacytidine greatly increased the rate of derepression of HPRT on the inactive X, while butyrate and dimethyl sulfoxide had smaller effects. Ethionine did not change the rate of derepression. Derepression of two other X-chromosomal loci, PGK and GPD, was also detected. The rate of derepression of PGKwas 20-fold higher than the rate for HPRT. Derepression events at the two loci appeared to be independent. Hybrids expressing derepressed X-chromosomal genes had more variable levels of human enzyme activities when compared to control hybrids. HPRT⁺ clones did not appear after transfer of purified DNA from a cell hybrid containing an inactive human X into HPRT^– recipients, but such clones did appear after transfer of DNA from derivative cells in which HPRT had been derepressed.     

Derepression ofHPRT locus on inactive X chromosome of human lymphoblastoid cell line

Human XX lymphoblastoid cells with a deletion in the HPRT locus on the active X were exposed to HPRT clone pHPT32. HPRT⁺ isolates GPT3 and GPT5 lacked pHPT32 DNA, suggesting that their HPRT⁺ phenotype resulted from expression of a cellular gene. GPT3 mutated to thioguanine resistance at least 100 times more frequently than cells in which the expressed HPRT locus was on the active X. Most GPT3- derived HPRT^}- had lost one entire X chromosome, indicating that the HPRT⁺ phenotype of GPT3 resulted from derepression of the HPRT locus on its inactive X. Virtually unchanged G6PD and PGK activities and the presence of a late-replicating X in GPT3 suggest that derepression of the inactive X was not general. Eleven of the GPT3-derived mutants had a tiny centric remnant that may result from a frequently operative mechanism of X chromosome loss. The detection of partial or complete loss of an X by direct selection presents unusual opportunities for genotoxicity detection with human cells.     

Transfer of genes to chinese hamster ovary cells by DNA-mediated transformation

We have transferred DNA to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk^– cells were transformed with the cloned gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk^– cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 × 10^–5 were obtained. CHO HSV- tk⁺ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10^–4 with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk^– cells could be transformed at a frequency of 10^–7 with cellular DNA isolated from CHO tk⁺ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.A preliminary account of this work has been presented at the twenty-first annual meeting of the American Society for Cell Biology, 1981 (Ref. 41).     

Reactivation of X-linked genes in human fibroblasts transformed by origin-defective SV40

To determine if expression of genes on the inactive X is inducible in human cells, we looked for reactivation events in a clone of fibroblasts transformed with origin-defective SV40. The karyotype of these cells was grossly heteroploid so that the aneuploidy associated with SV40 transformation occurs even in the absence of viral replication. This transformed clone, heterozygous for hypoxanthine phosphoribosyltransferase (HPRT), lacks HPRT activity, as the mutant allele is on the active X and the normal allele on the inactive X. Reactivation of the HPRT ⁺ allele on the inactive X was observed at a frequency of 6 x 10 ^–5 per cell and increased approximately eightfold following treatment with the cytidine analogs 5-azacytidine (5azaC) and 5-azadeoxycytidine. The fact that spontaneous reactivation is detectable in some clones, but not all, suggests that the environment of the SV40-transformed cell, although not sufficient to induce generalized derepression, increases the frequency of rare reactivation events. The methylation pattern at the HPRTlocus revealed transformation-associated alterations that may have predisposed these cells to reactivation events, spontaneous as well as 5azaC-induced.     

Isolation and characterization of HPRT-Deficient human hepatoma cells

Human hepatoma cells deficient in HPRT activity were isolated by challenging HepG2 cells with 6-thioguanine (6TG). Three 6TG-resistant isolates were plated in selective media, and each clonal line displayed an 8-azaguanine-resistant, HAT-sensitive phenotype. The HPRT-deficient phenotype of one of these clones. H30-1, was confirmed in genetic tests: the HAT-sensitivity of H30-1 cells was complemented by fusion by HPRT⁺ (Ltk⁻) but not HPRT⁻ (A9) cells. Furthermore, transfection of the bacterial xanthine-guanine phosphoribosyl transferase (gpt) gene into H30-1 cells rendered them HAT-resistant. H30-1 cells maintained the differentiated morphology, growth characteristics, fusion properties, and tranfection efficiencies typical of parental HepG2 cells, and they expressed several liver-specific genes. Finally, the H30-1 cell line contained a modal number of 50 chromosomes. Therefore, H30-1 cells represent an HPRT-deficient HepG2 derivative that retains its differentiated phenotypein vitro.     

Expression of human genes for adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase after genetic transformation of mouse cells with purified human DNA

Human DNA purified from HeLa cells and from three strains of skin fibroblasts was precipitated with calcium phosphate and added to mouse cells that were deficient in adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT). Selection for cells possessing either of the phosphoribosyltransferases was imposed by blocking de novo synthesis of purine nucleotides with azaserine in a medium supplemented with adenine and hypoxanthine. The frequency of colony formation after selection was 1.7×10^–7–3.3×10^–6. Excepting some azaserine-resistant colonies that appeared only in the first experiment and infrequent revertants expressing mouse APRT, all characterized clones expressed the human forms of APRT or HPRT according to the criteria of specific immunoprecipitation and electrophoretic mobility. The frequency of transfer of the human APRT gene was much greater than that of HPRT. Transfer efficiency was not significantly reduced when HeLa DNA was sheared to 6.5–13.5 kb size or when the donor DNA was isolated from a transferent that expressed human APRT.     

Expression of a human cDNA encoding a protein containing GAR synthetase,AIR synthetase,and GAR transformylase corrects the defects in mutant Chinese hamster ovary cells lacking these activities

The isolation of a human cDNA encoding the multifunctional protein containing GAR synthetase, AIR synthetase, and GAR transformylase by functional complementation of purine auxotrophy in yeast has been reported. Chinese hamster ovary (CHO) cell mutant purine auxotrophs deficient in GAR synthetase (Ade^–C) or AIR synthetase plus GAR transformylase (Ade^–G) activities were transfected with this human GART cDNA subcloned into a mammalian expression vector. This restored 49–140% of the activities of GAR synthetase, AIR synthetase, and GAR transformylase in transfected cells when compared to wild-type CHO K1 parental cells. Study of one stably expressing transfectant, AdeC2, revealed that the human GART cDNA was incorporated into the CHO genome. The enzyme activities appear to be associated with an expressed protein of 110 kDa, very similar to that of purified human GART trifunctional enzyme. The Ade^–C mutant shows reduced amounts of GART mRNA compared to CHO K1 and a protein of apparently reduced size, results consistent with the purine requirement and enzyme deficiency observed in the mutant. These experiments provide definitive evidence that the human GART cDNA encodes and can direct the production of active human GART trifunctional protein in mammalian cells. They also provide important evidence that the Ade^–C and Ade^–G mutants of CHO cells are defective in this gene.     

Bromodeoxyuridine resistance in CHO cells occurs in three discrete steps

Four independent mutants were isolated from mutagenized cultures of CHO cells by sib selection on the basis of resistance to a low concentration (2.6 × 10^–5M) of BrdU. All four lines were stable, but all had about 100% of the wild-type (WT) specific activity of thymidine kinase (TK). None of the four yielded derivatives resistant to a high level of BrdU (2 × 10^–4M) in one step even after mutagenesis, but variants resistant to 4–6 × 10^–5M BrdU could be isolated at frequencies of about 2 × 10^–5/cell. At frequencies of 10^–4–10^–5, the second-step mutants gave colonies resistant to 2 × 10^–4 M BrdU. The second and third steps of resistance were correlated with partial and complete reduction, respectively, in the specific activity of TK, suggesting that the variants may be genotypically heterozygous and homozygous-negative at the tk locus. The first step of BrdU resistance was dominant and appeared to result from a mutation in the gene for ribonucleotide reductase, since in vitro assays on partially purified preparations showed that the reductase activity in mutant cells was less sensitive to BrdUTP than that in WT cells.     

Inactivation and reactivation of sex-linked steroid sulfatase gene in murine cell culture

The murine X-linked steroid sulfatase gene (Sts) normally escapes X inactivation. However, we have observed that most long-term murine cell cultures are deficient in STS activity even though only the L cells are known to be derived from an STS^– mouse strain. To investigate this phenomenon, we developed a selective system whereby STS⁺ cells could be selected from STS^– populations. The system is based on making cells dependent on cholesterol-sulfate as the sole source of cholesterol, allowing only STS⁺ cells to grow. Two STS^– cell lines, after treatment with either 5-azacytidine (5AC) or ethyl methane sulfonate (EMS), yielded STS⁺ revertants, suggesting that their STS^– phenotype was due to hyper-methylation. To study the evolution of STS^– cell lines, we established XO and XX primary lines from STS⁺ strains; the XX cell line remained STS⁺ after more than 200 cell doublings whereas the XO became STS^– after about 100 doublings. Treatment of this STS^–XO cell line with 5AC produced clones with restored STS activity. All the revertants showed a growth disadvantage compared to their STS^– counterparts. It would appear that aberrant methylation is the basis for much of the STS deficiency observed in established murine lines and that its propagation is due to the growth advantage of STS^– over STS⁺ cells.     

Selection of highly transfectable variant from mouse mastocytoma P815

A tk^– cell line derived from mouse mastocytoma P815 was transfected with a plasmid carrying a thymidine kinase gene. The tk⁺ cells were obtained at a frequency of 10^–6. From some of these tk⁺ transfectants it was possible to select tk^– cells with BrdU so that these cells could be submitted again to transfection with the thymidine kinase gene. By repeating cycles of transfection and tk⁺ selection followed by reverse selection with BrdU, it was possible to obtain a stable variant having a 100-fold increase in transfection efficiency. This HTR (high transfection) variant shows a high efficiency of transfection (10^–4) for the neomycin-resistance gene as well as for the thymidine kinase gene.     

Chromosome-mediated gene transfer of HPRT and APRT in an intraspecific human cell system

Chromosome-mediated transfer of genes between human cell lines was accomplished using HeLa cells as chromosome donors and HT1080 fibrosarcoma lines as recipients. This report describes the intraspecific transfer of two genetic markers, hypoxanthine-guanine-phosphoribosyl-transferase (HPRT ⁺)and adenine phosphoribosyltransferase (APRT ⁺).The isolation and characterization of the necessary enzyme-deficient (HPRT ^? and APRT ^?)recipient HT1080 cell lines are also described. The chromosome-mediated gene transfer was carried out using a modification of the procedure of Miller and Ruddle, including treatment of the donor chromosomes with calcium phosphate and subsequent exposure of the recipient cells of dimethyl sulfoxide. In experiments to optimize this procedure for HT1080 cell recipients, we found that a brief (2-min) exposure to high DMSO concentration (20%) was effective for enhancing transfer efficiencies in this system. Transfer frequencies (transferents per recipient cells assayed) averaged approximately 1×10^?6 for HPRT ⁺ and were >2×10^?6 for APRT ⁺.     

UV mutagenesis,cytotoxicity and split-dose recovery in a human—CHO cell hybrid having intermediate (6−4) photoproduct repair

Somatic cell hybrids constructed between UV-hypersensitive Chinese hamster ovary cell line UV20 and human lymphocytes were used to examine the influence of a human DNA repair gene, ERCCI, on UV photoproduct repair, mutability at several drug-resistance loci, UV cytotoxicity and UV split-dose recovery. In hybrid cell line 20HL21-4, which contains human chromosome 19, UV-induced mutagenesis at the APRT, HPRT and Na⁺/K⁺-ATPase loci was comparable to that in repair-proficient CHO AA8 cells, whereas cell line 20HL21-7, a reduced human-CHO hybrid not containing human chromosome 19, exhibited a hypermutable phenotype at all 3 loci indistinguishable from that of UV20 cells. The response of 20HL21-4 cells to UV cytotoxicity reflected substantial but incomplete restoration of wild-type UV cytotoxic response, whereas responses of UV20 and 20HL21-7 cell lines to UV cytotoxicity were essentially the same, reflecting several-fold UV hypersensitivity. Repair of UV-induced (5–6) cyclobutane dimers and (6−4) photoproducts was examined by radioimmunoassay; (6−4) photoproduct repair was deficient in UV20 and 20HL21-7 cell lines, and intermediate in 20HL21-4 cells relative to wild-type CHO AA8 cells. UV split-dose recovery in 20HL21-4 cells was also intermediate relative to AA8 cells. These results show that the human ERCCI gene on chromosome 19 is responsible for substantial restoration of UV survival and mutation responses in repair-deficient UV20 cells, but only partially restores (6−4) UV photoproduct repair and UV split-dose recovery.     

Efficient modification of the APRT gene by FLP/FRT site-specific targeting

The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5×10^–5, which were 6–22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT⁺ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination.     

Impaired P2X and P2Y receptor-mediated signaling in HPRT-deficient B103 neuroblastoma cells

Defect of hypoxanthine phosphoribosyl transferase (HPRT) causes Lesch-Nyhan disease (LND), but the link between HPRT deficiency and the self-injurious behavior of LND is unknown. In a previous study (Pinto et al., J. Neurochem. 72 (2005) 1579-1586) we reported on a decrease in nucleotidase activity in membranes of several HPRT⁻ cell lines and fibroblasts from LND patients. Since nucleotidases are involved in ATP-induced signal transduction, in the present study, we tested the hypothesis that P2X and P2Y receptor-mediated signal transduction is impaired in HPRT deficiency. As model we studied rat B103 neuroblastoma cells. Compared to control cells, in HPRT⁻ cells, NTP and NDP-induced Ca²⁺ influx across the membrane and Ca²⁺ mobilization from intracellular stores were impaired. Both P2X and P2Y receptors were involved in the responses. Quantitative real-time PCR revealed reduced expression of receptors P2X₃, P2X₅, P2Y₂, P2Y₄, P2Y₁₂, P2Y₁₃ and P2Y₁₄ in HPRT deficiency. Collectively, HPRT deficiency is associated with abnormal purinergic signaling, encompassing P2X and P2Y receptors and nucleotidases.     

Spontaneous reversion of novel Lesch-Nyhan mutation byHPRT gene rearrangement

Molecular analysis of an unusual patient with the Lesch-Nyhan syndrome has suggested that the mutation is due to a partial HPRTgene duplication. We now report the cloning and sequencing of the mutant HPRTcDNA which shows the precise duplication of exons 2 and 3. This mutation is the result of an internal duplication of 16–20 kilobases of the gene. The structure of the mutant gene suggests that the duplication was not generated by a single unequal crossing-over event between two normal HPRTalleles. Growth of Epstein-Barr virus-transformed lymphoblasts from this patient in selective medium has permitted isolation of spontaneous HPRT⁺ revertants of this mutation. The reversion event involves a second major HPRTgene rearrangement where most or all of the duplicated portion of the mutant gene is deleted. The original mutation therefore has the potential for spontaneous somatic reversion. This may explain the relatively mild symptoms of the Lesch-Nyhan syndrome exhibited by this patient.     

Inhibition of sodium transport by angiotensin II in the main duct of the rabbit mandibular gland isolated and perfused in vitro

Summary The effect of angiotensin II on nett electrolyte transport by the main duct of the rabbit mandibular gland was investigated in vitro using a perfused duct preparation bathed in a Haemaccel^®-nutrient salt solution. In a bath concentration of 4×10^–10 M, angiotensin reduced nett absorption of Na⁺ and Cl^– by about 8% and depolarized the transepithelial electrical potential difference (P.D.) by about 13%; the drug had no effect on ductal transport of K⁺ and HCO ₃ ^– . In both lower (4×10^–11 M) and higher (4×10^–9 M) concentrations, angiotensin had qualitatively similar effects. After exposure to the hormone for about 30 min, Na⁺ transport and P.D. became unstable and gradually fell away towards zero. It is concluded that angiotensin in physiological concentrations has a specific inhibitory effect on Na⁺ absorption by salivary duct cells which could arise either from a change in the Na⁺ pump rate or from a conductance change in the apical or basal membrane of the epithelial cell.     

Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade−A mutant deficient in this activity

We report here the isolation of a human cDNA encoding the first step in de novo purine biosynthesis, amidophosphoribosyltransferase (PRAT). The human PRAT cDNA was isolated by complementation of a Saccharomyces cerevisiae ade4 mutant deficient in PRAT enzymatic activity. The identity of the isolated cDNA, designated pAdeA-3, was confirmed by several independent methods. Genomic DNA sequences homologous to pAdeA-3 show coordinate segregation with the hypoxanthine nutritional requirement in Chinese hamster ovary (CHO) cell Ade^–A-human hybrids, segregants of these hybrids, and irradiation reduction hybrids. The PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT enzyme deficiency in CHO Ade^–A mutants. This correction was monitored by both cell-free PRAT assays and in vivo phosphoribosylformylglycinamide (FGAR) accumulation studies. FGAR accumulation is a classic method for assessment of the early steps of purine nucleotide biosynthesis. Two of the isolated transformants, designated PRAT-1 and PRAT-2, exhibited 22% and 53%, respectively, of wild-type CHO K1 PRAT enzymatic activity using a cell-free enzyme assay. These same two transformants plus an additional transformant, designated PRAT-13, showed FGAR accumulations of 150%, 260%, and 140%, respectively, compared to the levels of accumulation seen in CHO K1. Transformants PRAT-1 and PRAT-2 both contained a mRNA species recognized by the PRAT cDNA of identical size to a mRNA species in human fibroblasts homologous to the PRAT cDNA. This observation, along with the functionality of the cDNA in both yeast and CHO cells deficient in PRAT activity, suggests the isolated cDNA is full length.     

Transfer of the human X chromosome to human-Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack HPRT activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in HAT medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of HAT-resistant clones was 32 × 10^–6 when 10⁷ cells were incubated with 10⁸ HeLa chromosomes. Potential reversion of the hybrid cells in HAT medium was less than 5 × 10^–7 The 16 isolated cell lines all contained activity of the human X-linked marker enzymes HPRT, PGK, -Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Production of transmitochondrial mouse cell lines by cybrid rescue of rhodamine-6G pre-treated L-cells

Treatment of mouse LMTK⁻ cells with the toxic mitochondrial dye rhodamine 6G (R-6G) at 2.5 μg/ml for 7 days prevented cell growth while maintaining viability, with less than 10⁻⁶ cells recovering to form colonies. Pre-treatment of LMTK⁻ cells with R-6G was followed by fusion with enucleated mouse 501–1 cells harboring a homoplasmic point mutation in the mitochondrial DNA (mtDNA) 16S rRNA gene conferring chloramphenicol resistance (CAP^R). Cybrids and any surviving unfused LMTK⁻ cells were selected in BrdU with or without CAP and their mtDNAs screened for the presence of the CAP^R marker. Approximately 1 colony per 2×10⁵ LMTK⁻ cells appeared in the fusion plates selected both with and without CAP. Most clones investigated were confirmed to be cybrids by showing the presence of the generally homoplasmic CAP^R mutation, whether or not CAP selection was used. Hence, R-6G pre-treatment permits construction of transmitochondrial cybrid cell lines carrying a variety of mtDNAs, without the need for ρ^o cell lines.