INCREASED CONCENTRATIONS OF GLYCINE IN HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE-DEFICIENT MOUSE NEUROBLASTOMA CELLS1

—The intracellular concentrations of a number of amino acids were compared in a parental line of mouse neuroblastoma cells, a line selected for gross deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and in a revertant line containing the enzyme selected from the deficient line. Most prominent was the increased concentration of free glycine and to a lesser extent glutamic acid in enzyme-deficient cells. The increase in glycine was eliminated after restoration of the HGPRT activity.     

Reversion in expression of hypoxanthine-guanine phosphoribosyl transferase following cell hybridization.

Hybridization of mutant cell lines deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C.: 2.4.2.8) from a variety of established rodent sources with HGPRT plus human cells yielded progeny cells which grew in selective medium containing hypoxanthine, aminopterin and thymidine (HAT). The same result was obtained when the human cell used was an HGPRT minus transformed line derived from a patient with the Lesch-Nyhan syndrome. Electrophoretic analysis indicated that all HAT-resistant progeny clones contained an active HGPRT enzyme which was indistinguishable from the wild type enzyme of the corresponding normal rodent cells. In contrast, no HAT-resistant cells have been obtained when the same HGPRT minus rodent cells were subjected to fusion processes in the absence of human cells or when they fused with similarly derived HGPRT minus mutant cells of other rodents. Reversion in expression of the rodent gene for HGPRT was detected in clones which retained one or more human chromosomes and in clones which contained no detectable human chromosomal material. The observed re-expression of rodent HGPRT in HAT-resistant clones suggests that HGPRT plus as well as HGPRT minus human cells contributed a factor which determined the expression of respective rodent structural genes for HGPRT. In contrast, HGPRT minus rodent cells were unable to induce the synthesis or normal HGPRT in the cells derived from the patient with the Lesch-Nyhan syndrome.     

Reversion in expression of hypoxanthine-guanine phosphoribosyltransferase in 6-thioguanine resistant neuroblastoma: evidence for reduced enzyme levels associated with unaltered catalytic activity.

Five clones of mouse neuroblastoma cells able to grow in hypoxanthine-aminopterin-thymidine containing medium were isolated from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8; IMP: pyrophosphate phosphoribosyltransferase) deficient cell line. These hypoxanthine-aminopterin-thymidine resistant revertant clone had 45-55% of wild-type cell HGPRT activity. Kinetic studies indicated that the HGPRT in revertant clones had a reduced maximal velocity as compared to wild type cells based on cell protein. Apparent Km values of HGPRT for hypoxanthine and 5-phosphoribosyl-1-pyrophosphate were similar in wild-type and revertant cells. Heat inactivation studies demonstrated a similar heat lability for HGPRT in revertant and wild-type cells. An antibody fraction prepared from serum of rabbits immunized with HGPRT partially purified from mouse liver was used to measure the amount of cross-reacting material in normal and revertant clones. The revertant clones had one-half the amounth of cross-reacting material present in wild-type cells, based on a given amount of cell protein. These data indicate that the revertant cells may contain fewer HGPRT molecules with unaltered catalytic activity.     

Presence of two active X chromosomes in hybrids between normal human and SV40-transformed fibroblasts from patients with the Lesch-Nyhan syndrome

Skin fibroblasts (LNSV) derived from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient patient with the Lesch-Nyhan syndrome, who has glucose-6-phosphate dehydrogenase (G6PD) type A, were transformed with SV40 and hybridized with WI38 human diploid fibroblasts derived from a female embryo which have normal HGPRT and G6PD type B activities. The hybrid clones selected in hypoxanthine, aminopterin and thymidine (HAT) medium, were essentially tetraploid and contained three X and one Y chromosomes. These hybrids contained HGPRT, types A and B and the AB heteropolymeric form of G6PD enzymes which were indicative that in these cells X linked genes of both parental cells were fully active. Hybrids back-selected in medium containing 8-azaguanine (8-AG) contained only two X chromosomes. They had no HGPRT activity and they contained only G6PD type A enzyme. It is concluded that the hybrid cells which grew in the presence of 8-AG retained the X chromosome of the LNSV parental cell and apparently the inactive X of the WI 38 cell.     

Lipochromosome mediated gene transfer: Identification and probable specificity of localization of human chromosomal material and stability of the transferents

Summary Using lipochromosomes (phospholipid-entrapped chromosomes) we have transferred the human HGPRT gene into HGPRT deficient mouse cells (A9) with a frequency of approximately 1×10⁻⁵ (Mukherjee et al., Proc. Natl. Acad. Sci. USA 75: 1361–1365; 1978). Two other genes located on the long arm of the human X-chromosome were also expressed in two independently derived populations of transferents (A9/GT3 and A9/GT4). We report here the chromosomal and enzymatic composition of human HGPRT-positive clones from each subpopulation analyzed in detail with alkaline Giemsa-11 staining. All the clones expressed human PGK and HGPRT, but one (A9/GT4C6) lacked human G6PD. In each of four clones examined microscopically, a small piece of presumptive human chromatin was visible in the karyotypes of most cells. The chromatin fragment was free or attached in each cell of an individual clone. When integrated, the human chromosomal fragment in each clone appeared associated with the centromere of the same telocentric A9 chromosome (No. 6 by Q-banding). These data suggest that: (a) substantial human chromosomal fragments can be transferred into recipient cells using the lipochromosome technique; (b) clones from human HGPRT positive A9 transferent subpopulations may or may not possess other human X-linked markers; (c) the stability of lipochromosomally transferred genes varied from clone to clone and stability is generally poor in the absence of continuous selection pressure (e.g., HAT); (d) when multiple X-linked human genes were transferred to mouse cells a cytologically detectable human chromosomal fragment was identified free or attached to a host chromosome; and (e) integration of transferred human chromosomal material into mouse chromosomes may occur at preferential site(s) in the recipient genome. This research was sponsored in part by the Office of Health and Environmental Research U.S. Department of Energy under Contract W-7405-eng-26 with the Union Carbide Corporation.     

Biological and biochemical characterisation of purine analogue resistant clones of V79 Chinese hamster cells

Purine analogue resistant clones have been selected from the closely related Chinese hamster lines V79A and V79S. Clones were of either spontaneous origin or induced by EMS or ultraviolet light. The majority of clones selected in 8-azaguanine showed stable cross resistance to 6-thioguanine. Clones derived from V79A and selected for 6-thioguanine resistance were cross resistant to 8-azaguanine: however a group of 6-thioguanine resistant mutants selected from V79S cells were 8-azaguanine sensitive. All clones except two were unable to grow in HAT medium. The two exceptions were 8-azaguanine resistant, showed partial sensitivity to 6-thioguanine, and also differed in other biochemical characteristics. HGPRT activity was measurable in extracts of all clones under standard conditions. In many clones, HGPRT activity increased as the hypoxanthine concentration was reduced. Whole cell uptake of [14C] hypoxanthine was low in all cases examined and was not modified by incubation in the presence of amethopterin. The heat sensitivity and electrophoretic mobility of HGPRT in extracts of some clones was compared to that in wild-type extracts. All clones tested except one, which was consistently HAT positive, showed enhanced heat sensitivity and reduced electrophoretic mobility. None of the mutants reverted spontaneously at detectable frequency but some could be induced to revert by EMS. The presence of measurable enzyme with altered properties in all clones suggests that these revertable drug resistant clones represent missense mutants.     

Genetic complementation in hybrid cells derived from mutagen-induced mouse clones deficient in HGPRT activity.

Cultured mouse clonal cells, H-5, were treated with two different mutagens, ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Then two selective procedures using 8-azaguanine (8-AZ) or 6-thioguanine (6-TG) were compared in an effort to isolate hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-deficient cells containing different gene alterations. While many 8-AZ resistant cells were induced by EMS treatment, considerably more 6-TG resistant cells were induced by the same treatment. MNNG treatment induced many 8-AZ resistant mutants but induced hardly any 6-TG resistant mutants. After a fusion experiment of 91 sets involving 13 HGPRT-deficient mouse clones, 7 of which were resistant to 8 AZ and 6 of which were resistant to 6TG with subsequent selection on HAT medium, complementation occurred only in those hybrid mixtures formed between 8-AZ- and 6-TG-resistant clones, while it did not occur at all in hybrid mixtures formed between different 8-AZ-resistant clones and mixtures formed between different 6-TG-resistant clones. The clonally isolated HGPRT-positive cells, characterized by tetraploid karyology, had an apparent activity of HGPRT ranging from 25 to 30% of that of the wild-type parental cells. Heat-inactivation of HGPRT at 65 °C revealed that HGPRT from wild-type cells was heat stable and HGPRT from some 8-AZ-resistant clones were heat labile, while HGPRT from hybrid cells had intermediate stability. These results indicate that there would be alterations in the structural gene of HGPRT in the 8-AZ- or 6-TG-resistant mutants, and also that two selective procedures with 8-AZ or 6-TG alone can isolate different alterations in the structural gene of HGPRT. Moreover, this indicates that some of these gene alterations were mutually complementary. It is most likely that there would be at least 3 cistrons in the locus responsible for HGPRT activity in the mouse cells.     

Characterization of hypoxanthine-guanine phosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electrophoretic method

The hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in a group of man-mouse somatic cell hybrids, produced by Sendai virus-mediated cell fusion and HAT selection, has been analyzed by a new electrophoretic technique. Evidence is presented which shows that the hybrid lines derived from fusion of a mouse fibroblast deficient in HGPRT with various human cell strains have an HGPRT activity that is characteristic of the human enzyme, whereas a hybrid line derived from a mouse fibroblast which is deficient in thymidine kinase has an HGPRT activity characteristic of the mouse. This new technique involves electrophoresis of cell extracts on cellulose acetate gel, followed by the localization of the enzyme activity by autoradiography.This research was supported in part by a research grant from the U.S. National Institutes of Health (No. GM-13415).     

The molecular nature of spontaneous and chemically induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene in rat liver epithelial cells

The molecular nature of mutations in 6-thioguanine-resistant hypoxanthine/guanine phosphoribosyl transferase (HGPRT)-deficient clones of an adult rat liver (ARL) epithelial cell line mutated by benzo[a]pyrene or aflatoxin B1 was studied. DNA from these clones or spontaneous HGPRT-deficient mutants was subjected to Southern blotting using an HGPRT probe following DNA digestion with the restriction enzymes BamH1, EcoR1, HindIII or XbaI. With either the chemically induced or spontaneous mutants, no difference in restriction fragment pattern was observed between any of the mutants and their wild-type parent. However, differences were found between two lines ARL 6 and ARL 14 and the lines ARL 18, ARL 19 and DNA from Fischer rat hepatocytes. Although the variants did not display loss of HGPRT activity. It is suggested that deletion or loss of a pseudogene sequence could be the basis for the alterations in restriction fragment patterns.     

The isolation and preliminary characterisation of 6-thioguanine-resistant mutants of human diploid fibroblasts

Mutant clones of human diploid fibroblasts deficient in the enzyme, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were selected by their ability to grow in medium containing the cytotoxic purine analogue, 6-thioguaninine (6TG). The optimal conditions for mutant selectiom were 6TG concentrations between 1 and 5 μg ml^?1 and cell plating densities ～ 10³ cells cm^?2.Nine spontaneous and four radiation-induced 6TG-resistant mutants had <2% of the parental strain HGPRT activity and were unable to grow in medium containing azaserine. These mutants were phenotypically stable during > 25 population doublings in non-selective medium and five mutants that were examined showed no gross change from the normal human karyotype.Evidence is presented to show that 6TG is a better selective agent than 8-azaguanine (8AG) for HGPRT-deficient mutants of human diploid fibroblasts.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9¹⁴-C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5–3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Suppressed expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in an irradiation-attenuated Plasmodium berghei XAT strain

Plasmodium berghei XAT (XAT) is a non-reversible, non-lethal type malaria parasite strain derived from the highly virulent lethal P. berghei NK65 (NK65) by X-irradiation. The difference in polypeptide expression between NK65 and XAT was examined in this study. Western blot patterns of the parasite polypeptides showed that a 30-kDa polypeptide was not detected in XAT. In the present paper, we focused the study on the difference in the expression of the 30-kDa polypeptide between XAT and NK65. Although several other significant differences were noted in the spots shown by two-dimensional gel electrophoresis, the 30-kDa polypeptide was isolated by means of preparative 2D-gel electrophoresis followed by HPLC, and N-terminal amino acid sequence of the polypeptide was eventually determined. Complementary DNA clones encoding the 30-kDa polypeptide were isolated and characterized. Full-length cDNA clones from XAT encoded a protein of 231 amino acid residues with a 693-bp open reading frame. The deduced amino acid sequence exhibited 67% identity with that for P. falciparum hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8), suggesting that this protein is P. berghei HGPRT. Northern blot analysis revealed that expression of HGPRT in XAT was only one-eighth of that in NK65. This finding indicates that HGPRT gene expression is markedly suppressed in XAT. The amino acid sequence of HGPRT from NK65 was identical to that from XAT. This finding showed that the amino acid sequence of XAT-HGPRT was not mutated and had not undergone deletion.     

THE CHARACTERIZATIO OF MONOAMINE OXIDASE IN CULTURED MAMMALIAN CELLS

The expression of monoamine oxidase (MAO) in a variety of mammalian cells has been investigated employing tryptamine as substrate. The enzyme present in those cell lines having sufficient activity for detailed analysis exhibited a monophasic response to the inhibitor clorgyline. On this basis the cell lines examined were found to express only A, or only B, type activity. Hypoxanthineguanine phosphoribosyl transferase (HGPRT) deficient derivatives of both MAO type A, or MAOtype B, expressing cells were examined. The HGPRT status of the cells appeared to have little influence on the expression of the enzyme.     

Studies on 1-beta-D-arabinofuranosyl-cytosine (Ara-C) resistant mutants of Chinese hamster fibroblasts. II. High resistance to Ara-C as a genetic marker for cellular hybridization

From a variety of independent Chinese hamster cell lines, stable variants resistant to 5 μg/ml of Ara-C were isolated via a single step selection; in contrast to variants selected at lower drug concentrations, the resistant clones appear to be uniformly deficient in Ara-C phosphorylation, an activity previously shown [14] to be carried out in hamster cells by a cytoplasmic dC-kinase (dC-kinase 2). These dC-kinase deficient (dCK^?) variants can be selected against because they are unable to divide in a medium containing dT (0.8 mM) and dC (0.01 mM), which supports the growth of wild type dCK⁺ cells. Plating of dCK^? cells in medium supplemented with both nucleosides yields only rare clones of pseudorevertants which escape the thymidine block through a secondary unknown defect; the growth of these clones can be prevented by further addition of dA to the selective medium. As expected from the complementation pattern for the deficient enzyme activities, hybrids between a dCK^? hamster line and TK^? lines of either mouse or hamster could be isolated in a modified HAT medium (HAT₅₀dC) containing dC and an increased dT concentration. In principle, the same selection can be used to isolate interspecific and intraspecific hybrids between Ara-C resistant variants obtained from a variety of mammalian species and azaguanine resistant lines deficient in HGPRT. The potential interest of this sytem for genetic mapping is discussed.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9-¹⁴C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5-3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Rapid determination of hypoxanthine-guanine-phosphoribosyl transferase in human fibroblasts and amniotic cells.

A micromodification of the method of HGPRT and APRT assay is described, which measures the incorporation of 14C hypoxanthine and 14C adenine into cultured skin fibroblasts and amniotic cells grown on microtiter plates. Only about 10000 cells are needed per assay. By this method HGPRT deficient cells can be easily distinguished from normal cells. Investigations with respect to the effect of substrate concentrations and time of incubation have been carried out on some normal fibroblast cell lines, amniotic cell lines and 3 Lesch-Nyhan cell lines. Another modified method is described for quantitative determination of HGPRT activity by means of radio thin-layer chromatography.     

HGPRT structural gene mutation in Lesch-Nyhan-syndrome as indicated by antigenic activity and reversion of the enzyme deficiency

Summary For three patients with the Lesch-Nyhan syndrome the existence of normal amounts of catalytically inactive hypoxanthine-guanine phosphoribosyltransferase (HGPRT) protein was demonstrated by using antibodies against the normal enzyme subunits. The lack of enzyme activity is reverted in virus transformed cells. Individual revertant cell clones contain different HGPRT enzymes as demonstrated here by isoelectric focusing. The data strongly support the idea of a structural gene mutation as the cause of enzyme deficiency in the Lesch-Nyhan syndrome.     

Immunological characterization of hypoxanthine-guanine phosphoribosyl transferase mutants of mouse L cells: Evidence for mutations at different loci in the HGPRT gene

A large collection (105) of mouse L cell mutants lacking hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT; E. C. 2.4.2.8) were analyzed for the presence of serologically cross reacting material (CRM). Antibody directed against highly purified mouse liver HGPRT was used for detecting CRM activity by two methods: (1) the standard precipitation-inhibition assay; and (2) a radioimmune-precipitation assay. The latter assay proved to have far greater sensitivity for the detection of altered forms of HGPRT. Approximately 40% of the HGPRT⁻ cell lines contain CRM activity (i.e., were CRM⁺). This indicates that a minimum of 40% of the HGPRT⁻ clones arose as a result of mutations in the HGPRT structural gene. The CRM⁺ cell lines were shown to contain different levels of CRM activity. Measurements of the heat sensitivity of CRM in the different HGPRT⁻ cell lines showed a broad spectrum of CRM heat inactivation kinetics. These latter two observations provide strong evidence that the mutations giving rise to the HGPRT⁻CRM⁺ phenotype occurred at different sites in the HGPRT structural gene.     

Osmoprotective effect of glycine betaine on thrombopoietin production in hyperosmotic Chinese hamster ovary cell culture: clonal variations

When 23 recombinant Chinese hamster ovary (rCHO) cell clones were cultivated in hyperosmolar medium resulting from NaCl addition (533 mOsm/kg), their specific thrombopoietin (TPO) productivity (q(TPO)) was increased. However, due to depressed cell growth at elevated osmolality, no enhancement in the maximum TPO titer was made in batch cultures of all 23 clones. To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved TPO production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures of 23 clones were performed in the presence of 15 mM glycine betaine. Glycine betaine was found to have a strong osmoprotective effect on all 23 clones. Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled 22 clones to grow at 542 mOsm/kg, where most clones could not grow in the absence of glycine betaine, but at a cost of reduced q(TPO). However, the relative decrease in q(TPO) varied significantly among clones. Thus, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among clones. Six out of 23 clones displayed more than a 40% increase in the maximum TPO titer in the hyperosmolar medium containing glycine betaine, compared with that in the standard medium with a physiological osmolality. Taken together, the results obtained here emphasize the importance of selection of clones for the successful use of hyperosmotic pressure and glycine betaine as an economical means to improve TPO production.     

Reversion of the 8-azaguanine resistant phenotype of variant Chinese hamster cells treated with alkylating agents and 5-brono-2'-deoxyuridine.

From cultures of V79 Chinese hamster cells, 10 independent clones of 8-azaguanine resistant cells were isolated and subcultured. Cells from all ten clones were resistant to 1 mg/ml levels of 8-azaguanine (8-AzG), contained less than 3% of the wild type levels of the enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT), and were unable to grow in HAT medium. The ten clones were classified according to the conditions under which they reverted to the wild type phenotype. Clones in classes I and II reverted spontaneously with frequencies of 40-10(-5) and about 3-10(-5) respectively, and the reversion frequency was independent of the density of cells of all but one of the clones in the culture medium used. Class II clones evinced increased reversion frequencies with ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and to a lesser extent with 5-bromo-2'-deoxyuridine (budR), suggesting that these clones contained point mutations in a locus which controls HGPRT activity. The processes of reversion and toxicity appeared to be associated. Class III clones did not revert spontaneously or with BUdR and MNNG, but did revert with EMS. The reversion frequency of class I clones was not increased after treatment with EMS, MNNG or BUdR.