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.     

Hypoxanthine transport in normal and hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient diploid human lymphoblasts

We have examined the possible relation between hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.7., HGPRT) activity and hypoxanthine transport in the normal human lymphoblast line MGL8 and two HGPRT- mutant lines derived from it. The mutant line MGL8A29 (L8A29) had considerable amounts of material cross-reacting immunologically to HGPRT, while mutant MGL8A18 (L8A18) had none. In the normal cells, hypoxanthine is taken up by both a saturable and non-saturable process. Kinetic studies show that the velocity of transport is much lower than HGPRT activity, while both have similar values of K_m. In the two mutant lines, we failed to demonstrate saturable transport, and the rates of hypoxanthine uptake by these cells were directly proportional to its concentration in the medium. Active HGPRT molecules appear to be related to the saturable transport process.     

Immunochemical identification of the chick HPRT gene transferred from chick erythrocytes to mammalian somatic cells

Immunochemical methods were used to identify the genetic origin of hypoxanthine phosphoribosyltransferase (HPRT) expressed in heteroploid, HPRT-deficient mouse (A9) cells and Chinese hamster ovary (K627) cells, after these cells were fused with chick embryo erythrocytes and selected for resistance to hypoxanthine-aminopterin-thymidine (HAT) medium. All of the HAT-selected clones produced HPRT activity which was immunoprecipitable by an antiserum specific for chick HPRT, but not by an antiserum specific for mouse and hamster HPRT. Furthermore, the HPRT activity in these clones was electrophoretically indistinguishable from chick liver HPRT and clearly different from mouse liver HPRT. These data provide evidence that the HPRT activity expressed in cell hybrids produced by the fusion of HPRT-negative mammalian cells and chick erythrocytes containing genetically inactive nuclei is indeed coded by the chick HPRT gene and that an avian gene can be stably incorporated and correctly expressed in a mammalian cells.     

Analysis of HGPRT- CRM+ human lymphoblast mutants.

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.     

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.     

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.     

Mutations affecting the antigenic properties of hypoxanthine-guanine phosphoribosyl transferase in cultured Chinese hamster cells.

Cells of the mutant Chinese hamster strain RJK10 do not contain either hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT) or protein that cross-reacts immunologically with HGPRT. HGPRT+ revertants have been isolated from RJK10 and those strains produce HGPRT with altered antigenic properties. HGPRT from the revertant cells is less reactive with anti-HGPRT serum than enzyme from the wild-type cells, and enzymes from the two sources are immunoprecipitated independently from mixtures of cell extracts. Thus one or more of the antigenic determinants present on Chinese hamster HGPRT are either missing or present in an altered form on HGPRT from revertants of RJK10. This indicates that RJK10 carries a mutation in the structural gene for HGPRT and that secondary mutations in the gene give rise to the revertants that produce the antigenically altered enzymes.     

Elevated intracellular glycine associated with hypoxanthine-guanine phosphoribosyltransferase deficiency in glioma cells

Abstract— The intracellular concentrations of a number of amino acids were measured in a normal clone of rat glioma cells, and in several independently derived clones selected for gross deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). A significant, approx 2-fold increase in the concentration of free glycine was observed in both mutagenized and non-mutagenized HGPRT deficient clones. The increase in glycine was independent of the phase of cell growth. A similar increase did not occur in HGPRT deficient lymphoblasts.     

Intraspecies transfer via total cellular DNA of the gene for hypoxanthine phosphoribosyltransferase into cultured mouse cells

Summary Under selective growth conditions a revertant of mouse cells, defective in hypoxanthine phosphoribosyltransferase activity (HPRT, EC-No. 2.4.2.8), was isolated, which contained an electrophoretically abnormal form of HPRT activity. The specific HPRT activity in crude extracts of the revertant cells is about 30% of the level determined in normal wild type cells. The variant HPRT reacts with antiserum against normal mouse HPRT but the rate of heat inactivation of the variant activity is different from the wild type form. By isozyme and karyotype analyses of somatic cell hybrids between the revertant mouse cells and Chinese hamster cells we found that the abnormal HPRT activity is coded for by the mouse X-chromosome as expected for a mutation in the structural HPRT gene.DNA has been purified from the abnormal HPRT revertant cells and incubated with mouse A9 cells (HPRT^-). After growth in selective medium one clone was isolated which expressed the electrophoretically abnormal form of HPRT. Six clones showed the normal form of HPRT due to reversion of the defective HRRT locus in A9 cells. This result indicates DNA-mediated transfer of the mouse HPRT gene at a frequency of about 0.5×10^-7. A similar frequency has been found for transfer of the variant HPRT locus via isolated metaphase chromosomes to A9 recipient cells. When placed in non-selective media the DNA-mediated transferent cells gradually lost their ability to express the HPRT transgenome at a rate of about 6% per average cell generation.     

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.     

Transfer of purine metabolites between cells through the medium and via cell contacts in cocultures of HGPRT+ and HGPRT- cells

Cells with and without hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity were used to examine the transfer of purine metabolites through the medium and via cell contacts. HGPRT- Chinese hamster and human fibroblasts were able to incorporate 3H-labeled purine metabolite(s) from medium in which mouse HGPRT+ B82 cells had been grown for 24 h with [3H]hypoxanthine, but mouse A9 fibroblasts that were deficient in HGPRT, adenine phosphoribosyltransferase (APRT), and methylthioadenosine phosphorylase (MTAP) were unable to incorporate these metabolites. This suggests that in recipient cells incorporation is due to [3H]MTA, which has been shown previously to be the major 3H-labeled purine metabolite to accumulate in B82 medium, being cleaved by MTAP to [3H]adenine, which is phosphoribosylated by APRT to [3H]AMP. Incorporation by recipient cells of metabolites from the medium is referred to as contact-independent metabolite transfer (CIMT). In autoradiograms of B82/A9 cocultures that were labeled with [3H]hypoxanthine, grains were found over A9 that were not in contact with B82, although A9 did not act as recipients of CIMT. This is termed proximity-dependent metabolite transfer (PDMT). Both CIMT and PDMT interfered with the assessment of nucleotide exchange between HGPRT+ and HGPRT- cells through cell contacts, which is referred to as contact-dependent metabolite transfer (CDMT). These problems were unique to HGPRT+ mouse L cells. However, HGPRT- mouse L cells, A9, could be used as potential recipients. A9 were positive recipients of CDMT with only one of five cell lines tested, which suggested that these cells were selective communicators. CDMT could not be studied with [3H]guanine because the nuclei of HGPRT- cells became labeled.     

Modulation of enzyme activity in azaguanine-resistant V79 cells selected by chronic drug exposure

Exposure of V79 cells to azaguanine (7-21 microM for 2-7 weeks) had little effect on growth or plating efficiency but resulted in gradual acquisition of resistance to 8-azaguanine (AZ) and 6-thioguanine (TG) and loss of ability to grow in HAT. The rate of evolution of the resistant phenotype was dependent on the concentration and duration of exposure to AZ. The increase in proportion of resistant cells was paralleled by a rise in phosphatase activity (pH optimum 7.0-7.5) expressed by intact cells and this preceded the fall in HGPRT activity. Elevated phosphatase activity and a resistant phenotype were stably expressed in clones isolated and cultured in the absence of AZ. Hypoxanthine guanine phosphoribosyl transferase (HGPRT) activity in cell extracts of three resistant clones ranged from 18 to 43% of wild-type levels but was unaltered with respect to substrate affinity and electrophoretic mobility. Mg2+-dependent activity dephosphorylated inosine 5'monophosphate (IMP), guanine 5'monophosphate (GMP), adenosine-5-monophosphate (AMP) and p-nitrophenylphosphate (PNPP) and was also elevated with respect to wild-type levels in resistant cell extracts. Purine nucleoside phosphorylase levels were similar in sensitive and resistant cell extracts. Cross-sensitivity studies with other purine analogues suggest that the elevated phosphatase activity does not contribute to the resistant phenotype. No karyotypic changes were observed in the resistant cell lines.     

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.     

Induction of supermelanin synthesis and morphological changes in interspecific reconstituted cells and its reversal by tumor promoter

Chloramphenicol-resistant (CAPr) reconstituted cells and cybrids were isolated by fusion of karyoplasts (or intact cells) of mouse amelanotic melanoma B16 cells with cytoplasts of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) -deficient, CAPr rat myoblastic cells, L6TG.CAPr, and double selection in HAT medium containing CAP. Reconstituted cells or cybrids exhibited unique cellular arrangement, and about one third of the isolated clones expressed high tyrosinase activity and marked melanin synthesis, although the parental mouse cells expressed low tyrosinase activity and the parental rat cells did not express tyrosinase activity. These phenotypic changes have been stable for more than a year. The phenotypic reversions of these clonal cells were induced by treatment with a tumor promoter. There were changes in the morphology of the treated cells to that of the mouse B16 cells and extinction of tyrosinase activity and melanin synthesis in pigmented clonal cells. These phenotypic changes and reversions induced by a promoter were repeatedly reversible.     

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.     

Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines.

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.     

Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.     

Synthesis and degradation of alcohol dehydrogenase in wild-type and Adh-null activity mutants of Drosophila melanogaster

Both the amount and the size of alcohol dehydrogenase-like cross-reacting material was determined in 14 ethyl methanesulfonate (EMS)-induced alcohol dehydrogenase-null activity mutants. In 11 mutants cross-reacting material of the same apparent molecular weight as alcohol dehydrogenase was detected, while in 3 mutants no cross-reacting material was found. In all cases, the amount of cross-reacting material found in the mutants was lower than that in wild-type flies. High, intermediate, and low cross-reacting material-producing mutants showed similar initial rates of incorporation of labeled amino acid into alcohol dehydrogenase-like protein, presumably reflecting similar rates of synthesis. If the rate of synthesis of cross-reacting material is the same in the mutants as in the wild type, then the different levels of cross-reacting material must be due to different rates of degradation.Supported by NIH Grants GM-18254 and ES-01527 and DOE Contract EY-76-S-2965.     

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.