Incorporation of hypoxanthine by PHA-stimulated HPRT-deficient lymphocytes

Phytohemagglutinin (PHA) markedly stimulates ³H-hypoxanthine incorporation by lymphocytes of normal subjects as revealed by radioautography. There is no corresponding increase in activity of hypoxanthine phosphoribosyltransferase (HPRT) in lysates but the level of phosphoribosylpyrophosphate (PRPP), the cosubstrate for the reaction, is higher. Lymphocytes from a patient with partial HPRT deficiency responded to PHA as did the normals, whereas the response in Lesch-Nyhan (LN) subjects was variable. PHA-stimulated lymphocytes from two LN patients showed some increase in ³H-hypoxanthine incorporation, while two others failed to respond. The observations provide further evidence of genetic heterogeneity among LN patients.     

Syndrome de Lesch-Nyhan

Résumé L'existence de deux populations cellulaires, l'une HGPRT⁺ et l'autre HGPRT^- a pu être démontrée en cultures de fibroblastes à partir de biopsies cutanées chez la soeur, la mère et une soeur de la mère d'un patient atteint du syndrome de Lesch-Nyhan. Ce but est aisément atteint en réalisant une sélection biochimique des cellules mutantes par la 8-azaguanine avant l'autoradiographie des cultures incubées avec l'³H-hypoxanthine; parallèlement les cellules normales sont isolées dans le milieu HAT.Ceci nous paraît être une méthode sûre et relativement peu compliquée, de diagnostiquer dans les familles atteintes, les femmes hétérozygotes dont la descendance mâle court un risque important de syndrome de Lesch-Nyhan. C'est précisément le cas de la jeune soeur du patient, dont toute grossesse éventuelle doit être suivie dès le début et exige une détection prénatale de cette affection.

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Lesch-Nyhan syndromeHeterozygote detection by biochemical selection of the mutant cells and autoradiography

Summary Two distinct cell populations (the first HGPRT⁺, the other HGPRT^-) were isolated in skin fibroblasts cultures from a sister, the mother and a maternal aunt of a patient with Lesch-Nyhan syndrome, an X-linked disorder.This result was achieved by means of a biochemical selection prior to autoradiography with ³H-hypoxanthine. Mutant cells were isolated in a medium containing 8-azaguanine, while normal cells were grown in HAT medium.This proved to be an easy and reliable method for detecting female heterozygotes in families where a patient with Lesch-Nyhan syndrome had been observed. A pre-pregnancy diagnosis of these female carriers is advisable because of the high probability of a full-blown syndrome in their male descendants. This was particularly the case of the 12 years old proband's sister in the family studied here.

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Zusammenfassung Zwei unterschiedliche Zellpopulationen (HGPRT+und HGPRT-) wurden aus den Hautfibroblasten einer Schwester, der Mutter und einer Tante mütterlicherseits eines Patienten mit X-chromosomalem Lesch-Nyhan-Syndrom isoliert.Das Ergebnis wurde mit Hilfe biochemischer Selektion vor der Autoradiographie mit ³H-Hypoxanthin erzielt. Mutierte Zellen wurden in einem Nährmedium mit 8-Azaguanin isoliert, während normale Zellen im HAT-Medium wuchsen. Diese erwies sich als eine einfache und zuverlässige Methode zur Erkennung von weiblichen Heterozygoten in Familien von Patienten mit Lesch-Nyhan-Syndrom. Wegen des hohen Risikos des Auftretens des Syndroms bei männlichen Nachkommen ist die pränatale Diagnostik bei weiblichen Trägern indiziert. In diesem Fall gilt das für die 12jährige Schwester des Probanden.

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Ce travail a reçu l'appui du Fonds de la Recherche Scientifique Médicale (Belgique).     

Intercellular communication and tissue growth

Summary Three cancer cell strains that fail to make permeable membrane junctions were tested for ability to transfer an endogenous hypoxanthine derivative from cell to cell. The cells of these strains, loaded with³H-hypoxanthine, were grown in contact with cells from a mutant line incapable of incorporating exogenous hypoxanthine. The transfer of the³H-hypoxanthine derivative to the mutant cells was determined by radio-autography and, in the same preparations, the presence of permeable membrane junctions was determined by intercellular fluorescein tracer diffusion and electrical measurement. The cells of the three strains showed no transfer of hypoxanthine derivative to contiguous mutant cells; the cells that make permeable junctions did show such transfer, under the same conditions.In contrast to this contact-requiring mode of transfer, a contact-independent transfer phenomenon was observed with these three cancer cell strains.     

Karyological characterization of a human lymphoblastoid cell line resistant to 6-thioguanine

Summary A mutant human lymphoblastoid cell line, Raji-TG, resistant to 10g 6-thioguanine (TG)/ml was produced from wild-type cells after exposure to ethylmethane sulfonate. The Raji-TG cells showed their failure to incorporate ³H-hypoxanthine, only 2% as much hypoxanthine guanine phosphoribosyl transferase (HPRT) activity as wild-type cells, and no revertant in HAT selective medium containing hypoxanthine, aminopterin, and thymidine. Raji-TG cells, which were maintained routinely in regular medium lacking TG for as long as 2 years, still retained resistance to the drug and inability to grow in HAT medium. A fusion of Raji-TG cells and mouse cells resistant to 5-bromodeoxyuridine and lacking thymidine kinase formed hybrids, and the resulting hybrid colonies proliferated in HAT medium. These observations strongly supported the hypothesis that Raji-TG line cells might be originated from a mutational event with deficiency of HPRT. Both parental and the mutant have a modal chromosome number of 49 with a remarkably stable karyotype. Excess chromosome materials are found in chromosomes 1, 5, 7, 14, and 16. Chromosome 8 is completely missing, but is represented by two respective isochromosomes of the short and long arms of No. 8. Five different marker chromosomes could be distinguished, and most of their origin has been determined. Isolation of Raji-TG X mouse hybrid clones which contained one of each marker chromosome is of considerable value in mapping human genes on regions within particular chromosomes.     

Interallelic complementation in hydrid cells derived from Chinese hamster diploid clones deficient in hypoxanthine-guanine phosphoribosyl-transferase activity

Activity of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) has been demonstrated in the hybrid cells formed from the fusion of clonal cells from four Chinese hamster diploid clones, Cl. 250, 252, 253, and 254 cells derived from clonal wild type cells, deficient in this enzyme activity, by the stepwise treatment with 8-azaguanine and 6-thioguanine. The HGPRT-positive cells, characterized by tetraploid karyology, were isolated clonally by the chemical selection (HAT) in 3 hybrid mixtures; Cl. 250 × 254, 252 × 254, and 253 × 254 cells, while none was isolated in another seven hybrid mixtures including 4 homologous mixtures. The enzyme activity was proved by enzymological as well as autoradiographical studies with the use of ³H-hypoxanthine. These results indicate that mutually complementable alterations rather than gene deletions were induced in the 2 cistrons of the locus responsible for the relevant enzyme activity in hamster cells by analog treatment. Interallelic complementation occurred in these hybrid cells.     

MONOAMINE OXIDASE ACTIVITY IN NORMAL and LESCH-NYHAN FIBROBLASTS

Monoamine oxidase (MAO) activity was studied in cultured skin fibroblasts from 10 Lesch-Nyhan patients, a Lesch-Nyhan variant and 11 controls matched for age. sex and race. Activity (predominantly type A) was measured in cell homogenates using tryptamine as the substrate. For each line activity varied with the conditions of culture. Activity increased 3-10 fold as cultures went from logarithmic to stationary phase of growth. When cultures were confluent, activity was lowered by frequent feedings or the use of fresh medium and serum. Activity for each line remained fairly stable during successive passages, but rose 3-8 fold as cultures became senescent. When comparing activity between control and Lesch-Nyhan lines, cells were cultured under standardized conditions. The mean value of MAO activity in Lesch-Nyhan lines was approximately one fourth of the mean activity in control lines (P < 0.012), In the control population, the distribution of activity appeared to be bimodal. Activities in the Lesch-Nyhan lines fell completely within the lower portion of the control distribution. Cells from a Lesch-Nyhan patient who lacked several of the neurologic symptoms of the disease (including self-mutilation) had an MAO activity 6 fold greater than the control mean. although his hypoxanthine phosphoribosyltransferase activity was <3% of control levels. It appears that: (1) MAO activity is low in fibroblasts from typical Lesch-Nyhan patients: (2) the severity of neurologic symptoms may be correlated with levels of MAO activity: and (3) some interaction between purine and catecholamine metabolism can affect nerve function.     

Alterations of alkaline phosphatase activity during adaptation of Escherichia coli to phosphite and hypophosphite

When Escherichia coli cells were grown in media containing either phosphite or hypophosphite as the sole source of phosphorus, they responded to this situation primarily in the same way as phosphatelimited cultures: The activity of alkaline phosphatase increased drastically, which under natural conditions would enable the cells to compklensatae for the shortage increased drastically, which under natural conditions would enable the cells to compensate for the shortage of phosphate. Subsequent transfers, however, resulted in a quite different response: While the phosphatase activity of phosphate-limited cells stays at a high derepressed level, its increase was followed by a gradual decline in organisms grown on phosphite or hypophosphite. After eight to ten transfers on these P-compounds, phosphatase activity was back to its initial, repressed, low level, indicating that the cells were fully adapted to these substrates. Adaptation to either PO ₃ ^3- or PO ₂ ^3- was completely abolished if the cells were again grown with PO ₄ ^3- as P-source, whereafter the entire process of adaptation had to be repeated. The observed adaptation pattern, reflected by the alterations of phosphatase activity, was qualitatively equal with PO ₃ ^3- and PO ₂ ^3- , but quantitatively different, because the response to hypophosphite gave much higher values than the increase obtained with phosphite.Phosphite-adapted cells are not simultaneously adapted to hypophosphite, but their response to the latter was less intense than observed after direct transfers from PO ₄ ^3- to PO ₂ ^3- . Adaptation to hypophosphite, however, led simultaneously to phosphite adaptation, so that these cells can utilize both P-compounds as a substitute for phosphate.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing aminopterin

When seeded in small numbers in medium containing 10^?6M aminopterin and fetal calf serum, V₇₉ Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10^?6M aminopterin and dialyzed serum, provided that the medium was supplemented with 10^?5M hypoxanthine and sufficient 5·10^?6M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V₇₉ cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT⁺ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT⁺ cells to prevent the growth of the latter in HAT medium. One of the HGPRT^? lines showed a high rate of spontaneous reversion (118/10⁵ cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to $\text{4}\text{10}{}^5\text{cells}$, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.     

Stage-Dependent Toxicity of N-Acetyl-Glucosamine to Plasmodium falciparum1

The effects of N-acetyl-glucosamine on growth of synchronized cultures of Plasmodium falciparum were assessed by morphological observations and by measurement of parasite incorporation of ³H-hypoxanthine. Inhibition of ³H-hypoxanthine incorporation was more marked during the later stages of the erythrocytic cycle. At concentrations of the sugar below 20 mM, however, the deleterious effects were mainly a result of failure of released merozoites to invade erythrocytes, rather than a failure of schizonts to mature or release merozoites. These results are compatible with the hypothesis that a lectin-like substance on the merozoite interacts with a surface glycoprotein on the red cell and that sugar residues on this glycoprotein may be involved in this recognition.     

Evaluation of the severity of hypoxanthine-guanine phosphoribosyltransferase deficiency using viable T cells

Peripheral T cells from 3 Lesch-Nyhan patients, 3 normal subjects, and 3 brothers with hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency but without Lesch-Nyhan syndrome (so-called partial deficiency) have been analyzed. Although these brothers contained HGPRT activities neither in the hemolysates nor in the T cell extracts at levels detectable by the regular radioenzyme assay, the enzyme deficiency had not caused any typical neurological symptoms of the Lesch-Nyhan syndrome. Although the T cells from these brothers were at least 10-fold more resistant to 6-thioguanine than normal T cells, they were more than 30-fold less resistant than the T cells from 3 Lesch-Nyhan patients indicating that there is a clear difference in the severity of the enzyme deficiency between the brothers and the Lesch-Nyhan patients. These data indicate that the long-term T cell culture in the medium containing a purine analog whose toxicity depends on a salvaging enzyme is useful for evaluating the severity of the enzyme deficiency in viable cells.     

Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant

Summary Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vg ^B/+ flies are reared on these substances (vg^B is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies.Our data suggest that the vg ⁺ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.     

The type and time of occurrence of aminopterin-induced chromosome aberrations in cultured Potorous cells

Many inhibitors of DNA synthesis have been found to induce chromosome aberrations. Our kinetic studies indicate that treatment of cellswith 10^?7M aminopterin in the presence of 10^?4M glycine, 10^?4M hypoxanthine, and 10^?4M thymidine allows continued normal cell growth. Omission of thymidine, a treatment which is known to inhibit DNA synthesis while allowing RNA and protein synthesis to continue, leads to cessation of cell growth. Treament of Potorous cell cultures with aminopterin in the presence of hypoxanthine and glycine without thymidine led to the following observations: (1) only non-exchange chromatid aberrations were formed after aminopterin treatment; (2) the aberrations were induced only in cells treated during S, and the breaks were associated with the replicating region of the chromosome; (3) breaks were observed at the first metaphase after the beginning of treatment; and (4) thymidine could reverse the chromosome-breaking action of aminopterin. A model for the molecular mechanism is suggested.     

A novel class of unstable 6-thioguanine-resistant cells from dog and human kidneys

Thioguanine-resistant primary clones were grown from single cell suspensions obtained from dog and human kidneys by enzymatic digestion. In medium containing a relatively high concentration (10g/ ml) of thioguanine, thioguanine-resistant primary clones arose from each source at frequencies ranging from 10^–4 to 10^–5. A reduction in total hypoxanthine uptake was found in the thioguanine-resistant primary clones which had developed in thioguanine medium, consistent with a reduction in hypoxanthine phosphoribosyltransferase activity. When these thioguanine-resistant primary clones were subsequently grown in the absence of thioguanine and assayed for the thioguanine-resistant phenotype and hypoxanthine phosphoribosyltransferase activity, it was found that most were now thioguanine-sensitive and yielded cell free extracts with substantial amounts of hypoxanthine phosphoribosyltransferase activity. In contrast, thioguanine-resistant human clones grown continuously in the presence of thioguanine yielded cell free extracts with little or no detectable hypoxanthine phosphoribosyltransferase activity. Southern blot analysis demonstrated no structural alterations in the hypoxanthine phosphoribosyltransferase gene in thioguanine-resistant primary human kidney clones. These results suggest that a novel mechanism(s) for thioguanine resistance and the control of hypoxanth phosphoribosyltransferase expression may occur in dog and human kidney cells.Abbreviations AG 8-azaguanine - APRT adenine phosphoribosyltransferase - DAPI 4-6 diamino-2-phenylindole - DV Dulbecco-Vogt - HAT hypoxanthine, aminopterin, thymidine - HPRT hypoxanthine phosphoribosyltransferase - PRPP 5-phosphoribosyl 1-pyrophosphate - TG 6-thioguanine - TG^r thioguanine-resistant - TG^s thioguanine-sensitive - TIP thymidine triphosphate     

Non-selective isolation of fibroblast-cybrids by flow sorting

Red- and green-fluorescing polystyrene beads were used to label different populations of cultured human fibroblasts. After enucleation of the green-fluorescing population, the cytoplasts were fused with red-fluorescing cells. Twenty-four hours after cell fusion the population of red-green heterofluorescent cells was isolated with a FACS II cell sorter. When Lesch-Nyhan fibroblasts (HPRT⁻) were fused with cytoplasts from control fibroblasts (HPRT⁺) more than 95% of the sorted cells were heterofluorescent and 90% of the sorted cells showed HPRT⁺ activity. Therefore almost all sorted heterofluorescent cells are true cybrids. With this procedure for cybrid isolation, earlier complementation studies using cybrids from different variants of β-galactosidase deficiency could be confirmed.     

Dihydrofolate reductase and aminopterin resistance in Pneumococcus

Summary Wild-type pneumococci derived from Avery's strain R36A are sensitive to extracellular concentrations of the folate antimetabolite aminopterin exceeding 1.0x10^-6 M. Three classes of resistant strains are phenotypically distinguishable: amiB-r, amiA-r and amiD-r strains are resistant to low (1.5x10^-6 M), intermediate (0.5–4.0×10^-5 M) and high (4.5x10^-4 M) aminopterin levels respectively. The amiA and amiB regions are weakly linked, but linkage has not been established between either of these loci and the amiD region.Consistent with the maximum resistance conferred by mutations in the amiA locus, dihydrofolate (FH₂) reductase in cell-free extracts (CFE) of amiA-r strains has a two- to six-fold greater affinity for the substrate than dose the enzyme in wild-type CFE (Table 1); FH₂ reductase from amiA-r strains may also have reduced affinity for aminopterin. Specific activity of the enzyme is not affected by mutation in the amiA locus (Table 1) and its affinity for the cofactor (NADPH) is probably unaffected by mutation in this locus (Table 4). Dihydrofolate reductase activity in amiA5 CFE is considerably more thermolabile than that in wild-type CFE (Table 2).The enzyme in CFE of the high resistance strain amiD1 has the same affinity for the substrate, cofactor and antimetabolite as FH₂ reductase in wild-type CFE (Figs. 1–4, 8 and 9; Table 4). However, specific activity of the enzyme in amiD1 CFE is 11-fold higher than that in wild-type CFE (Table 1) and it is much more heat stable (Table 2).Some properties of FH₂ reductase in CFE of the high resistance recombinant strain amiA5amiD1 are intermediate between those in CFE of wild-type and amiD1.Preliminary results suggest that CFE of wild-type and amiA5 contain a factor, which is neither dialyzable nor heat sensitive, that has an inhibitory effect upon activity and stability of FH₂ reductase in amiD1 CFE (Tables 2 and 3).     

Gene transfection of the HuLy-m2 (Leu-9) antigen into mouse L cells

Human DNA was transfected into mouse L cells and tk⁺ HuLy-m2⁺ (= CD7⁺) transfectants isolated after growth in hypoxanthine, aminopterin, thymidine medium and repeated cloning. After several cycles of transfection, > 90% of HuLy-m2⁺ L cells could be detected, by rosetting and by cytofluorography, which showed the transfectants to have a density of CD7 two to five times that found on peripheral blood lymphocytes. Despite this, the 37 kd CD7⁺ dimer could only be identified with difficulty using cell-surface radioiodination and sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques. An antiserum was produced (C3H anti-HuLy-m2⁺ L cells) which, after absorption, was shown to react with HuLy-m2 antigens present on human thymocytes and lymphocytes and on CD7⁺ transfected L cells.Abbreviations BSA bovine serum albumin - DME Dulbecco's modified Eagle's medium - EDTA ethylenediamine-tetraacetate - HAT hypoxanthine, aminopterin, thymidine - HSV herpes simplex virus - PBL peripheral blood lymphocyte - PBS phosphate-buffered saline - RFC rosette-forming cell - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - tk thymidine kinase     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.     

Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells.

HPRT mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., HAT or HAsT medium). However, it has been shown that some HPRT- clones are resistant to both HAT and HAsT medium. The present study was undertaken to investigate whether any common structural gene alteration exists for such 6TGr-HATr-HAsTr clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of MNU and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the HPRT gene, these 4 investigated 6TGr-HATr-HAsTr clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and L-azaserine suggesting that these clones carry mutations in the structural part of the HPRT gene. Sequence analysis of PCR-amplified HPRT cDNA from these mutants showed that the spontaneous and the 2 MNU-induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic DNA after digestion with BglII, EcoRI and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic DNA using exon 4-specific primers showed that all these 3 mutants had an AT to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the HPRT protein.     

The spontaneous azaguanine-resistant mutants of diploid human fibroblasts

Summary The range of incidences of azaguanine-resistant colonies in cultures of fibroblasts from 16 unrelated humans was 0.4×10^-6 to 19×10^-6 and the mean value was 4.1×10^-6. A fluctuation test showed that most or all of the mutant colonies derived from mutations that occurred during in vitro proliferation of the fibroblasts and before exposure to azaguanine. The estimated rate of spontaneous mutation was 0.45×10^-6 to 1.8×10^-6 per cell generation. At least ten independent mutants, comprising two general classes, were studied. Class I mutants were a minority and resembled cells from boys having the Lesch-Nyhan syndrome: they had very little HG-PRT activity, showed maximum resistance to azaguanine and could not utilize hypoxanthine for growth. At least 90% of the mutants were in Class II: their apparent HG-PRT activities ranged between normal and Lesch-Nyhan amounts, they were partially sensitive to azaguanine and they could utilize hypoxanthine. Some Class II mutants resembled cells cultured from a family having an X-chromosomal mutant gene that does not cause the Lesch-Nyhan syndrome but does confer resistance to azaguanine, although the quantity of HG-PRT activity is apparently normal and hypoxanthine can be utilized. Electrophoretic differences between the HG-PRT activities of normal and mutant strains were not detected but other qualitative alterations were observed in some mutants.Paper No. 1558 from the Laboratory of Genetics.Supported by N.I.H. Grants GM-06983 and GM-15422 and by a grant from the Food Research Institute of The University of Wisconsin, Madison, Wisconsin.Supported by Grant He 753-1 from Die Deutsche Forschungsgemeinschaft.     

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

Summary 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 10 000 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.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.