Molecular cloning of genes involved in purine biosynthetic and salvage pathways of Salmonella typhimurium

Summary Genes have been cloned from Salmonella typhimurium which when present on the multicopy plasmid pBR322 in the E. coli strain NT31 confer a Gua⁺ phenotype on this strain. NT31 is a purE gpt double mutant and it was expected that a Gua⁺ phenotype could be conferred on it by the cloning of either gpt or purE. It was, however found that in addition to these two loci the molecular cloning of another gene, which has been identified as hpt, in pBR322 confers a Gua⁺ phenotype on NT31. This result is explained by the overproduction of the hpt gene product, hypoxanthine phosphoribosyl transferase, which compensates for the lack of the gpt product guanine-xanthine phosphoribosyl transferase. Restriction analysis of the three loci, gpt, hpt and purE is also presented.Abbreviations Kb kilobase pairs - Tc tetracyline - m.o.i multiplicity of infection - 8AG 8-azaguanine     

Purine mutants of mammalian cell lines: III. Control of purine biosynthesis in adenine phosphoribosyl transferase mutants of CHO cells.

Spontaneous and mutagen-induced 2,6-diaminopurine-resistant mutants of Chinese hamster ovary (CHO-K1) cells were isolated. Such mutants fell into two classes: spontaneous and ethylmethane-sulfonate-induced mutants had approximately 5% wild-type adenine phosphoribosyl transferase (APRT) activity, whereas ICR-170G-induced mutants had barely detectable APRT activity. Since it has been reported that human hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Lesch-Nyhan syndrome) and APRT mutants over-produce purines, we examined the control and rate of purine biosynthesis in the Chinese hamster mutants. End product inhibition by adenine could not be demonstrated in such mutants, indicating that the active feedback inhibitor is a nucleotide rather than the free purine base, HGPRT activity was normal in all mutants examined except in one isolate. Purine biosynthesis as measured by the accumulation of the purine biosynthetic intermediate phosphoribosyl formylglycineamide was not elevated in the mutants as might have been predicted from work with Lesch-Nyhan cells. The data also suggest that our strain of CHO-K1 is physically or functionally haploid for the APRT locus.     

Profiles of hypoxanthine guanine phosphoribosyl transferase and adenine phosphoribosyl transferase activities measured in single preimplantation human embryos by high-performance liquid chromatography

The profiles of hypoxanthine guanine phosphoribosyl transferase (HGPRT) and adenine phosphoribosyl transferase (APRT) activities were examined in normally fertilized human embryos developing at the normal rate in vitro between the 2-4-cell stage on Day 2 and the blastocyst stage on Day 6 after insemination. The activities of both enzymes were assayed simultaneously in extracts of single embryos by measuring the rate of production of the reaction products, inosine monophosphate (IMP) and adenine monophosphate (AMP), separated by high-performance liquid chromatography (HPLC). The activity profiles of the two enzymes over this period showed marked differences. The activity of HGPRT, coded by the X chromosome, increased between Days 2 and 4 (P less than 0.01) but declined sharply by Day 6 (P less than 0.001), whereas autosome-coded APRT activity remained low between Days 2 and 5, but increased on Day 6 (P less than 0.05). The profile of HGPRT activity may reflect a combination of decreasing levels of maternal enzyme inherited from the oocyte and the initiation of embryonic gene expression followed by X inactivation at the blastocyst stage on Day 6.     

Sexing of preimplantation mouse embryos by measurement of X-linked gene dosage in a single blastomere

Single blastomeres were isolated from zona-free 8-cell mouse embryos and assayed for X-linked hypoxanthine phosphoribosyl transferase (HPRT) activity and autosome-linked adenine phosphoribosyl transferase (APRT) activity. At this stage of development both X chromosomes are active in female embryos. Hence, a bimodal distribution of HPRT: APRT ratios, corresponding to male (XY) and female (XX) biopsied samples, was observed due to the 2-fold difference in gene dosage for HPRT activity. Batches of putative male and female embryos identified in this way were transferred to pseudopregnant recipient females. Development of the seven-eighths embryos was equivalent to that of control zona-free intact embryos. Sex determination by measurement of X-linked gene dosage was accurate and rapid enough to allow transfer of embryos of known sex without the need for cryopreservation.     

Mutations affecting adenine phosphoribosyl transferase activity in Chinese hamster cells

Mutants of Chinese hamster ovary cells that were resistant to the adenine analog 2, 6-diaminopurine generally were deficient in the enzyme adenine phosphoribosyl transferase. Such mutants did not occur spontaneously, and were rare even after mutagenesis, presumably because two active genes in this pseudodiploid line must be affected for the recessive drug-resistant phenotype to be expressed. Revertants of one such double mutant were selected on the basis of their ability to utilize adenine as a purine source. These revertants contained reduced levels of enzyme activity and were presumed to be heterozygous for the enzyme structural gene. Forward mutation to diaminopurine resistance, starting with these heterozygous revertants, occurred at a rate 1,000 times greater than that found for the original homozygous cells. The results agree with predictions based on the idea that mutations in the structural gene for the enzyme are responsible for the drug-resistant character of these variants.     

Genes conferring resistance to 8-aza adenine in Neurospora crassa and the variability of resistant alleles in the aza-1 locus with respect to excretion of purines

Summary Strains inhibited by analogues of aromatic amino acids due to mutations in the 5mt locus were also sensitive to the purine analogues, 8-aza adenine, 8-aza guanine and 2,6-diaminopurine. Adenine or hypoxanthine and the respective ribosides relieved inhibition due to purine analogues but guanine or guanosine were not effective. Using resistance to 8-aza adenine and sensitivity to DL-4-methyl tryptophan as criteria aza-1 ^r and aza-2 ^r mutants were isolated and both loci were located in linkage group I the former was 10–20 units distal to the mating type and the latter approximately 2 units in the vicinity of mating type. The four allelic aza-1 ^r strains were resistant to 8-aza adenine and 2,6-diaminopurine but one of them showed increased resistance to 8-aza guanine also. The aza-2 ^r strain was resistant to both 8-aza analogues but was comparable to the parental aza-2 ^s strain in its sensitivity to 2,6-diaminopurine. One aza-1 ^r was distinguished by its ability to excrete hypoxanthine and/or inosine and this ability always segregated with the aza-1 ^r allele and appears to be a consequence of a single mutation in the aza-1 locus. In heterokaryon one aza-1 ^r and one aza-2 ^r alleles were found to be recessive to the respective wild type alleles. Uptake of exogenous adenine was reduced in germinating conidia of one aza-1 ^r and one aza-2 ^r strain when compared to a parental aza-1 ^s, aza-2^s strain but the low affinity of 8-aza adenine to compete with adenine even in the sensitive strain indicated a need for direct study of the transport and metabolic reactions of the analogue. If an altered purine phosphoribosyl transferase is involved in the aza-1 ^r phenotype, an analogy with the Lesch-Nyhan syndrome of man, with respect to analogue-resistance and altered regulation of purine metabolism, seems to be appropriate.     

Occurrence of a regulatory deficiency in purine biosynthesis among purA mutants of Salmonella typhimurium

Summary A defect in the repression of the de novo purine biosynthetic enzymes was detected among purA mutants of Salmonella typhimurium. We suggest that the defect is caused by an altered purine regulation gene (purR) which affects the response level of at least five of the de novo enzymes to repression by excess adenine. Thus the unlinked genes controlling these enzymes constitute a regulation controlled wholly or in part by a purR gene product. The regulation of the guanine operon is regulated by some other mechanism independent of purR.     

Renal hypertrophy in experimental diabetes. The activity of the ''de novo'' and salvage pathways of purine [corrected] synthesis.

Measurements were made of the activity of phosphoribosyl pyrophosphate amidotransferase (PPRibP-At, EC 2.4.2.14) and of adenine (APRT, EC 2.4.2.7) and hypoxanthine (HPRT, EC 2.4.2.8) phosphoribosyltransferases, representing the 'de novo' and salvage pathways respectively. PPRibP-At activity increased within 3 days of diabetes, whereas APRT and HPRT increased later. Incorporation of [14C]formate and of [8-14C]adenine into the nucleic acids of kidney slices showed that formate was incorporated earlier, and to a greater extent, than was adenine. These results indicate that, although the 'de novo' pathway for nucleotide synthesis is the main route in early diabetes, the salvage pathway assumes greater importance at later stages.     

Genetics of resistance to 4-aminopyrazolo-(3,4-d)-pyrimidine in Saccharomyces cerevisiae

Summary Thirty six mutants resistant to the purine analogue 4-aminopyrazolo(3,4-d)pyrimidine were isolated from a prototrophic strain of yeast carrying su-pur, a suppressor of purine excretion. The mutants were allocated to seven genes app1 to app7. Linkage was found between app3, app5, app6 and app7 but not close enough to suggest functional grouping. Some of the alleles of app1, app3 and app4 are dominant. None of the mutants excreted purine when out-crossed to remove su-pur, nor did they show allelism or linkage to any of the pur (purine excretion) genes. Two mutants, app6-30 and app7-31, are recessive in diploids homo-/or heterozygous for su-pur but are dominant in diploids homozygous for su-pur ⁺. The mutants exhibit gene specific, and in one case allele specific, patterns of cross-resistance to other analogues of adenine, hypoxathine and guanine. In the presence of su-pur all seven genes confer resistance to the adenine analogue 8-azaadenine; in addition mutants of app1, app3 (dominant alleles only) and app4 are resistant to 6-methylpurine, 6-mercaptopurine, 8-azaguanine and guanine. Combination with su-pur ⁺ confers resistance to all analogues tested except in the cases of app7, which remains sensitive to 8-azaguanine and app2 which is rendered sensitive to the five purine analogues. Exogenous adenine or hypoxanthine increases the growth rate of wild type (su-pur), app2, app6 and app7 but not app1, app3, app4 and app5. These effects of purine supplementation on strains of the genotype ade2 app sup-pur suggest that mutants of app1, app3, app4 and app5 are defective in the utilisation of exogenous purines. It is suggested that the other three genes may have defects in the control of de novo purine synthesis.Supported by a Medical Research Council Research Training Scholarship Awarded to W.R.P.     

General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum

Summary To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a supersuppressor selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HAT^r) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT mutants — selected by their resistance to 6-thioguanine (TG^r) — and TK^- mutants — selected by their resistance to 5-bromodeoxyuridine (BrdUrd^r) — are HAT^s. Therefore, from HPRT^- TK^- double nonsense mutants, whose phenotype would be TG^r BrdUrd^r (HAT^s), simultaneous HPRT⁺ TK⁺ double phenotypic revertants could be obtained by selecting HAT^r (TG^s BrdUrd^s) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TG^r variants, 53 of which were HAT^s and the rest HAT^r. Among 36 TG^r (HAT^s) variants tested, 23 did not revert to HAT^r, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT^- nonsense mutants because they were very stringent (had less than 2% of wild-type [³H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrd^r) in 7 of these strains allowed us to isolate 29 TG^r BrdUrd^r (HAT^s) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HAT^r (TG^s BrdUrd^s) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.     

Male sterility associated with APRT deficiency in Arabidopsis thaliana results from a mutation in the gene APT1

Four mutants of Arabidopsis thaliana that are deficient in adenine phosphoribosyl transferase (APRT) activity have been isolated by selecting for germination of seeds and growth of the plantlets on a medium containing 2,6-diaminopurine (DAP), a toxic analog of adenine. In all mutants, DAP resistance is due to a recessive nuclear mutation at a locus designated apt. The mutants are male sterile due to pollen abortion after meiosis. Furthermore, it has been shown that metabolism of cytokinins is impaired in the mutant BM3, which has the lowest level of APRT activity among the mutants tested. However, three different cDNAs encoding APRT have been isolated in A. thaliana and this raised the question of the nature of the mutation which results in low APRT activity. The mutation was genetically mapped to chromosome I and lies within 6 cM of the phenotypic marker dis2, indicating that the mutation affects the APT1 gene, a result confirmed by sequencing of mutant alleles. The mutation in the allele apt1-3 is located at the 5′ splicing site of the third intron, and eliminates a BstNI restriction site, as verified by Southern blotting and PCR fragment length analysis. Received: 20 February 1997 / Accepted: 28 August 1997     

Use of the HPRT gene and the HAT selection technique in DNA-mediated transformation of mammalian cells: first steps toward developing hybridoma techniques and gene therapy.

In 1956, I decided to apply my experience in microbial genetics to developing analogous systems for human cell lines, including the selection of mutants with either a loss or gain of a biochemical function. For instance, mutants resistant to azahypoxanthine showed a loss of the HPRT enzyme (hypoxanthine phosphoribosyl transferase), whereas gain of the same enzyme was accomplished by blocking de novo purine biosynthesis with aminopterin, while supplying hypoxanthine and thymine (HAT selection). Using HAT selection, we: (i) genetically transformed HPRT- mutant cells to HPRT+ wild type by using DNA extracted from HPRT+ cells, and (ii) selected HPRT+ hybrid cells by fusing HPRT- D98/AH2 cells with skin cells. These approaches, which we dubbed in 1962 as a 'first step toward gene therapy', contributed to the later development of (i) cell fusion techniques, (ii) the development of monoclonal antibodies, (iii) routine transformation of mammalian cells with cloned genes, and (iv) methods for creating transgenic organisms.     

The Isolation and Characterization of Saccharomyces Cerevisiae Mutants That Constitutively Express Purine Biosynthetic Genes

In response to an external source of adenine, yeast cells repress the expression of purine biosynthesis pathway genes. To identify necessary components of this signalling mechanism, we have isolated mutants that are constitutively active for expression. These mutants were named bra (for bypass of repression by adenine). BRA7 is allelic to FCY2, the gene encoding the purine cytosine permease and BRA9 is ADE12, the gene encoding adenylosuccinate synthetase. BRA6 and BRA1 are new genes encoding, respectively, hypoxanthine guanine phosphoribosyl transferase and adenylosuccinate lyase. These results indicate that uptake and salvage of adenine are important steps in regulating expression of purine biosynthetic genes. We have also shown that two other salvage enzymes, adenine phosphoribosyl transferase and adenine deaminase, are involved in activating the pathway. Finally, using mutant strains affected in AMP kinase or ribonucleotide reductase activities, we have shown that AMP needs to be phosphorylated to ADP to exert its regulatory role while reduction of ADP into dADP by ribonucleotide reductase is not required for adenine repression. Together these data suggest that ADP or a derivative of ADP is the effector molecule in the signal transduction pathway.     

Hypoxanthine-guanine phosphoribosyl transferase with altered substrate affinity in mutant mouse lymphoma cells

Cells with altered hypoxanthine-guanine phosphoribosyl transferase (HPRT) (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) have been selected. Compared to wild type, mutant enzyme has a reduced affinity for the substrate phosphoribosyl pyrophosphate and is more labile to heat inactivation. Mutant cells are resistant to 6-thioguanine at 33-39 degrees C and sensitive to hypoxanthine-aminopterin-thymidine at 37-39 degrees C, but not at 33 degrees C. We hypothesize that a single structural mutation of HPRT can explain these results.     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

Positive selection for male-sterile mutants of Arabidopsis lacking adenine phosphoribosyl transferase activity

Three mutants of Arabidopsis thaliana deficient in adenine phosphoribosyl transferase activity were isolated by selecting for germination of seeds on a medium containing 0.1 millimolar 2,6-diaminopurine. In each of the mutants, diaminopurine resistance was due to a recessive nuclear mutation at a locus designated apt. The mutants grow more slowly than wild type, and are male sterile due to abortion of pollen development after the meiotic divisions of the pollen mother cells. The reliability and ease with which the mutants can be selected should afford novel opportunities to investigate purine metabolism, pollen development, and genetic problems which require the ability to select for loss-of-function mutations.     

Cloning the complete human adenine phosphoribosyl transferase gene

We have isolated a clone from a human genomic lambda library which cross-hybridises with the cloned hamster adenine phosphoribosyl transferase gene (aprt). After restriction mapping and further hybridisation to the hamster gene, a series of putative human aprt-containing fragments has been isolated and tested for ability to transform adenine phosphoribosyl transferase-deficient (aprt-) strains of Chinese hamster ovary (CHO) cells to APRT proficiency. Transforming activity was detected in a 48-kb lambda clone, the 17.4-kb EcoRI insert, and an 8.6-kb HincII fragment. Smaller fragments have thus far shown no transforming activity. Transformants appear to be stable for the APRT+ phenotype, and human aprt DNA sequences are present in the hamster transformants. The 8.6-kb HincII fragment has been subcloned and the insert mapped. Nonrepetitive regions of this subclone have been identified, and should prove valuable for chromosome walking studies on human chromosome 16, familial studies of a human aprt- trait, the analysis of restriction fragment length polymorphisms (RFLPs) in the area surrounding the aprt gene, and the fine structure mapping of the mutations induced by chemical carcinogens and alkylating agents.     

Adenine phosphoribosyl transferase polymorphism in baboons

Two allelic isozymes of adenine phosphoribosyl transferase (APRT) were detected by starch gel electrophoresis of baboon hemolysates. Extensive family data verified autosomal codominant inheritance. The gene frequencies of five subspecies of baboons differed significantly. The activity of erythrocyte APRT is sufficiently high to enable the use of this enzyme as a sensitive marker for assessing chimerism in research involving bone marrow transplantation.This research was supported in part by NIH Grants HL28972, HG00336, and HV53030.     

Characterization of the biochemical basis of a complete deficiency of the adenine phosphoribosyl transferase (APRT)

Summary In order to study the biochemical basis of a complete deficiency of adenine phosphoribosyl transferase (APRT) the enzyme was purified to homogeneity, its properties were characterized, and antibodies raised. The enzyme is indirectly involved in adenine uptake. Apparently, by forming AMP the internal concentration of adenine is kept low allowing its diffusion.The same APRT is present in various tissues as was revealed by antibody inactivations employing anti-erythrocyte APRT as well as by direct enzyme assays in cells from the APRT deficient patient. In vitro cultured fibroblasts derived from this patient had less than 0.02% enzyme activity. No cross-reacting material was found in erythrocytes obtained from an APRT deficient child.     

Inhibition by 6-mercaptopurine of purine phosphoribosyltransferases from Ehrlich ascites-tumour cells that are resistant to this drug

1. A strain of Ehrlich ascites-tumour cells that showed little inhibition of growth in the presence of 6-mercaptopurine accumulated less than 5% as much 6-thioinosine 5′-phosphate in vivo, in the presence of 6-mercaptopurine, as did the sensitive strain from which it was derived. 2. Specific activities of the phosphoribosyltransferases that convert adenine, guanine, hypoxanthine and 6-mercaptopurine into AMP, GMP, IMP and 6-thioinosine 5′-phosphate were similar in extracts of the resistant and the sensitive cells. 3. As found previously with sensitive cells, 6-mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase from the resistant cells and does not inhibit the adenine phosphoribosyltransferase from these cells. Michaelis constants and inhibitor constants of the purine phosphoribosyltransferases from resistant cells did not differ significantly from those measured with the corresponding enzymes from sensitive cells. 4. Resistance to 6-mercaptopurine in this case is probably not due to qualitative or quantitative changes in these transferases.