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D Patterson

Publications and source records attributed to D Patterson.

At least 235 records · Page 13Linked to original sources
PubMed1979

Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells. II. Isolation and characterization of a mutant of Chinese hamster ovary cells with defective dihydroorotate dehydrogenase (E.C. 1.3.3.1) activity.

A mutant (A204) of Chinese hamster ovary cells (CHO-K1), which is deficient in dihydroorotate (DHO) dehydrogenase (E.C. 1.3,3.1) activity, has been isolated by a replica plating procedure. The mutant does not show a requirement for exogenously added pyrimidines. Examination of intact cells shows that the mutant accumulates a large amount of carbamyl aspartate and is markedly but not totally deficient in biosynthesis of orotate from earlier precursors of pyrimidine biosynthesis, including aspartate and dihydroorotic acid, when compared to wild-type cells. Analysis of cell-free extracts of mutant and wild-type cells shows that the mutant is deficient in DHO dehydrogenase activity, possessing ca. 5% of the wild-type activity. this evidence leads to the conclusion that this mutant, A204, is in fact partially deficient in DHO dehydrogenase, and that in these cells it is this enzyme which carries out the fourth step of de novo pyrimidine biosynthesis.

Animals↗
PubMed1979

Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: III. Association of carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase in mutants of cultured Chinese hamster cells.

Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3), the first three enzymes in de novo pyrimidine synthesis in Chinese hamster ovary cell strain Kl (CHO-Kl), cose diment through a glycerol gradient. When an extract from Urd- A, a pyrimidine-requiring auxotroph reduced in all three activities, is run on a glycerol gradient, the enzyme activities appear in two peaks higher in the gradient, a peak of aspartate transcarbamylase separated from a peak of carbamyl phosphate synthetase and dihydroorotase. Revertants of Urd- A have increased activity of all three enzymes and give glycerol gradient patterns similar to either CHO-Kl or Urd- A. The gradient pattern for Urd- A and some of its revertants can be mimicked by treating the CHO-Kl cell extract with trypsin. Hybrids made between a CHO-Kl purine-requiring auxotroph (Ade- C) and a Urd- A revertant gave a glycerol gradient pattern which is a composite of the CHO-Kl and revertant patterns. A model is presented for the structure of this multifunctional protein.

Amidohydrolases↗
PubMed1979

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: isolation and characterization of a mutant deficient in the activity of phosphoribosylaminoimidazole synthetase.

A new purine-requiring mutant of Chinese hamster ovary cells (CHO-Kl) is described. This mutant, Ade-G, grows on aminoimidazole carboxamide, hypoxanthine, or adenine. It complements all eight of our other previously described Ade- mutants. Biochemical analysis of de novo purine synthesis in whole cells suggests that Ade-G is capable of the first four reactions of de novo purine biosynthesis and that it synthesizes and accumulates phosphoribosylformylglycinamidine (FGAM). Direct enzyme assay in cell-free extracts confirms that Ade-G is defective in phosphoribosylaminoimidazole synthetase activity and does not convert FGAM to phosphoribosylaminoimidazole (AIR), the next intermediate in the de novo biosynthetic pathway.

Animals↗
PubMed1979

Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis.

A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-K1, the uridine-requiring auxotroph Urd(-)A, and selected Urd(-)A revertants. The enzymes of Urd(-)A and the Urd(-)A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-K1. The results presented permit the conclusion that (i) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (ii) the aspartate transcarbamoyltransferase activity is located on a portion ( approximately 20,000 daltons) at one end of the polypeptide; (iii) this portion may also be required for monomers to aggregate into the multimeric from present in mammalian cells; (iv) the mutations in Urd(-)A and the Urd(-)A revertants lie in the structural gene for this multifunctional protein; and (v) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.

Amidohydrolases↗
PubMed1979

A selective medium (GAMA) for the isolation of somatic cell hybrids from HPRT- and APRT- mutant cells.

A medium is described which can be used for the selection of somatic cell hybrids between HPRT- and APRT- mutant cells. This medium, called GAMA, contains as its relevant constituents the following: the purines guanine and adenine and the purine biosynthetic pathway inhibitors mycophenolic adic, which blocks conversion of adenine ribonucleotides to guanine ribonucleotides, and azaserine, which blocks de novo purine synthesis.

Adenine Phosphoribosyltransferase↗
PubMed1979

Effects of caffeine on purine metabolism and ultraviolet light-induced lethality in cultured mammalian cells.

Caffeine, at doses which enhance the killing action of ultraviolet light, inhibits both de novo synthesis and the utilization of exogenous purines in cultured CHO-K1, a Chinese hamster ovary cell line. The decrease in synthesis was measured as inhibition by caffeine of the accumulation of phosphoribosylformylglycineamide or of phosphoribosylaminoimidazolecarboxamide, the fourth and ninth intermediates, respectively, in the de novo biosynthetic pathway. The effect is dose dependent, with a caffeine concentration of 7.5 mM producing a 90% reduction in 15 min. Interference with utilization of exogenous purines was seen as a substantial decrease in the conversion of [14C]hypoxanthine, [14C]adenine, or [14C]guanine into their respective di- and triphosphates in the presence of caffeine. Purine deprivation either by starvation of purine-requiring mutants or by treatment of parental cells with methylmercaptopurine ribonucleoside, a known inhibitor of purine synthesis, results in a partial sensitization to killing by ultraviolet light which can be maximized by the addition of caffeine. Thus, one of the ways by which antimetabolites and caffeine act to enhance ultraviolet light killing may be by interference with the supply of purine nucleotides needed for repair.

Animals↗
PubMed1978

Characterization of a guanine-sensitive mutant defective in adenylo-succinate synthetase activity.

A contingent auxotrophic mutant of CHO-Kl cell is described. This mutant grows in minimal medium. Its growth is inhibited by the exogenous addition of guanine at levels which do not affect the wild type parent. Adenine reverses the guanine effect. This mutant does not complement ade-H (defective in adenylosuccinate synthetase) and has been denoted as ade-HG because of its guanine sensitivity. Some partial revertants of ade-H are found to be also sensitive to guanine, suggesting a close relationship between the ade-H locus and the guanine sensitivity. Studies of 14C-hypoxanthine incorporation into nucleotides indicated that ade-HG has some adenylosuccinate synthetase activity whether it is pre-exposed to guanine or not. Early de novo purine synthesis in ade-HG, however, is greatly inhibited when pre-exposed to guanine. This inhibition of purine synthesis by guanine is reversible and its recovery is facilitated by adenine.

Adenine↗
PubMed1977

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism. VI. Enzymatic studies of two mutants unable to convert inosinic acid to adenylic acid.

Ade-H and ade-I are two auxotrophic mutants of Chinese hamster ovary (CHO-K1) cells which specifically require adenine as the purine source to grow. The enzymatic defects of these mutants were examined in cell-free extracts. It was found that ade-H did not have any detectable adenylosuccinate synthetase activity and ade-I was defective in the adenylosuccinate lyase enzyme. The relevance of adenine-requiring mutants to the study of the regulation of purine metabolism in mammalian cells is discussed.

Adenine↗
PubMed1977

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: characterization of Chinese hamster cell mutants defective in phosphoribosylpyrophosphate amidotransferase and phosphoribosylglycinamide synthetase and an examination of alternatives to the first step of purine biosynthesis.

Activities of the first three enzymes in the de novo purine biosynthetic pathway have been measured in cell-free extracts of the Chinese hamster ovary cell (CHO-K1) and two purine-requiring auxotrophs of this cell. Ade-A has been found to be defective in phosphoribosylpryophosphate (PRPP) amidotransferase while Ade-C has been found to be defective in glycinamide ribonucleotide (GAR) synthetase. Neither enzyme deficiency is due to the presence of an excess of diffusible inhibitor, and mixed extracts of Ade-A and Ade-C are capable of performing both enzymatic steps in a coupled assay. Assays of GAR formyltransferase show that it is present in Ade-A and Ade-C, indicating that these cell types are defective in only one enzyme each of the early purine biosynthetic enzymes. Using the Ade-A mutant, analysis of alternatives to PRPP plus glutamine as substrates for the first step in the purine biosynthetic pathway showed that a common genetic unit must direct the synthesis for both PRPP plus glutamine and PRPP plus ammonia activities. Although ribose-5-phosphate plus ammonia can be used in cell-free extracts to perform the first step in purine biosynthesis, it is shown that this activity is apparently not used by intact CHO-K1 cells.

Amidophosphoribosyltransferase↗
PubMed1977

Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: I. Isolation of a mutant defective in the early steps of de novo pyrimidine synthesis.

The isolation and characterization of a new mutant of Chinese hamster ovary cells is described. This mutant, Urd-A, shows an absolute requirement for exogenously added pyrimidines for growth. Complementation analysis indicates that the lesion in this mutant is recessive. Revertants can be isolated at frequencies suggesting that it is a single gene alteration. Biochemical analysis of cell-free extracts of CHO-K1 (Urd+) and Urd-A revealed that Urd-A possesses no more than 10% of wild-type levels of carbamyl phosphate synthetase (EC 2.7.2.9) activity, no more than 1% of wild-type levels of aspartate transcarbamylase (EC 1.2.3.2) activity, and undetectable levels of dihydroorotase (EC 3.5.2.3) activity. Thus, this mutant appears simultaneously to possess marked or complete deficiencies in the activities of the first three enzymes of pyrimidine biosynthesis. Activities of the other enzymes of the pathway appear normal. The use of this mutant for biochemical-genetic studies of pyrimidine biosynthesis is discussed.

Aspartate Carbamoyltransferase↗
PubMed1976

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism. IV. Isolation of a mutant which accumulates adenylosuccinic acid and succinylaminoimidazole carboxamide ribotide.

The production, isolation, and characterization of a new complementation group (Ade-I) of adenine-requiring mutant of Chinese hamster cells (CHO-K1) is described. This mutant accumulates two intermediates of purine biosynthesis, both of which contain an aspartate moiety. One of these is shown to be adenylosuccinic acid (AMPS) by chromatographic analysis, while evidence is presented that strongly suggests the other intermediate is succinylaminoimidazole carboxamide ribotide (SAICAR). Thus, Ade-I is most likely lacking the activity of the enzyme adenylosuccinase (EC 4.3.2.2). The use of this and similar mutants for the analysis of regulation of purine biosynthesis in mammalian cells is discussed.

Adenine↗
PubMed1976

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism III. Isolation and characterization of a mutant unable to convert IMP to AMP.

The isolation and characterization of a new mutant of Chinese ovary cells (CHO-K1) is described. This mutant, Ade-H, has the following properties: (1) it forms a new genetic complementation group; (2) it specifically requires adenine for growth and will not grow on aminoimidazole carboxamide (AIC) or hypoxanthine; (3) it accumulates IMP; (4) it cannot synthesize adenine nucleotides; (5) its phenotype can be mimicked by treatment of CHO-K1 (the wild type parental strain) with hadacidin, an inhibitor of adenylosuccinate synthetase (E.C.6.3.4.4). Thus, the site of the defect in this mutant is presumed to involve the step in adenylate biosynthesis catalyzed by this enzyme. The usefulness of Ade-H for the study of regulation of purine biosynthesis in mammalian cells is discussed.

Adenine↗
PubMed1976

Isolation and characterization of temperature-sensitive mutants of Chinese hamster ovary cells after treatment with UV and x-irradiation.

The isolation of ten conditionally lethal temperature-sensitive mutants of the Chinese hamster ovary cell (CHO-Kl, pro-) by the BUdR-visible light selection procedure described. Treatment with radiation at doses known to cause single gene mutation in mammalian cells increases the mutation frequency by a factor of at least 14. These mutants will grow with normal plating efficiency at 34.5 degrees but will not grow at 39.5 degrees. Complementation analysis by two independent methods indicates that all mutants are recessive and allows the assignment of the mutants to six genetically independent complementation groups. Reversion analysis indicates that the TS-mutants are stable, spontaneous revertants arising at a frequency of less than 10(-6). Preliminary chromosome analysis revealed no systematic chromasomal abnormality in the mutants. Mitotic accumulation is used to study the generation time of the parental cells and representative mutants at 34.5 degrees and 39.5 degrees. The uses of these mutants for genetic analysis of mammalian cells in culture is discussed.

Cell Division↗
PubMed1976

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: isolation, selection, and characterization of a mutant lacking hypoxanthine-guanine phosphoribosyltransferase activity by nutritional means.

Mutants of the Chinese hamster ovary cell derived from CHO-K1 have been selected for lack of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) (HGPRT) without the use of a drug-resistance protocol. The procedure depends on the use of a parental strain carrying a mutation making it unable to synthetize purines and thus dependent upon exogenously added purines for growth. The standard "BUdR-visible-light" procedure is then used to select those cells which can use adenine but cannot use hypoxanthine as a purine source. These cells are shown to be thioguanine resistant, to be unable to incorporate exogenously added hypoxanthine into purine nucleotides, to complement our other adenine-specific purine auxotrophs, Ade-H and Ade-I but not to complement a cell isolated by virtue of thioguanine resistance, and to lack the activity of HGPRT. The use of such multiply marked mutants and cells related to them for further analysis of purine nucleotide biosynthesis and interconversion is discussed.

Animals↗