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Hereditary Orotic Aciduria: Evidence for a Structural Gene Mutation (6-Azauridine/Biochemical Genetics)

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Hereditary Orotic Aciduria: Evidence for a Structural Gene Mutation (6-Azauridine/Biochemical Genetics) Proc. Nat. Acad. Sci. USA Vol. 71, No. 8, pp. 3031-3035, August 1974 Hereditary Orotic Aciduria: Evidence for a Structural Gene Mutation (6-azauridine/biochemical genetics) THOMAS E. WORTHY, WOLFGANG GROBNER, AND WILLIAM N. KELLEY* Division of Rheumatic and Genetic Diseases, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Communicated by J. Edwin Seegmiller, May 6, 1974 ABSTRACT Orotic aciduria is a rare autosomal re- in man (5). This proposal was based on several observations. cessive disease in man due to a deficiency of orotate phos- (i) The double enzyme defect was difficult to explain as a phoribosyltransferase (EC 2.4.2.10; orotidine-5'-phos- phate: pyrophosphate phosphoribosyltransferase) and oro- structural gene mutation. Alternatively, some mutations in tidine-5'-phosphate decarboxylase (EC 4.1.1.23; orotidine- bacteria that affected the regulation of enzyme expression 5'-phosphate carbo.Ny-lyase). We have compared certain were known to reduce the levels of two or more enzymes in a physicochemical properties of orotidine-5'-phosphate pathway (6). In addition, OPRT and ODC levels were known decarboxylase from normal and mutant fibroblasts grown to under identical conditions. Orotidine-5'-phosphate de- be subject to genetic control in bacteria (7); more recently carboxylase from homozygous mutant cells was more this type of control has been demonstrated in yeast (8). thermolabile and exhibited at different electrophoretic (ii) In erythrocytes from heterozygotes, OPRT and ODC mobility when compared to the enzyme from normal cells; levels were often substantially less than 50% of normal. This orotidine-5'-phosphate decarboxylase from one hetero- was consistent with a trans dominant effect characteristic of zygous cell strain exhibited an intermediate thermplabil- ity while the other heterozygote displayed a thermal in- some regulatory mutations in prokaryotic cells (6). More activation curve indistinguishable from normal. The recently, Pinsky and Krooth noted that the addition of 6- enzyme from both normal and mutant cells exhibited bi. azauridine to normal cells in culture resulted in a modest in- phasic kinetics with the same apparent Michaelis con- crease in the activity of OPRT and ODC, whereas a marked stants. These data suggest that the molecular defect in the enzyme of this patient with orotic aciduria is due to a mu- increase in the activity of both enzymes was observed after the tation in a gene that affects the structure of either orotate addition of this pyrimidine analog to cells cultured from a phosphoribosyltransferase or orotidine-5'-phosphate de- patient with orotic aciduria (9). Although the mechanism carboxylase and cannot be Attributed to a mutation in responsible for this effect in either cell type was not clearly a regulatory gene, as previously suggested. elucidated, the findings were consistent with the hypothesis Enzymatic defects of pyrimidine biosynthesis in eukaryotic that 6-azauridine, or a metabolic intermediate accumulating organisms are exceedingly rare and, to date, are found only as the result of the presence of this analog, was capable of in- in a group of disorders that have in common the excretion of activating the product of a regulator gene that was presum- large quantities of orotic acid. In man the primary disorder ably preventing the synthesis of these enzymes in the mutant in this group is hereditary orotic aciduria, an autosomal reces- cells (10). sive genetic disease, characterized by megaloblastic anemia, Although these observations in hereditary -orotic aciduria leukopenia, retarded growth and development, and an in- were consistent with a regulatory defect, they could also be creased urinary excretion of orotic acid (1, 2). Biochemically, accounted for by a mutation in a structural gene(s) coding for this disorder is characterized by either of two phenotypes: one of the involved enzymes, and definitive data were not type I is most prevalent and exhibits deficient or decreased available to differentiate between these possibilities. In the activity of orotate phosphoribosyltransferase (OPRT; EC present study, we have used the 6-azauridine-augmented 2.4.2.10; orotidine-5'-phosphate:pyrophosphate phosphoribo- activity of ODC in fibroblasts in order to compare the syltransferase) and orotidine-5'-phosphate decarboxylase physicochemical properties of this enzyme from normal sub- (ODC; EC 4.1.1.23; orotidine-'-phosphate carboxy-lyase), jects and from a patient with hereditary orotic aciduria. The the enzymes that catalyze the conversion of orotic acid to results of these experiments suggest that the double enzyme uridine-5'-phosphate (3); type II is -characterized by a de- defect in hereditary orotic aciduria is due to a mutation in ficiency only in orotidine-5'-phosphate decarboxylase (4). either one or both of the structural genes coding for the two The genetic defect in hereditary orotic aciduria was pro- affected enzymes. A preliminary report of these findings has posed to be a consequence of a mutation in a regulator genet been presented (11). and has been cited as one of the few examples of such a defect MATERIALS AND METHODS Abbreviations: OPRT, orotate phosphoribosyltransferase; ODC. Materials. [7-'4C]Orotic acid (10.2 mCi/mmole) and [7- orotidine-5'-phosphate decarboxylase; OMP, orotidine-5'-mono- 14C]orotidine-5'-monophosphate (21 mCi/mmole) were from phosphate. New England Nuclear Corp.; 6-azauridine and tetrasodium 5- * To whom reprint requests should be addressed. phosphoribosyl-l-pyrophosphate were from Sigma Chemical t Defined by us, but not by the authors cited, as a gene that con- Co.; 6-azauridine-5'-monophosphate was from Calbiochem; trols the synthesis of an enzyme but has no effect on its structure. Sephadex G-25 was from Pharmacia; acrylamide, N,N'- 3031 Downloaded by guest on October 1, 2021 3032 Medical Sciences: Worthy et al. Proc. Nat. Acad. Sci. USA 71 (1974) methylenebisacrylamide, and N,N,N',N'-tetramethylethyl- mg/ml). The resultant solution was degassed for 15 min with enediamine were from Eastman Kodak Co.; tissue culture stirring. N,N,N',N'-Tetramethylethylene diamine (15 ul) materials were from Grand Island Biological Co.; and 10% was added, and 1.1 ml of the acrylamide solution was gently agarose was from Bio-rad. All other materials were the layered into gel tubes (70 mm X 5 mm inner diameter); the highest quality available and were purchased commercially. gels were then polymerized at room temperature for 60 min. of skin fibroblasts were ob- The gels were first subjected to electrophoresis in separation Cell Culture. Primary cultures gel buffer for 90 min at 170 at a constant current of 2.5 mA tained from explant cultures of punch biopsies from the inner of a for heredi- per gel. A stacking gel was added by layering 0.25 ml 4% forearm of normal subjects, a subject homozygous acrylamide solution, made with stacking gel buffer (45 mM tary orotic aciduria (strain 237) (12), and her heterozygous 7.1), on top of the separation gel and The cultures Tris, 32 mM\ H3PO4, pH parents (father, strain 241; mother, strain 242). polymerizing for 60 min. The gels were placed in the electro- were routinely maintained in a tissue culture medium de- mM Tris, 50 mM HCl, pH to contain 20% phoresis chamber, and anode (62 scribed by Silagi et al. (13) and modified 7.6) and cathode (42 mM Tris, 46 mM glycine, pH 9.0) (v/v) fetal-calf serum. Only cells between passages 2 and 15 buffers were added. Lysates (50-100 Al) containing brom- were used in the experiments. Fibroblasts were routinely tracking dye and sucrose (20%) before use in experiments; phenol blue (2 ,ug/ml) as the grown for 6-8 days after passage to increase the lysate density were gently layered on top of control experiments indicated that OPRT and ODC activities at were the gels, and the gels were subjected to electrophoresis 170 were stable between 6 and 9 days after passage. Cultures with a constant current of 2.5 mA per gel. Electrophoresis was fed every 3-4 days, and both normal and mutant cells were migrated to within 5 mm of the Medium containing stopped when the tracking dye refed, with Eagle's Minimal Essential end of the gels (about 90 min). The gels were removed from 10% fetal-calf serum, 2 days before use. the tubes; dye fronts and gel lengths were measured, and the Preparation of Cell Lysates. Cell lysates from confluent gels were frozen at -70°. The frozen gels were cut into 1.2- monolayers were prepared as reported (14). After centrifuga- mm slices, placed in assay tubes containing 100 M1A of 10 mM tion at 600 X g to remove cellular debris, the lysates were Trise HCl pH 7.4, and assayed for ODC activity. passed through individual Sephadex G-25 columns with a Determination of the Stokes Radius. The Stokes radius of bed volume of 1.5 ml and a void volume of 0.6 ml. Samples ODC was determined from the chromatographic behavior on (200 Il) of lysates were layered onto the top of the columns a 10% agarose column. The partition coefficient (Kay) of and allowed to enter the Sephadex; 600 ul of 10 mM Trise HCl ODC was determined by the method of Laurent and Killander (pH 7.4) was added and the column was allowed to drain. (18), with ovalbumin, alcohol dehydrogenase, and catalase as The desalted lysates were then collected by elution with 300 standards. Values of the Stokes radius of the standards were M1 of the same buffer. The desalting procedure had no ap- taken from published reports (19). The inverse error function parent effect on the specific activity of either enzyme. complement of the column partition coefficient (erfc -l) was Enzyme Assays. ODC was assayed according to Kelley and derived from the Kav as described by Ackers (20).
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