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Evidence for X-Linkage of Human Phosphoribosylpyrophosphate Synthetase (Genetic Mapping/Dosage Compensation) RICHARD C

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Evidence for X-Linkage of Human Phosphoribosylpyrophosphate Synthetase (Genetic Mapping/Dosage Compensation) RICHARD C Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 482-485, January 1978 Medical Sciences Evidence for X-linkage of human phosphoribosylpyrophosphate synthetase (genetic mapping/dosage compensation) RICHARD C. K. YEN, WILLIAM B. ADAMS, CHERI LAZAR, AND MICHAEL A. BECKER* Rheumatology Section, Department of Medicine, San Diego Veterans Administration Hospital; and University of California, San Diego, La Jolla, California 92161 Communicated by J. Edwin Seegmiller, October 31, 1977 ABSTRACT The mode of genetic transmission of human bers of these families (6-8) have not provided evidence for phosphoribosylpyrophosphate synthetase (ribosephosphate random X-chromosome inactivation as predicted by the Lyon pyrophosphokinase; ATP:D-ribose-5-phosphate pyrophospho- transferase; EC 2.7.6.1) was studied in fibroblasts cultured from hypothesis (9). However, in studies of fibroblasts cultured from members of a family with a structurally and electrophoretically the mother of an affected male patient described by Sperling altered phosphoribosylpyrophosphate synthetase that has in- et al. (2), values were found that were intermediate to those creased activity per enzyme molecule. Enzyme activity in fi- shown by cells from the affected son and normal individuals broblast lysates from the daughter of an affected male patient for PRPP synthetase activity, intracellular PRPP content, and was intermediate to the activities in lysates from her father (and purine synthetic rate (10). In addition, fibroblasts from this her affected paternal uncle) and from her mother and other normal individuals. Two bands of enzyme activity corre- woman showed rates of purine synthesis approximating those sponding to normal and mutant phosphoribosylpyrophosphate of her son after growth in a medium designed to select for cells synthetases were found in fibroblast lysates from the daughter with increased enzyme activity, thus providing indirect evi- after cellulose acetate strip electrophoresis. In contrast, only dence of X-linkage of PRPP synthetase (11). mutant enzyme was detectable in lysates derived from the male In the present study, more direct evidence for X-linkage of patients. Fibroblasts cloned from the daughter contained two the structural gene from PRPP synthetase is provided by phenotypically distinct (normal and mutant) populations of cells with respect to phosphoribosylpyrophosphate synthetase ac- demonstration of two distinct clonal populations with respect tivity and electrophoretic mobility. These studies support as- to this enzyme in fibroblasts from a female patient of the pre- signment of the structural gene for human phosphoribosylpy- viously described B. family (7) in which a structural alteration rophosphate synthetase to the X-chromosome. No evidence for in PRPP synthetase leading to increased activity per enzyme the presence of the normal enzyme was found in erythrocyte molecule has been established (5, 12). or lymphocyte lysates or in partially purified erythrocyte en- zyme preparations from the heterozygous daughter, suggesting either nonrandom X-chromosome inactivation in precursors of MATERIALS AND METHODS these cells or selection against hematopoietic cells bearing the normal enzyme after random X-chromosome inactivation. Cells. Fibroblast cultures were established from upper arm skin obtained by punch biopsy and were propagated in mo- 5-Phosphoribosyl-l-pyrophosphate (PRPP) synthetase (ATP: nolayer on 75-cm2 plastic flasks in Eagle's minimum essential D-ribose-5-phosphate pyrophosphotransferase; EC 2.7.6.1) medium supplemented with 2 mM L-glutamine, penicillin (100 catalyzes the reaction between ATP and ribose-5-P to form units/ml), streptomycin (100,ug/ml), and 10% fetal calf serum PRPP and AMP. PRPP is a rate-limiting substrate in purine (13). Clones were derived from fibroblast strains and isolated synthesis de novo as well as an allosteric activator of amido- with cloning rings as described by Ham and Puck (14). phosphoribosyltransferase (EC 2.4.2.14) (1), the first committed Lymphocytes were isolated from freshly drawn blood by enzyme in this pathway. In three families reported to date (2-4), Ficoll-Hypaque gradient centrifugation according to the affected male members with excessive PRPP synthetase activity method of Mendelsohn et al. (15) and incubated overnight at due to apparently different structural alterations in the enzyme 370 in RPMI 1640 medium containing 20% fetal calf serum and show increased intracellular PRPP synthesis, concomitant pu- 2 mM L-glutamine. After centrifugation, contaminating rine overproduction, and gout. Thus, increased PRPP synthetase erythrocytes were removed by resuspending the cell pellet for activity is a prototype for the association of a hereditary disease 10 min at 370 in 17 mM Tris-HCI with 140 mM ammonium state with a structurally aberrant protein of increased rather chloride (pH 7.4). than decreased catalytic activity. Preparation of Cell Lysates. Fibroblasts were harvested by The identification of an excessively active and electropho- treatment with trypsin as described (4), and an aliquot of each retically distinct form of PRPP synthetase (5) provides an op- cell suspension was counted with a Coulter Counter model ZBI. portunity to study the mode of inheritance of the enzyme. Further preparation of lysates was identical for lymphocytes Pedigree data from two of the families thus far reported (6, 7) and fibroblasts. Cells were washed twice in calcium-free Dul- are compatible with either autosomal dominant or X-linked becco's phosphate-buffered saline with 5.5 mM glucose. After transmission of the gene for PRPP synthetase. Father-to-son the final centrifugation, cell pellets were resuspended at a transmission of aberrant forms of the enzyme, a criterion es- density of 4-10 X 106 cells per ml in 8 mM sodium phosphate sential for the exclusion of X-linkage, has not been observed. buffer with 10 mM reduced glutathione/1 mM EDTA (pH 7.4), On the other hand, studies in erythrocytes from female mem- and the cells were lysed by freezing and thawing three times The costs of publication of this article were defrayed in part by the in liquid nitrogen. After centrifugation at 27,000 X g for 40 payment of page charges. This article must therefore be hereby marked "advertisemet" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviation: PRPP, 5-phosphoribosyl 1-pyrophosphate. this fact. * To whom reprint requests should be addressed. 482 Downloaded by guest on September 30, 2021 Medical Sciences: Yen et al. Proc. Natl. Acad. Sci. USA 75 (1978) 483 min, the supernatant layers were dialyzed for 2 hr against the buffer in which the cells were lysed. '-J. 5000 Cellulose Acetate Electrophoresis. A portion of each fi- broblast lysate (3-12 ,tg of protein) was applied to duplicate 400 cellulose acetate strips (Millipore Biomedica) with an applicator, after which electrophoresis was carried out at 40 for 45 min in 0 3000 a Phoroslide electrophoresis apparatus. The electrophoresis buffer was 250 mM tricine with 1 mM sodium phosphate/i mM 22000 magnesium chloride/3% Nonidet/50,M ribose-5-P (pH 8.5) L0. modified from the method of Lebo and Martin (16). After electrophoresis, one strip of each pair was immediately stained 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 at 370 for PRPP synthetase activity as described by Johnson et Relative density al. (17). The control strip was stained in the identical solution FIG. 1. PRPP synthetase activities in normal and B. family fi- from which PRPP was omitted. Comparison of the parallel broblast extracts prepared at various relative cell densities. Cell strips permitted the distinction of bands specific for PRPP numbers corresponding to a relative density of 1.0 varied from strain synthetase activity from those resulting from nonspecific to strain over a range of 8.25 X 105-3.45 X 106 cells per 75-cm2 flask. staining. Each point represents the mean value of three determinations. As previously described (5), electrophoresis of partially pu- Symbols for fibroblast strains are: ,, H.B.; 0, T.B.; A, C.B.; *, Y.B.; rified preparations of human erythrocyte PRPP synthetase was *, normal control; o, C.B. 3; 0, C.B. 2; , C.B. 9. carried out on duplicate Cellogel cellulose acetate strips in a 40 mM barbital/10 mM phosphate buffer solution containing 10 PRPP synthetase activities which were, however, intermediate mM reduced glutathione and 1 mM magnesium chloride (pH to those of her mother and her father or uncle. Although the 8.6). The duplicate gel strips were stained as described above activity of PRPP synthetase in fibroblasts from Y.B. was nearly to permit identification of bands specific for enzyme activi- twice that of the other control fibroblast strain shown in Fig. ty. 1, values for strain Y.B. were within the range of activities Other Methods. PRPP synthetase activity was measured by measured in lysates of fibroblasts cultured from the 10 other a two-step assay (7) in which the PRPP generated from ATP normal individuals (182-697 nmol/hr per mg of protein). and ribose-5-P at 32 mM inorganic phosphate in the first step Electrophoresis Studies. Comparison of purified prepara- was determined by the conversion of [14C]adenine to [14C]AMP tions of normal and B. family erythrocyte PRPP synthetases in the presence of highly purified adenine phosphoribosyl- have established structural and electrophoretic differences in transferase (EC 2.4.2.7) (18). Protein concentration was de- the enzymes (5, 12). PRPP synthetase in lysates of fibroblasts termined by the method of Lowry et al. (19), with bovine serum cultured from these patients also differed from the normal albumin as standard. enzyme in electrophoretic mobility on cellulose acetate strips (Fig. 2). Fibroblast extracts from each of five normal individ- RESULTS uals, including Y.B., showed PRPP synthetase activity in a single Fibroblast Enzyme Activities. Fibroblasts were cultured band with an identical electrophoretic mobility. In contrast, from skin biopsies obtained from B. family members, including the sole band of PRPP synthetase activity demonstrable in patient H.B., his daughter (C.B.), and his brother (T.B.), each lysates of fibroblasts from patients H.B.
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