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 phosphoribosylpyrophosphate synthetase (ribosephosphate pyrophosphokinase; ATP:D-ribose-5-phosphate pyrophosphotransferase; EC 2.7.6.1) was studied in fibroblasts cultured from members of a family with a structurally and electrophoretically altered phosphoribosylpyrophosphate synthetase that has increased activity per enzyme molecule. Enzyme activity in fibroblast lysates from the daughter of an affected male patient was intermediate to the activities in lysates from her father (and her affected paternal uncle) and from her mother and other normal individuals. Two bands of enzyme activity corresponding to normal and mutant phosphoribosylpyrophosphate synthetases were found in fibroblast lysates from the daughter after cellulose acetate strip electrophoresis. In contrast, only mutant enzyme was detectable in lysates derived from the male patients. Fibroblasts cloned from the daughter contained two phenotypically distinct (normal and mutant) populations of cells with respect to phosphoribosylpyrophosphate synthetase activity and electrophoretic mobility. These studies support assignment of the structural gene for human phosphoribosylpyrophosphate synthetase to the X-chromosome. No evidence for the presence of the normal enzyme was found in erythrocyte or lymphocyte lysates or in partially purified erythrocyte enzyme preparations from the heterozygous daughter, suggesting either nonrandom X-chromosome inactivation in precursors of these cells or selection against hematopoietic cells bearing the normal enzyme after random X-chromosome inactivation.

bers of these families (6-8) have not provided evidence for random X-chromosome inactivation as predicted by the Lyon hypothesis (9). However, in studies of fibroblasts cultured from the mother of an affected male patient described by Sperling et al. (2), values were found that were intermediate to those shown by cells from the affected son and normal individuals for PRPP synthetase activity, intracellular PRPP content, and purine synthetic rate (10). In addition, fibroblasts from this woman showed rates of purine synthesis approximating those of her son after growth in a medium designed to select for cells with increased enzyme activity, thus providing indirect evidence of X-linkage of PRPP synthetase (11). In the present study, more direct evidence for X-linkage of the structural gene from PRPP synthetase is provided by demonstration of two distinct clonal populations with respect to this enzyme in fibroblasts from a female patient of the previously described B. family (7) in which a structural alteration in PRPP synthetase leading to increased activity per enzyme molecule has been established (5, 12).

MATERIALS AND METHODS Cells. Fibroblast cultures were established from upper arm skin obtained by punch biopsy and were propagated in monolayer on 75-cm2 plastic flasks in Eagle's minimum essential medium supplemented with 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (100,ug/ml), and 10% fetal calf serum (13). Clones were derived from fibroblast strains and isolated with cloning rings as described by Ham and Puck (14). Lymphocytes were isolated from freshly drawn blood by Ficoll-Hypaque gradient centrifugation according to the method of Mendelsohn et al. (15) and incubated overnight at 370 in RPMI 1640 medium containing 20% fetal calf serum and 2 mM L-glutamine. After centrifugation, contaminating erythrocytes were removed by resuspending the cell pellet for 10 min at 370 in 17 mM Tris-HCI with 140 mM ammonium chloride (pH 7.4). Preparation of Cell Lysates. Fibroblasts were harvested by treatment with trypsin as described (4), and an aliquot of each cell suspension was counted with a Coulter Counter model ZBI. Further preparation of lysates was identical for lymphocytes and fibroblasts. Cells were washed twice in calcium-free Dulbecco's phosphate-buffered saline with 5.5 mM glucose. After the final centrifugation, cell pellets were resuspended at a density of 4-10 X 106 cells per ml in 8 mM sodium phosphate buffer with 10 mM reduced glutathione/1 mM EDTA (pH 7.4), and the cells were lysed by freezing and thawing three times in liquid nitrogen. After centrifugation at 27,000 X g for 40

5-Phosphoribosyl-l-pyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase; EC 2.7.6.1) catalyzes the reaction between ATP and ribose-5-P to form PRPP and AMP. PRPP is a rate-limiting substrate in purine synthesis de novo as well as an allosteric activator of amidophosphoribosyltransferase (EC 2.4.2.14) (1), the first committed enzyme in this pathway. In three families reported to date (2-4), affected male members with excessive PRPP synthetase activity due to apparently different structural alterations in the enzyme show increased intracellular PRPP synthesis, concomitant purine overproduction, and gout. Thus, increased PRPP synthetase activity is a prototype for the association of a hereditary disease state with a structurally aberrant protein of increased rather than decreased catalytic activity. The identification of an excessively active and electrophoretically distinct form of PRPP synthetase (5) provides an opportunity to study the mode of inheritance of the enzyme. Pedigree data from two of the families thus far reported (6, 7) are compatible with either autosomal dominant or X-linked transmission of the gene for PRPP synthetase. Father-to-son transmission of aberrant forms of the enzyme, a criterion essential for the exclusion of X-linkage, has not been observed. On the other hand, studies in erythrocytes from female memThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisemet" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviation: PRPP, 5-phosphoribosyl 1-pyrophosphate. * To whom reprint requests should be addressed. 482

Medical Sciences: Yen et al. min, the supernatant layers were dialyzed for 2 hr against the buffer in which the cells were lysed. Cellulose Acetate Electrophoresis. A portion of each fibroblast lysate (3-12 ,tg of protein) was applied to duplicate cellulose acetate strips (Millipore Biomedica) with an applicator, after which electrophoresis was carried out at 40 for 45 min in a Phoroslide electrophoresis apparatus. The electrophoresis buffer was 250 mM tricine with 1 mM sodium phosphate/i mM magnesium chloride/3% Nonidet/50,M ribose-5-P (pH 8.5) modified from the method of Lebo and Martin (16). After electrophoresis, one strip of each pair was immediately stained at 370 for PRPP synthetase activity as described by Johnson et al. (17). The control strip was stained in the identical solution from which PRPP was omitted. Comparison of the parallel strips permitted the distinction of bands specific for PRPP synthetase activity from those resulting from nonspecific staining. As previously described (5), electrophoresis of partially purified preparations of human erythrocyte PRPP synthetase was carried out on duplicate Cellogel cellulose acetate strips in a 40 mM barbital/10 mM phosphate buffer solution containing 10 mM reduced glutathione and 1 mM magnesium chloride (pH 8.6). The duplicate gel strips were stained as described above to permit identification of bands specific for enzyme activity. Other Methods. PRPP synthetase activity was measured by a two-step assay (7) in which the PRPP generated from ATP and ribose-5-P at 32 mM inorganic phosphate in the first step was determined by the conversion of [14C]adenine to [14C]AMP in the presence of highly purified adenine phosphoribosyltransferase (EC 2.4.2.7) (18). Protein concentration was determined by the method of Lowry et al. (19), with bovine serum albumin as standard. RESULTS Fibroblast Enzyme Activities. Fibroblasts were cultured from skin biopsies obtained from B. family members, including patient H.B., his daughter (C.B.), and his brother (T.B.), each of whom shows excessive urinary uric acid excretion and erythrocyte PRPP synthetase activity approximately 3-fold higher than the mean activity of normal individuals (7). Fibroblast strains were also developed from 11 clinically normal individuals, including H.B.'s wife (Y.B.). In order to compare specific activities of PRPP synthetase in these fibroblast strains, it was necessary to study the cultures at comparable stages of cell density (20). Therefore, in these experiments, internal controls for PRPP synthetase activity at various cell densities were prepared by plating each strain at three different cell densities (4 X 105, 1.3 X 105, and 0.4 X 105 cells per 75-cm2 flask). For each fibroblast strain, cells were permitted to grow until the culture plated at the intermediate density appeared confluent by microscopic examination. At that point, cells from all three flasks were harvested and enumerated, and lysates were prepared for enzyme assay. On the basis of the cell counts, relative densities were assigned to each of the flasks, with the flask plated at the highest density given a relative density of 1.0. Fig. 1 shows PRPP synthetase specific activities in dialyzed fibroblast lysates prepared at different cell densities from the three B. family patients and from normal individuals. A roughly inverse relationship between PRPP synthetase activity and cell density was observed for all cell strains. The activities of PRPP synthetase in the fibroblast extracts from patients H.B. and T.B. were consistently 3- to 6-fold higher than those in normal cells. Extracts of fibroblasts from patient C.B. also showed increased

Proc. Natl. Acad. Sci. USA 75 (1978)

'-J.

483

5000 400

0

3000 2 000

L0.

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Relative density

FIG. 1. PRPP synthetase activities in normal and B. family fibroblast extracts prepared at various relative cell densities. Cell numbers corresponding to a relative density of 1.0 varied from strain to strain over a range of 8.25 X 105-3.45 X 106 cells per 75-cm2 flask. Each point represents the mean value of three determinations. Symbols for fibroblast strains are: ,, H.B.; 0, T.B.; A, C.B.; *, Y.B.; *, normal control; o, C.B. 3; 0, C.B. 2; , C.B. 9.

PRPP synthetase activities which were, however, intermediate to those of her mother and her father or uncle. Although the activity of PRPP synthetase in fibroblasts from Y.B. was nearly twice that of the other control fibroblast strain shown in Fig. 1, values for strain Y.B. were within the range of activities measured in lysates of fibroblasts cultured from the 10 other normal individuals (182-697 nmol/hr per mg of protein). Electrophoresis Studies. Comparison of purified preparations of normal and B. family erythrocyte PRPP synthetases have established structural and electrophoretic differences in the enzymes (5, 12). PRPP synthetase in lysates of fibroblasts cultured from these patients also differed from the normal enzyme in electrophoretic mobility on cellulose acetate strips (Fig. 2). Fibroblast extracts from each of five normal individuals, including Y.B., showed PRPP synthetase activity in a single band with an identical electrophoretic mobility. In contrast, the sole band of PRPP synthetase activity demonstrable in lysates of fibroblasts from patients H.B. and T.B. migrated with diminished electrophoretic mobility. Mixtures of fibroblast lysates from H.B. (or T.B.) and normal individuals showed two enzyme activity-specific bands with mobilities corresponding to those found in the individual lysates. Electrophoresis of fibroblast extracts from patient C.B. produced a pattern of PRPP synthetase activity that was identical to the pattern observed for the mixture of normal and patient H.B. (or T.B.) extracts. Studies of Fibroblast Clones. Clones were derived from fibroblasts cultured from patients C.B., H.B., and T.B. and from three normal individuals. All of the clones derived from individuals other than C.B. (two from T.B., two from H.B., and eight from three normal individuals) showed PRPP synthetase activities similar to their respective uncloned parental populations. Of six putative clones developed from C.B.'s fibroblasts, two strains (C.B. 1 and C.B. 2) had PRPP synthetase activities intermediate to those of normal fibroblasts and those of H.B. or T.B. fibroblasts (Fig. 1). Strains C.B. 3 and C.B. 13 had PRPP synthetase activities identical in magnitude to the activity of this enzyme in cloned or uncloned H.B. or T.B. fibroblasts. In contrast, two additional strains (C.B. 9 and C.B. 16) had enzyme activities indistinguishable from that found in fibroblasts cultured from C.B.'s mother, Y.B. The electrophoretic mobility of PRPP synthetase in the putative clones derived from patient C.B.'s fibroblasts was also examined. Lysates of strains C.B. 1 and C.B. 2 showed two PRPP synthetase-specific bands identical in mobility to those

Proc. Natl. Acad. Sci. USA 75 (1978)

Medical Sciences: Yen et al.

484

+

(-)

NORMAL NORMAL T. B. NORMAL + T.B. C.B. C.B. 3

(+)

0D 0D

Table 1. PRPP synthetase activity in lysates of peripheral blood lymphocytes and erythrocytes from normal individuals and B. family members

Group Normal W.H. R.S. K.A. C.G.

0D 0D 0D

PRPP synthetase activity,* nmol/hr per mg protein Erythrocytes Lymphocytes 276 (266-286) 263 (244-281) 242 (224-259) 254(230-278)

77 (74-80) 72 (69-75) 64 (59-68) 83 (79-86)

B. family H.B.

C.B. 9

178 (173-182) 554(545-563) 182 (180-185) 565 (560-569) 183 (179-187) 536 (500-571) C.B. *Values are the means of two separate determinations of enzyme activity. The range is given in parentheses.

T.B.

NORMAL

NORMAL

T.B.

0i

NORMAL

j

T.B. C.B.

this patient showed only a single band of enzyme activity with the increased relative mobility previously described for similar preparations from patients H.B. and T.B. (5).

+

C.B. 3

0

C.B. 9 ORIGIN

FIG. 2. Cellulose acetate strip electrophoresis of normal and B. family fibroblast PRPP synthetase. PRPP was omitted from the staining solution for the top strip (e) which, therefore, shows only nonspecific staining. All of the other strips shown were developed withPRPP in the staining solution (@). Bands seen only after development in the presence of PRPP are PRPP synthetase. (Upper) Stained strips; (Lower) schematic drawing corresponding to the stained strips.

observed in lysates of uncloned C.B. cells. However, strains C.B. 3 and C.B. 13 showed only the slowly migrating band seen in lysates from H.B. or T.B. fibroblasts (Fig. 2). Strains C.B. 9 and C.B. 16 showed a single band with migration identical to that of the normal enzyme. These results indicated that strains C.B. 3 and C.B. 13 and strains C.B. 9 and C.B. 16 were clones with phenotypically distinct populations of cells representing fibroblasts with mutant and normal PRPP synthetases, respectively, and that strains C.B. 1 and C.B. 2 were mixed populations containing both types of cells rather than strains of clonal origin.

Studies in Lymphocytes and Erythrocytes. The activities of PRPP synthetase in lysates of peripheral blood lymphocytes isolated from patients H.B. and T.B. were 2-fold greater than those in identical preparations from normal individuals (Table 1). In contrast to the activities of this enzyme in fibroblasts from patient C.B. (which were intermediate to those of normal individuals and affected males patients), lymphocytes from this patient contained increased PRPP synthetase activity indistinguishable in magnitude from the lymphocytes of patients H.B. and T.B. Virtual identity in the magnitude of increased PRPP synthetase activity has previously been observed in erythrocyte lysates from these three patients (7), and this finding was confirmed in the present studies (Table 1). In contrast to the two bands of enzyme activity found after electrophoresis of fibroblast lysates from patient C.B., electrophoresis of partially purified erythrocyte PRPP synthetase from

DISCUSSION The Lyon hypothesis (9) of random X-chromosome inactivation in female cells during early embryogernesis predicts the existence of two populations of cells in females heterozygous for an X-linked gene: one population that expresses the wild-type allele and another that expresses the mutant allele. Demonstration of two such phenotypically distinct populations among the cells of heterozygous females has been used in the assignment of the genes for several biochemical markers, including glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (21), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) (22, 23), and a-galactosidase (EC 3.2.1.22) (24) to the human X chromosome. Similarly, the present studies provide support for the assignment of the structural gene for human PRPP synthetase to the X chromosome. A female member of the B. family, affected members of which have an inherited structurally altered PRPP synthetase with increased activity per enzyme molecule (12), showed in her fibroblast lysates enzyme activities intermediate to those of lysates from normal individuals and her affected father or uncle. Furthermore, extracts of fibroblasts from this female patient contained both normal and mutant PRPP synthetases, as shown by cellulose acetate strip electrophoresis. Irt contrast, only mutant enzyme was detectable after electrophoresis of fibroblast extracts from the affected male patients. Thus, both enzyme activities and the migration of the enzyme on electrophoresis of extracts of fibroblasts from the B. family were inconsistent with the previously proposed hypothesis (7) that the male patients T.B. and H.B. were heterozygotes bearing an autosomal mutation with dominant expression. In addition, these results could not be reconciled with an autosomal recessive transmission of PRPP synthetase, as was suggested from a study of a suspected primary PRPP synthetase deficiency by Wada et al. (25). In order to distinguish between X-linkage and autosomal codominant inheritance, either of which could account for the fibroblast activity and electrophoresis findings in the B. family, analysis of clonal fibroblast populations derived from the obligate heterozygote C.B. was undertaken. If hereditary transmission of PRPP synthetase were autosomal codominant, the

Medical Sciences: Yen et al. male patients H.B. and T.B. would necessarily expresboth mutant and normal genes. However, in any form of autosomal inheritance, all fibroblasts cultured from patient G.B. would be expected to manifest an identical phenotype. The isolation from patient C.B.'s fibroblasts of clones with normal (C.B. 9 and C.B. 16) and mutant (C.B. 3 and C.B. 13) phenotypes with respect to PRPP synthetase activity and electrophoretic mobility is inconsistent with autosomal inheritance of PTPP synthetase but is entirely compatible with X-linked transmission of this enzyme. Contrary to the findings in fibroblast extracts, lysates of erythrocytes (7) and lymphocytes (Table 1) from patient C.B. showed activities of PRPP synthetase equal in magnitude to those of the affected male patients and partially purified preparations of erythrocyte PRPP synthetase from all three B. family patients showed PRPP synthetase activity with the electrophoretic mobility of the mutant enzyme only. Absence of expression of the normal allele for PRPP synthetase could be due either to nonrandom inactivation of X chromosomes carrying the normal gene for the enzyme in hematopoietic precursor cells of this female or to selection against the population of cells with normal PRPP synthetase activity after random X-chromosome inactivation. Random inactivation resulting in suppression of X chromosomes bearing only the normal allele in hematopoietic precursors of this female heterozygote would seem an unlikely explanation, since in the only other family reported to date, the obligate heterozygote mother of a patient with excessive PRPP synthetase activity showed normal activity of this enzyme in her erythrocytes, despite increased enzyme activity in her fibroblasts (11). In this latter family, nonrandom X-chromosome inactivation or subsequent selection appears to operate against the mutant allele, thus resembling the situation observed in severe deficiency of hypoxanthine phosphoribosyltransferase (26), in which normal enzyme activity is found in the erythrocytes of obligate heterozygous females. Although distinction between nonrandom Xchromosome inactivation and subsequent selection is not presently possible, the observations in erythrocytes and lymphocytes of patient C.B. appear to represent an example in which the product of the mutant allele of an X-linked gene in human beings is expressed preferentially (27). While an X-linked mode of transmission of human PRPP synthetase appears to be established by the fibroblast studies reported here, confirmation of this finding may be obtained by the demonstration of synteny with other X-linked biochemical and antigenic markers in appropriate interspecific somatic cell hybrids, such as those between human and hamster cells (28). This work was supported in part by the Medical Research Service of the Veterans Administration, by a grant to Dr. Seegmiller from the

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20. Martin, D. W., Jr. & Maler, B. A. (1976) Science 193, 408411. 21. Davidson, R. G., Nitowsky, H. M. & Childs, B. (1963) Proc. Natl. Acad. Sci. USA 50,481-485. 22. Rosenbloom, F. M., Kelley, W. N., Henderson, J. F. & Seegmiller, J. E. (1967) Lancet ii, 305-306. 23. Migeon, B. R., Der Kaloustian, V. M., Nyhan, W. L., Young, W. J. & Childs, B. (1968) Science 160, 425-427. 24. Romeo, G. & Migeon, B. R. (1970) Science 170, 180-181. 25. Wada, Y., Nishimura, Y., Tanabu, M., Yoshimura, Y., Iinuma, K., Yoshida, T. & Arakwa, T. (1973) Tohoku J. Exp. Med. 113, 149-157. 26. Nyhan, W. L., Bakay, B., Connor, J. D., Marks, J. F. & Keele, D. K. (1970) Proc. Natl. Acad. Sci. USA 65,214-218. 27. Lyon, M. F. (1970) Phil. Trans. R. Soc. London Ser. B 259, 41-52. 28. Goss, S. J. & Harris, H. (1975) Nature 255, 680-684.

Evidence for X-linkage of human phosphoribosylpyrophosphate synthetase.

Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 482-485, January 1978 Medical Sciences Evidence for X-linkage of human phosphoribosylpyrophosphate sy...
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