Vol. 128, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 598-603 Copyright C 1976 American Society for Microbiology
Regulation of Hypoxanthine Transport in Neurospora crassa RICHARD L. SABINA,* JANE M. MAGILL, AND CLINT W. MAGILL Department of Plant Sciences, Genetics Section,* and Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
Received for publication 4 June 1976
Hypoxanthine uptake and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) were determined in germinated conidia from the adenine auxotrophic strains ad-1 and ad-8 and the double mutant strain ad-1 ad-8. The mutant strain ad-1 appears to lack aminoimidazolecarboximide ribonucleotide formyltransferase (EC 2.1.2.3) or inosine 5'-monophosphate cyclohydrolase (EC 3.5.4.10) activities, or both, whereas the ad-8 strain lacks adenylosuccinate synthase activity (EC 6.3.4.4). Normal (or wild-type) hypoxanthine transport capacity was found in the ad-1 conidia, whereas the ad-8 strains failed to take up any hypoxanthine. The double mutant strains showed intermediate transport capacities. Similar results were obtained for hypoxanthine phosphoribosyltransferase activity assayed in germinated conidia. The ad-1 strain showed greatest activity, the ad-8 strain showed the least activity, and the double mutant strain showed intermediate activity levels. Ion-exchange chromatography of the growth media revealed that in the presence of NH4+, the ad-8 strain excreted hypoxanthine or inosine, the ad-1 strain did not excrete any purines, and the ad-1 ad-8 double mutant strain excreted uric acid. In the absence of NH4+, none of the strains excreted any detectable purine compounds.
Previous studies in Bacillus subtilis (2), transport mechanisms, one resulting in inosine Escherichia coli (8), and Salmonella typhimu- 5'-monophosphate (IMP) accumulation and the rium (10) have indicated that the uptake of other in free hypoxanthine, has been reported purine bases may be mediated by the purine in vesicles (10). In humans, the loss of HPRTase activity has phosphoribosyltransferases (EC 2.4.2.7 and EC 2.4.2.8). In enteric bacteria (7, 10), it has been been demonstrated in persons with Lesch-Nypostulated that the mechanism of adenine and han disease or gout and results in an increase hypoxanthine uptake involves group transloca- in IMP catabolism (16). This apparently results tion in which the free bases are condensed with in an efflux of hypoxanthine (or a derivative), 5'-phosphoribosyl-1'-pyrophosphate (PRPP) by which contributes to excess uric acid formation membrane phosphoribosyltransferases, result- in the blood. In a similar situation, adenine ing in intramembranal accumulation of nucleo- auxotrophs of Neurospora crassa unable to utiside monophosphates. Similarly, work with lize IMP have been shown to efflux a breakcultured human fibroblasts and cultured cell down product, which is either inosine (13) or lines from patients with Lesch-Nyhan syn- hypoxanthine (12, 13), into the medium. A recent study with N. crassa (11) showed drome suggests that hypoxanthine-guanine phosphoribosyltransferase may catalyze the that adenine and hypoxanthine share one rate-limiting step in hypoxanthine transport transport system, whereas there is another (I). However, reduction of the activity of hy- transport system specific for adenine. It was poxanthine phosphoribosyltransferase (EC suggested that the uptake of hypoxanthine is in 2.4.2.8; HPRTase) did not inhibit uptake in some manner coupled to its utilization. In this other mammalian systems (5). study, an adenine double auxotroph (ad-1 adFree purine bases rather than purine nucleo- 8), which is blocked before and after the IMP tides have been found to accumulate in cells of intermediate in the purine pathway, was isoE. coli after very short incubation times (17), lated and used to show that the uptake of hyposuggesting that group translocation may not be xanthine and its utilization are related. Furthe only mechanism operative in purine base thermore, evidence is presented to support the transport. Although the free base might result hypothesis that the relative size of the intracelfrom breakdown of nucleotides formed during lular pool of IMP may be the basis for control of transport (2), evidence for two hypoxanthine the HPRTase activity. Additionally, ion-ex598
VOL. 128, 1976
REGULATION OF HYPOXANTHINE TRANSPORT
change chromatography was used to isolate and tentatively identify effluxed compounds from growth media. MATERIALS AND METHODS Chemicals. [8-'4C]hypoxanthine and Omnifluor were purchased from New England Nuclear Corp. 5'-Phosphoribosyl-1'-pyrophosphate and purine bases were purchased from Sigma Chemical Co. Glass-fiber filters (2.1 cm, grade 934AH) were obtained from Reeve-Angel Corp. Diethylaminoethylimpregnated paper circles (2.3 cm, DE81) were purchased from Whatman. Neurospora strains and media. The adenine auxotrophs ad-1,A (Y234M419), ad-8,A (E6), ad-8,a (E6), and ad-8,A (E80) of N. crassa were obtained from the Fungal Genetics Stock Center. Strain 73a (wild type) was obtained from V. W. Woodward. The adenine auxotrophs were maintained on Vogel's minimal medium (20) containing 20 mg of adenine per 100 ml. The ad-i strain appears to lack aminoimidaxole-carboximide ribonucleotide formyltransferase or IMP cyclohydrolase activities, or both (3). The ad-8 strain lacks adenylosuccinate synthetase activity (6). The double mutant ad-i ad-8,A was isolated from a cross between ad-i ,A and ad-8,a (E6) made on Difco cornmeal agar supplemented with 20 mg of adenine per 100 ml. Mutant spores were isolated on 3% agar, heat-shocked for 30 min at 60°C, and incubated at 30°C on Vogel's minimal medium containing 20 mg of adenine per 100 ml. Possible double mutants were analyzed by complementation tests with 'parent strains, by growth results on Vogel's minimal and Vogel's minimal plus hypoxanthine, and by backcrossing to ad-8,a (E6) and wild type (73a). Fries medium (19) containing 20 mg of adenine per 100 ml was used to support growth of the strains in experiments to determine excretion products. NH4+-free Fries indicates that the ammonium tartrate has been replaced by NaNO3. Preparation of conidia. Conidia harvested from 5- to 7-day cultures were shaken at 30°C for 5 h in Vogel's minimal medium (20), supplemented with 20 mg of adenine per 100 ml, plus 1.5% sucrose to initiate germination. Germinated conidia were counted on a hemocytometer, centrifuged in a clinical centrifuge, and resuspended in Vogel's minimal plus 1.5% sucrose to a final concentration of 4 x 106/ ml. Suspensions were shaken for 90 min at 30'C to diminish any existing pools of adenine before uptake was measured. Transport assay. The uptake was initiated by adding radioactive [8-14C]hypoxanthine to the shaking conidial suspensions to give a final concentration of 0.1 mM, with a specific activity of 0.25 juCi/ ,umol. At various intervals thereafter, 2-ml samples of conidial suspension were collected on a glass-fiber filter, washed with cold Vogel's minimal medium, and dried overnight. Dried filters were then added to 10 ml of Omnifluor scintillation fluid and counted for 5 min with a Beckman LS-250 liquid scintillation counter. HPRTase assay. The HPRTase assay was modi-
599
fied from that of Rubin et al. (18). Germinated conidia were collected on a membrane filter (Millipore Corp.) and ground in a tissue grinder (Ten Broeck) with 1 ml of 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.2. Samples (100 /ul) of whole-cell homogenate or washed precipitate, prepared by spinning the homogenate at 10,000 x g for 10 min at 4°C and then suspending the pellet in tris (hydroxymethyl) aminomethane - hydrochloride buffer (repeated twice), were added to the reaction mixture, which consisted of 20 ,ul of [8-'4C]hypoxanthine (2.6 ,g/10 ,l; 55 Ci/mol) and 20 ,dl of PRPP (0.5 mM). After incubation at room temperature, the reaction was stopped by boiling and the reaction mixture was centrifuged in a clinical centrifuge. Portions (100 ul) of the reaction mixture supernatant were placed on two diethylaminoethylimpregnated circles, placed on a rapid filtering apparatus, and washed with 10 ml each of 1 mM ammonium formate, deionized water, and absolute alcohol. The circles were dried overnight, added to 10 ml of Omnifluor scintillation fluid, and counted with a Beckman LS-250 liquid scintillation counter. As a control for nonspecific binding, an identical mixture was boiled before assaying enzyme activity. Also, to differentiate soluble from particulate enzyme fractions, samples of the pellet and supernatant were assayed separately. Spectrophotometric determination of excreted compound in growth medium. Strains ad-i, ad-8, and ad-i ad-8 were grown in 150 ml of Fries or NH,+free Fries liquid minimal medium (19) containing 20 mg of adenine per 100 ml, with shaking, at 30°C for 48 h. The Fries medium was chosen instead of Vogel's minimal medium because a component of the Vogel's medium absorbed strongly in the wavelength range (240 to 250 nm), which was critical for purine base determination. Fries medium has an absorption peak at 220 to 230 nm but much less absorbance than Vogel's medium, above 240 nm. Cells were removed from the liquid cultures using a Buchner funnel, and the filtrate was lyophilized to dry powder and suspended in 10 ml of deionized water. A 1.0-ml sample was added to a Dowex 50:H+ column (8.5 by 100 cm). Hypoxanthine or inosine was eluted from the column with 2 N HCl (4). The column was washed with several volumes of deionized water, and then adenine was eluted with 5% NH40H (4). The fractions having the largest absorption at 260 nm were scanned from 200 to 300 nm. Hypoxanthine was identified by its Xmax of 250 nm in acid, and uric acid was identified by its Xmaz of 280 nm in acid. All spectrophotometric data were obtained using a Beckman DBG-T spectrophotometer.
RESULTS Double mutant isolation. The ad-8 locus has been reported to be 20 units left of the centromere on linkage group VI (9), whereas the ad-i locus is located near the centromere on linkage group VI. From a cross between these two mutant strains, 16 of the 50 spores isolated germinated, and of these three failed to grow on
600
J. BACTERIOL.
SABINA, MAGILL, AND MAGILL
hypoxanthine as the purine supplement, demonstrating the presence of the ad-8 allele. (The ad-8 strain will not grow on hypoxanthine.) Complementation tests were made among these three isolates and the ad-i and ad-8 parental strains, and those that failed to complement either were selected as potential double mutants.
Final testing was done by crossing each double mutant strain to the ad-8 and wild-type strains. In the cross of one of the double mutant strains to the ad-8 strain, 80 spores were isolated. Of the 15 germinants, there were no wild-type progeny found, confirming the presence of the ad-8 mutation in the double mutant. In the cross of this double mutant strain to wild type, 45 spores were isolated. Of the 10 germinants tested, one adenine auxotroph was found to grow on hypoxanthine alone, confirming the presence of the ad-i mutation in the double mutant. Hypoxanthine uptake. Figure 1 shows the results of hypoxanthine uptake by the ad-i, ad8, and ad-i ad-8 strains in the presence and absence of NH4+. The ad-i strain served as the control since it accumulates hypoxanthine at approximately the same rate as wild type (11). The ad-8 strain did not take up hypoxanthine to any significant degree in either medium. The double mutant strain did take it up, but at a slower rate than the ad-i strain in NH4+-containing medium and at approximately the same rate in NH4+-free media.
25
20
HPRTase. HPRTase catalyzes the reaction: + PRPP -* IMP+ PP1. This activity was assayed using whole-cell homogenates of germinated conidia from the three adenine auxotrophic strains (ad-i, ad-8, and ad-i ad-8 double mutant) to establish whether there is a direct relationship between the ability of the cell to take up hypoxanthine and the rate at which it is converted to IMP. Table 1 shows the results of the assay, with relative activities expressed as picomoles of [14C]IMP formed per 4 min for 100 x 106 germinated conidia. There was intermediate activity in the ad-i ad-8 double mutant strain compared with the ad-i strain (greatest activity) and the ad-8 strain (very little activity) (Table 1). Boiled whole-cell homogenate from each strain was used as a control, and this base level activity was subtracted from the total counts/4 min of incubation for each strain. In the same experiment, the whole-cell homogenates of the ad-i and ad-8 strains were centrifuged at 10,000 x g for 10 min at 4°C, and 100 ,ul of the resuspended pellet and supernatant fractions was assayed separately. Most of the activity was found in the pellet fraction (see Table 1). Spectrophotometric data. Figure 2 shows the elution profile of the concentrated growth medium from the 48-h shaking cultures of the ad-8 strain from a Dowex 50:H+ column. Using 2 N HCl as eluant, fraction 2 absorbed most strongly at 260 nm, suggesting that this fraction contains hypoxanthine and/or inosine but not adenine (see Materials and Methods). To substantiate this, 200- to 300-nm scans of this fraction from each strain were made, and the results are shown in Fig. 3. Inosine and hypoxanthine were used in control experiments, and it was apparent that the two were inseparable by our methods. Both have Xmax values of 248 nm in acid. Fraction 42, eluted by 5% NH40H, gave strongest absorption at 260 nm. This absorption is attributed to adenine, which gave
hypoxanthine
TABLE 1. HPRTase activity Activity (pmol/4 min) Strain
5
10
15
20
25
30
35
40
TIME (min)
FIG. 1. Uptake of [8-'4C]hypoxanthine (0.10 mM; specific activity, 1.25 p,Cilmol) by germinated conidia of strains ad-i (-), ad-8 (A), and ad-i ad-8 (U) in the presence of NH4+. Results are also shown for identical experiments performed in the absence of NH4+ with strains ad-i (0), ad-8 (A), and ad-i ad-8 (C1).
Whole-cell
Washed Supernahomogeprecipitantb natea tateb ad-1 21.7 20.7 4.3 ad-8 2.1 3.0 2.6 ad-i ad-8 8.5 5.8 1.6 a A total of 100 x 106 conidia were used for homogenization. Data were taken from a regression line of 2-, 4-, and 8-min assays. b Data were taken from a single point at 4 min.
VOL. 128, 1976
REGULATION OF HYPOXANTHINE TRANSPORT
147
.7E
II
601 3b
1.2
0
05
,j A
.OF
z3 b
0* w
w
C)
CIO
10
20
30
40
50
FRACTION NUMBER
FIG. 2. Elution profile of concentrated growth medium from 48-h shaking liquid culture of the ad-8 strain. A 1 .0-ml sample was added to a Dowex 50:H+ ion-exchange column (8.5 by 100 cm). A hypoxanthine compound (a) was eluted with 2 N HCI, and adenine (b) was eluted with 5% NH40H. Fractions of 2 ml were collected at a flow rate of approximately 7 ml/min.
characteristic Xmax in base at 268 nm in control experiments. It is clear that in NH4+-containing Fries medium (Fig. 3a) the ad-8 and ad-i ad-8 strains have excreted different ultraviolet-absorbing compounds. In the presence of NH4+, the ad-i ad-8 double mutant strain excreted a compound having the spectral characteristics of uric acid (Xmaz = 280 nm in acid). In control experiments using 0.01 mM uric acid in NH4+containing Fries medium, uric acid was eluted from the Dowex 50:H+ column in fraction 2, the same fraction in which hypoxanthine or inosine is found under similar conditions. None of these strains, when grown in NH4+-free media, appear to excrete purines (Fig. 3b). DISCUSSION In a previous study (11) it was observed that [8-14C]hypoxanthine was not transported into germinated condia of the ad-8 mutant strain. Genetic studies (11), together with the results presented here, indicate that this lack of hypoxanthine transport is not due to a second mutation but is a consequence of the ad-8 lesion. The ad-8 locus is the structural gene for the enzyme adenylosuccinate synthetase (6), and lack of this enzyme (Fig. 4) probably causes cells to accumulate IMP or its breakdown product(s). It seemed that IMP (or a breakdown product of IMP) might be inhibiting hypoxanthine uptake in the ad-8 strain. To test this hypothesis, we synthesized the double adenine auxotroph ad-i ad-8. The ad-i strain appears to lack aminoimidazole carboxamide ribonucleotide formyltransferase or IMP cyclohydrolase activities, or both (3), and therefore cannot form IMP
.8
z 4t
m
m
0 C,)
cn4
cn
m
.6
4t
.4
.2 )
%
200 225 250 275
X,mI
200 225 250 275 300
x,mjL
FIG. 3. Ultraviolet spectra of fraction 2 eluted from a Dowex 50:H+ column from concentrated growth media of strains ad-8 (a), ad-i ad-8 (b), and ad-i (c). In (a) NH4+ was present in the growth media, whereas in (b) no NH4+ was present. In (a), the compound excreted by the ad-8 strain (a) appears as a shoulder at 250 nm and has been identified as hypoxanthine or inosine. The compound excreted by the ad-i ad-8 strain (b) appeared as a peak at 280 nm and has been identified as uric acid. The large peak at 230 nm represents a component of Fries minimal medium (see text).
de novo. Thus, the accumulation of IMP should not occur in the double mutant ad-i ad-8 strain to the extent that it does in the ad-8 strain. Some IMP probably is formed from adenosine 5'-monophosphate degradation and, if not utilized to form GMP (Fig. 4), may accumulate. The double mutant strain transported significantly more hypoxanthine than the ad-8 strain, as would be expected urider this hypothesis (Fig. 1). It has been shown that in some organisms (2, 8, 10) hypoxanthine transport is mediated by the enzyme HPRTase, possibly by a group translocation mechanism (8, 10). If this were the case in Neurospora, one would expect to find that changes in the levels of HPRTase activity would correspond to changes in the capacity for hypoxanthine transport. The HPRTase assays performed on germinated conidia from the ad-i, ad-8, and ad-i ad-8 mutant strains (Table 1) showed results similar to those found for hypoxanthine transport in these strains; i.e., the HPRTase activity was highest in ad-i conidial extracts, lowest in ad-8 conid-
602
J. BACTERIOL.
SABINA, MAGILL, AND MAGILL
I
synthesis
URflC ACID Ii
de novo
ad-I
Xn Ai
HxO(Ino) HPRTctm
f
AICAR
4ad-
FICAR
/~,VGMP AMP
t Id-8 0-IMP 11---
PRPP
ad-8
MP-S
'---
-M-AP
FIG. 4. Proposed scheme for hypoxanthine uptake and metabolism in N. crassa. The lesions in the ad-i and ad-8 strains are indicated. The vertical lines on the left-hand side represent the cytoplasmic membrane, with the solid line as the outer membrane surface. The abbreviations used in this figure are as follows: AICAR, 5-aminoimidazolecarboxamide ribonucleotide; FICAR, formyl AICAR; IMP, inosine monophosphate; XMP, xanthosine monophosphate; GMP, guanosine monophosphate; AMP, adenine monophosphate; AMP-S, adenylosuccinic monophosphate; Hx, hypoxanthine; Xn, xanthine; Ino, inosine; PRPP, 5'-phosphoribosyl-l '-pyrophosphate.
ial extracts, and intermediate in the conidial extracts from the ad-i ad-8 strain. Thus, the differences in HPRTase activities between the strains correspond to the differences found for hypoxanthine transport. However, this does not prove that HPRTase mediates hypoxanthine transport in this organism. Even though the HPRTase activity was lowest in the ad-8 strain, it was significantly greater than that of the boiled extract control, whereas transport of hypoxanthine by ad-8 conidia was essentially absent in all conditions, suggesting that intermediates accumulated in the cells of the mutant strains may regulate both hypoxanthine transport and HPRTase activity. In enteric bacteria (7, 10), it has been shown that IMP inhibits HPRTase activity and hypoxanthine transport. This could explain the lower activity in the whole-cell homogenate of ad-8, where the IMP concentration would be expected to be greater than in washed precipitates or the washed supernatant. Alternatively, compounds accumulating in these mutant strains may inhibit HPRTase activity which, in turn, may cause transported hypoxanthine to accumulate and prevent further hypoxanthine uptake. Evidence for the accumulation of IMP or an IMP derivative comes from analysis of the
NH4+-containing growth media (Fig. 3a), which shows that the ad-8 strain excretes fairly large quantities of hypoxanthine or inosine. This spectrophotometric data supports previous studies that suggested that the ad-8 strain does excrete hypoxanthine (12, 13) or inosine (13). If we assume that IMP breaks down to yield hypoxanthine (12), the hypoxanthine so formed may be further degraded to uric acid and then finally to NH4+. The ad-i ad-8 double mutant strain grown on adenine in NH4+-containing medium excreted uric acid, an intermediate in the degradative pathway from hypoxanthine to NH4+. Reinert and Marzluf (15) showed that uricase, the enzyme that breaks down uric acid, is repressed in the presence of NH4+ ions in the media. Since all these strains were grown in NH4+-containing Fries medium (Fig. 3a), uricase should be repressed in all the strains, causing uric acid or hypoxanthine to accumulate if formed. It is not clear at this time why the ad-8 strain excretes hypoxanthine and not uric acid when grown on NH4+-containing media. Neither uric acid nor hypoxanthine was excreted by ad-8 or the double mutant when NH4+-free media were used (Fig. 3b). Absence of NH,+ may allow excess hypoxanthine, resulting from IMP breakdown, to be degraded to NH3. The
VOL. 128, 1976
REGULATION OF HYPOXANTHINE TRANSPORT
ad-i strain presumably would never accumulate IMP or hypoxanthine (Fig. 4) and, therefore, would not be expected to excrete hypoxanthine or uric acid under either condition. If hypoxanthine or uric acid excretion indicates hypoxanthine accumulation, which inhibits hypoxanthine uptake, then the absence of NH4+ in the growth medium should allow degradation of excess hypoxanthine (to produce NH3) and thus stimulate hypoxanthine transport in the double mutant, as the only major course for transported hypoxanthine in this strain is through the degradative pathway. However, it was observed in this strain that hypoxanthine transport was decreased when NH4+ was absent (Fig. 1). Thus, it would appear that hypoxanthine is not the compound inhibiting hypoxanthine transport. The stimulation of hypoxanthine uptake by NH4+ in the ad-i strain does not appear to be a consequence of the genetic block, since similar results were obtained using wild type. This finding seems inconsistent with published results showing that NH4+ inhibits the transport of other potential sources of reduced nitrogen and amino acids (14) into Neurospora cells. The heritable disease gouty arthritis in humans is characterized by high levels of hypoxanthine and, consequently, uric acid in the blood stream of afflicted individuals. The primary lesion is thought to be a deficiency of HPRTase (16) or an aberration in the purine synthetic pathway that may lead to the excretion of excess hypoxanthine from cells into the blood stream. The lack of HPRTase activity and the excretion of hypoxanthine (or inosine) by the ad-8 strain suggests the similarity between the ad-8 auxotrophic strains in Neurospora and the phenotype of gouty arthritis in humans. ACKNOWLEDGMENTS This study was supported by Public Health Service Biomedical Support grant 55352 to J.M.M. and National Science Foundation grant GB41869 to C.W.M. LITERATURE CITED 1. Benke, P. L., N. Herrick, and A. Hebert. 1973. Transport of hypoxanthine in fibroblasts with normal and mutant hypoxanthine-guanine phosphoribosyl transferase. Biochem. Med. 8:309-323. 2. Berlin, R. D., and E. R. Stadtman. 1966. A possible role of purine nucleotide pyrophosphorylases in the regulation of purine uptake by Bacillus subtilis. J. Biol. Chem. 241:2679-2686. 3. Buchanan, F. 1954. The enzymatic synthesis of the
603
purine nucleotides. Harvey Lect. 54:104-130. 4. Cohn, W. E. 1949. The separation of purine and pyrimidine bases and of nucleotides by ion exchange. Science 109:377-378. 5. Dybing, E. 1974. Cycloheximide inhibition of hypoxanthine transport in cultured cells. Biochim. Biophys. Acta 373:100-105. 6. Giles, N. H., C. W. H. Partridge, and N. J. Nelson. 1957. The genetic control of adenylosuccinase in Neurospora crassa. Proc. Natl. Acad. Sci. U.S.A. 43:305317. 7. Hochstadt-Ozer, J. 1972. The regulation of purine utilization in bacteria. IV. Roles of membrane-localized and pencytoplasmic enzymes in the mechanisms of purine nucleoside transport across isolated Escherichia coli membranes. J. Biol. Chem. 247:2419-2426. 8. Hochstadt-Ozer, J., and E. R. Stadtman. 1971. The regulation of purine utilization in bacteria. III. Adenine phosphoribosyltransferase in isolated membrane preparations and its role in transport of adenine across the membrane. J. Biol. Chem. 246: 5304-5311. 9. Ishikawa, T. 1962. Genetic studies of ad-8 mutants in Neurospora crassa. I. Genetic fine structure ofthe ad8 locus. Genetics 47:1147-1161. 10. Jackman, L. E., and J. Hochstadt. 1976. Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium. J. Bacteriol. 126:312-326. 11. Magill, J. M., and C. W. Magill. 1975. Purine base transport in Neurospora crassa. J. Bacteriol. 124:149154. 12. Partridge, C. W. H., and N. H. Giles. 1959. Identification of major accumulation products of adenine-specific mutants of Neurospora. (Letters) Arch. Biochim. Biophys. 67:237-238. 13. Pendyala, L., and A. M. Wellman. 1975. Effect of histidine on purine nucleotide synthesis and utilization in Neurospora crassa. J. Bacteriol. 124:78-85. 14. Rao, E. Y. T., T. K. Rao, and A. G. DeBusk. 1975. Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity. Biochim. Biophys. Acta 413:45-51. 15. Reinert, W. R., and G. A. Marzluf. 1975. Regulation of the purine catabolic enzymes in Neurospora crassa. Arch. Biochem. Biophys. 166:565-574. 16. Rosenbloom, R. M., J. F. Henderson, I. C. Caldwell, W. N. Kelley, and J. E. Seegmiller. 1968. Biochemical basis of accelerated purine biosynthesis de novo in human fibroblasts lacking hypoxanthine-guanine phosphoribosyl transferase. J. Biol. Chem. 243:11661173. 17. Roy-Burman, S., and D. W. Visser. 1975. Transport of purines and deoxyadenosine in Escherichia coli. J. Biol. Chem. 250:9270-9275. 18. Rubin, C. S., J. Bancis, L. C. Yip, R. C. Nowinski, and M. E. Balis. 1971. Purification of IMP:pyrophosphate
phosphoribosyl-transferases: catalytically incompetent enzymes in Lesch-Nyhan syndrome. Proc. Natl. Acad. Sci. U.S.A. 68:1461-1464. 19. Ryan, F. J., G. W. Beadle, and E. L. Tatum. 1943. The tube method of measuring the growth rate of Neurospora. Am. J. Bot. 30:748-799. 20. Vogel, J. 1956. A convenient growth medium for Neurospora (medium N). Microb. Genet. Bull. 13:42-43.
JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 598-603 Copyright C 1976 American Society for Microbiology
Regulation of Hypoxanthine Transport in Neurospora crassa RICHARD L. SABINA,* JANE M. MAGILL, AND CLINT W. MAGILL Department of Plant Sciences, Genetics Section,* and Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
Received for publication 4 June 1976
Hypoxanthine uptake and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) were determined in germinated conidia from the adenine auxotrophic strains ad-1 and ad-8 and the double mutant strain ad-1 ad-8. The mutant strain ad-1 appears to lack aminoimidazolecarboximide ribonucleotide formyltransferase (EC 2.1.2.3) or inosine 5'-monophosphate cyclohydrolase (EC 3.5.4.10) activities, or both, whereas the ad-8 strain lacks adenylosuccinate synthase activity (EC 6.3.4.4). Normal (or wild-type) hypoxanthine transport capacity was found in the ad-1 conidia, whereas the ad-8 strains failed to take up any hypoxanthine. The double mutant strains showed intermediate transport capacities. Similar results were obtained for hypoxanthine phosphoribosyltransferase activity assayed in germinated conidia. The ad-1 strain showed greatest activity, the ad-8 strain showed the least activity, and the double mutant strain showed intermediate activity levels. Ion-exchange chromatography of the growth media revealed that in the presence of NH4+, the ad-8 strain excreted hypoxanthine or inosine, the ad-1 strain did not excrete any purines, and the ad-1 ad-8 double mutant strain excreted uric acid. In the absence of NH4+, none of the strains excreted any detectable purine compounds.
Previous studies in Bacillus subtilis (2), transport mechanisms, one resulting in inosine Escherichia coli (8), and Salmonella typhimu- 5'-monophosphate (IMP) accumulation and the rium (10) have indicated that the uptake of other in free hypoxanthine, has been reported purine bases may be mediated by the purine in vesicles (10). In humans, the loss of HPRTase activity has phosphoribosyltransferases (EC 2.4.2.7 and EC 2.4.2.8). In enteric bacteria (7, 10), it has been been demonstrated in persons with Lesch-Nypostulated that the mechanism of adenine and han disease or gout and results in an increase hypoxanthine uptake involves group transloca- in IMP catabolism (16). This apparently results tion in which the free bases are condensed with in an efflux of hypoxanthine (or a derivative), 5'-phosphoribosyl-1'-pyrophosphate (PRPP) by which contributes to excess uric acid formation membrane phosphoribosyltransferases, result- in the blood. In a similar situation, adenine ing in intramembranal accumulation of nucleo- auxotrophs of Neurospora crassa unable to utiside monophosphates. Similarly, work with lize IMP have been shown to efflux a breakcultured human fibroblasts and cultured cell down product, which is either inosine (13) or lines from patients with Lesch-Nyhan syn- hypoxanthine (12, 13), into the medium. A recent study with N. crassa (11) showed drome suggests that hypoxanthine-guanine phosphoribosyltransferase may catalyze the that adenine and hypoxanthine share one rate-limiting step in hypoxanthine transport transport system, whereas there is another (I). However, reduction of the activity of hy- transport system specific for adenine. It was poxanthine phosphoribosyltransferase (EC suggested that the uptake of hypoxanthine is in 2.4.2.8; HPRTase) did not inhibit uptake in some manner coupled to its utilization. In this other mammalian systems (5). study, an adenine double auxotroph (ad-1 adFree purine bases rather than purine nucleo- 8), which is blocked before and after the IMP tides have been found to accumulate in cells of intermediate in the purine pathway, was isoE. coli after very short incubation times (17), lated and used to show that the uptake of hyposuggesting that group translocation may not be xanthine and its utilization are related. Furthe only mechanism operative in purine base thermore, evidence is presented to support the transport. Although the free base might result hypothesis that the relative size of the intracelfrom breakdown of nucleotides formed during lular pool of IMP may be the basis for control of transport (2), evidence for two hypoxanthine the HPRTase activity. Additionally, ion-ex598
VOL. 128, 1976
REGULATION OF HYPOXANTHINE TRANSPORT
change chromatography was used to isolate and tentatively identify effluxed compounds from growth media. MATERIALS AND METHODS Chemicals. [8-'4C]hypoxanthine and Omnifluor were purchased from New England Nuclear Corp. 5'-Phosphoribosyl-1'-pyrophosphate and purine bases were purchased from Sigma Chemical Co. Glass-fiber filters (2.1 cm, grade 934AH) were obtained from Reeve-Angel Corp. Diethylaminoethylimpregnated paper circles (2.3 cm, DE81) were purchased from Whatman. Neurospora strains and media. The adenine auxotrophs ad-1,A (Y234M419), ad-8,A (E6), ad-8,a (E6), and ad-8,A (E80) of N. crassa were obtained from the Fungal Genetics Stock Center. Strain 73a (wild type) was obtained from V. W. Woodward. The adenine auxotrophs were maintained on Vogel's minimal medium (20) containing 20 mg of adenine per 100 ml. The ad-i strain appears to lack aminoimidaxole-carboximide ribonucleotide formyltransferase or IMP cyclohydrolase activities, or both (3). The ad-8 strain lacks adenylosuccinate synthetase activity (6). The double mutant ad-i ad-8,A was isolated from a cross between ad-i ,A and ad-8,a (E6) made on Difco cornmeal agar supplemented with 20 mg of adenine per 100 ml. Mutant spores were isolated on 3% agar, heat-shocked for 30 min at 60°C, and incubated at 30°C on Vogel's minimal medium containing 20 mg of adenine per 100 ml. Possible double mutants were analyzed by complementation tests with 'parent strains, by growth results on Vogel's minimal and Vogel's minimal plus hypoxanthine, and by backcrossing to ad-8,a (E6) and wild type (73a). Fries medium (19) containing 20 mg of adenine per 100 ml was used to support growth of the strains in experiments to determine excretion products. NH4+-free Fries indicates that the ammonium tartrate has been replaced by NaNO3. Preparation of conidia. Conidia harvested from 5- to 7-day cultures were shaken at 30°C for 5 h in Vogel's minimal medium (20), supplemented with 20 mg of adenine per 100 ml, plus 1.5% sucrose to initiate germination. Germinated conidia were counted on a hemocytometer, centrifuged in a clinical centrifuge, and resuspended in Vogel's minimal plus 1.5% sucrose to a final concentration of 4 x 106/ ml. Suspensions were shaken for 90 min at 30'C to diminish any existing pools of adenine before uptake was measured. Transport assay. The uptake was initiated by adding radioactive [8-14C]hypoxanthine to the shaking conidial suspensions to give a final concentration of 0.1 mM, with a specific activity of 0.25 juCi/ ,umol. At various intervals thereafter, 2-ml samples of conidial suspension were collected on a glass-fiber filter, washed with cold Vogel's minimal medium, and dried overnight. Dried filters were then added to 10 ml of Omnifluor scintillation fluid and counted for 5 min with a Beckman LS-250 liquid scintillation counter. HPRTase assay. The HPRTase assay was modi-
599
fied from that of Rubin et al. (18). Germinated conidia were collected on a membrane filter (Millipore Corp.) and ground in a tissue grinder (Ten Broeck) with 1 ml of 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.2. Samples (100 /ul) of whole-cell homogenate or washed precipitate, prepared by spinning the homogenate at 10,000 x g for 10 min at 4°C and then suspending the pellet in tris (hydroxymethyl) aminomethane - hydrochloride buffer (repeated twice), were added to the reaction mixture, which consisted of 20 ,ul of [8-'4C]hypoxanthine (2.6 ,g/10 ,l; 55 Ci/mol) and 20 ,dl of PRPP (0.5 mM). After incubation at room temperature, the reaction was stopped by boiling and the reaction mixture was centrifuged in a clinical centrifuge. Portions (100 ul) of the reaction mixture supernatant were placed on two diethylaminoethylimpregnated circles, placed on a rapid filtering apparatus, and washed with 10 ml each of 1 mM ammonium formate, deionized water, and absolute alcohol. The circles were dried overnight, added to 10 ml of Omnifluor scintillation fluid, and counted with a Beckman LS-250 liquid scintillation counter. As a control for nonspecific binding, an identical mixture was boiled before assaying enzyme activity. Also, to differentiate soluble from particulate enzyme fractions, samples of the pellet and supernatant were assayed separately. Spectrophotometric determination of excreted compound in growth medium. Strains ad-i, ad-8, and ad-i ad-8 were grown in 150 ml of Fries or NH,+free Fries liquid minimal medium (19) containing 20 mg of adenine per 100 ml, with shaking, at 30°C for 48 h. The Fries medium was chosen instead of Vogel's minimal medium because a component of the Vogel's medium absorbed strongly in the wavelength range (240 to 250 nm), which was critical for purine base determination. Fries medium has an absorption peak at 220 to 230 nm but much less absorbance than Vogel's medium, above 240 nm. Cells were removed from the liquid cultures using a Buchner funnel, and the filtrate was lyophilized to dry powder and suspended in 10 ml of deionized water. A 1.0-ml sample was added to a Dowex 50:H+ column (8.5 by 100 cm). Hypoxanthine or inosine was eluted from the column with 2 N HCl (4). The column was washed with several volumes of deionized water, and then adenine was eluted with 5% NH40H (4). The fractions having the largest absorption at 260 nm were scanned from 200 to 300 nm. Hypoxanthine was identified by its Xmax of 250 nm in acid, and uric acid was identified by its Xmaz of 280 nm in acid. All spectrophotometric data were obtained using a Beckman DBG-T spectrophotometer.
RESULTS Double mutant isolation. The ad-8 locus has been reported to be 20 units left of the centromere on linkage group VI (9), whereas the ad-i locus is located near the centromere on linkage group VI. From a cross between these two mutant strains, 16 of the 50 spores isolated germinated, and of these three failed to grow on
600
J. BACTERIOL.
SABINA, MAGILL, AND MAGILL
hypoxanthine as the purine supplement, demonstrating the presence of the ad-8 allele. (The ad-8 strain will not grow on hypoxanthine.) Complementation tests were made among these three isolates and the ad-i and ad-8 parental strains, and those that failed to complement either were selected as potential double mutants.
Final testing was done by crossing each double mutant strain to the ad-8 and wild-type strains. In the cross of one of the double mutant strains to the ad-8 strain, 80 spores were isolated. Of the 15 germinants, there were no wild-type progeny found, confirming the presence of the ad-8 mutation in the double mutant. In the cross of this double mutant strain to wild type, 45 spores were isolated. Of the 10 germinants tested, one adenine auxotroph was found to grow on hypoxanthine alone, confirming the presence of the ad-i mutation in the double mutant. Hypoxanthine uptake. Figure 1 shows the results of hypoxanthine uptake by the ad-i, ad8, and ad-i ad-8 strains in the presence and absence of NH4+. The ad-i strain served as the control since it accumulates hypoxanthine at approximately the same rate as wild type (11). The ad-8 strain did not take up hypoxanthine to any significant degree in either medium. The double mutant strain did take it up, but at a slower rate than the ad-i strain in NH4+-containing medium and at approximately the same rate in NH4+-free media.
25
20
HPRTase. HPRTase catalyzes the reaction: + PRPP -* IMP+ PP1. This activity was assayed using whole-cell homogenates of germinated conidia from the three adenine auxotrophic strains (ad-i, ad-8, and ad-i ad-8 double mutant) to establish whether there is a direct relationship between the ability of the cell to take up hypoxanthine and the rate at which it is converted to IMP. Table 1 shows the results of the assay, with relative activities expressed as picomoles of [14C]IMP formed per 4 min for 100 x 106 germinated conidia. There was intermediate activity in the ad-i ad-8 double mutant strain compared with the ad-i strain (greatest activity) and the ad-8 strain (very little activity) (Table 1). Boiled whole-cell homogenate from each strain was used as a control, and this base level activity was subtracted from the total counts/4 min of incubation for each strain. In the same experiment, the whole-cell homogenates of the ad-i and ad-8 strains were centrifuged at 10,000 x g for 10 min at 4°C, and 100 ,ul of the resuspended pellet and supernatant fractions was assayed separately. Most of the activity was found in the pellet fraction (see Table 1). Spectrophotometric data. Figure 2 shows the elution profile of the concentrated growth medium from the 48-h shaking cultures of the ad-8 strain from a Dowex 50:H+ column. Using 2 N HCl as eluant, fraction 2 absorbed most strongly at 260 nm, suggesting that this fraction contains hypoxanthine and/or inosine but not adenine (see Materials and Methods). To substantiate this, 200- to 300-nm scans of this fraction from each strain were made, and the results are shown in Fig. 3. Inosine and hypoxanthine were used in control experiments, and it was apparent that the two were inseparable by our methods. Both have Xmax values of 248 nm in acid. Fraction 42, eluted by 5% NH40H, gave strongest absorption at 260 nm. This absorption is attributed to adenine, which gave
hypoxanthine
TABLE 1. HPRTase activity Activity (pmol/4 min) Strain
5
10
15
20
25
30
35
40
TIME (min)
FIG. 1. Uptake of [8-'4C]hypoxanthine (0.10 mM; specific activity, 1.25 p,Cilmol) by germinated conidia of strains ad-i (-), ad-8 (A), and ad-i ad-8 (U) in the presence of NH4+. Results are also shown for identical experiments performed in the absence of NH4+ with strains ad-i (0), ad-8 (A), and ad-i ad-8 (C1).
Whole-cell
Washed Supernahomogeprecipitantb natea tateb ad-1 21.7 20.7 4.3 ad-8 2.1 3.0 2.6 ad-i ad-8 8.5 5.8 1.6 a A total of 100 x 106 conidia were used for homogenization. Data were taken from a regression line of 2-, 4-, and 8-min assays. b Data were taken from a single point at 4 min.
VOL. 128, 1976
REGULATION OF HYPOXANTHINE TRANSPORT
147
.7E
II
601 3b
1.2
0
05
,j A
.OF
z3 b
0* w
w
C)
CIO
10
20
30
40
50
FRACTION NUMBER
FIG. 2. Elution profile of concentrated growth medium from 48-h shaking liquid culture of the ad-8 strain. A 1 .0-ml sample was added to a Dowex 50:H+ ion-exchange column (8.5 by 100 cm). A hypoxanthine compound (a) was eluted with 2 N HCI, and adenine (b) was eluted with 5% NH40H. Fractions of 2 ml were collected at a flow rate of approximately 7 ml/min.
characteristic Xmax in base at 268 nm in control experiments. It is clear that in NH4+-containing Fries medium (Fig. 3a) the ad-8 and ad-i ad-8 strains have excreted different ultraviolet-absorbing compounds. In the presence of NH4+, the ad-i ad-8 double mutant strain excreted a compound having the spectral characteristics of uric acid (Xmaz = 280 nm in acid). In control experiments using 0.01 mM uric acid in NH4+containing Fries medium, uric acid was eluted from the Dowex 50:H+ column in fraction 2, the same fraction in which hypoxanthine or inosine is found under similar conditions. None of these strains, when grown in NH4+-free media, appear to excrete purines (Fig. 3b). DISCUSSION In a previous study (11) it was observed that [8-14C]hypoxanthine was not transported into germinated condia of the ad-8 mutant strain. Genetic studies (11), together with the results presented here, indicate that this lack of hypoxanthine transport is not due to a second mutation but is a consequence of the ad-8 lesion. The ad-8 locus is the structural gene for the enzyme adenylosuccinate synthetase (6), and lack of this enzyme (Fig. 4) probably causes cells to accumulate IMP or its breakdown product(s). It seemed that IMP (or a breakdown product of IMP) might be inhibiting hypoxanthine uptake in the ad-8 strain. To test this hypothesis, we synthesized the double adenine auxotroph ad-i ad-8. The ad-i strain appears to lack aminoimidazole carboxamide ribonucleotide formyltransferase or IMP cyclohydrolase activities, or both (3), and therefore cannot form IMP
.8
z 4t
m
m
0 C,)
cn4
cn
m
.6
4t
.4
.2 )
%
200 225 250 275
X,mI
200 225 250 275 300
x,mjL
FIG. 3. Ultraviolet spectra of fraction 2 eluted from a Dowex 50:H+ column from concentrated growth media of strains ad-8 (a), ad-i ad-8 (b), and ad-i (c). In (a) NH4+ was present in the growth media, whereas in (b) no NH4+ was present. In (a), the compound excreted by the ad-8 strain (a) appears as a shoulder at 250 nm and has been identified as hypoxanthine or inosine. The compound excreted by the ad-i ad-8 strain (b) appeared as a peak at 280 nm and has been identified as uric acid. The large peak at 230 nm represents a component of Fries minimal medium (see text).
de novo. Thus, the accumulation of IMP should not occur in the double mutant ad-i ad-8 strain to the extent that it does in the ad-8 strain. Some IMP probably is formed from adenosine 5'-monophosphate degradation and, if not utilized to form GMP (Fig. 4), may accumulate. The double mutant strain transported significantly more hypoxanthine than the ad-8 strain, as would be expected urider this hypothesis (Fig. 1). It has been shown that in some organisms (2, 8, 10) hypoxanthine transport is mediated by the enzyme HPRTase, possibly by a group translocation mechanism (8, 10). If this were the case in Neurospora, one would expect to find that changes in the levels of HPRTase activity would correspond to changes in the capacity for hypoxanthine transport. The HPRTase assays performed on germinated conidia from the ad-i, ad-8, and ad-i ad-8 mutant strains (Table 1) showed results similar to those found for hypoxanthine transport in these strains; i.e., the HPRTase activity was highest in ad-i conidial extracts, lowest in ad-8 conid-
602
J. BACTERIOL.
SABINA, MAGILL, AND MAGILL
I
synthesis
URflC ACID Ii
de novo
ad-I
Xn Ai
HxO(Ino) HPRTctm
f
AICAR
4ad-
FICAR
/~,VGMP AMP
t Id-8 0-IMP 11---
PRPP
ad-8
MP-S
'---
-M-AP
FIG. 4. Proposed scheme for hypoxanthine uptake and metabolism in N. crassa. The lesions in the ad-i and ad-8 strains are indicated. The vertical lines on the left-hand side represent the cytoplasmic membrane, with the solid line as the outer membrane surface. The abbreviations used in this figure are as follows: AICAR, 5-aminoimidazolecarboxamide ribonucleotide; FICAR, formyl AICAR; IMP, inosine monophosphate; XMP, xanthosine monophosphate; GMP, guanosine monophosphate; AMP, adenine monophosphate; AMP-S, adenylosuccinic monophosphate; Hx, hypoxanthine; Xn, xanthine; Ino, inosine; PRPP, 5'-phosphoribosyl-l '-pyrophosphate.
ial extracts, and intermediate in the conidial extracts from the ad-i ad-8 strain. Thus, the differences in HPRTase activities between the strains correspond to the differences found for hypoxanthine transport. However, this does not prove that HPRTase mediates hypoxanthine transport in this organism. Even though the HPRTase activity was lowest in the ad-8 strain, it was significantly greater than that of the boiled extract control, whereas transport of hypoxanthine by ad-8 conidia was essentially absent in all conditions, suggesting that intermediates accumulated in the cells of the mutant strains may regulate both hypoxanthine transport and HPRTase activity. In enteric bacteria (7, 10), it has been shown that IMP inhibits HPRTase activity and hypoxanthine transport. This could explain the lower activity in the whole-cell homogenate of ad-8, where the IMP concentration would be expected to be greater than in washed precipitates or the washed supernatant. Alternatively, compounds accumulating in these mutant strains may inhibit HPRTase activity which, in turn, may cause transported hypoxanthine to accumulate and prevent further hypoxanthine uptake. Evidence for the accumulation of IMP or an IMP derivative comes from analysis of the
NH4+-containing growth media (Fig. 3a), which shows that the ad-8 strain excretes fairly large quantities of hypoxanthine or inosine. This spectrophotometric data supports previous studies that suggested that the ad-8 strain does excrete hypoxanthine (12, 13) or inosine (13). If we assume that IMP breaks down to yield hypoxanthine (12), the hypoxanthine so formed may be further degraded to uric acid and then finally to NH4+. The ad-i ad-8 double mutant strain grown on adenine in NH4+-containing medium excreted uric acid, an intermediate in the degradative pathway from hypoxanthine to NH4+. Reinert and Marzluf (15) showed that uricase, the enzyme that breaks down uric acid, is repressed in the presence of NH4+ ions in the media. Since all these strains were grown in NH4+-containing Fries medium (Fig. 3a), uricase should be repressed in all the strains, causing uric acid or hypoxanthine to accumulate if formed. It is not clear at this time why the ad-8 strain excretes hypoxanthine and not uric acid when grown on NH4+-containing media. Neither uric acid nor hypoxanthine was excreted by ad-8 or the double mutant when NH4+-free media were used (Fig. 3b). Absence of NH,+ may allow excess hypoxanthine, resulting from IMP breakdown, to be degraded to NH3. The
VOL. 128, 1976
REGULATION OF HYPOXANTHINE TRANSPORT
ad-i strain presumably would never accumulate IMP or hypoxanthine (Fig. 4) and, therefore, would not be expected to excrete hypoxanthine or uric acid under either condition. If hypoxanthine or uric acid excretion indicates hypoxanthine accumulation, which inhibits hypoxanthine uptake, then the absence of NH4+ in the growth medium should allow degradation of excess hypoxanthine (to produce NH3) and thus stimulate hypoxanthine transport in the double mutant, as the only major course for transported hypoxanthine in this strain is through the degradative pathway. However, it was observed in this strain that hypoxanthine transport was decreased when NH4+ was absent (Fig. 1). Thus, it would appear that hypoxanthine is not the compound inhibiting hypoxanthine transport. The stimulation of hypoxanthine uptake by NH4+ in the ad-i strain does not appear to be a consequence of the genetic block, since similar results were obtained using wild type. This finding seems inconsistent with published results showing that NH4+ inhibits the transport of other potential sources of reduced nitrogen and amino acids (14) into Neurospora cells. The heritable disease gouty arthritis in humans is characterized by high levels of hypoxanthine and, consequently, uric acid in the blood stream of afflicted individuals. The primary lesion is thought to be a deficiency of HPRTase (16) or an aberration in the purine synthetic pathway that may lead to the excretion of excess hypoxanthine from cells into the blood stream. The lack of HPRTase activity and the excretion of hypoxanthine (or inosine) by the ad-8 strain suggests the similarity between the ad-8 auxotrophic strains in Neurospora and the phenotype of gouty arthritis in humans. ACKNOWLEDGMENTS This study was supported by Public Health Service Biomedical Support grant 55352 to J.M.M. and National Science Foundation grant GB41869 to C.W.M. LITERATURE CITED 1. Benke, P. L., N. Herrick, and A. Hebert. 1973. Transport of hypoxanthine in fibroblasts with normal and mutant hypoxanthine-guanine phosphoribosyl transferase. Biochem. Med. 8:309-323. 2. Berlin, R. D., and E. R. Stadtman. 1966. A possible role of purine nucleotide pyrophosphorylases in the regulation of purine uptake by Bacillus subtilis. J. Biol. Chem. 241:2679-2686. 3. Buchanan, F. 1954. The enzymatic synthesis of the
603
purine nucleotides. Harvey Lect. 54:104-130. 4. Cohn, W. E. 1949. The separation of purine and pyrimidine bases and of nucleotides by ion exchange. Science 109:377-378. 5. Dybing, E. 1974. Cycloheximide inhibition of hypoxanthine transport in cultured cells. Biochim. Biophys. Acta 373:100-105. 6. Giles, N. H., C. W. H. Partridge, and N. J. Nelson. 1957. The genetic control of adenylosuccinase in Neurospora crassa. Proc. Natl. Acad. Sci. U.S.A. 43:305317. 7. Hochstadt-Ozer, J. 1972. The regulation of purine utilization in bacteria. IV. Roles of membrane-localized and pencytoplasmic enzymes in the mechanisms of purine nucleoside transport across isolated Escherichia coli membranes. J. Biol. Chem. 247:2419-2426. 8. Hochstadt-Ozer, J., and E. R. Stadtman. 1971. The regulation of purine utilization in bacteria. III. Adenine phosphoribosyltransferase in isolated membrane preparations and its role in transport of adenine across the membrane. J. Biol. Chem. 246: 5304-5311. 9. Ishikawa, T. 1962. Genetic studies of ad-8 mutants in Neurospora crassa. I. Genetic fine structure ofthe ad8 locus. Genetics 47:1147-1161. 10. Jackman, L. E., and J. Hochstadt. 1976. Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium. J. Bacteriol. 126:312-326. 11. Magill, J. M., and C. W. Magill. 1975. Purine base transport in Neurospora crassa. J. Bacteriol. 124:149154. 12. Partridge, C. W. H., and N. H. Giles. 1959. Identification of major accumulation products of adenine-specific mutants of Neurospora. (Letters) Arch. Biochim. Biophys. 67:237-238. 13. Pendyala, L., and A. M. Wellman. 1975. Effect of histidine on purine nucleotide synthesis and utilization in Neurospora crassa. J. Bacteriol. 124:78-85. 14. Rao, E. Y. T., T. K. Rao, and A. G. DeBusk. 1975. Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity. Biochim. Biophys. Acta 413:45-51. 15. Reinert, W. R., and G. A. Marzluf. 1975. Regulation of the purine catabolic enzymes in Neurospora crassa. Arch. Biochem. Biophys. 166:565-574. 16. Rosenbloom, R. M., J. F. Henderson, I. C. Caldwell, W. N. Kelley, and J. E. Seegmiller. 1968. Biochemical basis of accelerated purine biosynthesis de novo in human fibroblasts lacking hypoxanthine-guanine phosphoribosyl transferase. J. Biol. Chem. 243:11661173. 17. Roy-Burman, S., and D. W. Visser. 1975. Transport of purines and deoxyadenosine in Escherichia coli. J. Biol. Chem. 250:9270-9275. 18. Rubin, C. S., J. Bancis, L. C. Yip, R. C. Nowinski, and M. E. Balis. 1971. Purification of IMP:pyrophosphate
phosphoribosyl-transferases: catalytically incompetent enzymes in Lesch-Nyhan syndrome. Proc. Natl. Acad. Sci. U.S.A. 68:1461-1464. 19. Ryan, F. J., G. W. Beadle, and E. L. Tatum. 1943. The tube method of measuring the growth rate of Neurospora. Am. J. Bot. 30:748-799. 20. Vogel, J. 1956. A convenient growth medium for Neurospora (medium N). Microb. Genet. Bull. 13:42-43.
