Selection of an adenine resistant strain of Drosophila melanogaster with an altered form of xanthine dehydrogenase

Insect Biochem. Vol. 19, No. 5, pp. 463-469, 1989 Printed in Great Britain. All rights reserved

0020-1790/89 $3.00+ 0.00 Copyright © 1989 Pergamon Press pie

SELECTION OF A N A D E N I N E R E S I S T A N T S T R A I N OF D R O S O P H I L A M E L A N O G A S T E R WITH A N A L T E R E D F O R M OF X A N T H I N E D E H Y D R O G E N A S E K. HAGOPIAN* and E. J. DUKE? Department of Zoology, University College Dublin, Belfield, Dublin 4, Republic of Ireland

(Received 5 April 1988; revised and accepted 3 March 1989) Abstract--A strain resistant to adenine (30 mM) has been selected from the wild-type Pac strain of Drosophila melanogaster. The resistant strain shows a prolonged life-cycle of 14 days, when cultured on adenine food, but reverts back to 9 days when cultured on normal food. The fecundity and fertility of the strain, when cultured on adenine food, are reduced by as much as 60%, but recover to the normal levels when the strain is cultured on normal food. Analysis of the stage distribution of mortalities show that the effective lethal phase (ELP) is larval. Analysis of the enzyme xanthine dehydrogenase from the adenine resistant strain revealed increases in the enzyme's activity, thermal stability and stability during dialysis, but no change in its molecular weight or electrophoretic mobility. The enzyme is also more stable with age and during prolonged incubation at 4°C. These alterations in the enzyme's properties were observed whether the flies were cultured on normal or adenine-containing food.

Key Word Index: Drosophila melanogaster, xanthine dehydrogenase, adenine, adenine resistance INTRODUCTION

Drosophila melanogaster is an important experimental model for evaluating the biological influences of environmental factors, such as diet (Ho et al., 1984a, b), insecticides (Reiss, 1975; M o r t o n and Singh, 1982) and naturally occurring mutagens (Kilbey et al., 1981; Clark, 1982). The selection in Drosophila for a wide range of characteristics and resistance to a variety of chemicals has been described and the effects of this selection, in many cases, on enzyme activities have been well documented. Keller and Glassman (1964) studied the selection for increased and decreased levels of xanthine dehydrogenase activity in Drosophila melanogaster. A n increase in activity was noted in the first two generations, but there was no further response towards higher enzyme activity, and there was a general increase in sterility due to inbreeding. The enzyme xanthine dehydrogenase (xanthine: N A D + oxidoreductase, EC 1.2.1.37) oxidises a wide range of purines, pteridines and aldehydes (Forrest et al., 1956; Glassman and Mitchell, 1959). The developmental effects of these purines and pyrimidines on Drosophila have been studied (Clynes and Duke, 1976; Ho et al., 1984a). Ho et al. (1984b) also reported the effects of a variety of 6-substituted purines on the development of D. melanogaster, with purine and 2,6-diaminopurine being very toxic to egg development, while larvae, when exposed to 0.23% purine, adenine, 2,6diaminopurine, guanine and xanthine showed 10, 50, 55, 90 and 95% survival respectively. Clynes (1976) reported that the presence of adenine (15 raM) in

*Present address: Department of Pharmaceutical Chemistry, School of Pharmacy, University of London, 29/39 Brunswick Square, London WCIN 1AK, England. tTo whom reprint requests should be addressed.

the medium resulted not only in a markcd decrease in the xanthine dehydrogenase activity, but also in increased stability of the remaining activity to overnight dialysis. Hagopian and Duke (1986) also reported that adenine increases the thermal stability of xanthine dehydrogenase, that it binds to the enzyme and inhibits its activity competitively. In this investigation, thc development of resistance to adenine in Drosophila melanogaster and the selection of an adenine resistant strain (ad r) is described. The properties of the enzyme in the selected strain are studied and compared with those of the wild-type. MATERIALS AND METHODS

Drosophila stocks and cultures The Pacific (Pac) wild-type strain of Drosophila melanogaster from which the adenine resistant strain was selected, is maintained at 25°C as previously described (Clynes and Duke, 1976). Adenine-containing food was prepared according to Hagopian and Duke (1986), except that the amount of adenine used was doubled for preparing 30 mM food. For cross-resistance studies, food containing the parent compound purine was prepared similarly to the adenine food, at concentrations of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 5.0 raM. Selection of the adenine-resistant (ad') strain of Drosophila Adult flies were allowed to lay eggs for 2-3 days on 15 mM adenine-containingmedia, then transferred to fresh 15 mM adenine-containing media again. At the end of the second period, they were removed to normal media for a further 48 h, to study possible sterilisation effects of the drug. When the first generation emerged from the adenine media, it was changed into fresh 15 mM adenine-containing cultures. This process was continued for 10 generations after which the emerging adults were changed to media containing 30 mM adenine. The first generation emerging from the 30 mM adenine media, and all the successive generations have been maintained ever since on this high level of adenine.

463

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K. HAGOPIANand E. J. DUKE

Collection of eggs Eggs were collected by shaking 20-30 breeding pairs of adult flies into empty bottles, which were then inverted over small Petri dishes containing normal food or an agar-acetic acid mixture, consisting of a 5% agar solution to which 2% acetic acid was added (Duke, 1964). Adults were left on the food for 2-3 h, after which the eggs were removed using a blunt needle. Fecundity and fertility Groups of flies (50 d'd' and 50 99) from the wild-type and resistant strains were pre-fed for 24 h on normal and 30 mM adenine-containing food respectively. They were then shaken into empty bottles and upturned over their respective media. In all cases, the females were allowed to oviposit for a fixed period of 6 h. They were then removed and eggs counted. The eggs were then allowed to develop and the number of emerging adults recorded. Stage distribution o f mortalities Groups of 100 eggs from both the wild-type and resistant strains were placed on small square pieces of black filter paper, already moistened with distilled water, and left inside vials containing normal and adenine food respectively. Also, another piece of filter paper carrying eggs of the resistant strain was placed in a vial containing normal food. The number of unhatched eggs was counted after 48 h and the development of the remainder monitored daily until adults emerged. The number of emerging adults and pupal cases was recorded. The distribution of mortalities was calculated using the formulae described by Wright (1973). Cross-resistance to purine Groups of 100 eggs from the resistant strain were placed inside vials containing different concentrations of the parent compound purine, and development monitored daily. Eggs from the wild-type strains were similarly monitored as controls, to compare the effects of this highly toxic compound on the two strains.

o

o

Preparation o f extracts and enzyme assays Unless otherwise stated, all extracts were prepared and experiments performed at 4°C. The enzyme was partially purified, as far as the Ultrogel AcA34 gel filtration step, as described by Hagopian and Duke (1988). All extracts were adjusted to contain equal amounts of protein. Xanthine dehydrogenase (XDH) activity was measured fluorometrically according to the method of Glassman (1962), as described by Hagopian and Duke (1986). Uric acid determination Uric acid production was measured according to Sigma Technical Bulletin, No. 292-UV (Sigma Chemical Co. 1977), using uricase enzyme (Urate:oxygen oxidoreductase, EC 1.7.3.3). Gel electrophoresis Polyacrylamide gel electrophoresis (Ornstein, 1964; Davis, 1964) was performed, at 4°C, using the recipes of Johnson (1983), in either slabs or rods. Gels were stained for enzyme activity according to Yen and Glassman (1965). Gel chromatography and isoelectric focusing Gel filtration using Ultrogel AcA34 and isoelectric focusing were performed as described by Hagopian and Duke (1988). Thermal stability Extracts were incubated in a water bath at 50°C and aliquots withdrawn at specific intervals, followed by an immediate cooling in ice-water and then centrifuged at 30,000 g for 20 min. Enzyme activity was then measured as described.

°i°i

Xanthine dehydrogenase in D. melanogaster

Chemicals All chemicalswere purchased from Sigma or BDH, except Ultrogel AcA34 from LKB-Bromma, Sweden. RESULTS

Isolation o f the adenine resistant (ad ~) strain Our initial attempt to select a resistant strain using 30 mM adenine was not successful. This problem was bypassed by initially using 15 mM adenine food. The emerging adults were collected and placed in one culture bottle containing 15 mM adenine food, and changed twice into new cultures to ensure maximum oviposition. This process was repeated for 10 generations. By this stage, the life cycle was reduced from 17 to 15 days. After the 10th generation the adults were changed to 30 mM adenine food and development lasted 20 days. The emerging adults were collected and placed on fresh 30 mM adenine food, changed twice to ensure maximum oviposition, and the stock has been kept on 30 mM adenine food ever since. The life cycle was also shortened to 17 days in the tenth generation and to 14 days in the twentieth and all subsequent generations. Stage distribution o f mortalities The results of the stage distribution of mortalities are shown in Table 1. The unhatched eggs were counted and these may be brown or white, the latter being considered as unfertilized eggs (Wright, 1973). No white eggs were detected in this study and all the unhatched eggs were brown. Therefore, the total fertilized eggs were equal to total eggs transferred minus unhatched fertilized (brown) eggs. The results show that the effective lethal phase (ELP) of the selected strain is larval. When the resistant strain flies were changed to normal food, the percent mortalities of different stages of the resulting generations were similar to those of the wild-type strain, but when these flies were changed back to 30 mM adenine food, after several generations on normal food, they reverted to the original stage distribution of mortalities of the selected strain. Fecundity and fertility A comparison of the actual number of eggs laid over a period of 6 h, by the resistant strain on 30 mM adenine food and normal food, and by the Table 2. Cross-resistanceof the adr strain to purine, compared to that of the wild-typestrain %Emergence of adults Concentration of purine ad' Wild-type (mM) strain strain 0.00 89 87 0.50 88 76 0.75 76 52 1.00

1.50 2.00 2.50 3.00 4.00 5.00

68

42

51 21 23 -2 -------The results of three replicates (which showed very little variation) were pooled, Each replicate contained 100 eggs.

465

1800

~ 1400 "__~1000

~< 60(

200

Time (hrs) Fig. l. Effect of ageing on xanthine dehydrogenase activity in adult wild-type ( - Q - ) and a d r (-(3-) strains of Drosophila melanogaster. Age is given in h from time of hatching. All values are means+ SEM of triplicates. wild-type strain on normal food, showed that the fecundity was severely affected by the drug. The number of eggs laid on the drug food, by the resistant strain, was approximately 35-40% of that laid on normal food by both strains. Fertility of the three groups was also checked by allowing the eggs from the fecundity tests to develop into adults. In this case the % emergence of the ad r strain on the adenine food was 41 4- 3%, compared with 86 _+4% and 85 + 3% for the ad r' and wild-type strains, respectively, on normal food.

Cross-resistance o f the selected strain to purine The results of the cross-resistance studies of the ad r strain to the compound purine are shown in Table 2. Wild-type strain was also used as a control. The results showed that % emergence of adults for every concentration was higher in the selected strain, when compared to those of the wild-type strain. Also, no wild-type adults emerged when media contained more than 1.5 mM purine, while emergence continued in the selected strain up to the 2.5 mM level, even though the numbers were greatly reduced. The life cycle for both strains on the purine food was 11 days. Xanthine dehydrogenase activity in the selected strain The XDH activity was not measured in the first stage (15 mM adenine) of selection, mainly due to the small number of adults available. Activity was measured in the second stage (30 m M adenine), and at the first generation the enzyme was found to be 55-60% less active than the controls. At the tenth generation the enzyme was 25-30% less active than the controls, while at the twentieth generation it was found to be the same as the controls. From the thirtieth generation onwards, the activity was found to be 20-25% higher than the controls and more stable during overnight dialysis, losing only 25-30% of its activity; as opposed to well over 50% in the controls. The enzyme in the ad' strain was more stable with age, losing little activity in flies over an ageing period of 120 h from hatching, when compared with wild-type controls (Fig. 1). This enzyme

K. HAGOPIAN and E. J. DUKE

466

T h e r m a l stability Results from heat treatment revealed that the e n z y m e f r o m a d r s t r a i n w a s m o r e r e s i s t a n t to h i g h t e m p e r a t u r e s (50°C) a n d lost j u s t u n d e r 3 0 % o f its o r i g i n a l a c t i v i t y a f t e r 1 h o f i n c u b a t i o n , as o p p o s e d to 9 0 - 9 5 % o f t h e a c t i v i t y in t h e c a s e o f t h e c o n t r o l s ( T a b l e 3). T h e half-life o f t h e a d r e n z y m e u n d e r t h e s e c o n d i t i o n s w a s c a l c u l a t e d as 131 h in c o m p a r i s o n to 18 h f o r t h a t o f t h e w i l d - t y p e . A t 3 5 ° C t h e r e s p e c t i v e h a l f - l i v e s were e s t i m a t e d to be 188 h a n d 50 h. B o t h enzymes were very unstable at the higher temperature o f 68°C.

od r

0

~2 >.

Electrophoresis, g e l f i l t r a t i o n a n d isoelectric f o c u s i n g

O~

1

2t* ~8

7'2

I

Time (hrs)

R e s u l t s f r o m gel e l e c t r o p h o r e s i s , gel f i l t r a t i o n a n d isoelectric f o c u s i n g s h o w e d n o c h a n g e s in t h e electrop h o r e t i c m o b i l i t y , t h e m o l e c u l a r w e i g h t (300,000) o r t h e isoelectric p o i n t (5.2) o f t h e e n z y m e f r o m e i t h e r t h e a d r o r wild-life s t r a i n s .

120

96

C h a r a c t e r i s t i c s o f the ad" strain c u l t u r e d on n o r m a l Fig. 2. Stability o f xanthine dehydrogenase during incubation at 4°C, of extracts from wild-type and ad r strains of Drosophila melanogaster over a period of 120 h. The activity of X D H is expressed on a log scale and the slopes drawn by regression line analysis.

was also more stable during overnight dialysis. The extracted enzyme from the a d r strain was also more stable than that of wild-type when incubated in the cold (4°C) for up to 120h (Fig. 2). From the slopes of this semi-log plot, half-lives of 64 h and 201 h were obtained for the wild-type and a d r enzymes respectively.

food

Adults from the selected strain were changed onto normal food, to investigate whether the enzyme would retain its altered properties or revert to those observed before selection. Table 4 shows that the enzyme did not revert to the wild-type form and still expressed higher activity and resistance to both dialysis and high temperatures. The duration of the life cycle reverted to 9 days and the number of adults emerging increased to the levels of that of the wildtype strain, as already shown. When these adults were changed back to 30mM adenine food, the first generation took 16 days to emerge, while the subse-

Table 3. Thermal stability of xanthine dehydrogenase from the ad r and wild-type strains of Drosophila melanogaster Enzyme activity (I.U./ml) at the indicated incubation times (min)* 30 60

Extracts

Temp.

0

Wild-type ad r Wild-type ad' Wild-type ad'

35 35 50 50 68 68

1170 ± 20 1550 ± 23 1166 ± 22 1540±25 1170 ± 21 1550 ± 23

650 ± 1380 ± 440 ± 1254± -250 ±

15 19 90 16 11

460 ± 1240 ± I l0 ± ll20± ---

12 18 50 12

Half-life in h

% activity lost after 1 h

50 188 18 131 ---

60.7 20 90.6 27.3 100 100

*Extracts were incubated at 35, 50 and 68°C and aliquots withdrawn at the end of each incubation period, cooled in ice-water, centrifuged and activity assayed. The ad' strain used in these experiments had been cultured on normal media. All values are means ± SEM of triplicates. The enzyme half-lives were calculated from the slopes of semi-log plots of activity versus time.

Table 4. Xanthine dehydrogenase activity in the ad' strain cultured on normal food for several generations Enzyme activity (I.U./ml)** Activity After lost Incubation at 50°C Generation* dialysis (%) 30 rain 60 min P 1760+ 14 1310± 11 25.6 1342+ 15 1210+ 15 Ft 1716± 14 1232± 12 28.3 1331 ± 14 1188 + 11 F3 1683 ± 13 1180± 11 30,0 1276± 13 1135± 13 Fe 1655±13 1166±12 29.6 1243± 15 1120±14 *Adults from the ad r strain were left on normal food for 2 days to oviposit, were designated as parents (P) and the enzyme's activity measured before and after heat treatment at 50°C. The first generation (F~) was changed to fresh bottles to oviposit for 2 days and then the enzyme's activity measured. This was repeated for several generations, of which Before dialysis

P, F~, F3 and F6 are shown here. **All values are means ± S E M of triplicates.

Xanthine dehydrogenase in D. melanogaster quent generations took 14 days. Also, the number of adults emerging was reduced to the levels previously observed for the selected strain on adenine food. Uric acid p r o d u c t i o n

Uric acid production was also measured in the second stage of selection and a reduction of 60% in the first generation was observed. As the X D H became more active in the tenth and twentieth generations, there was a gradual increase in the level of uric acid produced. Surprisingly, no increase in uric acid levels was detected in the thirtieth generation flies when the XDH activity increased by 25% over control levels. DISCUSSION

A strain of D r o s o p h i l a m e l a n o g a s t e r resistant to 30 mM adenine has been selected. The toxic effect of adenine was demonstrated when initial selection was attempted using a concentration of 30 mM. Not only did adults not emerge, but there were no second or third instar larvae and the food was not well worked, indicating death at an early larval stage. These results are in agreement with the previous findings of Clynes and Duke (1976). Selection was re-started using 15 mM adenine, since all of our previous experiments were performed using this concentration (Hagopian and Duke, 1986). A very small number of flies emerged in the initial experiment, which suggests the existence of a very small sub-population of resistant flies in the wild-type stock of D. m e l a n o g a s t e r . Nash and Henderson (1982) reviewed the biochemistry and genetics of purine metabolism in D. m e l a n o g a s t e r and reported that purine bases in some cases could stimulate growth in wild-type flies that had already been observed to be stimulated by R N A or its hydrolysis products, or in auxotrophs that had absolute growth requirements for one or another purine nucleotide. In our resistant a d ~ strain, adenine was neither utilized this way, since growth was not stimulated but rather inhibited, nor was the strain an auxotroph, since it could develop normally on control medium without adenine. These authors also reported that some purine bases were toxic to Drosophila. Although it is commonly presumed that toxicity first involves conversion of bases to nucleotides, studies in other systems have shown that this is not always the case (Henderson, 1980; Henderson and Scott, 1980; Henderson et al., 1980). Becker (1974a, b) reported that of the two enzymes involved in nucleotide synthesis from purine bases, the activity of adenine phosphoribosyltransferase (APRT) was the substantial one in the cultured D r o s o p h i l a embryo cell lines and extracts of adult flies, whereas no hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity could be detected. He selected cell lines resistant to 8-azadenine and 2-fluorouridine which lacked APRT activity. Johnson and Friedman (1981) reported that purine resistant mutants in D r o s o p h i l a m e l a n o g a s t e r were deficient in APRT activity, and the resistance was due to mutations in the APRT structural gene (Johnson and Friedman, 1983). In mammalian systems studies have shown that mutations within the structural gene for a specific purine base phosphoribosyltransferase IB t 9/5--B

467

enzyme can be the source of resistance to that particular purine base, because the defective enzyme could no longer convert it to a toxic nucleotide (Epstein et al., 1977; Fenwick et al., 1977; Wyss, 1979). It is possible, therefore, to suggest that resistance in the a d ~ strain could be due to some deficiency in the A P R T activity. Assays for this enzyme will form part of further detailed studies. In this study, adenine did not cause sterilisation, since eggs layed on normal food, by a d r flies that were transferred from the adenine medium, developed into adults. The life cycle of the a d r strain was lengthened, however, to 14 days when cultured on adenine medium. It appears that development is affected by the ingestion of adenine, since the life cycle reverts back to the normal 9 days when the a d ~ strain is cultured on normal medium. This is similar to the observations of Ho et al. (1984a). There was also a sharp decrease in the fecundity and fertility of the a d r strain when cultured on adenine food, but this was reversed on normal medium. When the fecundity results of the a d ~ strain cultured on adenine food are compared with those of the wild-type and a d r strain on normal food, they are statistically significant (P < 0.01), as are the fertility results (P < 0.005). The differences between the wild-type and a d ~ strains cultured on normal food are not significant. Results of stage distribution of mortalities show that the effective lethal phase (ELP) is larval, and in general, this stage is far more sensitive to drugs than the other stages (Clark, 1982). Significant differences were observed between the number of a d ~ adults emerging from adenine food and wild-type or a d r adults emerging from normal food (P <0.001). However, the difference between the number of a d ~ and wild-type adults emerging from normal food is not significant. Ho et al. (1984a) reported that adenine depressed the number of flies hatching by affecting the ovulation of parental flies and inhibiting larval development, probably due to changes in adenine metabolism at the cellular level. Our findings from selection, fecundity, fertility and stage distribution of mortalities tests agree with this. When adenine was absent from the culture medium, the levels of fecundity, fertility and stage distribution of mortalities were similar to the wild-type levels, indicating adenine's interference with developmental processes. After several generations on normal medium, these flies were returned to adenine-containing medium, and they reverted back immediately to the same characteristics of the a d ~ strain that is routinely cultured on adenine medium. Flies of the a d ~ strain also showed cross-resistance to purine which, according to Ho et al. (1984b), is the most toxic of all the purine bases and causes a sharp decrease in the number of adults emerging, while the wild-type strain was much more sensitive. At 0.5 mM purine, the difference in the number of emerging adults between the a d ~ and wild-life strains was not statistically significant but was significant at concentrations of 0.75, 1.0, 1.5, and 2.0mM (P <0.05, P < 0.05, P < 0.01, and P < 0.01 respectively). It is clear that different strains respond differently to a particular drug. Ho et al. (1984b), using 0.23% purine, which is the equivalent of just under 4 mM under our experimental conditions, reported a 9% survival rate, while in our case there were no

468

K. HAGOPIANand E. J. DUKE

survivors at this level in either strain. This crossresistance between purine and adenine would indicate a common basis for both. In selecting the a d ~ strain, the most important objective was to see if XDH in this strain would be affected. The effects of adenine on XDH from wildtype Drosophila melanogaster have been previously studied (Hagopian and Duke, 1986). Selection for XDH activity in D. melanogaster has been previously reported (Keller and Glassman, 1964). Two lines were selected, one for low enzyme activity which was due to gradual fixation of the l x d gene, and the second line for high enzyme activity. The latter strain, after several generations, showed increased sterility and, with the effects of inbreeding, resulted in a decline in the enzyme's activity and the loss of the strain. The ad r strain is neither sterile, since it has bred well for the last four years, nor has inbreeding had any detrimental effects on the enzyme's activity, since the strain is showing a constant 25% increase in its XDH activity. Our results would appear to indicate that some changes have taken place in the XDH molecule. The enzyme has become far more stable during overnight dialysis than the wild-type enzyme. Previous workers have used dialysis without any adverse effects on the enzyme's activity (Seybold, 1974; Andres, 1976) but they did not use the Pac strain of D. melanogaster, which was used in this study and contains a highly dialysis-sensitive XDH (Hagopian and Duke, 1986). Dialysis affects the enzyme by possibly changing the active sites, denaturation, loss of cofactors, proteolysis, conformational changes or a combination of effects. It is clear from this study that the XDH in the selected ad r strain is more stable and resistant to the above mentioned factors. It has also been purified (Hagopian and Duke, 1988) and shows decreased K,, values when compared with those of the wild-type. This could explain its higher catalytic activity and might indicate that it is a more active mutant form, with an altered structure. This is in contrast to previous studies (McCarron et al., 1979; Rushlow and Chovnick, 1984) where no significant differences in the enzyme's Km values from wild-type and several ry ÷ mutants were observed. Another possible reason for the increased activity could be an increased synthesis of the enzyme, although this point has not yet been investigated. The ad r enzyme's resistance to high temperature would, also, appear to indicate changes in its structure. Heat treatment is an important step in the purification procedure, and has been used before without any adverse effects (Parzen and Fox, 1964; Seybold, 1974; Andres, 1976; Hagopian and Duke, 1988). Schott et al. (1986) have reported that xanthine dehydrogenase from lxd mutants, produced by X-rays or ethyl methanesulphonate (EMS) has less thermostability than that of their wild-type counterparts and that this is due to modification of protein structure. Our gel filtration, electrophoresis and isoelectric focusing results, however, showed no changes in the XDH between the wild-type and mutant strains. This indicates that any changes in the a d r enzyme which might have occurred are not on a major structural scale, but are possibly due to a small mutation at the level of the structural gene.

It should also be noted that the enzyme from the ad r strain retained its altered properties, such as high

activity, stability during dialysis and resistance to high temperature, when cultured on normal food for several generations. This indicates yet again that a genetic selection has occurred rather than a general inductioh effect. The mutant enzyme is more stable during the prolonged incubation of extracts and with age as seen from the significant differences in the half-lives of the respective enzymes under these conditions. These two characteristics are retained by the enzyme when the strain is cultured on normal food. It is proposed at this stage that a mutation has taken place in the structural gene of XDH, resulting in an altered enzyme in the adenine resistant strain of Drosophila melanogaster further studies are being carried out to establish the genetic basis of the selection, the rate of enzyme synthesis, the nature of the differences, if any, of XDH from the resistant strain and whether any changes in the APRT system have occurred. Acknowledgement--K. Hagopian is the holder of a Boghos

Nubar Pacha Foundation grant, and wishes to thank the Foundation for this financial assistance. REFERENCES

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