T H9.“ *9 This is to certify that the dissertation entitled The Role of 20-Hydroxyecdysone and Dietary Protein in the Regulation of Urate Oxidase Activity During Development of the Third Instar of Drosophila Date presented by Leos G. Kral has been accepted towards fulfillment of the requirements for Ph. D. degreein Zoology and Genetics Major professor November 15, 1985 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 ‘IV1SSI_} RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from ” your record. FINES will be charged if book is returned after the date stamped below. THE ROLE OF 20-HYDROXYECDYSONE AND DIETARY PROTEIN IN THE REGULATION OF URATE OXIDASE ACTIVITY DURING DEVELOPMENT OF THE THIRD INSTAR LARVA OF DROSOPHILA. BY Leos George Kral A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program and Department of Zoology 1985 ABSTRACT THE ROLE OF 20-HYDROXYECDYSONE AND DIETARY PROTEIN IN THE REGULATION OF URATE OXIDASE ACTIVITY DURING DEVELOPMENT OF THE THIRD INSTAR LARVA OF DROSOPHILA. BY Leos George Kral The tissue specific enzyme urate oxidase is confined exclusively to the Malpighian tubules of Drosophila melanogaster and expressed only'in.the‘third instar larva and the adult. Shortly before pupariation urate oxidase activity declines precipitously and is not detectable 24 hours later. That 20—hydroxyecdysone is the factor that triggers the disappearance of urate oxidase in the late third instar larvaeris demonstrated using the temperature sensitive mutant ggdl which at the nonpermissive temperature of 29°C fails to accumulate a sufficient concentration of 20-hydroxyecdysone necessary for puparium formation. Both the life cycle and the temporal profile of urate oxidase activity in egdl larvae at 20°C is identical to that of the wild type. However, at 29°C egdl third instar larvae retain high urate oxidase activity; A precipitous decline in urate oxidase activity is observed when egdl larvae at 29°C are fed 20-hydroxyecdysone. These data implicate 20- hydroxyecdysone in the process that controls the rapid decline of urate oxidase activity at the time of puparium formation. Visualization of the urate oxidase peptide in SDS-polyacrylamide gels shows that the amount of the urate oxidase peptide is correlated with the amount of urate oxidase activity during development. A urate oxidase cDNA clone was isolated from a Malpighian tubule cDNA library and utilized in a Northern gel analysis of mRNA from Drosophila melanogaster larvae at various stages of development. This analysis shows that the amount of urate oxidase activity is correlated with the amount of urate oxidase mRNA present. Rearing third instar larvae»on diets containing different amounts of protein and purine compounds shows that urate oxidase activity is primarily induced by dietary protein. The inhibitors of de ngxg purine biosynthesis, azaserine and DON, inhibit induction of urate oxidase activity by dietary protein. ACNOWLEDGMENTS First and foremost I would like to extend sincere thanks to Dr. Thomas Friedman for his guidance, patience and encouragement during the course of this study; I would also like to thank Dr. James Asher, Dr. Lenny Robbins, Dr. Stephen Bromleyy Dr. Jerry Dodgson and Dr. Robert Robbins for their contributions to my development as a scientist and teacher. I would particularly like to thank Dr. Dan Johnson for his friendship, many helpfull discussions, and invaluable help during various phases of this study. Finally, I would like to thank my parents for both their moral and financial support and my wife, Deborah, for her understanding. ii TABLE OF CONTENTS CHAPTER ONE ...OOOOOOOOOOOOOOOOOOOO...0.00.00.00.00000000001 IntIOdUCtion I O I O O O O O I O O O O O O O O O O O O O I O I O O O I O O O O O O O O O O O O 1 Quantitative Regulation of Urate Oxidase .............4 Demonstartion of the Existence of a Diffusible Urate Oxidase Inducing Factor 4 Temporal Regulation of Larval Urate Oxidase ACtiVitY 0.00..O...OOOOOOOOOOOO0.0.0.000000000000000008 Evidence Suggesting a Humoral Repressor .........8 Evidence Implicating 20-Hydroxyecdysone as the Repressor 11 Effect of the Mutant £5311 on Urate OXidase ACtiVity COCOOOOOOOOOOOOOOOOOOO0...11 Effect of 20-Hydroxyecdysone on Larval Urate Oxidase Activity 16 Analysis of the Level of Urate Oxidase Regulation ...21 Temporal Distribution of Malpighian Tubule Proteins ...OOOOOOOOIOOOOOO00.0.0000...0.0.0.0000000021 Temporal Distribution of Urate Oxidase mRNA .........28 Does 20-Hydroxyecdysone Act Directly on the Genome Of Malpighian TUDuleS? OOOOOOOOOOIOOOOOOOO00.031 CODClUSionB ...ICOOOOOOOIOO0....00.00.00.00000000000036 CHAPTER Two COIOCOOOOOOOOO0.00.00.00.00...00.0.0000000000039 IntrOdUCtion 00.0.0000...0.....0...0.0.0....00000000039 Effect of Diet on Urate Oxidase Activity ............40 iii Role of Purine de nove Synthesis in Urate Oxidase IndUCtion 000......OOOOOOOOOOOOOOOO0.00.00.00.000000048 CODCIUSj-ons 0....0.0.0....00......00.0.0000000000000063 CHAPTER THREE I...OOOOOOOOOOOOOOO0.0000000000000000000000.66 Construction and Isolation of a Urate Oxidase CDNA Clone .00...OOOOOOOOOOIOOOOOOOOOO0.0.0....00...000.0066 Identification of in 11119, Translated Urate OXidase ...OOOOOOOOOOOOOCOOOOOCO00.0.0.0...0.0.0.....66 Protocol for Preparation of Malpighian Tubule CDNA Library OOOOOOOOOOOOOOOOOOOOO0.0...00.00.00.000069 Details of Malpighian Tubule cDNA Library Preparation O....OOOOOOOOOOOIOOOOOOO0.0.0.00.0..71 Isolation of Urate Oxidase cDNA From the Malpighian TUDUIe CDNA Library .0.00IOOOOOOOOOOOOOOOOO0.0.0.000083 Preparation of Plasmids ..83 Binding of cDNA to Nitrocellulose Filters ......84 Hybridization Selection of RNA 85 APPENDIX .................................................88 Materials and Methods ...88 Drosophila Strains .88 Timing and Rearing of Larvae .88 Determination of Urate Oxidase Activity .u.u.n.u.89 Transplants of Malpighian Tubules 91 SDS-Polyacrylamide Gel Electrophoresis u.u.u.u.u92 Immunoprecipitation of Urate Oxidase from Malpighian TUbUIeS OOOOOOOOOOOOOOOOOOOOOOOO0.0.0....093 Immunoprecipitation of Urate Oxidase peptide from in litLQ Tran81ate8 0.0.0...0....00.000.000.00000000094 Source of 20-Hydroxyecdysone........................94 iv Source and Preparation of.Azaserine and DON an.u.u95 Feeding Larvae on Defined Diets 95 RNA Extraction from Malpighian Tubules u.u.u.".u95 Northern Gel Analysis of RNA 98 Alkaline Agarose Gel Electrophoresis 9................9 REFERENCES ...OOOOOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOO0.0.0101 LIST OF TABLES Table I. Urate oxidase activity of adult donor and host tubules 24 hours after tansplantation ..............6 Table II. Urate oxidase activity of egdl larval and pupal donor Malpighian tubules cultured in newly eelosed adu1t hOStS 000.000.000.000...0.00.00.00.00...00.7 Table III. 1 Immunoprecipitation of urate oxidase activity from end larval Malpighian tubule homogenates .........24 Table IV. Urate oxidase activity of 9.9511 early third instar larvae reared on wet cellulose and supplements at 29°C for 24 hours 43 Table V. Urate oxidase activity of m1 early third instar larvae reared on wet cellulose and supplements at 29°C for 12 hours 45 Table VI. Urate oxidase activity of egdl early third instar larvae raised on low casein supplemented with purines and nucleic acids at 29 ..47 Table VII. Urate oxidase activity of egdl early third instar larvae raised on low casein supplemented with precursors of de novo purine synthesis at 29°C .........50 Table VIII. The effects of varying concentrations of azaserine on urate oxidase activity induced by dietary casein O...OOOOOOOOOOOOOOOOOO0.0.0.0.0000000000052 Table Ix. Inhibit n of casein induced urate oxidase activity by 10" oM azaserine at 12 and 24 hours of feeding at 29° C 0.00....CO...OOOOOOOOOOOOOOOOOOOOOOOOO0.53 Table X. Inhibitizo n of casein induced urate oxidase activigyc by 10" M DON at 12 and 24 hours of feeding at 29 0.0....OOCOOOQOOCOOOOOOO...0.0.00.0000000000000054 Table XI. The effect of dietary RNA on urate oxidase activity in larvae reared on casein and azaserine and the effect of azaserine on a yeast extract diet ........58 vi Table XII. Effect of dietary RNA and DON on casein induced urate oxidase .59 vii LIST OF FIGURES Figure 1. Urate oxidase activity during the development of two strains of Drosophila melanogaster ...............3 Figure 2. Uraie oxidase activity of £9511 largal tubules, and larval tubuleszcultured in ry. newly emerged adults, and the xx host tubules ...............10 Figure 3. Urate oxidase activity of egdl third instar larvae and pupae at 20° C and 29° C 0.0.00.00000000000000013 Figure 4. Effect of starvation onl urate oxidase activity of third instar larvae 15 Figure 5. Effect of feeding 20-hydroxyecdysone to egdl third inStar larvae at 29° C .0.0..0.00.00.00.0000000000020 Figure 6. SDS-polyacrylamide gel electrophoresis of Coomassie Blue siained proteins from Malpighian tubules of £251 third instar larvae 22 Figure 7. SDS-polyacrylamide gel electrophoresis of silver stained proteins from Malpighian tubules after removal of proteins bound to preimmune IgG and anti- Drosophila urate oxidase IgG 25 Figure 8. Northern gel analysis of urate oxidase mRNA during the larval stage of DrosoPhila development ......29 Figure 9. Urate oxidase activity of Malpighian tubules cultured in vitro at a time of maximum in £119. actiVity 0.00.00......OOOOOOOOOOOO...0.00.00.00.0000000033 Figure 10. Urate oxidase activity of Malpighian tubules cultured in _¥1_t_§% in Schneider's medium with and without 10" O-hydroxyecdysone .......................35 Figure 11. Urate oxidase activity of egdl third instar larvae feeding on cellulose saturated _4with 0.8% sucrose only and 0.8% sucrose with 10" M 20- hydroxyecdysone ....37 Figure 12. The effect on urate oxidase activity of feeding starved larve .41 viii Figure 13. Azaserine and DON sensetive site in de nggg purine biosyntheSis .0...O....OOOOOOOOOOOOOOOOO0.0.49 Figure 14. Catabolism of purines 57 Figure 15. Urate oxidase activity of pnzlzl and Amherst wild-type third instar larvae reared on yeast at 20°C 0......O...OOOOOOOOOOIOOOOO...OOOOOOOOOOOOOOOOO.62 Figure 16. Fluorographs of dried SDS-polyacrylamide gels separating in gitrg translated peptides synthesized in rabbit reticulocyte lysate systems .u.“68 Figure 17. Size distribution of double stranded cDNA synthesized from whole Drosophila larval polyA+ RNA ....75 Figure 18. Agarose gel of Pst I cut Malpighian tubule CDNA-pBRBZZ plsmids OOOOOIOOOOOOIOOOOOOO00.0.0000...0.0.82 Figure 19. SDS-polyacrylamide gel electrophoresis of in gitrg translated proteins from mRNA hybrid selected by Malpighian tubule cDNA 87 ix <3uuumm.ouE Introduction Urate oxidase activity in Dosophila has been shown to be tissue specific and highly regulated during development (Friedman, 1973). Urate oxidase [E.C.1.7.3.3] converts uric acid to allantoin and is confined to the Malpighian tubules, the excretory organ. Urate oxidase activity is detectable only during the third instar larval stage and in the adult (Figure 1 and Friedman, 1973). No activity is detected during the pupal period. During metamorphosis, many larval tissues in Dros0phila are histolized. The Malpighian tubules, however, are not histolized during metamorphosis but are carried over intact to the adult. In the adult, the reappearance of urate oxidase activity in the Malpighian tubules is synchronized with the emergence of the adult from the puparium. The temporal coordination of the reappearance of urate oxidase activity with emergence has been shown to be controlled in part by an autonomous clock-like mechanism (Friedman and Johnson, 1977; Friedman and Johnson, 1978). The temporal appearance and disappearance of proteins within a specific cell type during development is presumably due to differential gene expression. If the changes in urate oxiase activity during development of Drosophila can 2 be demonstrated to be due to differential levels of transcription of the urate oxidase gene, urate oxidase could be a very important model system for the study of complex regulation of eucaryotic genes. To this end, the regulation of urate oxidase during the third larval instar of Drosophila has been studied. Data presented in this dissertation demonstrate that the disapperance of urate oxidase prior to pupariation is dependent on high in vivo .levels of the steroid hormone 20-hydroxyecdysone and that the amount of urate oxidase activity during development is correlated with the amount of urate oxidase mRNA. 24hqwc i G l- /‘ : '\° E's . 8 \‘\. 35 4 . 2 O '- l 3 3 . 3 2 L M O Ill 2 , 9 s t \ . 0—- ~O o/ 0 -eo--co--eo-op 8—0—00— Euamro I" 2"" a" INSTAR PUPA ADULT Figure 1. Urate oxidase activity during the development of two strains ofz Drosophila melanogaster. Theurateoxidase actiyity of 11 :(O) and 913:3: (0) 1% expressed as the amount of [ C] uric acid converted to [ C] allantoin per minute of reaction time per set of Malpighian tubules from a single Drosophila. The bar in the upper right hand corner indicates the time scale at 25°C. The pattern is identical to that previously reported using a spectrophotometric assay for urate oxidase activity (Friedman, 1973). 4 Quantitative Regulation of Urate Oxidase Demonstartion of the Existence of a Diffusible Urate Oxidase Inducing Factor As illustrated in Figure 1, the urate oxidase activity of l-day-old ryz adults is five to tenfold higher than that of 1-day-old 933:3 adults (Friedman, 1973). The data in Table I (Kral et al., 1982) indicate that urate oxidase activity in Malpighian tubules from 913:3 is stimulated by a diffusible hemolymph factor that is present in high concentration in the hemolymph and Malpighian tubules of a newly emerged xanthine dehydrogenase deficient adult. Malpighian tubules from newly eclosed 933:3 adults were transplanted into newly eclosed ryz and 913:3 hosts. After 24 hours, the donor and host tubules were assayed for urate oxidase activity. The 913:3 donor tubules cultured in 132 hosts have on the average 1,683 units of urate oxidase activity, which is approximately four times the activity of the control 933:8 tubules (391 units). Malpighian tubules from newly eclosed xanthine dehydrogenase deficient adults mazljnxz, when transplanted into newly eclosed 933:3 hosts, stimulate urate oxidase activity almost fivefold in the host tubules (Table I). Presumably, the ryz donor Malpighian tubules excrete or release a factor into the blood which then acts upon the 913:3 tubules to stimulate urate oxidase activity. The hemolymph factor which induces urate oxidase appears to be 5 in higher concentrations in the hemolymph and Malpighian tubules of newly emerged xanthine dehydrogenase deficient adults as compared to the wild-type 913:3,(Tab1e I). Malpighian tubules from early third instar 33d1 larvae were transplanted into newly eclosed 112 and 913:3 hosts. After 24 hours of incubation at 25°C the urate oxidase activity of the donor larval tubules as well as the host tubules was determined. As shown in Table II, the urate oxidase activity of the donor larval tubules cultured in1y2 adults is about twofold higher than the activity of donor larval tubules cultured in 913:3 adults. It appears, therefore, that urate oxidase activity is stimulated in larval Malpighian tubules by the same factor that modulates urate oxidase activity in the adult. A tubule from an early third instar-instar 3391 larva transplanted into an 913:3 adult induces urate oxidase activity in the Malpighian tubules of the 913:3 adult host (Table II) to the same extent that a tubule from a xanthine dehydrogenase deficient newly emerged adult induces urate oxidase activity in the Malpighian tubules of an 913:3 adult host (Table I). These data indicate that an early third- instar larval Malpighian tubule also contains a considerable quantity of the diffusible factor that induces urate oxidase activity. TABLE 1. Urate Oxidase Activity of Adult Donor and Host Malpighian Tubules 24 Hours Alter Transplantation Urate oxidase activity“ Genotype of Control for transplanted Host Transplanted transplanted Host Control for tubule“ genotype” tubule tubule“ tubule host tubule“ Ore-r Ore-R 655 1: 54.9 292 i 93.0 855 :1: 253.4 292 :t 93.0 ma-l;ryz Ore-R 2.059 3 482 3.192 :1; 834.4 L852 1: 136.5 372 3; 64.4 Ore-R Ina-1:012 1.683 :1: 62.2 391 1 118.8 2.590 1 123.7 3.541 1 188.1 ‘Urate oxidase activity is expressed as the counts per minute of ("C)allantoin formed from ("C)uric acid per minute of reaction time for one set of Malpighian tubules. The numbers are means of two to four independent determinations with three replicas each. l’Malpighian tubules to be transplanted were obtained from newly emerged females and transplanted to newly emerged female hosts. The urate oxidase activity in the Malpighian tubules of newly emerged females of both genotypes is essentially zero. “Controls for the transplanted tubules and host tubules represent urate oxidase activity in Malpighian tubules of 24-hour-old female adults of the same genotype as the transplanted tubules and the host tubules. respectively. TABLE 2. Urate Oxidase Activity of «11' Larval and Pupal Donor Malpighian Tubules Cultured in Newly Eclosed Adult Hosts‘ Urate oxidase activity (cpml Transplantation protocol At time of transplantation 24 Hours after transplantation Donor 84-hour-old ecd' larva Donor: 293 : I63 Donor: 3.029 :- 1.214 Host: newly eclosed Ore—R female Host: 70 j; 55 Host: 2.!24 :t 796 Donor: 84-hour-old erd' larva Donor: 2|2 :1; I98 Donor: 6.241 1- l.085 Host: newly eclosed ry2 female Host: 268 :1: I78 Host: 5.9l6 :t |.566 Donor: («1' white pupa Donor: 1.42] i 649 Donor: 222 i 273 Host: newly eclosed ry’ female Host: 280 3 I69 Host: 5.633 i 1.527 The number of independent determinations ranges from 8 to IO. ‘ecd' larvae were raised at l9°C and Malpighian tubules were transplanted into newly eclosed adult hosts that were maintained at 25°C. 8 Temporal Regulation.of Larval Urate Oxidase Activity Evidence Suggesting a Eunoral Repressor Just prior to pupariation the activity of urate oxidase rapidly declines and is virtually undetectable 24 hours after puparium formation (Figure 1). To probe the mechanism which regulates this decline of urate oxidase activity, Malpighian tubules from 339; white prepupae raised at 20°C were transplanted into newly eclosed 1y2 adults. After 24 hours of in gigg_incubation the donor pupal tubules were assayed for urate oxidase activity. The data in Table II show that the urate oxidase activity of the donor tubules declined from 1,421 units to 222 units of activity despite the fact that the donor tubules are bathed in the urate oxidase-inducing environment of newly eclosed 112 adults. The tubules of the 1y2 host exhibited their normal increase in urate oxidase activity to 5,633 units. These data in Table II indicate that the scheduled decline of urate oxidase activity just prior to pupariation is not simply a reflection of a possible decline in the amount of the urate oxidase-inducing hemolymph factor at puparium formation. Rather, it appears that the Malpighian tubules are no longer competent to respond to the hemolymph factor regulating the quantity of urate oxidase activity. To test the hypothesis that the loss of competence to respond to the hemolymph factor is an autonomous property of the larval Malpighian tubules, the tubules from early 9 33511 third instar larvae were transplanted into newly eclosed 1y2 adults. The hosts were maintained on rich yeast-honey food at 20°C. The urate oxidase activity of the donor larval tubules and the host tubules was assayed periodically as was the activity of tubules from intact control sister 3331 larvae maintained on normal food at 20°C. In Figure 2, the urate oxidase activity profile of the sister control larvae is superimposed over the urate oxidase activity profile of the 11:2 host and the transplanted larval tubules. Note that the activity of the transplanted larval tubules closely parallels the activity of the host tubules. The urate oxidase activity of the donor larval tubules, although declining in parallel with the urate oxidase activity of the 84- and 92-hour-old 132 adult hosts, nevertheless remains high at a time when the urate oxidase activity in the tubules of the now pupariating sister 3391 larvae is almost extinguished. One interpretation of the data is that the failure of late third-instar Malpighian tubules to respond to the urate oxidase inducing hemolymph factor (Table II) and perhaps the decline of urate oxidase activity is caused by a factor exogenous to the tubules which acts upon the tubules in the mid to late third instar. 10 ACE OF THIRD INSTAR LARVA HMS FROM HATCHINO 96 IZO I44 I“ IOZ URATE OXIDASE ACTIVITY (crux 10-3) 24 48 72 90 AGE OF HOST HOURS FROM ECLOSION Figgre 2. Urate oxidase activity of 35:31 larval tubules“), 3351 larva tubules cultured in 13; newly emerged adults(D, and the 12, host tubulesal). Malpighian tubules from early third instar 3331 larvae were transplanted into 11 newly eclosed adults. Sister larvae and the hosts were maintained on food at 20°C. Urate oxidase activity was determined at times indicated. Each point is an average of four to six independent determinations. Error bars represent standard deviation. For clarity, the error bars were omitted from the from the urate oxidase activity of 120- to 156-hour old larvae( ). The magnitude of the standard deviations at these points is similar to those shown in Figure 3. The mean urate oxidase activity of all 339 Malpighian tubules cultured in 13 hosts for 96 hours was 6,544 1 7,024 cpm. This large standard deviation is due to the extremely high activity of one set of Malpighian tubules (20,328 cpm). This data point was eliminated as an outlier (P < 0.025) by the statistical test of Grubs and Beck (1972L. Therefore, the recalculated mean of urate oxidase activity without thfi outlier of the 339 tubules cultured for 96 hours in 1y hosts was 3,788 1 2,166 cpm. 11 Evidence Implicating 20-Hydroxyecdysone as the Repressor Effect of the Mutant god; on Urate Oxidase Activity The humoral agent which initiates pupariation in insects is the steroid hormone 20-hydroxyecdysone. The concentration of 20-hydroxyecdysone in the late third-instar larva of Drosophila increases rapidly just prior to puparium formation (Hodgetts et al., 1977; Garen et al., 1977). This increasing titer of 20-hydroxyecdysone coincides temporally with the decline of urate oxidase activity. Because of the correlation of these two events, it was hypothesized that 20-hydroxyecdysone is the exogenous factor which causes the decline of urate oxidase activity in the Malpighian tubules. This hypothesis was first tested by observing how the lack of endogenous 20-hydroxyecdysone in the late third instar larvae of the mutant m1 (Garen et al., 1977) affects the temporal profile of urate oxidase activity. A similar approach was used by Kraminsky et al. (1980) and Marsh and Wright (1980) to show that 20-hydroxyecdysone induces dopa decarboxylase in Drosophila larvae. The mutant egdl isolated by Garen et a1. (1977) is phenotypically wild type at the permissive temperature of 20°C. At the nonpermissive temperature of 29°C egdl larvae fail either to make or accumulate 20-hydroxyecdysone and as a consequence do not form pupae (Garen et al., 1977). Timed ggdl larvae were raised at 20°C. At 88 hours of postembryonic development, shortly after the second larval 12 molt, a group of the larvae were shifted up to 29°C. Malpighian tubules from larvae at 20°C and 29°C were assayed periodically for urate oxidase activity. At 20°C the temporal profile of urate oxidase activity in gcdl larvae (Figure 3) is similar to the wild type Qrgzk (Figure l). The activity increases continuously to a maximum and then drops precipitously at the time of puparium formation becoming undetectable shortly thereafter. At 29°C a similar temporal profileeof urate oxidase activity is observed in egdl larvae between 90 hours and 160 hours which includes a partial drop in activity at the time when egdl larvae form puparia at the permissive temperature (Figure 3). However, the urate oxidase activity of ggdl larvae at 29°C is never extinguished but remains relatively high until the death of the third-instar larva after 1-2 weeks. These data suggest a correlation between the presence of a high endogenous concentration of 20-hydroxyecdysone and the disappearance of urate oxidase activity in the larval Malpighian tubules. The partial drop in urate oxidase activity observed in late third-instar m1 larvae at 29°C (Figure 3) also temporally coincides with the wandering stage, a period of time during which larvae cease active feeding that precedes pupariation. 4Although egdl larvae do not pupariate at 29°C, they do cease active feeding and crawl out of the food onto the sides of the culture flask. Is it possible that the increase and maintenance of high urate oxidase activity in the third-instar larva is dependent upon continuous 13 I8. I4. our: oxnmr acrlvrrv (cm n :04) A A J 90 IIO I30 I50 I70 ISO 2IO AGE OF THIRD INSTAR LAM HOURS FROM HATCHINO Figure 3. Urate oxidase activity of egfil third instar larvae and cpupae at 20°C (.I and 29°C (0). Pupariation occurs at 20 C at about 170 hours. Each point is an average of six independent determinations. Error bars indicate standard deviations. l4 ingestion of food and that the partial loss of urate oxidase activity in egdl larvae at 29°C is due to starvation? To test this hypothesis, groups oflearly third instar egdl larvae, maintained on yeast, were shifted from 20°C to 29°C to eliminate endogenous 20-hydroxyecdysone. At the time of temperature shift (Figure 4, arrow b), one group of larvae was transferred from the yeast to inert Whatman CF11 cellulose powder saturated with water. Twelve hours later, (Figure 4, arrow a), another group of larvae was transferred from yeast to cellulose. Urate oxidase activity of all groups was determined periodically. When larvae with high urate oxidase activity are deprived of food, the activity declines (Figure 4, arrow a). Then early third-instar larvae with little activity are deprived of food, an increase of urate oxidase activity is not observed (Figure 4, arrow b). Presumably, the amount of hemolymph factor that induces urate oxidase activity in the Malpighian tubules is influenced by the amount of yeast ingested. Continuous ingestion of food appears to be necessary to maintain high urate oxidase activity and explains the partial drop in urate oxidase activity observed in the late third-instar ggdl larvae at 29°C (Figure 3). However, even though ecdl larvae at 29°C have stopped feeding, relatively high urate oxidase activity persists until death. There is additional discussion of the influence of diet on urate oxidase activity in chapter 2. 15 5'. Ti 5 K: 3 “--D m mm: OXIDASE ACTIVITY (cm 1: oh .3 I U “U N T Figure 4. Effect of starvation on urate oxidase activity of third instar egdl larvae. Early third instar god larvae were transfered to 10 ml beakers containing yeast paste (0) or moist cellulose powder (A) (arrow b). After 12 hours a portion of the larvae were transfered from the beakers with the yeast to to beakers containing moist cellulose (ED (arrow a). (All larvae were maintained at 29°C. Each point is an average of four to six independent determinations. Error bars represent standard deviations. 16 Effect of 20-Eydroxyecdysone on Larval Urate Oxidase Activity Late third-instar m1 larvae at 29°C contain no detectable 20-hydroxyecdysone (Garen et al., 1977; Berreur et al., 1984) and also maintain high urate oxidase activity (Figure 3). This suggests that 20-hydroxyecdysone is necessary for the disappearance of urate oxidase activity observed shortly after puparium formation. The primary defect of the egdl mutation has not yet been determined (Berreur et al., 1984). It is possible that this mutation may affect the temporal regulation of urate oxidase activity by some mechanism that does not involve the titer of 20- hydroxyecdysone. To implicate 20-hydroxyecdysone as the causal agent in the regulatory mechanism responsible for the decline of urate oxidase activity, it is necessary to show that exogenous 20-hydroxyecdysone can trigger the loss of urate oxidase activity in god; larvae at 29°C. Early third instar egdl larvae were shifted to 29°C to eliminate endogenous 20-hydroxyecdysone. At various times during the maturation of the third instar egdl larvae at 29°C, groups of larvae were transferred to either yeast with 20-hydroxyecdysone dissolved in ethanol or to yeast and ethanol alone. Urate oxidase activity was assayed at 12- hour intervals. ‘When 112- and 124-hour-old ggdl larvae were fed 20-hydroxyecdysone, a rapid loss of urate oxidase activity was observed (Figure 5, panels C and D). It might be argued that the observed decline of urate oxidase 17 activity may be a result of the larvae not eating the 20- hydroxyecdysone-supplemented yeast. However, note that the rate at which urate oxidase declines when larvae are starved is much slower (Figure 4) than when larvae of approximately the same age are fed 20-hydroxyecdysone (Figure 5, panel C). These experiments indicate that 20-hydroxyecdysone is necessary for the process that regulates the total loss of urate oxidase activity in the late third-instar larva. ‘It is interesting that the concentration of 20-hydroxy- ecdysone (0.4 mM) which triggers the decline of urate oxidase activity when fed to 112- and 124-hour old larvae (Figure 5, panels C and D) does not seem to be immediately effective in preventing the increase in urate oxidase activity when fed to early third-instar larvae shortly after the second larval molt (Figure 5, panels A and B). A similar phenomenon was observed by Lepesant et al. (1978) whO‘were unable to prematurely stimulate the synthesis of 20-hydroxyecdysone inducible fat body Pl protein by feeding 20-hydroxyecdysone to early third-instar larvae. There are a number of possible explanations for the failure of tissues in very early third-instar larvae to respond to exogenous 20-hydroxyecdysone. The molecular components which mediate the 20-hydroxyecdysone response may not as yet be functional. For example, the 20- hydroxyecdysone receptor protein (Maroy et al., 1978; Yund et al., 1978) may not be present in sufficient quantity in some tissues of the early‘third—instar larva. 18 Alternatively, the 20-hydroxyecdysone response may be suppressed by another humoral agent such as juvenile hormone which might be present in the early third instar larva just after the second larval molt. Chihara and Fristrom (1973) have reported that juvenile hormone can inhibit the 20- hydroxyecdysone induced evagination of imaginal discs in vitro as well as the 20-hydroxyecdysone induced [3H] uridine incorporation into imaginal disc cells. The endogenous levels of juvenile hormone during the development of Drosophila have not been published. However, Baehr et a1. (1979) have shown that in Locusta migratora a high concentration of juvenile hormone is present during the early part of the last larval instar. If a high concentration of juvenile hormone is present in the early third-instar Drosophila larva, then the feeding of 20- hydroxyecdysone to early third instar larvae may not be capable of prematurely inducing or repressing those 20- hydroxyecdysone dependent functions which are inhibited by juvenile hormone. It is possible to prevent the scheduled increase of urate oxidase activity in early third-instar larvae by feeding the larvae 20-hydroxyecdysone at concentrations much higher than is necessary to initiate a decline of urate oxidase activity in older larvae (Figure 5, panel B). These data are of questionable significance since concentrations of 20-hydroxyecdysone higher than 1 to 10 mM were lethal to the larvae within 24 hours after feeding. Furthermore, it 19 is impossible to conclude from these data (Figure 5, panel E) whether the putative inhibitory effect of juvenile hormone was overcome or whether a much higher concentration of 20-hydroxyecdysone is needed at a time when the intracellular concentration of 20-hydroxyecdysone receptors may be very low. 20 A 8 C D E file. 52 I gm. t: EIZD .— 2 9L :8 4 3 05. III E ,3.J OI2 24 OI2 24 OI2 24 OI2 24 OI2 24 TIME IN HOURS Figure 5. Effect of feeding 20-hydroxyecdysone to 1 third instar larvae at 29°C. 1) At the initial time ) larvae of approximate age 88 hours (panel A), 100 hours (panel B), 112 hours (panel C), and 124 hours (panel D) were transferred to 10-m1 beakers containing yeast paste with (D and without (0) 0.4 mM 20-hydroxyecdysone. Urate oxidase activity was determined at the time of transfer. 2) Panel E: 88 hour old 35d larvae (0) were transferred to 10-ml beakers containing yeast with no 20-hydroxyecdysone fl, 0.4mM '20-hydroxyecdysone (a), 1.6 mM 20-hydroxyecdysone (A), and 8 mM 20-hydroxyecdysone (4). Each point is an average of four to six independent determinations. Error bars represent standard deviations. 21 Analysis of the Level of Urate Oxidase Regulation Temporal Distribution of Malpighian Tubule Proteins Variations in the amount of enzyme activity in 1129 can be regulated in a number of ways. A particular enzyme may remain at the same concentration at all times but, 1) its activity can be modulated allosterically or 2) it can exist as an inactive proenzyme which is converted to active form as needed. 4Alternativelyu change in the amount of enzyme activity may be a direct reflection of the number of enzyme molecules. To test the hypothesis that the change in urate oxidase activity during development of Drosophila is due to a change in the concentration of the urate oxidase protein present in the Malpighian tubules, proteins from Malpighian tubules of ggdl early and mid third-instar larvae and white prepupae were fractionated on SDS-polyacrylamide slab gels. These gels show that the pattern of proteins in the Malpighian tubules is highly conserved during the third instar. The majority of proteins of the Malpighian tubules show no change in concentration between early and late third instar (Figure 6). The major exception to this observation is a protein band with an apparent molecular weight of 41,000 which comigrates with homogeneously pure Drosophila urate oxidase (Figure 6, lane 2). The changes in the relative amounts of this 41,000-dalton protein as judged by the density of Coomassie Blue staining correlate with the 22 -(D b 1? ' 3 8 5 3 2 I 3| i VIIIII‘Q'.” "I.” ’ 4m :3! wuwwuimw .. A, 5‘ I‘; 411’. Figure 6. SDS-polyacrylamide gel electrophoresis of Coomassie Blue stained proteins from Malpighian tubules of ggd third instar larvae. Lane 3 contains 30 tubules from 95 hour old larvae, lane 4 contains 15 tubules from 130 hour old larvae, and lane 5 contains 20 tubules from 170 hour old larvae. Lanes 3, 4, and 5 contained approximately equal quantities of protein. Pharmacia low molecular weight standards and purified Drosophila urate oxidase are in lanes 1 and 2 respectively. The molecular weights of the standards are a) 94,000; b) 67,000; c) 43,000; d) 30,000; and e) 20,100. The urate oxidase band in the spectrophotometric scans of lanes 3, 4, and 5 is indicated by an arrow. 23 changes of urate oxidase activity during the third instar (compare Figure 3 and Figure 6). In the early third instar, when urate oxidase activity is low, a relatively light staining band is present which comigrates with the purified urate oxides polypeptide. During the mid third instar, when urate oxidase activity is at its peak, the 41,000-dalton protein band is densely stained and is one of the major proteins in the Malpighian tubules. At pupariation, when urate oxidase activity is very low, the 41,000-dalton protein band is hardly visible. A direct correlation between urate oxidase activity and the density of the 41,000-dalton protein band that comigrates with purified urate oxidase is observed throughout development. The same correlation is observed in adult Drosophila (data not shown). In Q;e;R adults urate oxidase activity is low and the staining of the 41,000 dalton protein band is light. In 24-hour-old 112 adults, urate oxidase activity is relatively high (Figure l) and the 41,000-dalton protein band stains correspondingly dark (data not shown). The majority of the 41,000-dalton protein band (Figure 6) from high urate activity Malpighian tubules is recognized by rabbit anti-Drosophila urate oxidase antibody. A homogenatesof‘ggd1 larval Malpighian tubules with high urate oxidase activity was prepared and equal aliquots were added to preimmune IgG and anti-Drosophila urate oxidase antibody respectively. As shown in Table III, the rabbit anti-urate oxidase IgG inactivated more than 95% of the urate oxidase 24 Table I I. Immunoprecipitation of urate oxidase activity from ggd larval Malpighian tubule homogenates. Wofurateoxidasemmu Type of rabbit 196 before nits; additinn Di 199 preimmune 100 90.6 immune 100 3.5 25 . I 1; I III BS'III’I II“! I* t 2: .. I III JIM.) (III'II Ii, | Figure 7. SDS-polyacrylamide gel electrophoresis of silver stained proteins from Malpighian tubules after removal of proteins bound to preimmune IgG (lane 1) and anti-Drosophila urate oxidase IgG (lane 2). Spectrophotometric scans l and 2 are of gel lanes 1 and 2 respectively. The protein band that comigrates with purified urate oxidase is indicated by an arrow. : 26 activity, whereas no significant loss of activity occurred in the presence of control preimmune rabbit IgG. After removal of all 196 and IgG-Drosophila protein complexes by protein A-Sepharose, the remaining Malpighian tubule proteins were fractionated by SDS-polyacrylamide gel electrophoresis and stained with silver. As shown in Figure 7, more than 60% of the 41,000-dalton protein band in the control lane has been removed with rabbit anti-urate oxidase IgG. The data presented in Figure 3, Figure 6, and Figure 7 show that the amount of urate oxidase activity is a reflection of the amount of urate oxidase protein. Therefore, at pupariation, 20-hydroxyecdysone is involved in regulating the rapid decrease of the concentration of urate oxidase protein in Malpighian tubules. Steroid hormone mediated repression of the synthesis of some proteins is well documented. Exposure of hepatoma cells to the steroid hormone analogue dexamethasone causes the induction of the synthesis of tyrosine aminotransferase as well as the cesation of synthesis of a number of functionally unidentified proteins (Ivarie and OPFarrell, 1978). Roberts (1975) treated a Drosophila cell line with 20-hydroxyecdysone and reported the cesation of synthesis of several soluble proteins detected by immunoelectrophoresis. Using single dimensional SDS electrophoresis Savakis and coworkers (1980) occasionally detected reduced amounts of a 56,000-dalton and a 79,000-dalton protein in a 20- 27 hydroxyecdysone treated Drosophila cell. line. Coudere and Destugue (1975) also using cultured Drosophila cells identified two peptides whose synthesis appears to be inhibited by 20-hydroxyecdysone. There is, therefore, precedent for steroid hormone mediated cessation of synthesis of several functionally unidentified proteins in Drosophila and other organisms. Urate oxidase appears to belong to this small class of proteins whose synthesis is restricted in the presence of a high in 1119_titer of 20- hydroxyecdysone. 28 Temporal Distribution of Urate Oxidase mRNA The magnitude of urate oxidase activity is directly proportional to the amount of the urate oxidase enzyme present in the Malpighian tubules. This implies two possible modes of regulation. First, the amount of urate oxidase mRNA could remain constant and some kind of post - translational control mechanism'would regulate the amount of active urate oxidase. Alternatively, the amount of urate oxidase could be directly proportional to the amount of urate oxidase mRNA. To measure the urate oxidase mRNA levels at different stages of larval development, newly hatched egdl larvae were collected and maintained on yeast at 20°C. At various times during development, larvae (or prepupae) of specific age were collected and polyA+ RNA extracted from them. The mRNA was then fractionated on a denaturing formaldehyde agarose gel and transferred from the gel to a nitrocellulose filter. The RNA on the filter was then hybridized with [32?] labeled urate oxidase cDNAm The isolation of a Drosophila urate oxidase cDNA is discussed in detail in chapter three. As shown in Figure 8, the amounts of urate oxidase mRNA present at various stages of larval development correlate quite well with both urate oxidase activity (Figure 3) and the amounts urate oxidase enzyme present (Figure 6). There is no urate oxidase mRNA present in newly hatched first instar larvae. A miniscule amount of urate oxidase mRNA is present in the second instar larval RNA preparation. 29 ‘_ 1.69 kb ~ 1.55 kb -IJ3 kb I 2 3 4 5 6 Figure 8. Northern gel analysis of urate oxidase mRNA during the larval stage of Drosophila development. Total RNA was extracted from various stages of larval development. Poly A+ RNA was obtained from 200 ug of each of the total RNA preparations by a single pass through a micro oligo(dT)- cellulose collumn. Each of the poly A RNA samples was then denatured in a formamide-formaldehyde buffer and loaded onto a 1.2% denaturing formaldehyde agarose gel. After electrophoresis, the RNA was transfered from the gel to a sheet of 0.45 micron nitrocellulose by blotting. The RNA was then baked on to the nitrocellulose in a vacuum oven or 2 hours at 80°C and the filter Véas hybridiffd with 10 or» (specific activity of 1.6 x 10 CPM/ug) [ Plurate oxidase cDNA (pcDNAUOGB). After washing the filter was autoradiographed. Only one RNA band hybridized with the urate oxidase cDNA. Each lane contains approximately 5 ug of poly A RNA from 1:newly emerged first instar larvae; 2: second instar larvae; 3: 80 hour old third instar larvae; 4: 125 hour old third instar larvae; 5: 145 hour old third instar larvae; and 6: white prepupae. The nitrocellulose was stained with methylene blue after exposure to confirm that equal amounts of poly A RNA were transferred from the gel to the filter.(Additional details in the Appendix) 30 This small amount of urate oxidase mRNA present in the second instar RNA preparation does not necessarily mean that the urate oxidase gene is expressed in this developmental period. The population of second instar larvae may have been contaminated by a few early third instar larvae. Larvae were collected by age in hours from hatching. No attempt was made to visually inspect individual larvae for develOpmental stage. The levels of urate oxidase mRNA in third instar larvae (Figure»8) correlates precisely with the third instar levels of urate oxidase peptide (Figure 6). Both the urate oxidase peptide (Figure 6, lane 3) and mRNA (Figure 8, lane 3) are relatively low density bands seen in preparations from early third instar 95-hour-old larvae. During the mid-third instar, both the urate oxidase peptide (Figure 6, lane 4) and urate oxidase mRNA (Figure 8, lanes 4 and 5) are seen as very dense bands. At pupariation, the urate oxidase peptide band is extremely faint (Figure 6, lane 5) and the urate oxidase mRNA is virtually non existent (Figure 8, lane 6). These results show that urate oxidase activity and the amount of urate oxidase peptide are directly correlated with the level of urate oxidase mRNA. It appears, therefore, that changes in urate oxidase activity are modulated primarily at the level of urate oxidase mRNA transcription and/or post - transcriptional processing. 31 Does 20-Hydroxyecdysone Act Directly on the Genome of Malpighian Tubules? Data have been presented to show that 20- hydroxyecdysone is implicated in the mechanism by which urate oxidase mRNA, peptide and activity is repressed in the Malpighian tubules of the third larval instar of Drosophila. It has been shown in a number of tissues of Drosophila, including the Malpighian tubules, that 20-hydroxyecdysone acts directly on the genome to regulate gene transcription as evidenced by puffing changes in polytene chromosomes (Ashburner 1967, 1972, 1973,; Bonner and Pardue 1976, 1977; Berendes 1966, 1967). In particular, 20-hydroxyecdysone has been shown to directly repress the transcription of the salivary glue protein gene sgs-4 in Drosophila salivary glands (Beckendorf and Kafatos, 1976; Muskavitch and Hogness, 1980). To determine whether 20-hydroxyecdysone acts directly on the genome of Malpighian tubules to repress the urate oxidase gene, Malpighian tubules from egdl larvae raised at 29°C were cultured in vitro, in Schneider's medium with and without 20-hydroxyecdysone. In an initial experiment, egdl larvae were raised in the absence of endogenous 20- hydroxyecdysone at 29°C on yeast food to a point at which urate oxidase activity peaked. The Malpighian tubules were then dissected from a group of the larvae and each was transferred into 100 ul of Schneider's medium with or without 20-hydroxyecdysone. The cultured tubules as well as 32 the sister larvae were assayed for urate oxidase activity at 12 hour intervals. As the results in Figure 9 indicate, urate oxidase activity of the in vitro cultured Malpighian tubules declined at about the same rate as the activity in the tubules of the sister larvae regardless of the presence or absence of 20-hydroxyecdysone in the culture medium. On the average it seems that the tubules cultured in vitro in the presence of 20-hydroxyecdysone experienced a greater drop in urate oxidase activity then the tubules cultured in absence of the hormone but 1) the difference is not significant and 2) the rate of decline is not as rapid as that observed in Malpighian tubules of egdl larvae fed 20-hydroxyecdysone at the non-permissive temperature (see Figure 5). The data would indicate that 20-hydroxyecdysone does not repress urate oxidase activity in tubules cultured in vitro. However, it is possible that the Malpighian tubules may have some sort of autonomous developmental clock and that they are competent to respond to the hormone only within a narrow"developmental window\. It is possible that at 29°C, when the larvae experience the peak urate oxidase activity this ”window" is closed and the tubules are no longer competent to respond. To test this hypothesis, Malpighian tubules were obtained from goal larvae raised at 29°C at various times prior to peak urate oxidase activity; The tubules were then cultured in vitro in Schneider‘s medium with and without 20- 33 20 P -3) I6 ' I2 ’ urate oxidase activity (CPII x I!) 88 I00 IIZ 121 I36 I18 0 I2 21 36 hours from hatching hours in culture Figure 9. Urate oxidase activity of Malpighian tubules cultured in vitro at a time of maximfm in xixa.activity. Panel A: urate oxidase activity of egd third instar larvae reared on yeast at 29°C. Panel B: urate oxidase activity of Malpighian tubules cultured in Schneider's medium withm) and without (6) 10'4M 20-hydroxyecdysone. The tubules were transferredto Schneider's medium from 112 hour old and third instar larvae. 34 hydroxyecdysone and assayed for urate oxidase activity after 32 hours of culture. As shown in Figure 10, the urate oxidase activity of all in_1itrg cultured tubules declined slightly (but not significantly) during the 32 hours and there was no significant difference in activity between those tubules cultured in the presence of 20-hydroxyecdysone and those tubules cultured in the absence of 20- hydroxyecdysone. The decline in urate oxidase activity induced by 20-hydroxyecdysone in 1119_(Figure 6) was not observed in Malpighian tubules of the same age cultured in vitro in the presence of the hormone (Figure 10). These results indicate that l) Malpighian tubules obtained from larvae during a time when urate oxidase activity is being induced will maintain the level of urate oxidase activity which the tubules exhibit at the time of transfer to Schneider's medium; 2) tubules transferred to synthetic culture medium at a time when urate oxidase activity is no longer being induced will show a decline in urate oxidase activity in culture at about the same rate as the decline in activity exhibited by larvae in the absence of endogenous 20-hydroxyecdysone; and 3) 20- hydroxyecdysone does not cause a precipitous drop in urate oxidase activity in.in vitro cultured Malpighian tubules. One possible interpretation of the data is that the steroid hormone 20-hydroxyecdysone does not act on the Malpighian tubules directly to repress urate oxidase, but rather must act through some other cell type. This 35 -3) urate oxidase activity (CPfl x I0 88 I00 II2 I21I36I16 O 32 0 32 0 32 hours from hatching hours in culture Figure 10. Urate oxidase activity of Malpighian tubules culture in vitro, in Schneider's medium with (D) and without (00 10' M 20-hydroxyecdysone. The tubules w re transferred to Schneider's medium from 88 hour fld god third instar larvae (panel B); 100 hour 019 egd third instar larvae (panel C); and 112 hour old god third instafi larvae (panel 1». Panel A: urate oxidase activity of god third instar larvae, from which the tubules were obtained, reared on yeast at 29°C. 36 conclusion, however, may not be warranted in view of the following observations. Third instar m1 larvae reared at 29°C on yeast food were transferred to cellulose powder with and without 20- hydroxyecdysone for 24 hours at 29°C. At 12 hours and again at 24 hours, larvae from both treatment groups were assayed for urate oxidase activity. As the results presented in Figure 11 clearly show, the drop in urate oxidase activity of starving larvae is not in any way effected by the steroid hormone. That is, when ggdl larvae of the same age are fed yeast and 20-hydroxyecdysone the urate oxidase activity drops precipitously to almost zero (Figure 5, panel C), whereas starving larvae exhibit a drop in urate oxidase activity that is somewhat more gradual regardless of whether 20-hydroxyecdysone is ingested at the same time (Figure 11). Conclusions Collectively, all of the genetic and biochemical data presented in this chapter support the the notion that 20- hydroxyecdysone is implicated in the regulatory process by which urate oxidase activity declines precipitously at the time of puparium formation in Drosophila. There is no direct experimental evidence to support the hypothesis that 20-hydroxyecdysone acts directly on the genome of the Malpighian tubules causing the repression of transcription of the urate oxidase gene. 37 .. 10L 9? ”IA So» )1 57a 8 '3 :5' U U 3" U E 33* 0 22' 3 I I00 II2 I21 hours from hatching Figure 11. Urate oxidase activity of egdl third instar larvae feeding on cellulose sat rated with 0.8% sucrose (a) only and 0.8% sucrose with 10" M 20-hydroxyecdysone (a). The larvae were transferred from yeast food at 100 hours and reared at 29°C for the duration of the experiment. 38 The hypothesis that 20-hydroxyecdysone directly represses transcription of the urate oxidase gene is not ruled out by the fact that a substantial drop in urate oxidase activity can not be initiated by 20-hydroxyecdysone in larval Malpighian tubules in vitro. .As has been shown, the in gixg_induced decline in urate oxidase activity is dependent on a specific physiological state of the larva. It may very well be that 20-hydroxyecdysone acts directly on the Malpighian tubules to repress the urate oxidase gene, but some other humoral agent absent in the starving larvae and also absent in Schneider's medium may be necessary for the synthesis and degradation of the urate oxidase peptide and mRNA. CHAPTER TWO Introduction One goal of studying the urate oxidase gene will be to understand how the structure of the gene and its flanking sequencesinteracts with various effector molecules in‘the Malpighian tubules to modulate the gene's expression. To this end, it is imperative to identify all factors which act upon the tubules to induce and repress urate oxidase. The data presented in the previous chapter support the hypothesis that urate oxidase activity is induced in both Drosophila larvae and adults by the same diffusible factor. A further correlation has been drawn between the induction of urate oxidase activity and food intake by the larvae. The same observation was reported by Friedman (1973) for adult wild-type Qrezfi flies. .Adult wild-type Drosophila raised on yeast have significantly higher urate oxidase activity than those starved for 24 hours. This chapter will further explore the effect of diet on urate oxidase activity in the third instar Drosophila larva. The purpose of the experiments presented here is to obtain a better understanding of the physiological processes which control the induction of urate oxidase. This information may prove usefull in identifying the diffusible urate oxidase inducing factor. 39 40 Effect of Diet on Urate Oxidase Activity Data presented in Table II of chapter one demonstrated that Malpighian tubules obtained from white prepupae are no longer competent to respond to the urate oxidase inducing factor. The decline of urate oxidase activity at the time of puparium formation has been shown to be due to the presence of 20-hydroxyecdysone. However, it has also been demonstrated that a weaker decline in urate oxidase activity can be initiated in the larvae by starvation. The following experiment was performed to determine whether the decline of urate oxidase activity in starving larvae can be reversed by returning the larvae to food, or whether the activity is permanently repressed once a decline is initiated. Early third instar ggdl larvae raised on yeast food were shifted to 29°C. When urate oxidase activity was at about half of the maximum possible activity, a group of the larvae were transferred to wet cellulose powder. After 12 hours of starvation, a group of the starved larvae was transferred to yeast food. Larvae were assayed for urate oxidase activity at 12 and 24 hours after the initial transfer to cellulose. As Figure 12 shows, urate oxidase activity declines during the initial 12 hours of starvation. But once the larvae are transferred to food, the urate oxidase activity is once again induced at the same rate as in the sister larvae which were never starved. These results further confirm that the increasing level of urate oxidase activity in third instar larvae is directly 41 I5 ’ ‘7 I3 ' c b x =. II I O. 8 r 9 b urate oxidase activity in I00 II2 I21 hours from hatching Figure 12. The effect on urate oxidase activity of feeding stafved larve. At 100 hours of development, third instar egd larvae were transferred from yeast food to cellulose powder saturated with 0.8% sucrose (CD. At 112 hours of development, a group of the starved larvea was transferred back to yeast food for 12 hours MD. The larvae were reared at 29°C for the duration of the experiment. 42 dependent on food intake. Presumably, the quantity of the diffusible urate oxidase inducing factor to which Malpighian tubules respond is modulated by the nutritional status of the larvae. Drosophila, like many other insects and birds are uricotelic organisms in that they excrete nitrogenous wastes in the form of uric acid and/or its metabolites. Urate oxidase converts the waste product uric acid to allantoin. Allantoin is the major excretion product in the butterfly Papilio xuthus (Tojo and Yushima, 1972). There is also some evidence that allantoin may be the major excretion product in Drosophila when urate oxidase activity is puesent (Johnson et al., l980a,b,c). A number of experiments were designed to test which nitrogenous component of the larval diet is important for urate oxidase induction. In all of the previous experiments, larvae were reared on live yeast. In order to determine if live yeast was necessary for the induction of urate oxidase, ecdl larvae were raised on cellulose powder saturated with a solution of yeast extract. As the results in Table IV show, the induction of urate oxidase, which is inhibited by rearing the larvae on moist cellulose powder alone, proceeds normally on the yeast extract diet. To determine if an increase in the nitrogen content of the diet will cause a greater induction of urate oxidase activity, yeast extract was supplemented with 0.4% yeast RNA (Sigma). This amount of RNA was chosen because it was found 43 Table IV. Urate oxidase activity of ggdl early third instar larvae reared on wet cellulose and supplements at 29°C for 24 hours. Supplements Urate oxidase activity (CPM) 0.8% sucrose 4,257 i 530 6.0% yeast extract 24,726 1 5,410 0.8% sucrose 6.0% yeast extract 29,163 1 3,582 0.8% sucrose 0.4% RNA *initial urate oxidase activity at 100 hours - 11,847 i 533 The number of independent determinations c 4. 44 to be optimum in a defined diet (Burnet and Sang, 1963). A group of larvae was raised on yeast extract and another group was raised on yeast extract with 0.4% RNA. Both groups of larvae were assayed for urate oxidase activity after 24 hours. As data in Table IV shows, urate oxidase activity is induced on both diets and there is no statistically significant difference in activity on the RNA supplemented yeast diet as compared to the yeast diet without added RNA. RNA is a component of yeast and yeast extract. It may be>that the amount of dietary nitrogen present in yeast is enough to cause the maximum possible rate of urate oxidase induction. To explore this possibility, it was necessary to prepare a diet which was defined with respect to nitrogen containing compounds. The major source of dietary nitrogen is probably amino nitrogen in the form of amino acids and nitrogen from purine and pyrimidine compounds. Early third instar ggdl larvae were divided into three groups. One group was reared on moist cellulose, the second on cellulose saturated with 5% casein, and the third on cellulose-casein supplemented with 0.4% RNA. After 12 hours at 29°C, the larvae were assayed for urate oxidase. As the results in Table V indicate, the casein diet alone is capableof inducing urate oxidase activity at about the same rate as a live yeast diet (Figure 3). There is no significant increase in urate oxidase activity when the casein diet is supplemented with RNA. Table y. Urate oxidase activity of egdl early third instar 45 larvae reared on wet cellulose and supplements at 29°C for 12 hours. Supplements Urate oxidase activity (CPM) 0.8% sucrose 3,878 1 822 6.0% casein 10,725 i 1,498 0.8% sucrose 6.0% casein 12,114 i,l,527 0.8% sucrose 0.4% RNA * initial urate oxidase activity at 88 hours - 2696 i 331 The number of independent determunations = 4. 46 Based on these results it would appear that amino nitrogen is reponsible for urate oxidase induction. However, since urate oxidase was maximally induced, any possible contribution by nucleic acid nitrogen would have been masked even if nucleic acid nitrogen can induce urate oxidase. To control for this possibility, early third instar egdl larvae were reared on a number of diets of reduced (1.5% casein) amino nitrogen each supplemented with a different concentration of purine or nucleic acid. The larvae were assayed for urate oxidase activity after 12 hours at 29°C. As the results in Table VI show, low levels of casein (1.5%) induce urate oxidase at a slower rate than high levels of dietary casein (6.0%) (Table V). Supplementing the protein diet with other nitrogenous compounds at levels greater than that of casein did cause a slight increase in activity, but this increase was neither significant nor reproducible. Even if the apparent increase in urate oxidase activity on.a low protein, high nucleic acid diet were significant, the level of induction of urate oxidase is minimal in comparison to that observed in larvae reared on a high protein diet. ‘Therefore, the most likely explanation for the dietary dependence of urate oxidase activity is that the waste nitrogen excreted by Drosophila as uric acid and allantoin is primarily the excess amino nitrogen from amino acids. 47 Table VI. Urate oxidase activity of ecdl early third instar larvae raised on low casein supplementeduwith purines and nucleic acids at 29°C. Diet Urate oxidase activity (CPM) 1.5% casein 4,182 1 1,623 0.8% sucrose 0.06% uridine 0.8% sucrose 2,699 i 345 0.06% uridine 1.5% casein 5,828 1 2,052 0.8% sucrose 0.06% uridine 0.2% RNA 1.5% casein 5,019 i 684 0.8% sucrose 0.06% uridine 2.0% RNA 1.5% casein 5,685 i 1,110 0.8% sucrose 0.06% uridine 0.2% hypoxanthine 1.5% casein 6,539 i 1,534 0.8% sucrose 0.06% uridine 2.0% hypoxanthine 1.5% casein 5,637 1 1,212 0.8% sucrose 0.06% uridine 0.2% inosine 1.5% casein 6,076 1 1,690 0.8% sucrose 0.06% uridine 2.0% inosine The number of independent‘determinations = 4. 48 Role of Purine de nove Synthesis in Urate Oxidase Induction Any waste nitrogen which is to be excreted via the uricotelic pathway other than excess purines has to pass through the amino acid pool. The amino acids glycine, glutamine, aspartate, and serine (Figure 13) are then utilized in the de novo purine synthesis pathway which leads through the formation of inosine monophospahte to the formation of uric acid and allantoin, the excretory products. To test the hypothesis that the induction of urate oxidase is somehow effected by the d: nova synthesis of purines, early third instar ggdl larvae were raised at 29°C on moist cellulose supplemented with very low dietary protein and a high concentration of the amino acids involved in de ngxg purine synthesis. After 24 hours, urate oxidase activity was determined. As the data in Table VII show, urate oxidase activity of larvae which ingested the precursors of purine de noxg synthesis (Figure 13) was almost twice the activity of the larvae which were fed casein alone. Interestingly, feeding the larvae the precursors alone without additional casein failed to induce urate oxidase activity. As indicated in Figure 13, there is one step in the de novo synthesis of purines which can be blocked by the glutamine analogs azaserine and 6-diazo-5-oxo-L-norleucine (DON). The effects of inhibition of purine biosynthesis by these compounds on urate oxidase activity was determined. 49 Ribose-S-phosphate 1 ATP 5-Phosphoribosyl-l-pyrophosphate 1 Glutamine 5-Phosphoribosylamine L Glycine, ATP Glycinamide ribotide l FormleHFA Formylglycinamide ribotide Azaserine and DON ----> 1 Glutamine, ATP Formylglyiinamidine ribotide 5-Amino-4§imidazole-carboxylic acid ribotide Aspartate, ATP 5-Amino-4-imidazole-N-succinocarboxamide ribotide l P s f: I Figure 13. Azaserine and DON sensetive site in de nogo purine biosynthesis. (Pittillo and Hunt, 1967). This site has been determined in bacteria and mammalian cells. Whether the same enzymatic step is inhibited by these antibiotics in Drosophila has not been confirmed. Table VII. instar 50 Urate oxidase activity of egdl early third larvae raised (n1 low casein supplemented with precursors of de novo purine synthesis at 29 . Diet Urate oxidase activity (CPM) 0.5% casein 0.8% sucrose 0.2% glycine 0.2% glutamine 4,227 i 957 (n=8) 0.2% aspartate 0.2% serine 0.0003% folate 0.057% uridine 0.5% casein 0.8% sucrose 2,488 i 493 (n=8) 0.057% uridine 0.8% sucrose 0.2% glycine 0.2% glutamine 0.2% aspartate 2,355 i 531 (n=4) 0.2% serine 0.0003% folate 0.057% uridine n a number of independent determinations. 51 Initially, early third instar esdl larvae were transferred to cellulose soaked with a solution of 5% casein supplemented with different concentrations of azaserine. Urate oxidase activity was determined after 24 hours at 29°C. Azaserine at a concentration of 10’2M in the diet proved effective in inhibiting a further casein induced urate oxidase increase (Table VIII). Azaserine at 10'4M and lO'GM had no effect on urate oxidase induction (Table VIII). Data in Table IX show the effect of 10'2M azaserine on urate oxidase activity at 12 and at 24 hours after feeding as a supplement in a 5% casein diet. The inhibition of urate oxidase activity is not complete at this concentration of azaserine. The activity more than doubled in the presence of the inhibitor. However, the induction of activity in the presence of azaserine may be due to physiological factors influencing the levels of the urate oxidase inducing factor which are independent of the inhibitory effects of azaserine. This interpretation is supported by the observation that very little if any increase in urate oxidase activity occurs in the presence of azaserine between 12 and 24 hours of the experiment (Table IX). The effects of 10'2M DON on urate oxidase activity at 12 and at 24 hours after feeding as a supplement in a 5% casein diet are shown in Table x. The inhibitory effect of DON is very much like that of azaserine. That there is no increase in activity after 12 hours on the DON supplemented 52 Table VIII. The effects of varying concentrations of azaserine on urate oxidase activity induced by dietary casein. azaserine in diet urate oxidase activity (CPM) none 9,649 1 1,410 10'224 5,620 1 1,048 10"»: 9,790 3; 1,745 10“»: 10,728 i 1.759 Early third instar end]- 1arvae were rerared on wet cellulose supplemented with casein and azaserine as indicated at 29° C for 24 hours. Number of independent determinations = 6. 53 Table IX. Inh' ition of casein induced urate oxidase acgivity by 10" M azaserine at 12 and 24 hours of feeding at 29 C. urate oxidase activity (CPM) diet 12 hours 24 hours 5.0% casein 3,763 i 1,106 9,144 1 1,566 0.8% sucrose 5.0% casein 3,139 i 499 3,481 1 1,146 0.8% sucrose 0.2% azaserine Urate oxidase activity of early third instar ecdl larvae prior to transfer to casein diet was 1,660 i 256. Number of independent determinations = 6. 54 Tab1e_§. Inhibition of casein induced urate oxidase activity by 10 M DON at 12 and 24 hours of feeding at 29°C. urate oxidase activity (CPM) diet 12 hours 24 hours 5.0% casein 13,846 1 2,506 16,456 1 1,673 0.8% sucrose 5.0% casein 5,510 1 1,290 8,240 1 969 0.8% sucrose 0.2% DON Urate oxidase activity of early third instar goal larvae prior to transfer to casein diet was 5,577 1 2,106. Number of independent determinations = 6. 55 diet is probably due to the fact that this experiment was initiated with larvae somewhat older than those used in the experiment with azaserine as the inhibitor (Table IX). In the experiment where azaserine was used as the inhibitor the initial activity of urate oxidase was 1660 units whereas in the one where DON was tested, the initial activity was 5510 units. When early third instar larvae are starved at a time when urate oxidase activity is very low (2500 units) the activity increases slightly during the first 12 hours of starvation (Figure 4, arrow b). But when older larvae which have high activity are starved, urate oxidase activity drops during the first 12 hours of starvation (Figure 4, arrow a). An explanation for this observation may be that since there is no urate oxidase activity present in the second instar, the excretion of waste nitrogen may be below optimum. This may cause an increase in the nitrogen pool which may, in turn, cause an increase in the amount of the urate oxidase inducing factor. Between 12 hours and 24 hours of feeding on a DON supplemented diet, the larvae show a slight increase in activity (Table X). This may be due to metabolic degradation of some of the DON. Regardless of any minor differences that may exist, urate oxidase activity induction by dietary protein is inhibited by both azaserine and DON. In both cases only the increase of activity is prevented at the maximum concentration of inhibitors tested. These results suggest 56 that urate oxidase is induced in the Malpighian tubules by some metabolite made as a result of de novo purine synthesis or that the level of the diffusible urate oxidase inducer is modulated in response to the activity or pool size of metabolites of the de novo purine synthesis pathway. Various nucleotides, nucleosides, and purines can be ingested by Drosophila larvae and utilized in the formation of all the catabolites of inosine monophospahte (Figure 14), the end product of de novo purine synthesis (Johnson et al, l980L. To test the hypothesis that any excess purine nitrogen ingested by larvae will also induce urate oxidase activity, larvae were fed aicasein.diet supplemented‘with various concentrations of RNA, and azaserine or DON. The results of feeding larvae 2% RNA.at the same time de novo synthesis is inhibited by azaserine are shown in Table XI. Although the larvae fed RNA have a slightly higher level of activity than the larvae fed casein and azaserine only, the increase is not significant and certainly is substantially less than the increase observed in larvae fed casein only without the inhibitor. Results in Table XI also show that the effect of azaserine on a yeast extract diet is not significantly different from its effect on a casein diet. In both cases the increase of urate oxidase activity is inhibited. The effect of adding various concentrations of RNA to the diet of casein and the inhibitor DON is shown in Table XII. Early third instar ggdl larvae were transferred to 57 de novo synthesis I AMP ---------- > IMP --------- > XMP ------- > GMP adenosine ----- > inosine xanthosine guanosine I I I hypoxanthine ---> xanthine <-- guanine I uric acid allantoin Figure 14. Catabolism of purines. AMP, adenosine-S'-mono- phosphate; IMP, inosine-5'-monophosphate; XMP, xanthosine- 5'-monophosphate; GMP, guanosine-5'-monophosphate. (Johnson et. al., 1980a,b,c) Table XI. 58 The effect of dietary RNA.