1EVELOPMENTAL
BIOLOGY
53,
241-249
(1976)
The Effects of cinnamon on Xanthine Dehydrogenase, Aldehyde Oxidase, and Pyridoxal Oxidase Activity during Development in Drosophila LEON Department
of Biology,
W. BROWDER’ The
University Accepted
melanogasterl H. WILLIAMSON’
AND JOHN of Calgary, June
Calgary,
Alberta,
Canada
TZN
IN4
23,1976
The cinnamon (tin) eye color mutant of Drosophila melanogaster was characterized to determine biochemical correlations with another mutant, maroon-like. As with maroon-like, cinnamon flies lack three enzymatic activities: xanthine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase. Xanthine dehydrogenase (XDH) is subject to a maternal effect in both mutants; i.e., mutant progeny of heterozygous mothers have XDH activity, resulting in wildtype eye color. However, the maternal effect is stronger in cinnamon than in maroon-like. Whereas maternally affected cinnamon show a large increase in XDH activity during larval stages, and XDH activity is still detectable after eclosion, the magnitude of increase in XDH activity is less in maroon-like, and activity is no longer detectable in second-day pupae and all later stages. The large increase in XDH activity in maternally affectedcinnamon suggests that there is de nouo synthesis of enzymatically active XDH during development in the absence of the tin+ gene. Cinnamon is also unique in that maternally affected flies retain isoxanthopterin (IXP), the product of XDH activity. These flies appear to be deficient in some aspect of either pteridine metabolism or excretion.
1962). The structural gene for A0 (Aldox) maps at 3-57~. Electrophoretic variants of A0 map there, and the Aldox locus exhibits a gene dosage effect on A0 activity (Dickinson, 1970). The presumed structural gene for PO also maps at 3-57* (1~0 = low pyridoxal oxidase activity; Collins et al., 1969. There is no method to detect electrophoretic variants of PO, but the lpo locus does show a dosage effect on PO activity. Three additional loci affect the enzymatic activities of the products of these structural genes. The sex-linked mutant maroon-like (mal, 1-64.8) produces a brown eye color, indistinguishable from ry eye color, and simultaneously eliminates XDH, AO, and PO activities (Glassman, 1965a,b). The ma1 phenotype is a classic example of a maternally affected phenotype in that ma1 progeny of heterozygous females (+/mal) have normal eye color and detectable (but low) levels of XDH activity (Glassman and McLean, 1962). The recessive mutant lxd (low xanthine
INTRODUCTION
In Drosophila melanogaster, xanthine dehydrogenase (XDH), aldehyde oxidase (AO), and pyridoxal oxidase (PO) activities are subject to multiple genetic control (Glassman 1965a,b; Dickinson and Sullivan, 1975; Fig. 1). At least six genetic loci are involved in the control of the activities of these three enzymes, making this the most extensive gene-enzyme system described in an eukaryotic organism. The structural genes for the three enzymes map within five units of each other in the right arm of the third chromosome. The rosy locus (ry, 3-52) is considered to be the structural gene for XDH, since null mutants and electrophoretic variants map there (Yen and Glassman, 19651,and there is a direct relationship between XDH activity and the number of copies of the normal allele of ry in the genome (Grell, ’ The authors wish to acknowledge the excellent technical assistance of Ms. Lindis Tucker. 2 Supported by the National Research Council of Canada. 241 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
242
DEVELOPMENTAL
BIOLOGY
VOLUME
53, 1976
GENETIC CONTROL OVER XANTHINE OEHYOROGENASE, ALOEHYOE OXIOASE AND PYRIOOXAL OXIDASE
W&TURAL
A&ku(3-57) Ipa (3-57)
ry (3-52)
CHROMOSOME
ADDITIONAL CONTROL
FIG. Al&x,
“control”
ma/ (I -64.6 Ird (3-33) tin (1-O)
1. The salivary aland chromosome and lpo have been localized (from genes are also indicated.
MATERIALS
AND
)
region of the right Lindsleq and Grell,
dehydrogenase actiuity, 3-33) has no visible phenotype, but reduces XDH activity to approximately 20% of wild-type levels, while reducing A0 and PO activities to even lower levels (Courtright, 1967; Glassman 1965a,b). XDH activity is also affected by cinnamon (tin, l-O), recently described by Baker (1973) as having three phenotypic components. The eye color component is comparable to that of the mutant mal; i.e., tin flies have eye color similar to ma1 and ry individuals. As with mal, the tin phenotype i-s maternally affected, with tin progeny of heterozygous ( +/tin) females having normally colored eyes. However, tin is unique among these mutants that affect XDH activity, since tin progeny of tin mothers die during embryogenesis. The similarities between the phenotypes of tin and mal prompted us to make further comparisons of these two mutants. to determine Specifically, we sought whether maternally affected tin flies have any XDH activity and whether tin also has an effect on A0 and PO activities. METHODS
Stocks. Oregon-R. A wild-type strain with normal XDH, AO, and PO activities. Urbana-S. A wild-type stock deficient for A0 activity due to the Aldox” allele.
3
arm of the third chromosome 1968). The genetic locations
to which ry, of the three
mal. Deficient for XDH, AO, and PO activities. ry2. Deficient for XDH activity. tin. Cinnamon eye color, deficient for XDH activity. This mutant is routinely kept in balanced stocks to avoid the lethal component of its phenotype. All stocks and crosses were reared on a standard cornmeal-yeast-sugar culture medium (Lewis, 1960). All crosses were incubated at 25 + 0.5”C and 40 + 3% relative humidity. All wild-type stocks and mutants, except tin (Baker, 19731, are fully described by Lindsley and Grell (1968). Maternally
Affected
Progeny
Maternally affected ma1 progeny were obtained from a stock culture of C (I )DX, y f/Y females and mallY males. Females of this stock have yellow body color, forked bristles, and normal eye color. Although males are genotypically mal, they are phenotypically wild type in eye color due to a maternal effect. Maternally affected tin progeny were obtained from a stock culture of C(1 IRM, ltsE90/Y and yciniY males. Females of this stock are phenotypically wild type (PE90 is a temperaturesensitive lethal used for other purposes), while males have yellow body color and wild-type eye color, the latter due to a maternal effect. C (1 )DX and C (I )RM are
BRO~VDER
AND
WILLIAMSON
attached X chromosomes that are inherited from female parent to female progeny, while the mutant X chromosome in males of each stock is inherited from male parent to male progeny. Developmental
Stage Determination
Eggs were collected over a 3-hr period from mass matings on cornmeal-yeastsugar medium in quarter-pint (120 ml) bottles and incubated at 25°C. Timing of development was begun at the midpoint of the egg collection period (1.5 hr). Organisms were collected at the midpoint of each developmental stage. Larvae were collected 34.5, 58.5, 82.5, and 106.5 hr after the egg collection period, corresponding to first-instar, second-instar, and early and late third-instar larvae, respectively. Each sample was taken from a separate culture bottle by floating larvae off the culture medium in a 20% sucrose solution. Larvae were sexed by observing the fifth abdominal segment for the presence or absence of testes (Bodenstein, 1950) and/or the difference in pigmentation of larval mouthparts that distinguishes between wild-type and yellow phenotypes. Pupae were collected at 130.5, 154.5, 178.5, and 202.5 hr after the egg collection period. The sex of pupae was known by isolating third-instar male and female larvae into separate culture vials. XDH
Assays
Fluorometric assay. Extracts were prepared by homogenizing 10 individuals in 1 ml of cold (4°C) 0.1 M Tris-Cl buffer (pH 8.0). The extracts were then treated with Norit A, centrifuged, and assayed as described by Sayles et al. (1973). Activity was shown to be proportional to the amount of extract used. Assay by thin-layer chromatography. Although the standard fluorometric assay of XDH is extremely sensitive, it is unsuitable for detection of XDH activity from a single larva. We therefore developed a thin-layer chromatographic technique that allowed the detection of the conver-
Enzyme
Activities
in the tin
Mutant
243
sion of microquantities of 2-amino-4-hydroxypteridine (AHP) to isoxanthopterin (IXP). 1. Preparation of extracts. Individual larvae were homogenized in wells of Falcon microtest tissue culture plates containing 0.015 ml of the reaction mixture (6 x 10. 5 M AHP and 0.02% p-nicotinamide adenine dinucleotide in 0.1 M Tris-Cl, pH 8.0). Pupae and adults were homogenized in 0.005 ml of 0.1 M Tris-Cl (pH 8.0) containing 0.75% (w/v) Norit A to adsorb endogenous pteridines. The homogenates were kept on ice for 10 min for pteridine adsorption, drawn into a O.Ol-ml capillary micropipet that was sealed with Dade Miniseal and centrifuged for 10 min at 5000 rpm in an International Model HN centrifuge fitted with an hematocrit head. Following centrifugation, the sealed end of the micropipet was broken off, and the supernatant was blown into a microtest plate well containing 0.015 ml of reaction mixture. In addition to the extracts prepared for enzyme assays, extracts were prepared in Tris-Cl buffer (a) without charcoal for determination of endogenous levels of AHP and IXP and (b) with charcoal to determine whether such treatment completely removed endogenous pteridines. 2. Assay procedures. The microtest plates were incubated at 30°C for 3 hr in the dark. Water-soaked cotton maintained humidity within the plates to prevent desiccation of the reaction mixture. Controls lacking homogenates were incubated concurrently to monitor nonenzymatic conversion of AHP to IXP. At the completion of the incubation period, 0.005 ml of absolute ethanol was added to the reaction mixture to facilitate spotting on chromatograms by speeding evaporation. The reaction mixture was drawn into a 0.025-ml capillary micropipet, sealed, and centrifuged as above. Following centrifugation, the end of the tube opposite the sealed end was drawn to a fine point. The sealed end of the micropipet was broken off and the
244
DEVELOPMENTAL
BIOLOGY
drawn end touched to the chromatography medium (MN-300 cellulose on Brinkman plastic TLC film) to deliver the supernatant. Development in a Gelman Instant TLC chamber was for 10 cm with 5% acetic acid. After drying, the chromatograms were observed and photographed using long wavelength (365 nm) ultraviolet light. Aldehyde
Oxidase
Assay
Single organism assay. Single larvae, pupae, or adults were homogenized with 0.005 ml of 0.02 M Tris-Cl (pH 8.0), 0.001 M EDTA. The homogenate was drawn into a O.Ol-ml micropipet, one end of the pipet was sealed, and the homogenate was then centrifuged at 5000 rpm for 10 min. The supernatant was assayed quantitatively for enzyme activity in a mixture consisting of 0.04 mg/ml of phenazine methosulphate (PMS), 0.02 mg/ml of dichlorophenolindophenol (DCPIP), 0.05 M acetaldehyde, 0.1 M Tris-Cl (pH 7.5), and 0.001 M EDTA (Courtright, 1967; Dickinson, 1970). PMS, DCPIP, and redistilled acetaldehyde were made up in concentrated stock solutions and mixed prior to use. Assays were done at 30°C by following the linear decrease in optical density at 600 nm on a Beckman Acta III recording spectrophotometer. All assays were run against a reference of identical composition, except that the extract (total volume, 0.005 ml) was omitted. Activity was shown to be proportional to the amount of extract used. Specific actiuity. Ten flies were homogenized with 0.1 ml of 0.02 M Tris-Cl (pH 8.0), 0.001 M EDTA. The homogenate was drawn into a O.l-ml micropipet, one end of the pipet was sealed, and the homogenate was centrifuged at 5000 rpm for 10 min. The supernatant was blown out into a small test tube. Four aliquots (0.005 ml each) were taken for the enzyme assay, and two aliquots (0.01 ml each) were used for protein determination by the technique of Lowry et al. (1951).
VOLUME
Pyridoxal
53, 1976
Oxidase
Assay
Extracts were prepared by homogenizing 100 flies in 1.0 ml of 0.1 M Tris-Cl (pH 7.5), treated with Norit A, and assayed for pyridoxal oxidase according to the procedure of Collins and Glassman (1969). RESULTS
Xanthine
Dehydrogenase
Maternal
Effects
The results of TLC assays (Figs. 2 and 3) show that both maternally affected ma1 and tin progeny have readily detectable XDH activity during development. A low level of activity was detected in individual first-instar maternally affected mal larvae (Fig. 2B-1). Second-instar larvae (Fig. 2B2) had more activity than their first-instar counterparts, while activity in early thirdinstar larvae (Fig. 2B-3e) was no higher than in second-instar larvae. In late thirdinstar mal larvae and pupae (Fig. 2B-P) XDH activity was reduced and was undetectable in individual adults. Maternally affected tin individuals exhibited a different pattern and higher levels of XDH activity than did maternally affected ma1 progeny. There was easily detected activity in individual first-instar larvae (Fig. 2D-l), with increasing activity in subsequent stages of development. In late thirdinstar larvae (Fig. 2D-31) and early pupae (Fig. 2D-P), the higher levels of activity were maintained, but no activity was detected in the week-old adult. Determinations of endogenous pteridines revealed that tin larvae and pupae accumulate high levels of IXP (Fig. 2D-E). This is in contrast to wild types (Figs. 2A-E and 2C-E), which do not accumulate significant amounts of either AHP or IXP, and mal (Fig. 2B-E), which accumulates AHP. Newly eclosed maternally affected tin progeny retain IXP. The IXP begins to accumulate in the testes within 12 hr and is retained in aged (10 days old) adults. The charcoal treatment step (Ch) was effective in removing all endogenous pterdines, confirming that they did not interfere with interpretation of the enzyme assays.
BROWDER
AND
Enzyme
WILLIAMSON
Actiuities
in
the
tin
245
Mutant
FIG. 2. Chromatographic assays of XDH activity for single organisms. (A), C(1 )DX, y f/Y females (mal’l; (B), maternally affected ma1 males (sibs of A); (Cl, C(1 IRM, Z’xE90/Y females c&n+); CD), maternally affected tin males (sibs of Cl. 1, 2, 3e, 31, First-, second-, early third-, and late third-instar larvae, respectively; P, 4day pupa; E, endogenous pteridines; Ch, charcoal-treated extract of 4-day-old pupa; AHP and IXP, standards. GENOTYPE
INSTAR 181
FIG.
affected amounts symbols
2nd
3rd e
3rd 1
1
PUPAE
(Day)
2
3
ADULT 4
STANDARDS AHP
IXP
3. Diagrammatic representation of conversion of AHP to IXP in wild-type females, maternally mal males, and maternally affected tin males. The intensity of shading is indicative of the relative of substrate and end product. Open symbols refer to definite, though small amounts; broken refer to equivocal chromatographic spots.
Our results suggest quantitative differences in the extent of the XDH maternal effects in tin and ma1 progeny of nonmutant females. Accordingly, we conducted fluorometric assays to compare more accurately the maternal effects of tin+ and mal+ on XDH activity in mutant progeny.
Data from these assays are presented in Fig. 4. The activities are expressed on a per organism basis. This mode of data presentation was selected since maternal effects are mediated by deposition in the egg of a finite amount of a substance that cannot be synthesized by the zygote. Any
246
DEVELOPMENTAL
BIOLOGY
FIG. 4. XDH activities (2 standard error) of maternally affected tin and ma1 males compared with Oregon-R males. Activity was determined on extracts of 10 organisms and expressed as picomoles of AHP oxidized per organism. Each point is the average of three determinations.
change in activity must then be a consequence of developmental processing of a nonrenewable resource. XDH activity in both mutant classes is significantly less than for the wild-type controls (Oregon-R) at all developmental stages assayed. Both maternally affected tin and ma1 progeny have detectable XDH activities as early third-instar larvae, but the tin progeny show a significant rise in activity in the late third-instar larval stage not shown by mal progeny. Detectable levels of XDH activity continued in maternally affected tin progeny throughout all developmental stages, including adults less than 24 h of age. In aged tin adults (collected immediately after eclosion and aged for 7 days on standard culture medium at room temperature, 19-20°C) no detectable XDH activity remained. On the other hand, XDH activity is not detectable in maternally affected ma1 progeny past the first-day pupal stage. Aldehyde
Oxidase
Activity
Aldehyde oxidase specific activity determinations were made on adults of several genotypes (Fig. 5). The Urbana-S wildtype strain lacks A0 activity due to the Aldox” allele and demonstrates a dosage effect in the heterozygote in accordance
VOLUME
53. 1976
FIG. 5. A0 specific activities for adults of several genotypes. Specific activities were calculated from four A0 determinations and two protein determinations per genotype (Materials and Methods). M.A., maternally affected.
with Dickinson’s data (1970). A0 specific activity is dramatically reduced in both lrd and ma1 stocks, although in neither is A0 activity completely missing. Neither of these loci shows a dosage effect on A0 specific activity in the heterozygote. These observations also agree with Dickinson’s data; however, Courtright (1967) reported that +/lxd flies had a reduced level of A0 activity. A0 specific activity is also reduced in tin flies. By necessity, these data were obtained from tin males derived from nonmutant mothers. Similar specific activity determinations were not made for tin progeny of tin mothers. We have, however, determined A0 activity per organism for a small number (five females) of newly eclosed tin progeny of tin mothers. Enzyme activity was slightly higher thari for tin progeny of nonmutant mothers (8.5 ? 1.0 vs 2.75 ? 0.25). Specific activity of +/tin flies, while lower than that of Ore.gon-R, is not low enough to indicate a dosage effect on A0 activity. Thus, both the tin and ma1 genes are analogous in that each affects A0 activity, but neither has a dosage effect. These two genes, therefore, affect the activity of the enzyme produced by another structural gene (Aldox). Since the A0 specific activity data for tin and ma1 were obtained on adults, we wanted to know whether the effects of tin and ma1 were stage specific; i.e., do the genes affect A0 activity only in the adult
BROWDER
AND
Enzyme
WILLIAMSON
or throughout larval, pupal, and adult life? Therefore, we compared A0 activity of Oregon-R males with that of tin and rr& males for these developmental stages (Pig. 6). As before, the tin males were derived from nonmutant females, but the mal males were from mutant stock. Both tin and ma1 contain detectable levels of A0 activity from the late third-instar larval stage on. The activities are significantly less than for the wild type at all stages but not significantly different from one another. Pyridoxal
Oxidase
Activity
The insensitivity of the fluorometric assay for PO activity precludes detailed comparisons among the various genotypes. We were able to measure PO activity in Oregon-R using 20-100 organisms per assay, but in no case were we able to detect PO activity in maternally affected tin males (100 organisms), either as adults or as third-instar larvae. Confirmation That Effects on Enzyme tivities Are Due to Cinnamon
Ac-
The assumption inherent in the experiments reported in this paper is that the effects on the enzymes are due to the cinnamon mutation itself. However, since tin was mutagen induced (Baker, 19731, it is possible that another mutation that affects the activities of XDH, AO, and PO was also induced on the X chromosome. To test
FIG. 6. A0 activities (+ standard error) of tin and ma1 males compared with Oregon-R males. Each point is the average of four determinations.
Activities
in the tin
Mutant
247
this possibility, we produced flies with a y tin/y+ Y genotype. The y+ Y chromosome has the tip of In (1) SC* attached to the tip of the Y. This segment of the X chromosome contains the small number of loci distal to UC+, including y+ and tin+. Homogenates of the y tin/y+ Y flies were assayed for XDH, AO, and PO activity, all of which were at wild-type levels. Our observations confirm Baker’s localization of XDH deficiency to the SC” duplication and extend this definitive genetic test to the other two enzymes of this system. One can conclude that the y+ Y chromosome contains the wild-type allele of a gene that affects these enzyme activities. That gene is either tin or another gene (for which there is no evidence) that maps at 0.0. DISCUSSION
Data presented here strengthen the analogy between maroon-like and cinnamon but reveal certain essential differences. The basic similarity between the mutants is their effect on the three enzymes xanthine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase. In this respect, ma1 and tin are also similar to lxd, each being a control gene that in some way regulates the activities of the three enzymes. There are several models concerning the interactions among these loci, and these are adequately reviewed by Glassman (1965a,b) and Dickinson and Sullivan (1975). The maternal effects of mal+ and tin+ differ considerably. Maternally affected tin males show a large increase in XDH activity during larval stages, and this activity remains in pupae and in newly eclosed adults. A complete deficiency of XDH activity is observed only in aged adults. Data not included in Fig. 4 indicate that by 2 days of age, XDH activity is undetectable. In maternally affected ma1 males, XDH activity of late third-instar larvae is significantly less than in their tin counterparts and is undetectable after the day 1 pupal stage. Our data on mater-
248
DEVELOPMENTAL
BIOLOGY
nally affected ma1 males are consistent with those of Glassman and McLean (1962). The data are also consistent with the hypothesis that some maternally synthesized enzyme or enzyme component(s) is deposited in the ooplasm and is utilized by the ma1 progeny during development (Sayles et al., 1973). By early pupal stages this maternally synthesized material is no longer functional. We suggest that in the case of tin the relatively large increase in XDH activity in maternally affected late third-instar male larvae and the long-term retention of that activity is due to de nouo synthesis of enzymatically active XDH, although at lower levels than in the wild type. Another unique aspect of the maternal effect of tin is the retention of large amounts of IXP. It is ironic that individuals deficient in XDH activity would retain the end product of one of the reactions catalyzed by that enzyme. In addition to IXP, maternally affected tin flies also accumulate a number of other pteridines in much greater amounts than do wild types. The amounts of biopterin and some other unidentified pteridines increase dramatically following eclosion of these flies (Browder and Williamson, unpublished). These pteridines could be metabolites that are produced from AHP that accumulates since it is not converted to IXP. However, neither ma1 nor ry flies, which also accumulate AHP, accumulate these pteridines. Hence, we conclude that tin is deficient in some aspect of either pteridine metabolism or excretion. The most unusual component of the tin phenotype is conditional female sterility; i.e., tin progeny of tin females do not survive. This has been difficult to understand since neither XDH, AO, nor PO is essential for viability. It is interesting to speculate that the lethality may be related to the accumulation of pteridines by maternally affected tin individuals. The failure of these tin females to produce viable progeny could result from deposition of pteri-
VOLUME
53. 1976
dines in their oocytes. This possibility is attractive because of observations in the literature that correlate pteridines with lethality and defects in RNA synthesis. Counce (1957) has reported that the female-sterile mutant deep orange (dor) accumulates IXP. Puckett and Snyder (1975) have recently demonstrated that IXP, which has a high affinity for DNA, is an effective inhibitor of RNA synthesis in Drosophila. Two observations indicate that IXP itself is not the cause of cinnamon female sterility. We have constructed +lcin stocks that are also homozygous for ry. Maternally affected tin, ry flies also fail to produce tin, ry progeny. Since ry prevents the formation of IXP, this provides a direct test of the effects of IXP on fecundity. In addition, one of us (JHW) has isolated a new cinnamon allele (tin’) that is not female-sterile. However, maternally affected tin’ individuals do accumulate IXP. Although IXP itself cannot be responsible for the failure of maternally affected tin females to produce tin progeny, it is possible that one or more of the pteridines that accumulate in tin flies are responsible. We are presently investigating this possibility. REFERENCES B. S. (1973). The maternal and zygotic control of development by cinnamon, a new mutant in Drosophila melanogaster. Develop. Biol. 33, 429440. BODENSTEIN, D. (1950). The postembryonic development of Drosophila. In “Biology of Drosophila” (M. Demerec, ed.), pp. 275-367. Wiley, New York (reprinted by Hafner, 1965). COLLINS, J. F., DUKE, E. J., and GLASSMAN, E. (1971). Multiple molecular forms of xanthine dehydrogenase and related enzymes. IV. The relationship of aldehyde oxidase to xanthine dehydrogenase. Biochem. Genet. 5, 1-13. COLLINS, J. F., and GLASSMAN, E. (1969). A third locus (lpo) affecting pyridoxal oxidase in Drosophila melanogaster. Genetics 61, 833-839. COUNCE, S. J. (1957). A female-sterile mutant (deep orange 1 of Drosophila melanogaster increasing isoxanthopterine content. Enperientia 13,354-356. COURTRIGHT, J. B. (1967). Polygenic control of aldehyde oxidase in Drosophila. Genetics 57, 25-39. BAKER,
BROWDER
AND
WILLIAMSON
Enzyme Activities
W. J. (1970). The genetics of aldehyde oxidase in Drosophila melanogaster. Genetics 66, 487-496. DICKINSON, W. J., and SULLIVAN, D. T. (1975). “Gene-Enzyme Systems in Drosophila.” SpringerVerlag, New York. GLA~~MAN, E. (1965a). Genetic regulation of xanthine dehydrogenase in Drosophila melanogaster. Fed. Proc. 24, 1243-1251. GLAMHAN, E. (1965b). Xanthine dehydrogenase of Drosophila melanogaster. J. Elisha Mitchell Sci. Sot. 81, 42-54. CLASSMAN, E., and MCLEAN, J. (1962). Maternal effect of ma1 on xanthine dehydrogenase of Drosophila melanogaster. II. Xanthine dehydrogenase activity during development. Proc. Nat. Acad. Sci. USA 48, 17121718. C~RELL,, E. H. (1962). The dose effect of mal’ and ry+ on xanthine dehydrogenase activity in Drosophila melanogaster. Z. Vererbungslehre 93, 371-377. DICKINSON,
in the tin Mutant
249
LEWIS, E. B. (1960). A new standard culture medium. Drosophila Informat. Sew. 34, 117-118. LINSLEY, D. L., and GRELL, E. H. (1968). “The Genetic Variations ofDrosophila melanogaster” Carnegie Institution (Washington), Publ. 627, Washington, D.C. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (19511. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. PUCKETT, L. D., and SNYDER, L. A. (1975). Inhibition of RNA synthesis in Drosophila embryos by isoxanthopterin. Biochem. Genet. 13, l-6. SAYLES, C. D., BROWDER, L. W., and WILLIAMSON, J. H. (1973). Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster. Deuelop. Biol. 33, 213-217. YEN, T. T., and GLASSMAN, E. (1965). Electrophoretie variants of xanthine dehydrogenase in Drosophila melanogaster. Genetics 52, 977-981.
BIOLOGY
53,
241-249
(1976)
The Effects of cinnamon on Xanthine Dehydrogenase, Aldehyde Oxidase, and Pyridoxal Oxidase Activity during Development in Drosophila LEON Department
of Biology,
W. BROWDER’ The
University Accepted
melanogasterl H. WILLIAMSON’
AND JOHN of Calgary, June
Calgary,
Alberta,
Canada
TZN
IN4
23,1976
The cinnamon (tin) eye color mutant of Drosophila melanogaster was characterized to determine biochemical correlations with another mutant, maroon-like. As with maroon-like, cinnamon flies lack three enzymatic activities: xanthine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase. Xanthine dehydrogenase (XDH) is subject to a maternal effect in both mutants; i.e., mutant progeny of heterozygous mothers have XDH activity, resulting in wildtype eye color. However, the maternal effect is stronger in cinnamon than in maroon-like. Whereas maternally affected cinnamon show a large increase in XDH activity during larval stages, and XDH activity is still detectable after eclosion, the magnitude of increase in XDH activity is less in maroon-like, and activity is no longer detectable in second-day pupae and all later stages. The large increase in XDH activity in maternally affectedcinnamon suggests that there is de nouo synthesis of enzymatically active XDH during development in the absence of the tin+ gene. Cinnamon is also unique in that maternally affected flies retain isoxanthopterin (IXP), the product of XDH activity. These flies appear to be deficient in some aspect of either pteridine metabolism or excretion.
1962). The structural gene for A0 (Aldox) maps at 3-57~. Electrophoretic variants of A0 map there, and the Aldox locus exhibits a gene dosage effect on A0 activity (Dickinson, 1970). The presumed structural gene for PO also maps at 3-57* (1~0 = low pyridoxal oxidase activity; Collins et al., 1969. There is no method to detect electrophoretic variants of PO, but the lpo locus does show a dosage effect on PO activity. Three additional loci affect the enzymatic activities of the products of these structural genes. The sex-linked mutant maroon-like (mal, 1-64.8) produces a brown eye color, indistinguishable from ry eye color, and simultaneously eliminates XDH, AO, and PO activities (Glassman, 1965a,b). The ma1 phenotype is a classic example of a maternally affected phenotype in that ma1 progeny of heterozygous females (+/mal) have normal eye color and detectable (but low) levels of XDH activity (Glassman and McLean, 1962). The recessive mutant lxd (low xanthine
INTRODUCTION
In Drosophila melanogaster, xanthine dehydrogenase (XDH), aldehyde oxidase (AO), and pyridoxal oxidase (PO) activities are subject to multiple genetic control (Glassman 1965a,b; Dickinson and Sullivan, 1975; Fig. 1). At least six genetic loci are involved in the control of the activities of these three enzymes, making this the most extensive gene-enzyme system described in an eukaryotic organism. The structural genes for the three enzymes map within five units of each other in the right arm of the third chromosome. The rosy locus (ry, 3-52) is considered to be the structural gene for XDH, since null mutants and electrophoretic variants map there (Yen and Glassman, 19651,and there is a direct relationship between XDH activity and the number of copies of the normal allele of ry in the genome (Grell, ’ The authors wish to acknowledge the excellent technical assistance of Ms. Lindis Tucker. 2 Supported by the National Research Council of Canada. 241 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
242
DEVELOPMENTAL
BIOLOGY
VOLUME
53, 1976
GENETIC CONTROL OVER XANTHINE OEHYOROGENASE, ALOEHYOE OXIOASE AND PYRIOOXAL OXIDASE
W&TURAL
A&ku(3-57) Ipa (3-57)
ry (3-52)
CHROMOSOME
ADDITIONAL CONTROL
FIG. Al&x,
“control”
ma/ (I -64.6 Ird (3-33) tin (1-O)
1. The salivary aland chromosome and lpo have been localized (from genes are also indicated.
MATERIALS
AND
)
region of the right Lindsleq and Grell,
dehydrogenase actiuity, 3-33) has no visible phenotype, but reduces XDH activity to approximately 20% of wild-type levels, while reducing A0 and PO activities to even lower levels (Courtright, 1967; Glassman 1965a,b). XDH activity is also affected by cinnamon (tin, l-O), recently described by Baker (1973) as having three phenotypic components. The eye color component is comparable to that of the mutant mal; i.e., tin flies have eye color similar to ma1 and ry individuals. As with mal, the tin phenotype i-s maternally affected, with tin progeny of heterozygous ( +/tin) females having normally colored eyes. However, tin is unique among these mutants that affect XDH activity, since tin progeny of tin mothers die during embryogenesis. The similarities between the phenotypes of tin and mal prompted us to make further comparisons of these two mutants. to determine Specifically, we sought whether maternally affected tin flies have any XDH activity and whether tin also has an effect on A0 and PO activities. METHODS
Stocks. Oregon-R. A wild-type strain with normal XDH, AO, and PO activities. Urbana-S. A wild-type stock deficient for A0 activity due to the Aldox” allele.
3
arm of the third chromosome 1968). The genetic locations
to which ry, of the three
mal. Deficient for XDH, AO, and PO activities. ry2. Deficient for XDH activity. tin. Cinnamon eye color, deficient for XDH activity. This mutant is routinely kept in balanced stocks to avoid the lethal component of its phenotype. All stocks and crosses were reared on a standard cornmeal-yeast-sugar culture medium (Lewis, 1960). All crosses were incubated at 25 + 0.5”C and 40 + 3% relative humidity. All wild-type stocks and mutants, except tin (Baker, 19731, are fully described by Lindsley and Grell (1968). Maternally
Affected
Progeny
Maternally affected ma1 progeny were obtained from a stock culture of C (I )DX, y f/Y females and mallY males. Females of this stock have yellow body color, forked bristles, and normal eye color. Although males are genotypically mal, they are phenotypically wild type in eye color due to a maternal effect. Maternally affected tin progeny were obtained from a stock culture of C(1 IRM, ltsE90/Y and yciniY males. Females of this stock are phenotypically wild type (PE90 is a temperaturesensitive lethal used for other purposes), while males have yellow body color and wild-type eye color, the latter due to a maternal effect. C (1 )DX and C (I )RM are
BRO~VDER
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attached X chromosomes that are inherited from female parent to female progeny, while the mutant X chromosome in males of each stock is inherited from male parent to male progeny. Developmental
Stage Determination
Eggs were collected over a 3-hr period from mass matings on cornmeal-yeastsugar medium in quarter-pint (120 ml) bottles and incubated at 25°C. Timing of development was begun at the midpoint of the egg collection period (1.5 hr). Organisms were collected at the midpoint of each developmental stage. Larvae were collected 34.5, 58.5, 82.5, and 106.5 hr after the egg collection period, corresponding to first-instar, second-instar, and early and late third-instar larvae, respectively. Each sample was taken from a separate culture bottle by floating larvae off the culture medium in a 20% sucrose solution. Larvae were sexed by observing the fifth abdominal segment for the presence or absence of testes (Bodenstein, 1950) and/or the difference in pigmentation of larval mouthparts that distinguishes between wild-type and yellow phenotypes. Pupae were collected at 130.5, 154.5, 178.5, and 202.5 hr after the egg collection period. The sex of pupae was known by isolating third-instar male and female larvae into separate culture vials. XDH
Assays
Fluorometric assay. Extracts were prepared by homogenizing 10 individuals in 1 ml of cold (4°C) 0.1 M Tris-Cl buffer (pH 8.0). The extracts were then treated with Norit A, centrifuged, and assayed as described by Sayles et al. (1973). Activity was shown to be proportional to the amount of extract used. Assay by thin-layer chromatography. Although the standard fluorometric assay of XDH is extremely sensitive, it is unsuitable for detection of XDH activity from a single larva. We therefore developed a thin-layer chromatographic technique that allowed the detection of the conver-
Enzyme
Activities
in the tin
Mutant
243
sion of microquantities of 2-amino-4-hydroxypteridine (AHP) to isoxanthopterin (IXP). 1. Preparation of extracts. Individual larvae were homogenized in wells of Falcon microtest tissue culture plates containing 0.015 ml of the reaction mixture (6 x 10. 5 M AHP and 0.02% p-nicotinamide adenine dinucleotide in 0.1 M Tris-Cl, pH 8.0). Pupae and adults were homogenized in 0.005 ml of 0.1 M Tris-Cl (pH 8.0) containing 0.75% (w/v) Norit A to adsorb endogenous pteridines. The homogenates were kept on ice for 10 min for pteridine adsorption, drawn into a O.Ol-ml capillary micropipet that was sealed with Dade Miniseal and centrifuged for 10 min at 5000 rpm in an International Model HN centrifuge fitted with an hematocrit head. Following centrifugation, the sealed end of the micropipet was broken off, and the supernatant was blown into a microtest plate well containing 0.015 ml of reaction mixture. In addition to the extracts prepared for enzyme assays, extracts were prepared in Tris-Cl buffer (a) without charcoal for determination of endogenous levels of AHP and IXP and (b) with charcoal to determine whether such treatment completely removed endogenous pteridines. 2. Assay procedures. The microtest plates were incubated at 30°C for 3 hr in the dark. Water-soaked cotton maintained humidity within the plates to prevent desiccation of the reaction mixture. Controls lacking homogenates were incubated concurrently to monitor nonenzymatic conversion of AHP to IXP. At the completion of the incubation period, 0.005 ml of absolute ethanol was added to the reaction mixture to facilitate spotting on chromatograms by speeding evaporation. The reaction mixture was drawn into a 0.025-ml capillary micropipet, sealed, and centrifuged as above. Following centrifugation, the end of the tube opposite the sealed end was drawn to a fine point. The sealed end of the micropipet was broken off and the
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drawn end touched to the chromatography medium (MN-300 cellulose on Brinkman plastic TLC film) to deliver the supernatant. Development in a Gelman Instant TLC chamber was for 10 cm with 5% acetic acid. After drying, the chromatograms were observed and photographed using long wavelength (365 nm) ultraviolet light. Aldehyde
Oxidase
Assay
Single organism assay. Single larvae, pupae, or adults were homogenized with 0.005 ml of 0.02 M Tris-Cl (pH 8.0), 0.001 M EDTA. The homogenate was drawn into a O.Ol-ml micropipet, one end of the pipet was sealed, and the homogenate was then centrifuged at 5000 rpm for 10 min. The supernatant was assayed quantitatively for enzyme activity in a mixture consisting of 0.04 mg/ml of phenazine methosulphate (PMS), 0.02 mg/ml of dichlorophenolindophenol (DCPIP), 0.05 M acetaldehyde, 0.1 M Tris-Cl (pH 7.5), and 0.001 M EDTA (Courtright, 1967; Dickinson, 1970). PMS, DCPIP, and redistilled acetaldehyde were made up in concentrated stock solutions and mixed prior to use. Assays were done at 30°C by following the linear decrease in optical density at 600 nm on a Beckman Acta III recording spectrophotometer. All assays were run against a reference of identical composition, except that the extract (total volume, 0.005 ml) was omitted. Activity was shown to be proportional to the amount of extract used. Specific actiuity. Ten flies were homogenized with 0.1 ml of 0.02 M Tris-Cl (pH 8.0), 0.001 M EDTA. The homogenate was drawn into a O.l-ml micropipet, one end of the pipet was sealed, and the homogenate was centrifuged at 5000 rpm for 10 min. The supernatant was blown out into a small test tube. Four aliquots (0.005 ml each) were taken for the enzyme assay, and two aliquots (0.01 ml each) were used for protein determination by the technique of Lowry et al. (1951).
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Pyridoxal
53, 1976
Oxidase
Assay
Extracts were prepared by homogenizing 100 flies in 1.0 ml of 0.1 M Tris-Cl (pH 7.5), treated with Norit A, and assayed for pyridoxal oxidase according to the procedure of Collins and Glassman (1969). RESULTS
Xanthine
Dehydrogenase
Maternal
Effects
The results of TLC assays (Figs. 2 and 3) show that both maternally affected ma1 and tin progeny have readily detectable XDH activity during development. A low level of activity was detected in individual first-instar maternally affected mal larvae (Fig. 2B-1). Second-instar larvae (Fig. 2B2) had more activity than their first-instar counterparts, while activity in early thirdinstar larvae (Fig. 2B-3e) was no higher than in second-instar larvae. In late thirdinstar mal larvae and pupae (Fig. 2B-P) XDH activity was reduced and was undetectable in individual adults. Maternally affected tin individuals exhibited a different pattern and higher levels of XDH activity than did maternally affected ma1 progeny. There was easily detected activity in individual first-instar larvae (Fig. 2D-l), with increasing activity in subsequent stages of development. In late thirdinstar larvae (Fig. 2D-31) and early pupae (Fig. 2D-P), the higher levels of activity were maintained, but no activity was detected in the week-old adult. Determinations of endogenous pteridines revealed that tin larvae and pupae accumulate high levels of IXP (Fig. 2D-E). This is in contrast to wild types (Figs. 2A-E and 2C-E), which do not accumulate significant amounts of either AHP or IXP, and mal (Fig. 2B-E), which accumulates AHP. Newly eclosed maternally affected tin progeny retain IXP. The IXP begins to accumulate in the testes within 12 hr and is retained in aged (10 days old) adults. The charcoal treatment step (Ch) was effective in removing all endogenous pterdines, confirming that they did not interfere with interpretation of the enzyme assays.
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Enzyme
WILLIAMSON
Actiuities
in
the
tin
245
Mutant
FIG. 2. Chromatographic assays of XDH activity for single organisms. (A), C(1 )DX, y f/Y females (mal’l; (B), maternally affected ma1 males (sibs of A); (Cl, C(1 IRM, Z’xE90/Y females c&n+); CD), maternally affected tin males (sibs of Cl. 1, 2, 3e, 31, First-, second-, early third-, and late third-instar larvae, respectively; P, 4day pupa; E, endogenous pteridines; Ch, charcoal-treated extract of 4-day-old pupa; AHP and IXP, standards. GENOTYPE
INSTAR 181
FIG.
affected amounts symbols
2nd
3rd e
3rd 1
1
PUPAE
(Day)
2
3
ADULT 4
STANDARDS AHP
IXP
3. Diagrammatic representation of conversion of AHP to IXP in wild-type females, maternally mal males, and maternally affected tin males. The intensity of shading is indicative of the relative of substrate and end product. Open symbols refer to definite, though small amounts; broken refer to equivocal chromatographic spots.
Our results suggest quantitative differences in the extent of the XDH maternal effects in tin and ma1 progeny of nonmutant females. Accordingly, we conducted fluorometric assays to compare more accurately the maternal effects of tin+ and mal+ on XDH activity in mutant progeny.
Data from these assays are presented in Fig. 4. The activities are expressed on a per organism basis. This mode of data presentation was selected since maternal effects are mediated by deposition in the egg of a finite amount of a substance that cannot be synthesized by the zygote. Any
246
DEVELOPMENTAL
BIOLOGY
FIG. 4. XDH activities (2 standard error) of maternally affected tin and ma1 males compared with Oregon-R males. Activity was determined on extracts of 10 organisms and expressed as picomoles of AHP oxidized per organism. Each point is the average of three determinations.
change in activity must then be a consequence of developmental processing of a nonrenewable resource. XDH activity in both mutant classes is significantly less than for the wild-type controls (Oregon-R) at all developmental stages assayed. Both maternally affected tin and ma1 progeny have detectable XDH activities as early third-instar larvae, but the tin progeny show a significant rise in activity in the late third-instar larval stage not shown by mal progeny. Detectable levels of XDH activity continued in maternally affected tin progeny throughout all developmental stages, including adults less than 24 h of age. In aged tin adults (collected immediately after eclosion and aged for 7 days on standard culture medium at room temperature, 19-20°C) no detectable XDH activity remained. On the other hand, XDH activity is not detectable in maternally affected ma1 progeny past the first-day pupal stage. Aldehyde
Oxidase
Activity
Aldehyde oxidase specific activity determinations were made on adults of several genotypes (Fig. 5). The Urbana-S wildtype strain lacks A0 activity due to the Aldox” allele and demonstrates a dosage effect in the heterozygote in accordance
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53. 1976
FIG. 5. A0 specific activities for adults of several genotypes. Specific activities were calculated from four A0 determinations and two protein determinations per genotype (Materials and Methods). M.A., maternally affected.
with Dickinson’s data (1970). A0 specific activity is dramatically reduced in both lrd and ma1 stocks, although in neither is A0 activity completely missing. Neither of these loci shows a dosage effect on A0 specific activity in the heterozygote. These observations also agree with Dickinson’s data; however, Courtright (1967) reported that +/lxd flies had a reduced level of A0 activity. A0 specific activity is also reduced in tin flies. By necessity, these data were obtained from tin males derived from nonmutant mothers. Similar specific activity determinations were not made for tin progeny of tin mothers. We have, however, determined A0 activity per organism for a small number (five females) of newly eclosed tin progeny of tin mothers. Enzyme activity was slightly higher thari for tin progeny of nonmutant mothers (8.5 ? 1.0 vs 2.75 ? 0.25). Specific activity of +/tin flies, while lower than that of Ore.gon-R, is not low enough to indicate a dosage effect on A0 activity. Thus, both the tin and ma1 genes are analogous in that each affects A0 activity, but neither has a dosage effect. These two genes, therefore, affect the activity of the enzyme produced by another structural gene (Aldox). Since the A0 specific activity data for tin and ma1 were obtained on adults, we wanted to know whether the effects of tin and ma1 were stage specific; i.e., do the genes affect A0 activity only in the adult
BROWDER
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WILLIAMSON
or throughout larval, pupal, and adult life? Therefore, we compared A0 activity of Oregon-R males with that of tin and rr& males for these developmental stages (Pig. 6). As before, the tin males were derived from nonmutant females, but the mal males were from mutant stock. Both tin and ma1 contain detectable levels of A0 activity from the late third-instar larval stage on. The activities are significantly less than for the wild type at all stages but not significantly different from one another. Pyridoxal
Oxidase
Activity
The insensitivity of the fluorometric assay for PO activity precludes detailed comparisons among the various genotypes. We were able to measure PO activity in Oregon-R using 20-100 organisms per assay, but in no case were we able to detect PO activity in maternally affected tin males (100 organisms), either as adults or as third-instar larvae. Confirmation That Effects on Enzyme tivities Are Due to Cinnamon
Ac-
The assumption inherent in the experiments reported in this paper is that the effects on the enzymes are due to the cinnamon mutation itself. However, since tin was mutagen induced (Baker, 19731, it is possible that another mutation that affects the activities of XDH, AO, and PO was also induced on the X chromosome. To test
FIG. 6. A0 activities (+ standard error) of tin and ma1 males compared with Oregon-R males. Each point is the average of four determinations.
Activities
in the tin
Mutant
247
this possibility, we produced flies with a y tin/y+ Y genotype. The y+ Y chromosome has the tip of In (1) SC* attached to the tip of the Y. This segment of the X chromosome contains the small number of loci distal to UC+, including y+ and tin+. Homogenates of the y tin/y+ Y flies were assayed for XDH, AO, and PO activity, all of which were at wild-type levels. Our observations confirm Baker’s localization of XDH deficiency to the SC” duplication and extend this definitive genetic test to the other two enzymes of this system. One can conclude that the y+ Y chromosome contains the wild-type allele of a gene that affects these enzyme activities. That gene is either tin or another gene (for which there is no evidence) that maps at 0.0. DISCUSSION
Data presented here strengthen the analogy between maroon-like and cinnamon but reveal certain essential differences. The basic similarity between the mutants is their effect on the three enzymes xanthine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase. In this respect, ma1 and tin are also similar to lxd, each being a control gene that in some way regulates the activities of the three enzymes. There are several models concerning the interactions among these loci, and these are adequately reviewed by Glassman (1965a,b) and Dickinson and Sullivan (1975). The maternal effects of mal+ and tin+ differ considerably. Maternally affected tin males show a large increase in XDH activity during larval stages, and this activity remains in pupae and in newly eclosed adults. A complete deficiency of XDH activity is observed only in aged adults. Data not included in Fig. 4 indicate that by 2 days of age, XDH activity is undetectable. In maternally affected ma1 males, XDH activity of late third-instar larvae is significantly less than in their tin counterparts and is undetectable after the day 1 pupal stage. Our data on mater-
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DEVELOPMENTAL
BIOLOGY
nally affected ma1 males are consistent with those of Glassman and McLean (1962). The data are also consistent with the hypothesis that some maternally synthesized enzyme or enzyme component(s) is deposited in the ooplasm and is utilized by the ma1 progeny during development (Sayles et al., 1973). By early pupal stages this maternally synthesized material is no longer functional. We suggest that in the case of tin the relatively large increase in XDH activity in maternally affected late third-instar male larvae and the long-term retention of that activity is due to de nouo synthesis of enzymatically active XDH, although at lower levels than in the wild type. Another unique aspect of the maternal effect of tin is the retention of large amounts of IXP. It is ironic that individuals deficient in XDH activity would retain the end product of one of the reactions catalyzed by that enzyme. In addition to IXP, maternally affected tin flies also accumulate a number of other pteridines in much greater amounts than do wild types. The amounts of biopterin and some other unidentified pteridines increase dramatically following eclosion of these flies (Browder and Williamson, unpublished). These pteridines could be metabolites that are produced from AHP that accumulates since it is not converted to IXP. However, neither ma1 nor ry flies, which also accumulate AHP, accumulate these pteridines. Hence, we conclude that tin is deficient in some aspect of either pteridine metabolism or excretion. The most unusual component of the tin phenotype is conditional female sterility; i.e., tin progeny of tin females do not survive. This has been difficult to understand since neither XDH, AO, nor PO is essential for viability. It is interesting to speculate that the lethality may be related to the accumulation of pteridines by maternally affected tin individuals. The failure of these tin females to produce viable progeny could result from deposition of pteri-
VOLUME
53. 1976
dines in their oocytes. This possibility is attractive because of observations in the literature that correlate pteridines with lethality and defects in RNA synthesis. Counce (1957) has reported that the female-sterile mutant deep orange (dor) accumulates IXP. Puckett and Snyder (1975) have recently demonstrated that IXP, which has a high affinity for DNA, is an effective inhibitor of RNA synthesis in Drosophila. Two observations indicate that IXP itself is not the cause of cinnamon female sterility. We have constructed +lcin stocks that are also homozygous for ry. Maternally affected tin, ry flies also fail to produce tin, ry progeny. Since ry prevents the formation of IXP, this provides a direct test of the effects of IXP on fecundity. In addition, one of us (JHW) has isolated a new cinnamon allele (tin’) that is not female-sterile. However, maternally affected tin’ individuals do accumulate IXP. Although IXP itself cannot be responsible for the failure of maternally affected tin females to produce tin progeny, it is possible that one or more of the pteridines that accumulate in tin flies are responsible. We are presently investigating this possibility. REFERENCES B. S. (1973). The maternal and zygotic control of development by cinnamon, a new mutant in Drosophila melanogaster. Develop. Biol. 33, 429440. BODENSTEIN, D. (1950). The postembryonic development of Drosophila. In “Biology of Drosophila” (M. Demerec, ed.), pp. 275-367. Wiley, New York (reprinted by Hafner, 1965). COLLINS, J. F., DUKE, E. J., and GLASSMAN, E. (1971). Multiple molecular forms of xanthine dehydrogenase and related enzymes. IV. The relationship of aldehyde oxidase to xanthine dehydrogenase. Biochem. Genet. 5, 1-13. COLLINS, J. F., and GLASSMAN, E. (1969). A third locus (lpo) affecting pyridoxal oxidase in Drosophila melanogaster. Genetics 61, 833-839. COUNCE, S. J. (1957). A female-sterile mutant (deep orange 1 of Drosophila melanogaster increasing isoxanthopterine content. Enperientia 13,354-356. COURTRIGHT, J. B. (1967). Polygenic control of aldehyde oxidase in Drosophila. Genetics 57, 25-39. BAKER,
BROWDER
AND
WILLIAMSON
Enzyme Activities
W. J. (1970). The genetics of aldehyde oxidase in Drosophila melanogaster. Genetics 66, 487-496. DICKINSON, W. J., and SULLIVAN, D. T. (1975). “Gene-Enzyme Systems in Drosophila.” SpringerVerlag, New York. GLA~~MAN, E. (1965a). Genetic regulation of xanthine dehydrogenase in Drosophila melanogaster. Fed. Proc. 24, 1243-1251. GLAMHAN, E. (1965b). Xanthine dehydrogenase of Drosophila melanogaster. J. Elisha Mitchell Sci. Sot. 81, 42-54. CLASSMAN, E., and MCLEAN, J. (1962). Maternal effect of ma1 on xanthine dehydrogenase of Drosophila melanogaster. II. Xanthine dehydrogenase activity during development. Proc. Nat. Acad. Sci. USA 48, 17121718. C~RELL,, E. H. (1962). The dose effect of mal’ and ry+ on xanthine dehydrogenase activity in Drosophila melanogaster. Z. Vererbungslehre 93, 371-377. DICKINSON,
in the tin Mutant
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LEWIS, E. B. (1960). A new standard culture medium. Drosophila Informat. Sew. 34, 117-118. LINSLEY, D. L., and GRELL, E. H. (1968). “The Genetic Variations ofDrosophila melanogaster” Carnegie Institution (Washington), Publ. 627, Washington, D.C. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (19511. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. PUCKETT, L. D., and SNYDER, L. A. (1975). Inhibition of RNA synthesis in Drosophila embryos by isoxanthopterin. Biochem. Genet. 13, l-6. SAYLES, C. D., BROWDER, L. W., and WILLIAMSON, J. H. (1973). Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster. Deuelop. Biol. 33, 213-217. YEN, T. T., and GLASSMAN, E. (1965). Electrophoretie variants of xanthine dehydrogenase in Drosophila melanogaster. Genetics 52, 977-981.
