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Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster
DEVELOPMENTAL
BIOLOGY
33, 213-217
(1973)
Expression
of Xanthine
Dehydrogenase
Embryonic
Development
of Drosophila
CYRIL D. SAYLES,* LEON W. BROWDER, Department
of Biology.
University Accepted
Activity
of Calgary, Januam
during
melanogaster’
AND JOHN H. WILLIAMSON Calgary,
Alberta,
Canada
25, 1973
The contributions of oogenesis and zygotic genome expression to xanthine dehydrogenase activity during embryogenesis were investigated utilizing the mal and p2 mutants. In vitro complementation experiments demonstrated the presence of the mal+ complementation factor in the oocyte, suggesting an explanation f’or the mal maternal effect. The p+ complementation factor synthesized from paternal template was detected at gastrulation. This is the e,:liest detection of a paternal enzyme during nonmammalian embryonic development. INTRODUCTION
Oogenesis is a time of intense synthetic activity that is essential to prepare the unfertilized egg for initial embryonic development prior to the expression of the zygotic genome (Gross and Cousineau, 1964; Lockshin, 1966; Davidson et al., 1966; Raff et al., 1972). The synthetic products of oogenesis are also implicated in influencing later embryonic development directed by the zygotic genome. The contributions of these products are dramatically illustrated by maternal inheritance, which is characterized by an influence of the maternal genotype over the zygotic phenotype. It is usually apparent when the mother is heterozygous at a given locus and produces homozygous mutant progeny with wild-type phenotypic characteristics. The maroonlike (mal) eye color gene in Drosophila melanogaster is an intriguing case of maternal inheritance. Homozygotes for ma1 are deficient in xanthine dehydrogenase (XDH) activity (Forrest et al., 1956). However, Glassman and Mitchell (195913) showed that mallmal and mal/Y progeny of heterozygous females exhibit 1 Supported by National Research Council of’ Can ada Grants A-6209 (to L.W.B.) and A-5860 (ta J.H.W.). * Present address: Department of Anatomy, University of Colorado Medical Center, 4200 E. 9th Ave., Denver, Colorado 80220.
MATERIALS
0 1973 hy Academic Press. Inc. of reproduction in any form reserved
AND
METHODS
Stocks
1. Oregon-R (Ore-R)-wild-type with normal XDH activity 213
Copyright All rights
low levels of XDH activity in larval and adult forms and have wild-type eye color. XDH activity is dependent upon complementation between the mal+ factor and the rosy+ (ry+) factor. The ry+ gene is assumed to be the structural gene for XDH (Yen and Glassman, 1965; Glassman et al., 1962; Grell, 1962). The exact role of mul+ in XDH activity is not understood. It might code for a polypeptide subunit or it might be involved in controlling the synthesis or attachment of a cofactor to the enzyme. One aim of this investigation was to determine whether the mul+ complementation factor is a normal constituent of Drosophila oocytes. Its presence would suggest an explanation for the ma1 maternal effect. We also sought to monitor the changes that occur in XDH activity during development of wild-types and mutants with respect to time and developmental stage. By assaying for XDH activity in progeny of v/v females that had been fertilized by +/+ males, it was possible to estimate the time of de nouo synthesis of q+ complementation factor from paternal template.
strain
214
DEVELOPMENTAL
BIOLOGY
2. mal-maroonlike eye color, deficient in XDH activity 3. ryy2-rosy eye color; an allele of ry, deficient in XDH activity 4. X/O-males having a wild-type X chromosome but no Y chromosome; infertile due to the absence of the Y-linked fertility factors. These males will mate and stimulate females to oviposit; obtained by crossing C(l)RM, y u bbl0 virgin females with +/BsY males All stocks are described in Lindsley and Grell (1968) and were maintained on a standard cornmeal-yeast-sugar-agar-propionic acid medium in ‘4 pint milk bottles at 25.5 + 05°C and 40 f 3% relative humidity. Both ma1 and ry2 stocks were obtained from the Drosophila Stock Center, Pasadena, California; the ry2 stock used did not show the temperature sensitivity as described in Lindsley and Grell. Embryo
Collections
Embryos of known developmental age were obtained by utilizing a modification of the method of Mitchell and Mitchell (1964). A 12 x 12 x 24 inch plastic population cage was partially saturated with flies of the desired genotypes. Embryos were collected over l- or 2-hr periods by replacing the 5 x 8 inch food trays at these intervals. The first collection was discarded, thus increasing developmental uniformity of subsequent collections. The embryos were collected by suspending them in a freshly prepared 40% (w/v) sucrose solution, using a light camel’s hair brush to loosen the embryos from the surface of the food. The embryos were decanted into a small separatory funnel and washed with several volumes of Drosophila Ringer solution (Ephrussi and Beadle, 1936). A Nitex nylon bolting cloth circle (106 pm pore size; B. and S. H. Thompson) was placed in a glass funnel, and the embryos were poured in and rewashed several times with Ringer solution. Wet weight was approximated after plac-
VOLUME
33.
1973
ing the cloth circles on filter paper and allowing the embryos to air dry for 15 min. The cloth and embryos were weighed, the embryos were removed with a small spatula, and the cloth was reweighed. Extracts were prepared for immediate use in enzyme assays. XDH
Assay
The enzyme assay was a modification of Glassman and Mitchell’s procedure (1959a). Extracts were prepared by homogenizing embryos or whole flies in precooled (less than 5°C). Ten Broeck tissue grinders using 2 ml of cold 0.1 M Tris buffer, pH 8.0. Four milligrams of Norit A (Matheson, Coleman and Bell Corp.) was added to the homogenate to reduce endogenous fluorescence. The homogenate was shaken vigorously, then allowed to settle for 10 min before centrifugation at 12,000~ in a Sorvall RCB-B at 0°C for 20 min. The extract was then filtered through a sintered-glass funnel to remove any remaining particulate matter (Glassman, 1962b). The assay for XDH activity used 2amino, 4-hydroxypteridine (AHP; Calbiothem) as the substrate and followed its conversion to isoxanthopterin (IXP). The assay mixture consisted of 0.5 ml of the enzyme extract, 0.48 ml of 0.1 M Tris buffer (pH 8.0), 0.01 ml of 8-nicotinamide adenine dinucleotide (NAD; Sigma). This was warmed to 3O”C, 0.01 ml of AHP (3.0 x lo-‘M) was added, and the reaction was monitored until linearity was lost on an Aminco-Bowman spectrophotofluorometer (365 nm excitation and 405 nm emission wavelengths) with a Beckman 10” recorder. The fluorometer was calibrated prior to use so that 1.0 x lo-’ M quinine sulfate (Matheson, Coleman and Bell Corp.) in 0.1 N sulfuric acid had a reading of 30 units. A mixed-dilution curve (decreasing increments of AHP and increasing increments of IXP; 3.0 x low4 M, Calbiochem) was done in order to correlate the linear increase in fluorescence to picomoles AHP
215
BRIEF NOTES
oxidized. A change of one fluorescent unit was equivalent to oxidation of 9.6 picomoles of AHP. Protein was estimated using the Folin-phenol method of Lowry et al. (1951). The rate of XDH activity was proportional to the amount of extract used. Verification of the reaction product as IXP was done by thin-layer chromatography. A reaction mixture containing a boiled extract produced no IXP. In Vitro Complementation
Complementation of ma1 and ry extracts yields detectable levels of XDH activity, demonstrating the presence of the mal+ factor in ry extracts and the ry+ factor in ma1 extracts (Glassman, 1962a). In order to determine whether the mal+ complementation factor is a normal oocyte constituent, unfertilized ryz egg extracts were complemented with adult ma1 extracts, which provided ry+ factor. Unfertilized eggs were obtained by mating virgin ry* females with Xl0 males for 5 days. Wet weights of egg samples were measured after a 4-hr collection period, and equal wet weights of ma1 flies were used in the complementation reaction. Several 4-hr collections of eggs were obtained and stored at -20°C until a suitable amount (185 mg) was available for complementation. The existence of the mal+ factor during early embryogenesis in the ry* mutant was investigated by complementation of extracts of embryos at successive stages of development with ma1 adult extracts. Extracts were prepared by homogenizing whole eggs, embryos, or adults in 2 ml of cold 0.1 M Tris buffer pH 8.0 in precooled tissue grinders. The resultant homogenates were adjusted to pH 5.2, centrifuged at 12,000 g for 20 min, the precipitate discarded, and the supernatant filtered through a sintered-glass funnel. The pH was then readjusted to 8.0. The complementation reaction used equal volumes of ry2 and ma1 extracts, 50 units of penicillin and 50 pg of streptomycin per milliliter (to
prevent bacterial contamination) and was incubated in a 30°C water bath for 12 hr. The complementation mixture was then concentrated to 1 ml using Gelman Lyphogel and refiltered prior to assaying for XDH activity. Mutant extracts that were not complemented were subjected to the same treatment and were utilized as controls. The complementation procedure yields only qualitative information that establishes the presence of the mal+ and v+ complementation factors. Without purification of these factors and estimates of the efficiency of complementation, it is impossible to quantitate the reaction. RESULTS
malt Complementation Factor in Unfertilized Eggs and Early Embryos
The existence of an active mal+ factor in unfertilized ry* eggs was investigated by in uitro complementation with adult ma1 extracts and assaying for XDH activity. XDH activity (3.8 pmoles/min) was detected. The continued presence of the malt factor during early embryogenesis of the ry* mutant was demonstrated by in uitro complementation of extracts prepared from ryz embryos at 2, 4, 5, and 7 hr of development with ma1 adult extracts. XDH activity was detected in each case. No activity was detected in either ryz or ma1 extracts alone. XDH Activity
during Development
In the Ore-R strain, XDH activity (Fig. 1) is detectable at low levels in preblastoderm embryos (-2 hr). No determination prior to 2 hr was possible because of the collection procedure. An increase in specific activity is concomitant with gastrulation (~4 hr) and at 8-9 hr has increased approximately lo-fold. Extracts of ma1 and ry2 mutants showed no activity at any of the times tested. Zygotic Genome-Directed Synthesis
To estimate
the initial
appearance of the
216
DEVELOPMENTAL
BIOLOGY
VOLUME
33,
1973
08
J 0
4
8
12
DEVELOPMENT
FIG. 1. The specific activity ry’/+ Drosophila melanogaster. The mal, rya, and paI+ stocks
16
20
28
(hours)
of XDH as a function of developmental Each point on the curve for Ore-R were assayed once.
IY’+ factor from zygotic genome-directed synthesis, virgin $ females were mated with Ore-R males, and extracts derived from their progeny (ry”/+) were assayed at hourly intervals following oviposition (Fig. 1). XDH activity, reflecting the expression of the wild-type paternal allele, was detectable at 4 hr of development, coincident with gastrulation. Assays of activity past 7 hr were not done. Presumably activity would parallel that of Ore-R with approximately one half the specific activity due to the gene-dosage effect (Glassman et al., 1962; Grell, 1962).
24
time (in hours) of Ore-R, mal, ry*, and is the average of two independent assays.
factor at gastrulation is the earliest demonstration of a paternal enzyme during nonmammalian embryogenesis. The correspondence between time of synthesis and time of detection is a function of the sensitivity of the assay technique. Fluorometry is considerably more sensitive than electrophoretic or spectrophotometric techniques that have been utilized previously to detect zygotic synthesis in Drosophila (Courtright, 1967; Wright, 1970; Wright and Shaw, 1969, 1970; Dickinson, 1971). More sensitive assay techniques may establish an even earlier appearance of XDH or other enzymes.
DISCUSSION
The functional mul+ complementation factor is a normal constituent of unfertilized ry2 eggs, and it persists after fertilization. Since complementation reactions could not be treated quantitatively, it is unknown whether there is a net increase in the mal+ product (suggesting synthesis) during embryonic development. XDH activity does increase dramatically at gastrulation. Our data suggest that this increase is due, at least in part, to interaction of the mal+ factor with ry+ factor that is synthesized de nouo from zygotic template. The detection of newly synthesized ~JJ+
REFERENCES COURTRIGHT, J. B. (1967). Polygenic control of aldehyde oxidase in Drosophila. Genetics 57, 25-39. DAVIDSON, E. H., CRIPPA, M., KRAMER, F. R., and MIRSKY, A. E. (1966). Genomic function during the lampbrush chromosome stage of amphibian oogenesis. Proc. Nat. Acad. Sci. U.S. 56, 856-863. DICKINSON, W. J. (1971). Aldehyde oxidase in Drosophila melanogaster: A system for genetic studies on developmental regulation. Deuelop. Bid. 26,77-86. EPHRUSSI, B., and BEADLE, G. W. (1936). A technique of transplantation for Drosophila. Amer. Natur. 70, 218-225. FORREST, H. S., GLASSMAN, E., and MITCHELL, H. K. (1956). The conversion of 2-amino-4-hydroxypteridine to isoxanthopterin in Drosophila melanogaster. Science 124, 725-726.
BRIEF GLASSMAN, E. (1962a). In vitro complementation between nonallelic Drosophila mutants deficient in xanthine dehydrogenase. Proc. Nat. Acad. Sci. U.S 48, 1491-1497. GLASSMAN, E. (1962b). Convenient assay of xanthine dehydrogenase in single Drosophila melanogaster. Science 137, 990-991. GLASSMAN, E., and MITCHELL, H. K. (1959a). Mutants of Drosophila melanogaster deficient in xanthine dehydrogenase. Genetics 44, 153-162. GLASSMAN, E. and MITCHELL, H. K. (1959b). Maternal effect of ma/+ on xanthine dehydrogenase of Drosophila melanogaster. Genetics 44, 547-554. GLASSMAN, E., KARAM, J. D., and KELLER, E. C., Jr. (1962). Differential response to gene dosage experiments involving the two loci which control xanthine dehydrogenase of’ Drosophila melanogaster. 2. Verebungslehre 93, 399-403. GRELL, E. H. (1962). The dose effect of ma!+ and r.y+ on xanthine dehydrogenase activity in Drosophila melanogaster. 2. Verebungslehre 93, 371-377. GROSS, P. R., and COUSINEAU, G. H. (1964). Macromolecule synthesis and the influence of actinomycin on early development. I&p. Cell Res. 33, 368-395. LINDSLEX, D. L., and GRELL, E. H. (1968). “Genetic Variations of Drosophila melanogaster.” Carnegie Inst. Wash., Publ. No. 627. LOCKSHIN, R. A. (1966). Insect embryogenesis:
NOTES
217
Macromolecular synthesis during early development. Science 154, 775-776. LOWRY, 0. H., ROSEBROU~H, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MITCHEI.L, H. K., and MITCHELL, A. (1964). Mass culture and egg selection in Drosophila. Drosophila Inform. Sew. 39, 135-137. MITCHF.I,L, H. K., GLASSMAN, E., and HADORN, E. (1959). Hypoxanthine in rosv and maroon-like mutants of’ Drosophila melanogaster. Science 120, 268-269. RAFF, R. A., COLOT, H. V., SELVIC., S. E., and GROSS, P. R. (1972). Oogenetic origin of messenger RNA for embryonic synthesis ofmicrotubule protein. Nature (London) 235, 211-214. WRIGHT, D. A., and SHAW, C. R. (1969). Genetics and ontogeny of a-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster. Biochem. Genet. 3, 343-353. WRIGHT, D. A., and SHAW, C. R. (1970). Time of expression of genes controlling specific enzymes in Drosophila embryos. Biochem. Genet. 4, 385-394. WRIGHT, T. R. F. (1970). The genetics of embryogenesis in Drosophila. Aduan. Genet. 15, 261-395. YEN, T. T. T., and GLASSMAN, E. (1965). Electrophoretie variants of xanthine dehydrogenase in Drosophila melanogaster. Genetics 52, 977-981.
BIOLOGY
33, 213-217
(1973)
Expression
of Xanthine
Dehydrogenase
Embryonic
Development
of Drosophila
CYRIL D. SAYLES,* LEON W. BROWDER, Department
of Biology.
University Accepted
Activity
of Calgary, Januam
during
melanogaster’
AND JOHN H. WILLIAMSON Calgary,
Alberta,
Canada
25, 1973
The contributions of oogenesis and zygotic genome expression to xanthine dehydrogenase activity during embryogenesis were investigated utilizing the mal and p2 mutants. In vitro complementation experiments demonstrated the presence of the mal+ complementation factor in the oocyte, suggesting an explanation f’or the mal maternal effect. The p+ complementation factor synthesized from paternal template was detected at gastrulation. This is the e,:liest detection of a paternal enzyme during nonmammalian embryonic development. INTRODUCTION
Oogenesis is a time of intense synthetic activity that is essential to prepare the unfertilized egg for initial embryonic development prior to the expression of the zygotic genome (Gross and Cousineau, 1964; Lockshin, 1966; Davidson et al., 1966; Raff et al., 1972). The synthetic products of oogenesis are also implicated in influencing later embryonic development directed by the zygotic genome. The contributions of these products are dramatically illustrated by maternal inheritance, which is characterized by an influence of the maternal genotype over the zygotic phenotype. It is usually apparent when the mother is heterozygous at a given locus and produces homozygous mutant progeny with wild-type phenotypic characteristics. The maroonlike (mal) eye color gene in Drosophila melanogaster is an intriguing case of maternal inheritance. Homozygotes for ma1 are deficient in xanthine dehydrogenase (XDH) activity (Forrest et al., 1956). However, Glassman and Mitchell (195913) showed that mallmal and mal/Y progeny of heterozygous females exhibit 1 Supported by National Research Council of’ Can ada Grants A-6209 (to L.W.B.) and A-5860 (ta J.H.W.). * Present address: Department of Anatomy, University of Colorado Medical Center, 4200 E. 9th Ave., Denver, Colorado 80220.
MATERIALS
0 1973 hy Academic Press. Inc. of reproduction in any form reserved
AND
METHODS
Stocks
1. Oregon-R (Ore-R)-wild-type with normal XDH activity 213
Copyright All rights
low levels of XDH activity in larval and adult forms and have wild-type eye color. XDH activity is dependent upon complementation between the mal+ factor and the rosy+ (ry+) factor. The ry+ gene is assumed to be the structural gene for XDH (Yen and Glassman, 1965; Glassman et al., 1962; Grell, 1962). The exact role of mul+ in XDH activity is not understood. It might code for a polypeptide subunit or it might be involved in controlling the synthesis or attachment of a cofactor to the enzyme. One aim of this investigation was to determine whether the mul+ complementation factor is a normal constituent of Drosophila oocytes. Its presence would suggest an explanation for the ma1 maternal effect. We also sought to monitor the changes that occur in XDH activity during development of wild-types and mutants with respect to time and developmental stage. By assaying for XDH activity in progeny of v/v females that had been fertilized by +/+ males, it was possible to estimate the time of de nouo synthesis of q+ complementation factor from paternal template.
strain
214
DEVELOPMENTAL
BIOLOGY
2. mal-maroonlike eye color, deficient in XDH activity 3. ryy2-rosy eye color; an allele of ry, deficient in XDH activity 4. X/O-males having a wild-type X chromosome but no Y chromosome; infertile due to the absence of the Y-linked fertility factors. These males will mate and stimulate females to oviposit; obtained by crossing C(l)RM, y u bbl0 virgin females with +/BsY males All stocks are described in Lindsley and Grell (1968) and were maintained on a standard cornmeal-yeast-sugar-agar-propionic acid medium in ‘4 pint milk bottles at 25.5 + 05°C and 40 f 3% relative humidity. Both ma1 and ry2 stocks were obtained from the Drosophila Stock Center, Pasadena, California; the ry2 stock used did not show the temperature sensitivity as described in Lindsley and Grell. Embryo
Collections
Embryos of known developmental age were obtained by utilizing a modification of the method of Mitchell and Mitchell (1964). A 12 x 12 x 24 inch plastic population cage was partially saturated with flies of the desired genotypes. Embryos were collected over l- or 2-hr periods by replacing the 5 x 8 inch food trays at these intervals. The first collection was discarded, thus increasing developmental uniformity of subsequent collections. The embryos were collected by suspending them in a freshly prepared 40% (w/v) sucrose solution, using a light camel’s hair brush to loosen the embryos from the surface of the food. The embryos were decanted into a small separatory funnel and washed with several volumes of Drosophila Ringer solution (Ephrussi and Beadle, 1936). A Nitex nylon bolting cloth circle (106 pm pore size; B. and S. H. Thompson) was placed in a glass funnel, and the embryos were poured in and rewashed several times with Ringer solution. Wet weight was approximated after plac-
VOLUME
33.
1973
ing the cloth circles on filter paper and allowing the embryos to air dry for 15 min. The cloth and embryos were weighed, the embryos were removed with a small spatula, and the cloth was reweighed. Extracts were prepared for immediate use in enzyme assays. XDH
Assay
The enzyme assay was a modification of Glassman and Mitchell’s procedure (1959a). Extracts were prepared by homogenizing embryos or whole flies in precooled (less than 5°C). Ten Broeck tissue grinders using 2 ml of cold 0.1 M Tris buffer, pH 8.0. Four milligrams of Norit A (Matheson, Coleman and Bell Corp.) was added to the homogenate to reduce endogenous fluorescence. The homogenate was shaken vigorously, then allowed to settle for 10 min before centrifugation at 12,000~ in a Sorvall RCB-B at 0°C for 20 min. The extract was then filtered through a sintered-glass funnel to remove any remaining particulate matter (Glassman, 1962b). The assay for XDH activity used 2amino, 4-hydroxypteridine (AHP; Calbiothem) as the substrate and followed its conversion to isoxanthopterin (IXP). The assay mixture consisted of 0.5 ml of the enzyme extract, 0.48 ml of 0.1 M Tris buffer (pH 8.0), 0.01 ml of 8-nicotinamide adenine dinucleotide (NAD; Sigma). This was warmed to 3O”C, 0.01 ml of AHP (3.0 x lo-‘M) was added, and the reaction was monitored until linearity was lost on an Aminco-Bowman spectrophotofluorometer (365 nm excitation and 405 nm emission wavelengths) with a Beckman 10” recorder. The fluorometer was calibrated prior to use so that 1.0 x lo-’ M quinine sulfate (Matheson, Coleman and Bell Corp.) in 0.1 N sulfuric acid had a reading of 30 units. A mixed-dilution curve (decreasing increments of AHP and increasing increments of IXP; 3.0 x low4 M, Calbiochem) was done in order to correlate the linear increase in fluorescence to picomoles AHP
215
BRIEF NOTES
oxidized. A change of one fluorescent unit was equivalent to oxidation of 9.6 picomoles of AHP. Protein was estimated using the Folin-phenol method of Lowry et al. (1951). The rate of XDH activity was proportional to the amount of extract used. Verification of the reaction product as IXP was done by thin-layer chromatography. A reaction mixture containing a boiled extract produced no IXP. In Vitro Complementation
Complementation of ma1 and ry extracts yields detectable levels of XDH activity, demonstrating the presence of the mal+ factor in ry extracts and the ry+ factor in ma1 extracts (Glassman, 1962a). In order to determine whether the mal+ complementation factor is a normal oocyte constituent, unfertilized ryz egg extracts were complemented with adult ma1 extracts, which provided ry+ factor. Unfertilized eggs were obtained by mating virgin ry* females with Xl0 males for 5 days. Wet weights of egg samples were measured after a 4-hr collection period, and equal wet weights of ma1 flies were used in the complementation reaction. Several 4-hr collections of eggs were obtained and stored at -20°C until a suitable amount (185 mg) was available for complementation. The existence of the mal+ factor during early embryogenesis in the ry* mutant was investigated by complementation of extracts of embryos at successive stages of development with ma1 adult extracts. Extracts were prepared by homogenizing whole eggs, embryos, or adults in 2 ml of cold 0.1 M Tris buffer pH 8.0 in precooled tissue grinders. The resultant homogenates were adjusted to pH 5.2, centrifuged at 12,000 g for 20 min, the precipitate discarded, and the supernatant filtered through a sintered-glass funnel. The pH was then readjusted to 8.0. The complementation reaction used equal volumes of ry2 and ma1 extracts, 50 units of penicillin and 50 pg of streptomycin per milliliter (to
prevent bacterial contamination) and was incubated in a 30°C water bath for 12 hr. The complementation mixture was then concentrated to 1 ml using Gelman Lyphogel and refiltered prior to assaying for XDH activity. Mutant extracts that were not complemented were subjected to the same treatment and were utilized as controls. The complementation procedure yields only qualitative information that establishes the presence of the mal+ and v+ complementation factors. Without purification of these factors and estimates of the efficiency of complementation, it is impossible to quantitate the reaction. RESULTS
malt Complementation Factor in Unfertilized Eggs and Early Embryos
The existence of an active mal+ factor in unfertilized ry* eggs was investigated by in uitro complementation with adult ma1 extracts and assaying for XDH activity. XDH activity (3.8 pmoles/min) was detected. The continued presence of the malt factor during early embryogenesis of the ry* mutant was demonstrated by in uitro complementation of extracts prepared from ryz embryos at 2, 4, 5, and 7 hr of development with ma1 adult extracts. XDH activity was detected in each case. No activity was detected in either ryz or ma1 extracts alone. XDH Activity
during Development
In the Ore-R strain, XDH activity (Fig. 1) is detectable at low levels in preblastoderm embryos (-2 hr). No determination prior to 2 hr was possible because of the collection procedure. An increase in specific activity is concomitant with gastrulation (~4 hr) and at 8-9 hr has increased approximately lo-fold. Extracts of ma1 and ry2 mutants showed no activity at any of the times tested. Zygotic Genome-Directed Synthesis
To estimate
the initial
appearance of the
216
DEVELOPMENTAL
BIOLOGY
VOLUME
33,
1973
08
J 0
4
8
12
DEVELOPMENT
FIG. 1. The specific activity ry’/+ Drosophila melanogaster. The mal, rya, and paI+ stocks
16
20
28
(hours)
of XDH as a function of developmental Each point on the curve for Ore-R were assayed once.
IY’+ factor from zygotic genome-directed synthesis, virgin $ females were mated with Ore-R males, and extracts derived from their progeny (ry”/+) were assayed at hourly intervals following oviposition (Fig. 1). XDH activity, reflecting the expression of the wild-type paternal allele, was detectable at 4 hr of development, coincident with gastrulation. Assays of activity past 7 hr were not done. Presumably activity would parallel that of Ore-R with approximately one half the specific activity due to the gene-dosage effect (Glassman et al., 1962; Grell, 1962).
24
time (in hours) of Ore-R, mal, ry*, and is the average of two independent assays.
factor at gastrulation is the earliest demonstration of a paternal enzyme during nonmammalian embryogenesis. The correspondence between time of synthesis and time of detection is a function of the sensitivity of the assay technique. Fluorometry is considerably more sensitive than electrophoretic or spectrophotometric techniques that have been utilized previously to detect zygotic synthesis in Drosophila (Courtright, 1967; Wright, 1970; Wright and Shaw, 1969, 1970; Dickinson, 1971). More sensitive assay techniques may establish an even earlier appearance of XDH or other enzymes.
DISCUSSION
The functional mul+ complementation factor is a normal constituent of unfertilized ry2 eggs, and it persists after fertilization. Since complementation reactions could not be treated quantitatively, it is unknown whether there is a net increase in the mal+ product (suggesting synthesis) during embryonic development. XDH activity does increase dramatically at gastrulation. Our data suggest that this increase is due, at least in part, to interaction of the mal+ factor with ry+ factor that is synthesized de nouo from zygotic template. The detection of newly synthesized ~JJ+
REFERENCES COURTRIGHT, J. B. (1967). Polygenic control of aldehyde oxidase in Drosophila. Genetics 57, 25-39. DAVIDSON, E. H., CRIPPA, M., KRAMER, F. R., and MIRSKY, A. E. (1966). Genomic function during the lampbrush chromosome stage of amphibian oogenesis. Proc. Nat. Acad. Sci. U.S. 56, 856-863. DICKINSON, W. J. (1971). Aldehyde oxidase in Drosophila melanogaster: A system for genetic studies on developmental regulation. Deuelop. Bid. 26,77-86. EPHRUSSI, B., and BEADLE, G. W. (1936). A technique of transplantation for Drosophila. Amer. Natur. 70, 218-225. FORREST, H. S., GLASSMAN, E., and MITCHELL, H. K. (1956). The conversion of 2-amino-4-hydroxypteridine to isoxanthopterin in Drosophila melanogaster. Science 124, 725-726.
BRIEF GLASSMAN, E. (1962a). In vitro complementation between nonallelic Drosophila mutants deficient in xanthine dehydrogenase. Proc. Nat. Acad. Sci. U.S 48, 1491-1497. GLASSMAN, E. (1962b). Convenient assay of xanthine dehydrogenase in single Drosophila melanogaster. Science 137, 990-991. GLASSMAN, E., and MITCHELL, H. K. (1959a). Mutants of Drosophila melanogaster deficient in xanthine dehydrogenase. Genetics 44, 153-162. GLASSMAN, E. and MITCHELL, H. K. (1959b). Maternal effect of ma/+ on xanthine dehydrogenase of Drosophila melanogaster. Genetics 44, 547-554. GLASSMAN, E., KARAM, J. D., and KELLER, E. C., Jr. (1962). Differential response to gene dosage experiments involving the two loci which control xanthine dehydrogenase of’ Drosophila melanogaster. 2. Verebungslehre 93, 399-403. GRELL, E. H. (1962). The dose effect of ma!+ and r.y+ on xanthine dehydrogenase activity in Drosophila melanogaster. 2. Verebungslehre 93, 371-377. GROSS, P. R., and COUSINEAU, G. H. (1964). Macromolecule synthesis and the influence of actinomycin on early development. I&p. Cell Res. 33, 368-395. LINDSLEX, D. L., and GRELL, E. H. (1968). “Genetic Variations of Drosophila melanogaster.” Carnegie Inst. Wash., Publ. No. 627. LOCKSHIN, R. A. (1966). Insect embryogenesis:
NOTES
217
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