Genetic Localization and Biochemical Effect in Drosophila Characterization O F a Trans-acting Regulatory

A region-specific, trans-acting regulatory gene that alters in vivo protein levels of a-glycerophosphate dehydrogenase (a-GPDH) has been mapped to position 55.4 on the third chromosome of Drosophila melanogaster. The gene has been found to affect the in vivo stability of a-GPDH in adult thoracic tissue but has no effect on a-GPDH levels in the abdomen. Although no other thoracic proteins were found to be influenced by the locus, it appears to modify the level of one additional abdominal protein. The action of the gene over development and its possible mode of control are discussed. substantial body of evidence has accumulated over the last several years A indicating that there are eukaryotic genes that influence the timing or expression of the products of other nonlinked loci (RAWLS and LUCCHESI 1974; LUSIS and PAICEN 1975; BOUBELIK et al. 1975; ABRAHAM and DOANE 1978; SCANDALIOS et al. 1980). Although such genes have been operationally defined as “regulatory,” it is recognized that there are a variety of molecular mechanisms by which they may exert their control (MCDONALD 1983; MACINTYRE 1982). In Drosophila, such regulatory genes and their controlled “producer genes”* have, in some instances, been associated with different chromosomes (MCDONALD and AYALA 1978a; LAURIE-AHLBERC et al. 1980; TEPPER et al. 1982; HORI et al. 1982). One producer gene recently shown to be subject to this type of interchromosomal control is a-gpdh in D. melanogaster (2-17.8, GRELL, JACOBSON and MURPHY 1965). a-gpdh codes for the soluble sn -glycerol 3 -phosphate dehydrogenase (GPDH:NAD+ oxidoreductase; EC 1.1.1.8), an enzyme known to play a key role in the insect’s cytosol-mitochondrial shuttle system (SACTOR 1965). An aglycerophosphate dehydrogenase (a-GPDH) regulatory effect that alters the level of a-GPDH activity has recently been associated with the third chromosome in D. melanogaster (LAURIE-AHLBERC et al. 1980; WILSON and MCDONALD 1981). We report here the results of a genetic localization and biochemical ’ To whom requests for reprints and all correspondence should be addressed: Department of Molecular and * Loci that specify cellular products directly involved in metabolic or structural functions (BRITTEN and Population Genetics, Biological Sciences Building, University of Georgia, Athens, Georgia 30602. DAVIDSON 1969; HEDRICK and MCDONALD 1980). Genetics 105 55-69 September, 1983 56 J. J. KING AND J. F. MCDONALD characterization of a region-specific, trans-acting gene that modifies the levels of a-GPDH in D. inelanogaster. MATERIALS AND METHODS Drosophilri strti ins A wild-type strain (F2) made completely homozygous for the first, second and third chromosomes was derived from a single wild-caught male collected from a Napa County, California, population (MCDONALD, ANDERSON and SANTOS 1980). A substituted strain (F2, M M 3 ) was constructed to have the same second (and thus the same a-gpdh genotype) and X chromosome constitution as the wild-type strain but homozygous for the multiply marked third chromosome carrying roughoid ( r u , 3-0.00), hairy ( h , 3-26.5), thread (th, 3-43.2), curled (cu , 3-50.0), stripe (sr, 3-62.0), ebony-sooty (e’, 3-70.7) and claret (ca, 3-1 00.7). Our method of substituted strain construction utilizes the balancer stock Cy/B12, SbSer/el’ and has been fully described earlier (MCDONALD and AYALA 1978b). An a-GPDH CRM(i.e., lacking detectable levels of a-GPDH cross-reacting material) strain (N-5-4) was kindly provided by GLENN BEWLEY (LEE, NIESEL and BEWLEY 1980). Biochemical techniques Enzyme a c h i t y a s s q : a-GPDH activity was measured spectrophotometrically according to a modification of the procedures of MCDONALD and AVISE (1976). Whole fly extracts of ten adults (48 days posteclosion), early pupae (160-180 hr postoviposition; eyes and wing pads not visible), late pupae (150-160 hr postoviposition; eyes and wing pads visible) or third instar larvae (155-145 hr after hatching; “roaming stage” with guts visibly free of media) were homogenized in 0.5 ml of 100 mM Tris-HCI (pH 8.6), 8 mM EDTA. Adult tissue-specific expression of a-GPDH, alcohol dehydrogenase (ADH) and phosphoglucose isomerase (PGI) enzyme activities were investigated by homogenizing adult thorax and abdomen sections separately (20 sections/ml). All homogenates were centrifuged at 5” at 12,000 X g for 20 min, and the supernatant was recovered for enzymatic analysis. a-GPDH activity was measured on either a Beckman ACTA I1 (adding 100 pl of crude extract to 900 rl of the reaction mix) or a Du-8 spectrophotometer (via an “improved enzyme assay’’ that extended the period of time for which the reaction (AOD) was linear: 10 pl of crude extract to 990 pl of the reaction mix). The reaction mix is 1 mM NAD+, 90 mM D,L-a-glycerophosphate made up in 100 mM Tris-HCI, (pH 8.6). ADH and PGI activites were monitored on a Du-8 spectrophotometer by previously published techniques (MCDONALD and AVISE 1976; AVISE and MCDONALD 1976). All flies examined in this study were determined to be insignificantly different in weight per individual and milligrams of protein per individual. Electrophoresis: Polyacrylamide gel electrophoresis was carried out in 7% gels according to the method of SMITH (1968). Gels were specifically stained for a-GPDH as described by AYALA et al. (1 972). Gel sieving: Polyacrylamide gel sieving was as described by JOHNSON (1975) using bovine hemoglobin as a standard. Isoelectric focusing: Isoelectric focusing was performed according to LKB, Inc. manual no. 1804. Samples were homogenized in distilled H 2 0 (six adult 66/0.1 ml) and focused in a thin layer acrylamide gel (72 X 24 cm) containing an ampholine pH gradient (pH 5.5-8.5). The gradient was set up in the long direction to maximize separation. Focusing was at 11 watts of constant power for 2 hr. The gel was stained for a-GPDH activity and sliced into strips, and the bands were scanned on a Beckman Du-8 spectrophotometer at 560 nm. The relative areas under each a-GPDH activity peak were computed for each sample. a-GPDH antisera. a-GPDH antisera were prepared from purified a-GPDH (40-60% ammonium sulfate cut followed by electrophoretic separation in a 7% polyacrylamide gel). a-GPDH bands were cut out of the polyacrylamide gel, homogenized in distilled H 2 0 and subcutaneously injected into New Zealand white rabbits. The antisera were otherwise prepared according to the method of McDonald rt al. (1977). The specificity of the prepared a-GPDH antisera was verified by the radioimmunological procedures of ANDERSON and MCDONALD (1981). The results presented in Figure 1 demonstrate that more than 80% of the precipitable [“C] counts associate with a-GPDH protein separated on a 7% sodium dodecyl sulfate polyacrylamide gel.