Genetics of Acetylcholinesterase in Drosophila Melanogasteri

Genes in Drosophila melanogaster that control acetylcholinesterase (AChE) were searched for by segmental aneuploidy techniques. Homogenates of flies containing duplications o r deletions for different segments were assayed for enzyme activity. A region on the third chromosome was found for which flies having one dose consistently gave lower AChE activity than euploid flies, which in turn had lower activity than flies with three doses. The activity differences were in the approximate ratio 1:2:3. Fine structure deletion mapping within this region revealed a very small segment for which one-dose flies have approximately half-normal activity. To obtain putative AchE-null mutations, lethal mutations within this region were assayed. Four allelic lethals have approximately half-normal activity in heterozygous condition. These lethals probably define the structural locus (symbol: Ace) for AChE. EUROLOGICAL mutations in Drosophila hold promise as tools for studying the development and function of the nervous system (reviewed by BENZER 1973; GROSSFIELD 1975; PAK 1975). Most of the existing ones have been isolated on the basis of defective behavior. Another approach is to choose a gene that controls a specific protein involved in nervous system function, isolate mutations in the gene, and analyze the defective gene product’s effects on the development, neurophysiology, and behavior of the organism. Potentially, such mutations can also be used as markers for nervous system tissues in genetic mosaic experiments (e.g. HOTTA and BENZER 1973; KANKEL and HALL 1976). Acetylcholinesterase (AChE) is a favorable enzyme in Drosophila for this type of analysis. The enzyme hydrolyzes acetylcholine, which is an important excitatory central nervous system neurotransmitter in insects (reviewed by PITMAN 1971). The enzyme is a good histochemical marker for nerve cells. Using segmental aneuploidy, the genome was screened for loci controlling the enzyme. This technique is based on the principle that three doses of the structural gene for a given enzyme could result in increased enzyme activity, compared to that in flies having the normal two doses; and one dose could lead to decreased Supported by Public Health Service Fellowsbps to J C H and D R K , by American Cancer Society Institutional Grant Number IN 104 to Brandels University, by Public Health Service grants NS-11788 (awarded to D R K ), and NS-12346 (to D R K and J C H ), and by National Science Foundation research grant GB-27228 to DR. SEYMOUR BENZER * Current address Department of Biology, Brandels University, Waltham, Massachusetts 02154. Current address Department of Biology, Yale University, New Haven, Connecticutt 06520. Genetics 83: 517-535 July, 1976 518 J. C. H A L L A N D D. R. KANKEL activity. This has been confirmed for several Drosophila enzymes: Xanthine dehydrogenase ( GRELL 1962) , glucose-6-phosphate dehydrogenase ( SEECOF, KAPLAN and FUTCH 1969; MARONI and PLAUT 1973; BOWMAN and SIMMONS 1973; STEWART and MERRIAM 1974), 6-phosphogluconate dehydrogenase (SEECOF, KAPLAN and FUTCH 1969; BOWMAN and SIMMONS 1973; STEWART and MERRIAM 1974) , tryptophan oxygenase (TOBLER, BOWMAN and SIMMONS 1971; BAILLIE and CHOVNICK 1971 ) , isocitrate dehydrogenase (STEWART and MERRIAM 1974; RAWLS and LUCCHESI 1974) and the soluble form of a-glycerophosphate dehydrogenase (RAWLS and LUCCHESI 1974). Several investigators have constructed duplications (three doses) for series of second and third chromosome regions in searches for previously unlocalized enzyme-controlling segments ( O’BRIEN and GETHMANN 1973; RAWLS and LUCCHESI 1974; HODGETTS 1975). Most of the duplications were generated using the Y-autosome translocations of LINDSLEY et aZ. (1972). In the present study, a similar screen has been carried out with respect to AChE. Three chromosome regions have been found that give lower activity for one-dose flies than two-dose flies, which in turn have lower activity than threedose flies. Lethal mutations within one of the regions were found to cause reduced AChE activity in flies heterozygous for the mutations. These lethals are candidates for mutations that eliminate the activity of this enzyme, and are likely to be mutations in the structural gene (symbol: Ace) for AChE. MATERIALS AND METHODS A. Construction of aneuploid genotypes The principal means of generating flies aneuploid for various portions of the genome was through the use of translocations involving the Y chromosome, and either the X , second, or third chromosome (Figure 1). For second or third chromosome segmental aneuploids (which comprise the bulk of the screening), deletions are recovered in one sex, and duplications in the other (Figure 1). AChE assays of male homogenates (see below) gave more reproducible kinetics; so in re-tests of promising segments, and in all further work on segments that gave reproducible dosage effects, activity in deleted us. duplicated males was compared. For Y-2 and Y-3 translocations (called T(Y;Z)’s and T(Y;3)’s, respectively) reciprocal crosses were carried out for a given chromosome segment, so that deleted males and duplicated males could be obtained (e.g., deleted males from the cross in Figure 1, and duplicated males from the reciprocal cross). When using two different T(Y;Z)’s o r T(Y;3)’s, crosses between two translocations with Y chromosome breakpoints in different arms (as in Figure 1) allows all euploid and aneuploid genotypes unambiguously to be recognized among the progeny (LINDSLEY et al. 1972). Crosses were made between translocations with different Y-arm breakpoints whenever availability of stocks with appropriate autosomal breakpoints made this possible. For some regions (totalling 16% of the autosomal complement), crosses had to be made between two given translocations with the same Y-arm breakpoints (see LINDSLEY et al. 1972). Here, one can be reasonably sure of the deleted us. duplicated genotype of a given progeny since meiotic nondisjunction of homologous centromeres is rare in the translocation-bearing parents (O’BRIEN and GETHMANN 1973). Using these crosses to generate deletions, about 80% of the second-third chromosome complement could be screened for effects on AChE activity. However, virtually the entire autosomal complement was screened with respect to duplicated segments (Figure 4A). The only region that cannot be tested is 83D-E on chromosome 3, which causes lethality in one-dose and three.dose flies (LINDSLEY et al. 1972). DROSOPHILA ACETYLCHOLINESTERASE 519 X CHROMOSOME DUPLICATIONS T(I;Y)B26 stock of Stewart and Merriam(1973) with X c h r o " e breakpoint in 9 C a. X̂ X. y w f 2 euploid b. fi. y w f 0 hyperploid for distal half of X 7 B' C. f X . y w f p hyperploid for proximal half of X 2nd AND 3rd CHROMOSOME DUeLICATK)NS AND DELETIONS (cf. Lindsley et al. 1972) B. Y Dom A v y * Y I,. 'Y' duplicated deleted 4 t h CHROMOSOME T R I M AND MO-Y euploid = C(4)RM 10 = attached-4 = &I / 0