genetic localization and action of regulatory genes … · 2003. 8. 1. · both amy4 and amy6...

Copyright 0 1986 by the Genetics Society of America

GENETIC LOCALIZATION AND ACTION OF REGULATORY GENES AND ELEMENTS FOR TISSUE-

SPECIFIC EXPRESSION OF &-AMYLASE IN DROSOPHILA MELANOGASTER

A. J. KLARENBERG, A. J. S. VISSER, M. F. M. WILLEMSE AND W. SCHARLOO

Department of Population and Evolutionary Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

Manuscript received September 9, 1985 Revised copy accepted August 20, 1986

ABSTRACT Regulation of tissue-specific a-amylase (Amy) expression in Drosophila melano-

gaster was investigated with a newly developed method that detects the distribution of a-amylase allozymes in midguts of single adults or third-instar larvae. Trans regulation was found for a-amylase production in the posterior midgut (PMG) of adults, whereas cis regulation was demonstrated for the larval midgut. Independent regulation of components of the duplicated Amy locus was found in larvae. Recombination between the components of the Amy locus did not result in separation of the putative, very closely linked (less than 0.1 cM) cis- acting regulatory elements for a-amylase expression in the anterior midgut (AMG) of larvae. The expression of one of the components of the duplicated Amy locus in the AMG of larvae was influenced by a regulatory gene that was mapped at 2-79.1. a-Amylase expression in the adult PMG was controlled by a trans-acting regulatory gene localized at 2-79.0, in agreement with the data of ABRAHAM and DOANE.

URING the past decade there has been a growing interest in regulation D of developmental, quantitative and tissue-specific expression of structural genes in higher eukaryotes. Especially in maize, Drosophila and mice it has been possible to characterize gene regulatory systems (review SCANDALIOS 1982; MACINTYRE 1982; LAURIE-AHLBERG 1985; PAICEN 1979). It has been proposed that evolutionary change may result predominantly from changes in regulatory genes (e.g., WHITT 1983; WILSON 1976). Moreover, regulatory gene variation may have significance for adaptation.

Concerning the chromosomal position of these control systems obtained with classical mapping techniques, they can be divided into two groups. First, there are regulatory genes located very close to, or even adjacent to, the structural gene. Second, there are regulatory genes situated at a greater distance from the structural gene, or even in another chromosome. In general, the first group are cis regulators, whereas the second group are trans regulators.

Identification of regulatory variants that influence differential tissue-specific Genetics 114: 1131-1 145 December. 1986

1132 A. J. KLARENBERG E T AL.

expression of a-amylase (1.4-a-D-glucan glucanohydrolase, EC3.2.1.1) enabled ABRAHAM and DOANE (1 978) to localize a trans-acting regulatory gene, map (midgut activity pattern), in adults of Drosophila melanogaster. The map-PMG locus was placed on the genetic map at 2-80f, approximately two crossover units distal of the structural gene for a-amylase (Amy, 2-77.8, BAHN 1967; DOANE 1969a; KIKKAWA 1964). Homozygous strains bearing two different a- amylase variants carry duplicated Amy genes between which a distance of 0.008 cM was found (BAHN 1967). This was confirmed by molecular cloning of the Amy region (LEVY, GEMMILL and DOANE 1985).

POWELL and LICHTENFELS (1 979) showed that Drosophila pseudoobscura has similar midgut patterns. They could not localize regulatory genes similar to map, and it was concluded that a-amylase midgut expression was determined pol ygenica I 1 y .

We studied large numbers of strains isogenic for the second chromosome, which had been isolated from different populations of D. melanogaster. We confirmed that map variation is determined by the second chromosome. Mid- gut pattern variation of several other carbohydrases was found to be independent of the a-amylase patterns (KLARENBERG and SCHARLOO 1982). In addition, Amy and map variants showed considerable nonrandom association, which could be interpreted as a linkage disequilibrium between Amy and map.

In this paper we present a method that simultaneously provides for determination of Amy and map variation by means of agarose gel electrophoresis of an intact midgut of a single adult or larva. In applying midgut electrophoresis we were able to demonstrate cis- and trans-regulated tissue-specific gene expression of structural Amy variants in D. melanogaster. In recombination experiments with Amy and midgut pattern variants in which the Amy region was enclosed between outside markers, a regulatory gene for larval a-amylase midgut activity patterns was localized. Chromosomes originating by recombination in this region were made homozygous with a suitable inversion chromosome. Thereby, problems arising from variable expression due to environmental effects could be excluded by testing the same chromosome of individual larvae or adults. Moreover, we could corroborate ABRAHAM and DOANE'S (1978) localization of map-PMG in adults. Finally, rare recombination events within the structural Amy region of the Amy' and Amy4r6 genes were informative for putative, closely linked, cis-acting regulatory elements affecting a-amylase midgut activity patterns in third-instar larvae of D. melanogaster.

MATERIALS AND METHODS Fly stocks: Amy', Amy3,6 and Amy4,6 stocks were isolated from Dahomey, Kaduna and

Pacific cage populations of D. melanogaster. The AmynuL' stock was provided by D. A. HICKEY (Ottawa, Canada). Stocks with recessive morphological mutants of the second chromosome, curved (c, 2-75.5) and welt (wt, 2-82), were obtained from the Californian Institute of Technology (Pasadena, California) and the Mid-American Stock Center (Bowling Green, Ohio). The recessive mutant speck (sp, 2-107.0) was from our labora- tory. For a detailed description of these morphological mutants and the balancer stock CyO/Pu, see LINDSLEY and GRELL (1968). By crossing a multiply marked stock, c Amy' wt sp was obtained. Strains were made isogenic for the second chromosome. After analysis of a-amylase midgut activity patterns of 4- to 6-day-old adults, a c Amy' wt sp

Amy REGULATORY GENES AND ELEMENTS 1133

stock was selected, the adults of which had only a-amylase activity in the anterior midgut (map-AMG 123); the posterior midgut of this strain showed no a-amylase activity (map- PMG 00). In 120-hr third-instar larvae, the c Amy' wt sp strain had only activity in the PMG. The other stock, used for the genetic localization of regulatory genes and elements, was an Amy4s6 wild-type strain isogenic for the second chromosome derived from a Pacific cage population. Both Amy4 and Amy6 enzymes produced by the duplicated Amy locus were present in the anterior and posterior midguts of 4- to 6-day-old adults and 120-hr third-instar larvae. The nomenclature of DOANE (1980) was followed for designating the a-amylase midgut patterns.

Midgut activity determination: Methods for a-amylase midgut pattern determination were already described by ABRAHAM and DOANE (1 978). Recombinant male and female adults were dissected for histochemical staining of midguts for a-amylase at an age of 4-6 days. For the analysis of larvae of strains isogenic for recombinant second chromosomes, midguts of 120-hr third-instar larvae were used.

Midgut electrophoresis: Midguts of 4- to 6-day-old adults or 120-hr third-instar larvae (feeding stage) were dissected in cold Ringer's solution. Freshly prepared midguts were immediately transferred to glass covers (20 X 40 mm) and were stretched to dry for 10 min at room temperature. The glass covers were not coated with albumin, because the midguts adhere very well. With adults, four female or five male midguts could be stretched in the middle of the glass cover. In experiments in which a large number of midguts had to be dissected, the glass covers with the midguts were directly frozen at -20 O .

For detection of a-amylase enzyme variants, agarose gel electrophoresis was used on homogenates of adults and larvae (DE JONC et al. 1972). For midgut electrophoresis, a whole organ was submitted to electrophoresis. The intact midgut, which was fixed on a glass cover, was placed on the surface of the gel. Agarose gels were made of 0.9 g of agarose in 100 ml of 41 mM Verona1 (5,5-diethylbarbituric acid sodium salt) buffer, pH 8.4. After the agarose had been dissolved on a stirring heater, the solution was poured on a glass plate and cooled at room temperature for 30 min. To check progress of electrophoresis, a small well was punched out of the gel and filled with the tracking dye Brome Phenol Blue. Electrophoresis was performed in a Gelman unit at 60 mA and 300 V for 3 hr at 4". The gel buffer was identical with electrophoresis buffer. After electrophoresis, the glass covers were removed and the gel was stained for a- amylase activity, following the methods of DE JONC et al. (1972). Activity was recorded as clear spots in the dark blue field of the starch-iodine complex. Both the tissue-specific distribution of a-amylase and the expression of the Amy variants in the midgut was determined.

Enzyme activity assay and protein determination: a-Amylase activity was measured spectrophotometrically according to a modified DNSA method of NOELTINC and BERN- FELD (1948). Further details of this method are given by HOORN and SCHARLOO (1978). Protein content was measured using the Bio-Rad protein assay reagent. All measure- ments were made of two separate midgut homogenates of 50 120-hr third-instar larvae. The Amy""" of Hickey (HAJ-AHMAD and HICKEY 1982) was taken as a reference for absence of a-amylase activity.

Localization: Virgin female adults of the progeny of the wild-type Pacific Amy4.6 and c Amy' wt sp strains were mated with the multiply marked Amy' stock. From the progeny of this cross, all male recombinants between curved and welt were individually mated with 4-6 CyO/Pu females. In this way a large number of lines isogenic for recombinant second chromosomes were obtained.

Adults: After mating with CyO/Pu, the 5- to 6-day-old male adults were used for midgut electrophoresis. Of each recombinant line, two male and three female adults were used for midgut electrophoresis to determine the Amy and adult map variants. Adults were reared on a standard cornmeal medium (with '7.7% sucrose; see THORIG, SCHOONE and SCHARLOO 1975) at 25" and 60% relative humidity (RH), except larvae of the progeny of the backcross with the multiply marked stock that, for isolation of

1134 A. J. KLARENBERG ET AL.

the recombinants, were bred at 27" to provide optimal expression of the mutant welt (see LINDSLEY and GRELL 1968).

Larvae: Localization of larval map genes was only possible by the production of a large number of strains isogenic for recombinant second chromosomes for the interval between curued and welt. For each strain, 6-10 midguts of 120-hr third-instar larvae were used for midgut electrophoresis. Larvae were reared on an agar-1.8% dead yeast food medium (with no added sugars) at 25" and 60% RH.

RESULTS

Trans regulation of Amy in the PMG of adults: To demonstrate trans action of map-PMG in adults, we used Amy' and Amy4.' stocks with different adult a- amylase midgut pattern variants reared on standard cornmeal medium. Be- cause these strains were isogenic for their second chromosome, both Amy and map loci were homozygous, and all 4- to 6-day-old adults of the same stock had the same a-amylase midgut patterns as revealed with the histochemical method.

As illustrated in Figure la-c, the Amy' stock (from Kaduna) had only a- amylase activity in the adult AMG (map-AMG 123), whereas the adult PMG showed no a-amylase activity (map-PMG 00). In contrast, the Amy416 stock (from Pacific) had a-amylase activity in both AMG and PMG of adults (map-AMG 123; map-PMG 12). Midgut electrophoresis shows that Amy4 and Amy6 have a similar expression in the AMG and PMG of the adult. Heterozygous adults of the Amy' and Amy4z6 stocks, investigated with the histochemical method, show the same midgut patterns as the parental Amy436 stock. Midgut electrophoresis of the A m y ' / A m ~ ~ , ~ adults reveals that all three Amy variants are expressed in the AMG and PMG. These observations clearly demonstrate a trans action of the adult map-PMG 12 allele associated with the Amy426 chromosome, because Amy' of the homologous chromosome with the adult map-PMG 00 allele is activated in the PMG of the heterozygous adult. T h e heterozygotes obtained by reciprocal crosses show no differences. To test whether trans action of map- PMG in adults occurred independent of the structural Amy variants, an Amy' stock (from Dahomey) with a-amylase in the adult AMG and PMG (map-AMG 123; map-PMG 12) was crossed with an Amy416 stock (from Kaduna) with only a-amylase activity in the adult AMG (map-AMG 123; map-PMG 00). Analysis of the heterozygous adults showed again a trans action of the adult map-PMG 12 allele, now located on the Amy' chromosome, inducing activity of Amy4" in the adult PMG (Figure Id-f). Crossing of the Amy' and Amy4,' stocks with a- amylase activity only in the adult AMG (map-ilMG 123; map-PMG 00) revealed (Figure lg-i) that the heterozygous adults had identical midgut patterns to those of their parental stocks. Crossing of the Amy' and Amy's6 stocks with a- amylase activity in both AMG and PMG produced heterozygous adults with the same adult pattern as their parents.

Effects of the food medium on Amy and map in larvae: a-Amylase midgut preparations of 120-hr third-instar larvae revealed that the expression of a- amylase depends on the Amy variant and the kind of food medium on which the larvae a re reared (KLARENBERG et al. 1983). When larvae are grown on a standard cornmeal medium, all strains showed a reduced larval a-amylase mid-


FIGURE 1 .-Regulation of tissue-specific a-amylase expression in midguts of 4- to 6day-old adults. Each section (a-i) shows the a-amylase zymogram obtained with midgut electrophoresis and, underneath, the a-amylase midgut pattern found by histochemical staining. In a-c and d-f: trans action of map-PMG on Amy' and Amy'.' in hybrids (c and f). Parents had the adult map-PMG 00 (a and e) or the adult map-PMG 12 allele (b and d). Hybrids had expression of all three Amy variants in the adult AMG and PMG (c and f). In g-i, the parental and hybrid adult midguts show expression of Amy' and Amy'.' in the AMG only, because they had all adult map-PMG 00 alleles (see text for further explanation).

1136

0

A

A. J. KLARENBERG E T AL.

FIGURE 2.-Effects of different food media on a-amylase midgut expression in 120-hr third- instar larvae of Amy’,6. Each section (a-b) shows the a-amylase zymogram obtained with midgut electrophoresis and, underneath, the a-amylase midgut pattern found by histochemical staining. a, The a-amylase midgut pattern of Amy’.6 larvae on agar-1.8% dead yeast food medium. Both the larval AMG and PMG have @-amylase; note differential expression of Amy’ and Amy6. b, The a- amylase midgut pattern of larvae on standard cornmeal food medium. Amy’ and Amy6 are only expressed in the anterior part of the larval PMG.

gut pattern if compared with larvae grown on an agar-1.8% dead yeast food medium. Midgut electrophoresis of the Amy’*6 strain (from Dahomey) as given in Figure 2a and b, demonstrates that the food medium has a very dramatic effect on the tissue-specific expression of the Amy’ and Amy6 structural variants. The larvae reared on the agar-1.8% dead yeast have both a-amylase activity in the larval AMG and PMG, whereas larvae of the standard cornmeal medium have only a-amylase activity in a small region of the larval PMG that borders on the larval MMG (middle midgut) in which a-amylase is never detected. Therefore, the larval PMG shows two types of activity with respect to a- amylase: PMG 10, with activity in only the smaller part of the PMG, and PMG 12, with a-amylase activity also in the remaining part. Figure 2a shows, in addition, that there is an unequal expression of Amy variants in the larval midgut, as Amy3 is not present in the larval AMG, in contrast to Amy6. Both genes are expressed in the larval PMG.

From these observations it is obvious that the tissue-specific expression of a- amylase may be strongly influenced by the food medium. Moreover, there is differential activation by food conditions of the two genes in the duplicated Amy locus.

Cis action on Amy in the AMG and PMG of larvae: The expression of a- amylase in the midguts of Amy’ and Amy4*6 third-instar larvae was maximal when they were reared on the agar-1.8% dead yeast food medium. Amy’ strains, however, oiily showed activity in the larval PMG. Most of the strains have a-amylase activity in the larval AMG and PMG. The larval MMG never showed a-amylase activity. To determine whether the tissue-specific expression of the structural Amy variants in the AMG of third-instar larvae is controlled in a cis or trans fashion, an Amy’ strain (from Kaduna) with only a- amylase activity in the larval PMG was crossed with an strain (from


- FIGURE .!%.-Cis regulation of tissue-specific a-amylase expression in the AMG of a 120-hr third-

instar hybrid Amy'/Amy'.6 larvae reared on an agar-1.8% dead yeast food medium (see Figure 2 and text).

Pacific) with Amy' expressed in the larval AMG, and Amy' and Amy6 in the larval PMG. If there is trans action, we would expect that the hybrid larvae had both Amy' and Amy' expressed in the larval AMG. However, the Amy'/

larvae had only Amy4 expressed in the larval AMG, whereas Amy', Amy' and Amy6 were expressed in the same region of the larval PMG (Figure 3). When the Amy' strain was crossed with an Amy4s6 strain (from Pacific) with both Amy' and Amy6 expression in the larval AMG and PMG, the hybrid larvae again showed no expression of Amy' in the larval AMG, whereas in the larval PMG all three Amy variants were expressed.

When the Amy' larvae were grown on a standard cornmeal medium, the a- amylase activity was limited to a small region of the anterior part of the larval PMG (map-PMG IO), whereas the larvae had equal expression of Amy' and Amy6 in a larger part of the larval PMG (map-PMG 12). The larval AMG and MMG showed no a-amylase activity. If hybrid A m ~ ' / A m y ' * ~ larvae, reared on the same food medium, are investigated with midgut electrophoresis (Figure 4), a differential expression of Amy' and in the larval midgut is found that corresponds to their parental patterns. Consequently, the expression of Amy' in the PMG is not affected by the

These experiments show that the tissue-specific expression of these Amy variants is cis-regulated in both the larval AMG and PMG.

Recombination within the structural Amy region: Recombination was studied in the region between the morphological mutants curued and welt that encloses the structural Amy loci and, according to ABRAHAM and DOANE (1978), the map locus for regulation of a-amylase production in the posterior midgut of adults (map-PMG). According to LINDSLEY and GRELL (1968) the map distance between the mutants curved and welt is 6.5 crossover units. As given in Table 1, a lower recombination frequency for this segment of the second chromosome was found. Based on a total of 13,480 adults, a recombination

chromosome.

1138 A. J. KLARENRERG ET AL.

FIGURE 4.--Cis regulatioii of tissue-specific- a-amylase expression i n the PMG of 120-hr third- instar larvae reared on standard cornmeal food medium (see Figure 2 and text). a, Larva of parental strain with Amy' expression in the anterior part of the PMG. b, Larva of parental strain with Amy',6 expression in a larger part of the PMG. c, Hybrid larva of parental strains with a cis- regulated pattern of Av~y'/Amy'.~ expression in the PMG.

TABLE 1

Recombination between the mutants curved and welt

Experiment A Experiment B

Recombinant genotypes Males Females Males Total

Curved I I7 108 217 442 Welt 84 98 171 353

Total no. of recombinants 20 1 206 388 795

Total no. of adults counted 3,343 3,522 6,615 13,480 Recombination percentage 6.0 5.9 5.9 5.9

In experiment A, both male and female adults were counted; in experiment B, only male adults were counted.

frequency of 5.9% was obtained. T h e deficit in numbers of recombinants recovered can be attributed to a lower fitness of the mutants curved and welt in comparison to the wild type (see BAHN 1967). Because it was practically unfeasible to give a reliable estimate of the viabilities of all genotypes generated in the recombination experiments, we used the map distance of 6.5 cM for the chromosomal interval between curved and welt.

In Table 2 the results of the recombination experiments, in which crossing over within the structural regions of Amy' and Amy4*6 occurred, are summa- rized. In contrast to earlier experiments with Amy' and of BAHN (1967), we were able to detect three recombinants, all + Amy4 wt sp. As the most simple explanation, it can be deduced from the combination of Amy4 with the markers welt and speck that the Amy' locus has to be considered as a single gene, located opposite the Amy' locus on the homologous chromosome. T h e Amy6 locus is distal from Amy4, forming together a gene duplication, with a distance between the components of 0.04 cM. Recent results of molecular


TABLE 2

Recombination within the structural Amy region ___ - ~ ~

Region c and Amy

+ Amy'wt 79 c Amy' + 166 + Amy'wt 3 c Amy'.6 + 80 + Amy4s6 rut 118

Total 159 284 3

Total no. of adults counted: 7559 Total no. of recombinant adults: 446

Region Amy and wt Within Amy region

Determination of the distance between the Amy' and Amy6 loci and of the position of Amy on the map of the second chromosome. Distance between Amy' and Amy6: 0.04 cM; position of Amy: 77.8.

analysis of the Amy region of Amy',' by restriction enzyme mapping (GEMMILL, LEVY and DOANE 1985; LEVY, GEMMILL and DOANE 1985) revealed that both enzyme variants are encoded by separate Amy genes. Strains with a single Amy variant, e.g., Amy', also contain duplicated Amy genes (W. W. DOANE and J. N . LEVY, personal communication). Thus, it is possible that the Amy' strain we used also contains duplicated Amy genes. It is important, however, whether only one or both Amy' genes are active. In the first instance, we have to deal with a silenced or pseudo Amy gene. Because recombination of Amy' and Amy4p6 produced recombinants with only Amy' expression, we conclude that there is no active Amy' gene in these recombinants that is associated with Amy4. Whether one or both Amy genes are active in the c Amy' wt sp stock is not known. Only a molecular analysis of the DNA region for a-amylase could give a final answer. Our estimate of the position of Amy at 2-77.8 is in agreement with the localization of Amy by BAHN (1967), who used the same markers. Mapping data of Amy with other markers deviate to a minor degree (2-78.1, KIKKAWA 1964; 2-77.3, DOANE 1969a).

Localization of map-PMG in adults: In Table 3 the results of a recombination analysis for two different adult map-PMG variants, map-PMG 00 and map-PMG 12 (Figure l a and b) are given. The c wt sp strain contained Amy', whereas the wild-type second chromosome had a duplicated Amy locus with Amy4 and Amy6. In the first instance, recombinant progeny of the second chromosome segment between the mutants curved and welt, which includes Amy (2-77.8) and, according to ABRAHAM and DOANE (1978), the map gene for regulation of a-amylase expression in the adult PMG (map-PMG; 2-SO+), was investigated with midgut electrophoresis. Later, a similar analysis was made of adults from strains isogenic for recombinant second chromosomes. Three recombinants within the Amy structural region, all + Amy4 wt sp, gave direct evidence for the position of map-PMG for adults distal of Amy, because they were associated with an adult map-PMG 00 variant (Figure 5b and c). This was confirmed by the recombinants of the interval between curved and Amy, because no recombination between Amy and map-PMG was detected in + Amy' wt and c Amy4v6 + recombinant adults. Therefore, recombinants for the 4.2- cM interval between Amy and welt were used for the localization of map-PMG


TABLE 3

Recombination analysis for map-PMG in adults

Recombinant genotypes FI progeny ISO-II stocks ~

c Amy' map-PMC 00 + c Amy' map-PMG 12 + c Amy'.6 map-PMC 12 + + Amy' map-PMC 00 wt + Amy'.6 map-PMG 12 wt + Amy'*6 map-PMC 00 wt + Amy' map-PMC 00 wt

Total no. of recombinants

128 38 80 79 76 42

3

446

72 15 56 46 37 25

2

253 Locali7ation of map-PMG in adults

Region Amy and map Region map and rut

F, ISO-II FI ISO-I1

c Amy' map-PMC 12 + 38 15 c Amy' map-PMC 00 + 128 72 + Amy'*6 map-PMC 00 wt 42 25 + Amy'.6 map-PMG 12 wt 76 37

Total no. of recombinants 80 40 204 109

Position of map-PMG: 79.0.

--- FIGURE 5.--a-Amylase midgut expression in 4- to Ciday-old adults obtained by recombination

between Amy and the map-PMG gene for adults (see Figure 1 and text). a, Adult with Amy'*6 expression in the AMG. b, Adult with Amy' expression in the AMG. c, Original heterozygous male adult with Amy'lAmy' expression in the AMG from which the Amy' strain (b) was isolated.

for adults as indicated in Table 3. This regulatory gene for tissue-specific production of a-amylase in the posterior midgut of adults was localized at 2- 79.0.

Evidence for &-acting regulatory elements for a-amylase in the AMG of larvae: Localization of map genes, active in larvae, depends on the production of a large number of stocks isogenic for second chromosomes recombinant for the interval between curved and welt. The parental c Amy' wt sp stock had a- amylase activity only in the larval PMG, in contrast to the wild-type stock with both Amy4 and Amy6 in the larval AMG and PMG (Figures 4a and 6c). A total of 152 isogenic stocks (two Amy4 recombinant lines and 150 ran- domly chosen out of the 253 isogenic lines) reared on the agar-1.8% dead yeast food medium were used for the analysis of map-AMG in third-instar larvae with midgut electrophoresis. In Table 4 the frequencies of the different re-


E -1 .. . - - -~ . . .. .

FIGURE B.--a-Amylase midgut e, pression in 1 'LO-hr third-instar larvae reared on agar-I .8% dead yeast food medium. Genotypes obtained by recombination between Amy and map-AMG for larvae, and by recombination within Amy',6 gene duplication (see Figure 2 and text). a, Larva with Amy' expression in the AMG and PMG. b, Larva with Amy' expression in the AMG and both Amy' and Amy6 expression in the PMG. c , Larva with Amy' and Amy6 expression in the AMG and PMG (parental Amy'.6 strain).

TABLE 4

Recombination analysis of regulatory genes that influence the expression of structural Amy variants in the anterior midgut of third-instar larvae

Recombinant genotypes' I s d l stocks

c Amy' map-AMG 0 + c Amy's6 map-AMG B + + Amy' map-AMG A wt

+ map-AMG B wt 33

48 28

+ Amy' map-AMG 0 wt 27 2

+ map-AMG A wt 14

Total no. of recombinants 152

Localization of a regulatory locus for Amy6 in the AMG of larvae

Region Amy' and map Region map and vf

Iso-11 Iso-I1

+ Amy'.6 map-AMG A wt 14 + Amy'" map-AMG B wl 33

Position of map-AMG for Amy6: 79.1. a map-AMG 0 shows no a-amylase expression in the AMG (see Figure 4a); map-AMG A shows

only Amy' expression in the AMG (see Figure 6a and b); map-AMG B shows Amy' and Amy6 expression in the AMG (see Figure 6c).

combinant genotypes are given. With respect to the absence or presence of a- amylase activity in the larval AMG, no recombination was detected for the whole region between the mutants curved and welt. Even in the + Amy' wt sp recombinants, no change was observed; they had Amy' expression in the larval AMG and PMG (Figure 6a). Based on 152 crossovers between curved and welt, the putative cis-acting regulatory elements for larval AMG expression of a- amylase are located at a distance of less than 0.1 cM from the Amy loci.

Localization of a map gene with differential effects on Amy in the larval A M G Further investigation of the recombinant stocks revealed that a number of these had only Amy' expression in the larval AMG, whereas the

1142 A. J. KLARENBERG E T AL.

PMG of these larvae had expression of both Amy4 and Amy6 (Figure 6b and c). However this new pattern in larvae has only been found when recombination occurred between Amy4 and welt (Table 4). This implies that, in larvae, Amy6 is affected by a regulatory gene that is located somewhere between Amy4 and welt. Because only Amy6, and not Amy4 and Amy', is controlled by this regulatory gene, only + Amy4v6 wt recombinants could be used for mapping this gene, which controls Amy6 expression in the AMG of third- instar larvae; it was localized at 2-79.1. Comparison of recombination for the map-PMG gene of adults in the same + Amy436 wt stocks showed that this regulatory gene is proximal of 2-79.1. Because of the close linkage of the adult and larval map genes, we suggest that they may belong to a single gene.

DISCUSSION

Genetic analysis of tissue-specific a-amylase expression in D. melanogaster requires investigation of individual larvae or adults. We used midgut electrophoresis to identify both regulatory and structural genotypes of tissue-specific a-amylase expression in the midgut. With this method we confirmed trans action of map-PMG on Amy genes in adults, as was earlier found by ABRAHAM and DOANE (1978). Moreover, our localization of this regulatory gene at 2- 79.0 corroborates their analysis (map-PMG, 2-80f). In contrast to adults, we found cis action for the tissue-specific expression of Amy variants in AMG and PMG of third-instar larvae. Our recombination data suggest that cis-acting regulatory elements, very closely linked to the structural Amy region (less than 0.1 cM), are responsible for genetic differences in Amy' and Amy4'6 expression of the AMG of third-instar larvae. Isolation of rarely occurring crossing over products of duplicated Amy genes gives an important clue for understanding the action and function of very closely linked cis-acting regulatory elements associated with the structural Amy regions. Our recombination experiments show that recombination within the structural Amy region could not separate these elements from their structural Amy components, indicating that these control elements are indeed very tightly linked.

Evidence was presented for a distant regulatory map locus active in larvae that modulates the expression of the Amy6 enzyme in the AMG of AmyJv6 larvae. The agreement of the position of this locus at 2-79.1 and that of the map locus for control of a-amylase expression in the PMG of adults (map-PMG) at 2-79.0 suggests that they belong to the same regulatory locus.

Differences in regulatory properties between stocks homozygous for Amy and Amy4,6, respectively, were also observed in their developmental profiles of a-amylase activity and their reaction to food components (HOORN and SCHAR- LOO 1980, 198 1). Independent regulation of enzyme production by duplicated Amy genes is found during development, or as a response to environmental factors (HICKEY and BENKEL 1982). Our data with midgut electrophoresis of

third-instar larvae confirm earlier results of DOANE (1969b) on differential expression of these duplicated Amy genes at the tissue level in response to diet. We have demonstrated similar phenomena for Amy4f6. Moreover, we have shown also that genetic variation occurs for a regulatory gene determin-

Amy REGULATORY GENES AND ELEMENTS

TABLE 5

Midgut a-amylase activities of 120-hr third-instar larvae with different Amy variants

1143

~~~~ ~~ ~ ~

Midgut pattern a-Amylase activity"

Stock AMG MMG PMG a b

+ Amy""" + + 0.09 0.0

+ wt sp 4 4, 6 6.54 97.3 + Amy4.6 wt sp 4, 6 4, 6 6.72 100.0

+ Amy' wt sp 1 1.64 24.4 + Amy4 wt sp 4 4 5.24 78.0

The midgut pattern variant is indicated for each stock. The Amy""" of HICKEY (HAJ-AHMAD and

a a, Gives a-amylase activity in micrograms of maltose.min-' per microgram of midgut protein;

ing independently the activity of the separate genes of an Amy duplication in different parts of the larval midgut. Our results with duplicate Amy genes demonstrate that one has to proceed with caution in deriving Amy genotypes from parts (e.g., the gut) of Drosophila, because each gene may be differently expressed in different tissues during development. Moreover, the expression of Amy genes is strongly affected by the diet.

To what extent putative cis-acting regulators influence the quantity of a- amylase produced is not known. Investigation of midgut extracts of third-instar larvae (Table 5 ) show a strong nonrandom association between a-amylase activity and Amy variants, indicating that closely linked cis-acting regulatory elements or structural gene differences are responsible.

Closely linked cis-acting regulatory elements have been identified in D. melanogaster for a number of gene-enzyme systems, such as alcohol dehydrogenase (THOMPSON, ASHBURNER and WOODRUFF 1977; MARONI and LAURIE-AHL- BERG 1983; GOLDBERG, POSAKONY and MANIATIS 1983) and xanthine dehydrogenase (SPRADLING and RUBIN 1983; CLARK et al. 1984). Evidence for quantitative and tissue-specific cis-acting regulatory elements has also been found for mouse a-amylase (HJORTH 1979; BLOOR, MEISLER and NIELSEN 1981; SCHIBLER et al. 1983) and maize alcohol dehydrogenase (WOODMAN and FREELING 1981).

Our results, together with DOANE'S (DOANE et al. 1983), show that both near and distant regulators participate in the tissue-specific expression of a-amylase in the larval and adult stages of D. melanogaster. The genetic variation of regulatory genes and elements, the independent regulation of parts of the Amy locus in different tissues and in different life stages, and the production of new regulatory patterns by recombination suggest a large potential for rapid evolution of regulatory systems in D. melanogaster.

HICKEY 1982) was taken as a reference.

b, denotes a-amylase activity relative to the highest value found.

We wish to thank B. L. A. DE RUITER and B. TUINSTRA for technical assistance. Further, we are much indebted to P. W. H. HEINSTRA, G. DE JONG, S. M. C. SCHOUTEN, G. E. W. THORIG, M. M. DE WAAL-MALEFIJT and J. J. ZUETENHORST for help in some experiments. We thank P. BROUWER and D. SMIT for photographs. The investigations were supported by the Foundation for


Biological Research (BION), which is subsidized by the Netherlands Organization for the Advance- ment of Pure Research (Z.W.O.).

LITERATURE CITED

ABRAHAM, I. and W. W. DOANE, 1978 Genetic localization of tissue-specific expression of amylase

Crossing over in the chromosomal region determining amylase isozymes in Dro-

Genetic determination of amylase synthesis

structural genes in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 75: 4446-4450.

BAHN, E., 1967 sophila melanogaster. Hereditas 58: 1-12.

BLOOR, J. H., M. H. MEISLER and J. T. NIELSEN, 1981 in the mouse. J. Biol. Chem. 256 373-377.

Tissue- specific and pretranslational character of variants of the rosy locus control element in Droso- phila melanogaster. Genetics 108: 953-968.

Frequencies of amylase

DOANE, W. W., 1969a Amylase variants in Drosophila melanogaster: linkage studies and charac-

Drosophila amylases and problems in cellular differentiation. pp. 73-109.

CLARK, S. H., S. DANIELS, C. A. RUSHLOW, A. J. HILLIKER and A. CHOVNICK, 1984

DE JONG, G., A. J. W. HOORN, G. E. W. THORIG and W. SCHARLOO, 1972 variants in Drosophila melanogaster. Nature 238: 453-454.

terization of enzyme extracts. J. Exp. 2001. 171: 321-342.

DOANE, W. W., 196913 In: RNA in Development, Edited by E. W. HANLEY. University of Utah Press, Salt Lake City.

DOANE, W. W., 1980 Midgut amylase activity patterns in Drosophila: nomenclature. Drosophila

DOANE, W. W., L. G. TREAT-CLEMONS, R. M. GEMMILL, J. N. LEVY, S. A. HAWLEY, A. M. BUCHBERC and K. PAIGEN, 1983 Genetic mechanism for tissue-specific control of alpha- amylase expression in Drosophila melanogaster. pp. 63-90. In: Isozymes: Current Topics in Bio- logical and Medical Research, Vol. 9, Edited by M. C. RATTAZZI, J. G. SCANDALIOS and G. S. WHITT. Alan R. Liss, New York.

Molecular cloning of the a-amylase genes from Drosophila melanogaster. I. Clone organization by use of a mouse probe. Genetics 110:

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germline. Cell 34: 59-73.

A molecular explanation of frequency-dependent selec-

Regulation of amylase activity in Drosophila melanogaster:

Genetic variation in mouse salivary amylase rate of synthesis. Biochem.

The functional signficance of amylase polymorphism in Drosophila melanogaster. I. Properties of two amylase variants. Genetica 4 9 173-180.

Functional significance of amylase polymorphism in Drosophila melanogaster. 111. Ontogeny of amylase and some a-glucosidases. Biochem. Genet. 18: 51-63.

The functional significance of amylase polymorphism in Drosophila melanogaster. VI. Duration of development and amylase activity in larvae when starch is a limiting factor. Genetica 55: 195-20 1.

An electrophoretic study on amylase in Drosophila melanogaster. Jpn. J. Genet.

Inform. Serv. 55: 36-39.

GEMMILL, R. M., J. N. LEVY and W. W. DOANE, 1985

299-3 12.

GOLDBERG, D., J. POSAKONY and T. MANIATIS, 1983

HAJ-AHMAD, Y. and D. A. HICKEY, 1982

HICKEY, D. A. and B. BENKEL, 1982

tion in Drosophila. Nature 299: 350-352.

effects of dietary carbohydrates. Biochem. Genet. 20: 1 1 17-1 129.

Genet. 17: 665-682. HJORTH, J. P., 1979

HOORN, A. J. W. and W. SCHARLOO, 1978



KIKKAWA, H., 1964 39: 401-411.


Environmental and genetic control of larval midgut pattern variation of a-amylase in Drosophila melanogaster. Genetics 104 (Suppl.): s43.

Midgut activity pattern variation in a-amylase and other carbohydrases in Drosophila melanogaster. Genetics 100 (Suppl.): s36-37.

Genetic variation affecting the expression of enzyme coding genes in Drosophila: an evolutionary perspective. pp. 33-88. In: Isozymes: Current Topics in Biological and Medical Research, Vol. 12, Edited by M. C. RATTAZZI, J. G. SCANDALIOS and G. S. WHITT. Alan R. Liss, New York.

Molecular cloning of the a-amylase genes from Drosophila melanogaster. 11. Clone organization and verification. Genetics 110: 31 3-324.

Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627.

Regulatory genes and adaptation: past, present and future. Evol. Biol.

Genetic control of Adh expression in Drosophila

Sur les enzymes amykolytiques. 111. La &amylase: dosage

Acid hydrolases as models of genetic control. Annu. Rev. Genet. 13: 417-466.

Population genetics of Drosophila amylase. 1. Genetic

KLARENBERG, A. J., P. J. M. JACOBS, C. VERMEULEN and W. SCHARLOO, 1983

KLARENBERG, A. J. and W. SCHARLOO, 1982

LAURIE-AHLBERG, C. C., 1985

LEVY, J. N., R. M. GEMMILL and W. W. DOANE, 1985

LINDSLEY, D. L. and E. H. GRELL, 1968

MACINTYRE, R. J., 1982

MARONI, G. and C. C. LAURIE-AHLBERG, 1983

NOELTING, G. and P. BERNFELD, 1948

PAIGEN, K., 1979

POWELL, J. R. and J. W. LICHTENFELS, 1979

SCANDALIOS, J. G., 1982

SCHIBLER, U,, 0. HAGENBUCHLE, P. K. WELLAUER and A. C. PITTET, 1983

15: 247-285.

melanogaster. Genetics 105: 92 1-933.

d’activit; et contr6le de I’absence d’a-amylase. Helv. Chim. Acta 31: 286-290.

control of tissue-specific expression in D. pseudoobscura. Genetics 92: 603-6 12.

Developmental genetics of maize. Annu. Rev. Genet. 16: 85-112.

T w o promotors of different strengths control transcription of the mouse alpha-amylase gene Amy’“ in the parotic gland and the liver. Cell 33: 501-508,

The effect of the chromosomal position on the expression of the Drosophila xanthine dehydrogenase gene. Cell 3 4 47-57.

Presumptive control mutation for alcohol dehydrogenase in Drosophila melanogaster. Nature 270: 363,

Variation between electrophoreti- cally identical alleles at the alcohol dehydrogenase locus in Drosophila melanogaster. Biochem. Genet. 13: 721-731.

Isozymes as probes and participants in developmental and evolutionary genetics. pp. 1-4. In: Isozymes: Current Topics in Biological and Medical Research, Vol. 10, Edited by M. C. RATTAZZI, J. G. SCANDALIOS and G. S. WHITT. Alan R. Liss, New York.

Gene regulation and evolution. In: Molecular Evolution, Edited by F. J. AYALA. Sinauer Associates, Sunderland, Massachusetts.

Identification of a genetic element that controls the organ-specific expression of Adh’ in maize. Genetics 98: 357-378.

SPRADLING, A. C. and G. M. RUBIN, 1983

THOMPSON, J. N., M. ASHBURNER and R. C. WOODRUFF, 1977

THORIG, G. E. W., A. A. SCHOONE and W. SCHARLOO, 1975

WHITT, G. S., 1983

WILSON, A. C., 1976

WOODMAN, J. C. and M. FREELING, 1981

Communicating editor: J. R. POWELL

genetic localization and action of regulatory genes … · 2003. 8. 1. · both amy4 and amy6...

Documents