Complex developmental regulation of the Drosophila affinidisjuncta alcohol dehydrogenase gene in Drosophila melanogaster

DEVELOPMENTAL

BIOLOGY

125, 64-74

(1988)

Complex Developmental Regulation Alcohol Dehydrogenase Gene MARK *Biochemistry

Depurtment and TDepartment

Program,

Received

June

D.

BRENNAN*.’

of the Drosophila afli~idisjuncta in Drosophila melanogaster AND

of Biology, University of Biology, University 29, 1987; accepted

W. J.

DICKINSON?

of Alabu~mu, Box 1927, Tusculoosa, oj. Utah, Salt Lake City, Utah 84112

in revised

form

September

Alabama

35486,

8, 1987

During development, the alcohol dehydrogenase genes of Drosophila melarmgaster and D. a.nidisjuncta are expressed in similar, yet distinct, tissue- and stage-specific patterns. Transcripts from both of thcsc genes arise from two promoters (distal and proximal) that also display tissue and stage specificity. We used P-element-mediated transformation to introduce the D. afinidisjunxtn Adh gene into the germ line of I1. mehnogastrx We show that the D. a&Gdisjuncta Adh gene is expressed at comparable overall levels in both species and that the tissue- and stage-specific expression for this gene (including promoter utilization) is similar in the donor and the host species. However, in some details, the expression of the D. ufinidisjuncta gene in D. melanogaster resembles the host pattern, and one novel tissue-specific expression phenotype is displayed by transformants. In general, our results suggest that there has been strong conservation of cis- and trans-acting regulatory factors since the divergence of the two species but that this conservation has not been perfect. 6 19x8 Academic PWSS. 1”~ INTRODUCTION

est to the structural gene (the proximal promoter) is responsible for the majority of Adh transcripts seen in larvae, while a further upstream (distal) promoter is responsible for most of the adult Adh, transcripts (Rowan et al., 1986). Moreover, there are as many as eight different transcripts, representing different RNA 3’ ends, that are detectable both in larvae and in adults (Rowan et ul., 1986; Rowan and Dickinson, 1987). Regulatory variation has been proposed to play an important role in evolution (Wilson, 1976; Whitt, 1983). Also, relatively little is known about the factors governing the expression of a single gene in multiple tissues. Thus there is both evolutionary and mechanistic interest in developing an assay system for the analysis of Adh genes from the Hawaiian Drosophila. In principle, transformation of Hawaiian Drosophila might be used to assay the developmental regulation of cloned Adh genes (Brennan et ah, 1984a). However, the long generation time (72 weeks) and poor survival of the Hawaiian flies in the laboratory make this an awkward approach. The similarity of the Hawaiian Drosophila Adh genes to the Adh gene of D. melanogaster and the ability of P-element-mediated transformation (Spradling and Rubin, 1982; Rubin and Spradling, 1982) to provide an in vivo assay for the correct expression of cloned genes from D. melanogaster (e.g., Goldberg et al., 1983) prompted us to determine if D. melanogaster could be used as a host species for functional analysis of cloned Adh genes from the Hawaiian Drosophila. This would provide an assay system for analysis of evolved differences in the cis-dominant regulation of Adh genes.

The evolved regulatory variation observed for the alcohol dehydrogenase (ADH) loci within the Hawaiian picture-winged Drosophila provides a model system for study of the differential developmental expression of eukaryotic genes. In this group of closely related flies, species-specific patterns of ADH production are observed (Dickinson, 1980a). The observed regulatory differences between species are complex. Some tissue-specific variations are essentially qualitative, whereas others are more subtle. The genetic and molecular bases for this variation are not well understood. However, experiments involving interspecific hybrids have shown that all analyzed regulatory differeces are attributable to &s-dominant regulatory information (Dickinson and Carson, 1979; Dickinson, 1980b; Rowan and Dickinson, 1986). Despite these regulatory differences, molecular characterization of Adh genes from Hawaiian Drosophila indicates that their major structural and functional features are similar to one another and to those of the Adh gene from the distantly related species, D. melanogaster (Benyajati et al., 1981; Benyajati et al., 1983; Brennan et ah, 1984b; Rowan et al., 1986; Rowan and Dickinson, 1986). Of the Adh genes from various Hawaiian Drosophila, the D. aflnidisjuncta gene has heen the most thoroughly characterized (see Fig. 1). Specifically, this gene has two promoters. The promoter clos‘To whom dressed. 0012-1606/88 Copyright All rights

correspondence

and

$3.00

0 1988 hy Academic Press, Inc. of reproduction in any form reserved.

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64

BRENNAN

AND DICKINSON

distal 3

-

2I.M bp

FIG. 1. Transcriptional organization of the D. afinidisjuncta Adh, gene (see Rowan et al., 1986; Rowan and Dickinson, 1987). Transcription is from left to right as drawn. Boxes above and below the line represent the exons corresponding to transcription from the distal and proximal promoters, respectively. The right-most exon shown is that which is found for longest Adh RNA. The vertical lines indicate the transcription initiation points for the two promoters. The arrow shows the position and size of the end-labeled primer used for primer extension analysis (see Results).

Accordingly, we introduced the Adh gene from D. uf jinidisjuncta into the germ line of D. melanogaster and compared the resulting pattern of expression to that observed for the endogenous Adh genes in the host and the donor species. Analysis of tissue-specific gene expression in larval and adult transformants indicates that in most respects, including an unusual case of tissue-specific promoter utilization, the qualitative pattern of expression follows that of D. afinidisjuncta. These observations indicate that D. melanogaster is indeed a useful host for the analysis of evolved regulatory variation. During the course of this study, we noted that, in some details, Adh gene expression in transformants resembles the D. melanogaster pattern and that one tissue (the larval salivary gland) in transformants exhibits expression that is not characteristic of either the donor or the host. We address the possible implications of these unpredicted results. MATERIALS

AND

METHODS

Drosophila strains and genetic analysis. The D. afinidisjuncta stock S36Gl (Dickinson, 1980a), from which the Adh gene used in this study was isolated (Brennan et al., 1984b), was used as the source for all D. afinidisjuncta RNA, protein, and tissue preparations. The D. afinidisjuncta stock was maintained according to the method of Wheeler and Clayton (1965), and all D. melunogaster stocks were maintained on Formula 4-24 instant Drosophila medium (Carolina Biological Supply Co.) at 25°C. For both D. a&nidisjuncta and D. melanogaster, all larval samples were from feeding third instar larvae and adult samples were from flies aged 4 to 7 days after eclosion. All analyses on transformants employed lines homozygous for the introduced transposons. Because Adh null embryos survive microinjection relatively poorly, the ry506 strain of D. melanogaster was used for microinjection. Integrated genes were subse-

Regulation

of Drosop/~,hiln

Adh

65

quently introduced into the background, Adhfn6cn;ry5’“, by employing an Adhfn6cn;ry506 strain (constructed and kindly provided by J. Posakony). The Adhf”’ allele produces a low level of incompletely processed Adh RNA but no detectable ADH protein (Benyajati et al., 1982). Individual, transformant females were crossed to Adhf”6cn;ry506 males and the ry+Fl progeny (males and females separately) were test-crossed with Adhfn6cn;ry 506flies. For integrations on the first and third chromosomes, cn;ry+ progeny were obtained from crosses in which the ry+ parents were males. For integrations on the second chromosome, cn;ry’ progeny necessarily were obtained from crosses in which the female parents were ry+. The cn;ry+ progeny were mated individually to Adhf”6cn;ry506 flies. After this cross resulted in offspring, the cn;ry+ parent was sacrificed, and the presence of D. afinidisjuncta, ADH and the absence of functional ADH from the host were confirmed by gel electrophoresis (below). All subsequent genetic manipulations were carried out by using these stocks as starting material. This scheme for the introduction of genes into an Adhfn6 background was limited because it required that the integrated gene was not closely linked to the host’s Adh gene and that the integration did not map between cn and Adh on the second chromosome. However, these requirements were met by all of the integrations that we obtained. The results of the above crosses indicated whether the integration was X-linked or autosomal and, if autosomal, whether it was located on the second chromosome (as evidenced by linkage to en+). Unambiguous assignment of integrations to the third chromosome was obtained by employing the balanced lethal stock CyO/Pm;In(3LR)CxD/Sb (Stock jlS8 from the MidAmerica Drosophila Stock Center, Bowling Green, OH). For third chromosome integrations, Cy/Pm flies but not D/Sb flies carrying the introduced Adh gene (as evidenced by gel electrophoresis) could be obtained. The balanced lethal stock was used to prepare lines homozygous for integrations on the second and third chromosomes. Male progeny of a cross of single cn;ry+ females to jl88 males were back-crossed individually to jl88 females. Males from those lines in which siblings expressed the D. afinidisjuncta Adh gene (determined electrophoretically) were mated to CyO;In(3LR)CxD females from the same line. Homozygous lines were started from pair matings of offspring not displaying the dominant markers, and homozygosity was verified by test crosses of their progeny. The X-linked integrations were made homozygous by employing a stock carrying the Base chromosome (Spencer and Stern, 1948) in an Adhfn6cn;ry506 background. Individual cn;ryc males were crossed to

66

DEVELOPMENTAL BIOLOGY

Basc;Adhfn6cn;ry 506females and ry+ female progeny were back-crossed to cn;ry+ males from the original stock. Homozygous lines were started from pair matings of non-bar-eyed sibling offspring from the previous cross, and their progeny were test-crossed to verify homozygosity. Con&m&ion and introduction of P-element transposons. A 5.4-kb genomic DNA fragment carrying the Adh gene of D. afinidisjuncta was obtained by partially digesting DNA from Xs36Gl (Brennan et al., 1984b) with SalI. In addition to cleaving 1.4 kb downstream of the ultimate polyadenylation site (the desired cleavage) Sal1 cuts within 20 base pairs of the distal RNA initiation point and approximately 2 kb further upstream. The partially digested DNA was treated with Sl nuclease, and the DNA ends were repaired by the large fragment of Escherichia coli DNA polymerase I in the presence of all four deoxyribonucleotide triphosphates as described (Maniatis et al., 1982). XhoI linkers were ligated to the DNA, and the DNA was digested with BglII and XhoI. BglII cleaves 2.4 kb upstream of the distal Adh promoter. The resulting BglII to XhoI fragment was purified by agarose gel electrophoresis and inserted into the corresponding sites on pBRXB2. The latter is a derivative of pBR322 that has a XhoI linker inserted into the EcoRI site and a BolII linker inserted into the PvuII site (M. Brennan, unpublished data). The resulting construction, llBXB2, was digested with BglII. The DNA ends were repaired as above, and an XhoI linker was inserted to produce llBXX5. Following digestion with X’hoI, the 5.4-kb fragment carrying the Adh gene was isolated by agarose gel electrophoresis and ligated to Carnegie 20 (Rubin and Spradling, 1983) that had been cleaved at the unique Sal1 site and treated with calf intestine alkaline phosphatase (Boehringer Mannheim). The desired products, pC2OAAl and pC20AA2, were identified by mapping restriction endonuclease cleavage sites. Plasmids used for microinjection were purified by centrifugation in CsCl gradients in the presence of ethidium bromide. Embryos were injected with a mixture consisting of either pC2OAAl or pC20AA2, at 375 pg/ml, and the helper plasmid pr25.7~~ (Karess and Rubin, 1984), at 100 pg/ml, as described (Spradling and Rubin, 1982; Rubin and Spradling, 1982). The ry+ progeny of injected individuals were mated to flies from the ry506 stock prior to further genetic analysis (above). DNA axd RNA preparation a,nd analysis. Total and poly(A)-containing RNAs from whole larvae and adults and total nucleic acids from adult midguts were prepared as previously described (Brennan et al., 198413; Rowan and Dickinson, 1986). Preparation of genomic DNAs from adult flies was also as described previously (Brennan et al., 1984a).

VOLUME 125.1988

Radiolabeled probes for whole genome Southern analysis (Southern, 1975) and for RNA analysis were prepared by nick translation (Rigby et al., 1977) in the presence of cu-[32P]dCTP(New England Nuclear, >3000 Ci/mM). Specific activities ranged from 5 X lo* to 5 X 10’ cpm/pg DNA. Gel electrophoresis of RNA and genomic DNA, transfer to nitrocellulose and hybridization and washing of nitrocellulose filters were as previously described (Thomas, 1980; Brennan et al., 198413). Where indicated in the text, autoradiograms (or Auorograms for primer extension analysis) were scanned and quantitated with a recording densitometer (E-C Apparatus Corp.). The 45-bp H&c11 to BstEII fragment from the cloned D. afirGdisjuncta Adh gene (see Rowan and Dickinson, 1987, for the D. ufinidisjuncta gene sequence) was used as the primer for primer extension analysis. To prepare this primer, the plasmid pUC8-9 (Rowan et al., 1986) was digested with BstEII and treated with calf intestine alkaline phosphatase followed by incubation with bacteriophage T4 polynucleotide kinase in the presence of y-[32P]ATP (New England Nuclear, >3000 Ci/mlll) as described by Maxam and Gilbert (1980). The specific activities were approximately lo7 cpm/pg DNA. Following digestion with HincII, the desired fragment was isolated by polyacrylamide gel electrophoresis as described (Maxam and Gilbert, 1980). The primer (5 X lo4 cpm per reaction) was annealed to RNA in 80% formamide at 47 to 48°C for 12 to 15 hr in the presence of 10 pg yeast carrier RNA as described (Favoloro et al., 1980). DNA-RNA duplexes were recovered, and the primer was extended by reverse transcriptase as described by Rowan et al. (1986). Reactions were terminated by ethanol precipitation and the resulting products were analyzed on 5% denaturing polyacrylamide gels (Maxam and Gilbert, 1980). Dried gels were subjected to fluorography using intensifying screens at -80°C. ADH activity measuremen& gel electrophoresis, and histochemical stai?zing. In all cases, tissues were handdissected in ice-cold PBR (129 mM NaCl, 4 mM KCl, 2 mM CaCle, 20 mM sodium phosphate, pH 6.5). For preparation of extracts used for activity measurements or gel electrophoresis, whole flies or dissected tissues were homogenized in ice-cold water and insoluble material was removed by centrifugation (12,000 rpm for 1 min at 4°C in an Eppendorf microcentrifuge). Protein determination was by the micro-Biuret method, using bovine serum albumin as a standard (Legget-Bailey, 1962). ADH activity was determined as described by Sofer and Ursprung (1968) using 2-propanol as a substrate. Gel electrophoresis on 1% agar noble gels and ADH activity staining of gels was as described previously (Ursprung and Leone, 1965; Dickinson and Carson, 1979).

67 Histochemical localization of ADH was performed on dissected tissues fixed in a freshly prepared solution of glutaraldehyde in PBR. Fixation was either for 1 hr in 2%’ glutaraldehyde (standard fixation) or for 5 min in 0.5% glutaraldehyde (minimal fixation), as noted in the legend to Fig. 5. The former procedure yielded better structural preservation but decreased sensitivity. Fixed tissues were washed extensively (at least 1 hr with four to six buffer changes) in PBR and stained at 37°C in the dark for lengths of time as noted in the legend to Fig. 5. The staining solution contained 0.4 mg/ml NAD, 0.5 mg/ml nitro blue tetrazolium, 0.04 mg/ml phenazine methosulfate, and 2.8% (v/v) 2-propanol in PBR adjusted to pH 7.5. Stained tissues were washed in PBR (pH 6.5) and mounted in a water-soluble mounting medium (CMCP-10, Masters Chemical Co.). IT/, situ hybridixution. The plasmid llBXB2 (above) was used to prepare a biotinylated probe for i?r situ hybridization. Synthesis of the biotinylated probe, hybridization to polytene chromosome, and detection with a streptavidine-alkaline phosphatase conjugate were carried out as previously described (Engels et al., 3986). RESIJLTS

The Adh Gene of D. afinidisjunctu Is Expressed at Sirnilur Levels in the Donor und Host The P-element transposon carried on the plasmid C20AAl is diagrammed in Fig. 2. In this construction, and in the related pC20AA2 (see legend), the Adh structural gene has been inserted into the unique S&I site of the vector Carnegie 20 (Rubin and Spradling, 1983). This vector, which carries the structural gene for xanthine dehydrogenase (XDH) from D. wwlano~guster, allows efficient recovery and analysis of transformants due to the dominance of the wild-type Xdh (ry’) gene in a homozygous ry background. The inserted D. ufi71idisjuncta DNA contains, in addition to the entire structural gene for ADH, approximately 2.4 kb of sequence upstream of the distal promoter and 1.4 kb downstream of the ultimate polyadenylation site (Fig. 2). D. melanoyaster transformants were recovered by virtue of the wild-type eye color conferred by the Xdh gene. Of the many transformed lines displaying a pattern of inheritance suggesting single transposon integrations, six were chosen for further analyses. In total, three integrations (two autosomal and one sex-linked) of each transposon were analyzed. Additional experiments were carried out in order to ensure that the lines chosen for study carried single, intact transposons. Genomic DNAs were prepared and probed with a cloned fragment of DNA that hybridizes to one of the flanking P-element sequences in the transposons (see Fig. 2). Following digestion of genomic DNA

Adh 0

Xdh P

-t ‘Ikb’ FIG. 2. The P element transposon of pC2OAAl. Only the longer of the two flanking P element sequences (light box) is shown. The lincl and the dark box represent the AtUt and Xdh sequences, respectively. The direction of transcription for the Xdh gene is indicated by the horizontal rrow (Cot6 et ul., 1986). The letters (D) and (P) designate the distal and proximal promoters of the Adh gene, respectively. The vertical arrow shows the location of the ultimate polyadenylation site for the Adh gene. The transposon of pC20AA2 differs in that the 4dh sequences are inverted relative to the remainder of the transposon. The bar below the transgoson represents the 0.X4-kh HircdIII frag ment from the P element of ~~25.1 (Bingham et ul., 1982). This fragment was used as a probe for the genomic Southern analysis shown in Fig. 3. P element sequences to the right of the vertical line on the hat are not present in Carnegie 20.

with EcoRI, this probe recognizes a genomic fragment having a minimum size of 0.59 kb. The actual size of the recognized fragment will depend upon the particular genomic context of the transposon. As can be seen in Fig. 3, genomic DNAs from each of the lines contain a DNA fragment of only one size when analyzed in this manner. This observation is consistent with the interpretation that each line has only a single integration of the transposon in question. Similarly designed experiments with other restriction endonucleases and this, as well as other probe fragments, also failed to detect multiple integrations in these lines. Further, whole genome Southern analysis provided no evidence that any of the transposons had been rearranged (data not presented). As a final confirmation, i)r sitz~ hybridization to polytene chromosomes reveals only a single hybridization site in each line (Table 1). Thus by all available criteria, the lines carry single integrations. In D. rnelunoguster, ADH production is characterized by a relatively high larval level and a somewhat higher adult level (Ursprung et (il., 1970) (Table 1). In contrast, in D. c~~?lidisj~l?lctcx, ADH activity (and Ad/r RNA abundance) is relatively low in both stages, but is about twofold to threefold higher in larvae than in adults (Brennan et nl.. 1984b) (Table 1). Interestingly, Table 1 clearly shows that, for transformants, ADH specific activity is consistently higher in larvae than in adults. However, with the possible exception of line l-6, the difference between larvae and adults is more pronounced in transformants than it is in I). (~.fi~idjsju mfu. Table 1 also addresses the question of dosage compensation of the introduced Adh gene. Within each line, the male and female larval values are quite similar,

68

DEVELOPMENTAL 1

23

456

BIOLOGY

78

FIG. 3. Genomic Southern analysis of transformants. DNAs were digested with EcoRI and electrophoresed on a 1% agarose gel. Following transfer to nitrocellulose, the DNAs were probed with the 0.84.kb Hind111 fragment of ~~25.1 (see Fig. 2). Lanes (1) through (4) and (6) through (8) contain genomic DNAs as follows: (1) line l-6; (2) line l-29; (3) line 1-31; (4) Adhfn6,cn;ry 5o6 host; (5) line 2-3; (6) line 2-35; (7) line 2-41. Lane (5) contains size markers consisting of the plasmids AXS2 and AXS4 (described in Brennan et al., 1984a) digested with various enzymes.

regardless autosomal.

of whether the integration is X-linked or The results for adults are less clear. For D. melanogaster and for all lines carrying autosomal integrations, the ADH specific activity per gene in males

RELATIVE

Strain” D. nfinidisjuncta AdhF;rym l-6 l-29 l-31 2-3 2-35 2-41

Transposon integrationb

3 2 1 2 3 1

(65B-D) (46BC) (11-12) (25CD) (64CD) (17C)

Larval female 2.6 11.5 2.0 2.3 4.0 2.7 3.2 4.7

(k.07) (+-1) (2.1) (2.3) (k.1) (k.2) (+.l) 0.4)

ADH

TABLE SPECIFIC

Larval male 2.2 10 2.4 2.6 4.1 3.0 3.3 5.5

(5.5) (f.5) (k.4) (k.2) (k.2) (k.3) (1.2) 0.3)

VOLUME

125, 1988

divided by the same value for females ranges from 1.25 to 1.75. The same calculation for the X-linked integragene gives a value of 1.8 tions of the D. aflnidisjuncta for line 1-31 and 2.4 for line 2-41. Although these values are smaller than the 2.5 to 3.5 expected for full dosage compensation, they are slightly higher than those obtained for the autosomal integrations. Analysis of total poly(A)-containing RNA from the transformed lines gives results that agree well with the activity measurements (Fig. 4). Similar results are obtained with total cellular RNA (not shown). Not surprisingly, the samples which have a relatively high abundance of Adh RNA (e.g., 1-31 and 1-41 larvae) also have high specific activities. Consistently, adult RNA abundance is lower in all cases than that in the corresponding larval sample. Figure 4 documents well that transcripts of various lengths are produced by the introduced Adh gene. Note that the Adh transcript from migrates as a relatively wild-type D. melanogaster sharp band, in agreement with the detection of only one 3’ end observed for Adh RNA in this species (Benyajati et ah, 1981; Benyajati et al., 1983). As is true for the the transcripts of the Adh gene in D. afinidisjuncta, lengths of the detectable transcripts produced by the D. gene in transformants range from apujinidisjuncta proximately 1050 to 1300 nt. The transcripts from transformants fall into two major, perhaps discrete, size classes of approximately 1250 and 1050 nt (arrows, Fig. 4). We have not yet assigned the transcripts in transformants to particular polyadenylation sites, nor have we determined if all of the 3’ ends observed for D. afinidisjuncta Adh RNAs are detectable in transformants. 1 ACTIVITY

PER GENE

Adult female 1.0 15 0.7 0.4 0.5 0.4 0.6 0.25

(k.2) (k1.5) (k.05) (k.03) (Jr.04) (2.05) (f.1) (k.04)

Adult male 1.3 26 1.2 0.5 0.9 0.7 0.9 0.6

(k.1) (&l) (k.2) (k.04) (k.03) (f.03) (f.2) (1.03)

Larval/adult female

Larval/adult male

2.6 0.77 2.9 5.7 8.0 6.8 5.3 18.8

1.7 0.38 2.0 5.2 4.6 4.3 3.7 9.2

Note. Extracts were prepared from larval and adult males and females and ADH specific activity (units per milligram of protein per gene) was determind (see Materials and Methods) and normalized to the value obtained for D. afinidisjuncta adult females. Each entry represents the mean specific activity obtained for at least two independent extracts. Protein and activity measurements were done twice on each extract. The numbers in parentheses represent the ranges of values obtained. “Transformed strains carrying the transposons of pC2OAAl (transcription toward theXdh gene) and of pCZOAA2 (transcription away from the X&z gene) are indicated by the prefix numerals 1 and 2, respectively. *The chromosome carrying the integrated transposon is given followed, in parentheses, by the cytological assignment based on in situ hybridization.

BRENNAN I-29 2-3 1-31 --ALALALFHALALALAL

1-6

c-m-

2-35

2-41

AND DICKINSON aff.

FIG. 4. Analysis of poly(A)-containing RNAs from transformants. RNAs were electrophoresed on a 1.2% agarose gel containing formaldehyde. Following transfer to nitrocellulose, the RNAs were probed with radiolabeled p328El. This plasmid carries the entire Adh structural gene (Brennan et al., 198413). For all transformed lines and for D. afinidisjuncta (aff.), 100 ng of RNA was loaded. The letters (A) and (L) correspond to adult and larval RNAs, respectively. For D. melaw ogaster AdhF (F) and for the Adh’“’ host (H), 1 Kg of adult RNA was loaded. The arrows indicate the two major size classes of Adh transcripts seen in transformants. Based on the relative migration of single-stranded DNA standards (not shown), these size classes are approximately 1250 and 1050 nt.

Tissue-Specific

Production

of ADH

The tissue distribution of ADH is generally similar in D. melanogaster and D. afinidisjuncta, but there are reproducible differences in detail. Transformants preserve the major shared features but, interestingly, display some details characteristic of each species and one novel phenotype. Thus in both species and in transformants, fat body is the major source of ADH, with lesser amounts localized in regions of the gut, in Malpighian tubules, and in the larval hypodermis. The larval midgut stains strongly for ADH activity in both species and in transformants. In D. melanogaster, the gastric caeca have markedly lower activity than the adjacent main axis of the anterior midgut, and there is a sharp line of demarcation between the two regions. In contrast, D. afinidisjuncta, has a higher activity in the gastric caeca. Generally, transformants resemble D. melanogaster with respect to this feature (Fig. 5, top row). There is some individual variability observed for transformants-about 10% of the sampled larvae from each line show staining of one or more of the gastric caeca that is as strong as or stronger than that seen for the body of the anterior midgut. In adults, there is another quantitative difference between D. afinidisjuncta and transformants. Transforin having relatively mants resemble D. mezanogaster more histochemical staining in the adult hindgut and rectum where D. aflnidisjuncta normally has little activity (not shown).

Regulation

of Drosophila

Adh

69

For other tissues, transformants display a staining pattern that closely resembles the D. afinidisjuncta pattern. D. afinidisjuncta stains strongly for ADH throughout most of the anterior half of the adult midgut, beginning just posterior to the cardia, while n. melanogaster has a prominently staining band on the cardia but only scattered activity elsewhere in the midgut (mostly in the posterior half). Transformants have weaker midgut activity than D. afinidisjuncta but with similar distribution (Fig. 5, middle row). Transformants also resemble D. afinidisjuncta in having easily detected activity in the larval hypodermis and no detectable activity in the adult male genital apparatus (not shown). The only example of a novel tissue-specific feature in transformants is the detectable, but weak, expression in the larval salivary glands of most individuals. This is seen in all transformed lines examined, although often light or variable from cell to cell. In this tissue, neither endogenous gene produces histochemically detectable ADH (Fig. 5, bottom row). The production of D. afixidisjuncta ADH by various tissues in transformants was analyzed also by ADH activity gel electrophoresis. To do this, transformed males were crossed to Oregon R females, and ADH production in the dissected tissues of Fl individuals was analyzed by electrophoresis. Representative results are shown in Fig. 6. This analysis confirms production of D. afinidisjuncta ADH by the adult midgut, Malpighian tubules, crop and hindgut, and by the larval salivary gland. Of the various adult tissues, the midgut appears to be exceptional in having as much or more D. afinidisjuncta ADH as ADH from the host. This is particularly striking given that overall ADH activity is 20 to 30 times higher for the D. melanogaster gene (see Table 1). Densitometric scans of activity gels on adult midgut samples from the six lines shows that the ratio of D. afinidisjuncta activity to D. melanogaster activity in this tissue varies from 0.6 (in line 2-41) to 1.9 (in line l-6). The apparent absence of the heterodimeric enzyme in the adult midgut (Fig. 6A, lane 2) is consistent with the histochemical observation that D. afinidisjuncta ADH and D. melanogaster ADH are produced largely by different cell populations in the adult midgut. Table 2 summarizes the main features of ADH distribution in both species and in transformants. For all of these features, there is appreciable individual variability in the exact patterns and the relative staining intensities but no case of tissue-specific differences between transformed lines. Promoter Utilization in Transformants As described above, the Adh genes of D. melanogaster and D. a&nidis.juncta are similarly organized. In both

FIG. 5. Histochemical localization of ADH in selected larval and adult tissues. In each horizontal row, the samples from left to right are G ~~~~~~~s~~~c~u, L). ?~e~~r~~~~~ wild type (Oregon R), null control (~~~fn6,~~;r~~), and a transformant. Top row: larva1 proventriculous (P) gastric caeca (G), and anterior midgut (standard fixation and 30min staining). Middle row: adult crop (Cr), cardia (C), and anterior midgu (minimal fixation and 2O-min staining (wild types) or 60-min staining (null control and transformant)). Bottom row: larval salivary gland (S and fat body (F) (minimal fixation and 30-min staining). 70

BRENNAN A

1

2

AND

Regulation

DICKINSON

4

1

2

r

bet.-

ub

aff;

*

*

I) -

I,

FIG. 6. ADH activity gel analysis of adult and larval tissue extracts. Transformants homozygous for the introduced Adh gene were outcrossed to Oregon R flies (which are homozygous for the D. melancgaster AdhF allele), and the Fl progeny were analyzed. Extracts were prepared from hand-dissected tissues, electrophoresed on 1% agar noble gels, and stained for ADH activity. The various ADH isozymes are labeled as follows: F, D. melanogaster ADHF; het., interspecific heterodimer; aff., D. aflnidisjuncta ADH. Tissues were pooled from the same group of larval or adult individuals, but they were homogenized in various volumes so that the amount of D. afinidisjunctu ADH activity loaded would be approximately equal for each lane. (A) Adult tissue extracts from line 1-29 as follows: (1) fat body from one individual; (2) midguts (cardias and crops removed) from 15 individuals; (3) Malpighian tubules from 30 individuals; (4) crops from 15 individuals. (B) Larval tissue extracts from line 2-3 as follows: (I), larvae lacking only salivary glands (mostly fat body activity) from one individual; (2) salivary glands from 50 individuals.

species, transcripts derived from the proximal promoter predominate in larvae, while those from the distal promoter predominate in adults. Given these observations, it is of considerable interest to determine if this developmental switch is displayed by the D. afinidisjuncta gene when introduced into D. melanogaster. In order to address this, a 45-nt, end-labeled primer (arrow, Fig. 1) was hybridized to larval and adult RNAs from each line, and the primer was extended using reverse transcriptase. The expected lengths of the major TABLE ADH

PRODUCTION

71

Adh

products resulting from “proximal” and “distal” RNA templates are 223 and 249 nt, respectively (Rowan et al., 1986). As can be seen in Fig. 7, the proximal transcript is the predominant form in larval transformants, while the distal transcript is the major form in adults. In D. afinidisjuncta and in all transformed lines, there are actually two detectable, but poorly resolved, extension products corresponding to both the proximal (sizes 223 -t 1 and 219 * 1 nt) and the distal (sizes 249 + 1 and 244 it_ 1 nt) promoters. The larger product of each pair is the more abundant whether D. afinidisjuwta RNAs or RNAs from any of the transformed lines are used as templates. Multiple, closely spaced transcription initiation points have been described previously for the ADH gene of D. afinidisjuncta (Rowan and Dickinson, 1987). Within the limits of resolution of the gel system, the transcripts resulting from the D. afinidisjuncta ADH gene in the host and in the donor species are identical in this regard. The relative amounts of distal and proximal transcripts in adult D. afinidisjuncta and in adult transformants are slightly different. Densitometric scans of various exposures of the film indicate that the proximal transcript in D. afinidisjuncta adults represents 670 of the total, but in transformants, the proximal transcript represents 11 to 14% of the total. In contrast, the level of distal transcript in larval samples is quite similar in D. afinidisjuncta and in five of the six transformed lines (less than 1% of the total). Line 2-41 is exceptional, because approximately 3% of the larval Adh RNA in this line is from the distal promoter. The tissue specificity of promoter utilization has been described for D. afinidisjuncta and for three other Hawaiian Drosophila species (Rowan and Dickinson, 1986). Promoter utilization in the adult midgut is of particular interest. ADH production by this tissue is unusual in three regards. First, significant ADH production by the adult midgut is a relatively rare phenotype among Drosophila species (Dickinson, 1980a; and unpublished observations). D. afinidisjuncta is one of only a few spe-

0

3

of Drosophilu

2

BY LARVAL

AND

ADULT

TISSIJES

Larval

D. afinidisjuncta D. melanogaster Transformant

Adult

FB

MT

MG

SG

H

FB

MT

MG

HG

c

GA

++ ++ ++

+ + +

+ + +

-

+ + +

++ ++ ++-

+ t f

t + *

-?I t f

+ + +

+ .-

+

-

Note. The distribution of ADH activity was determined by histochemical staining and gel electrophoresis (see text). Abbreviations used: FB, fat body; MT, Malpighian tubules; MG, midgut; SG, salivary gland; H, hypodermis; HG, hindgut; C, crop; GA male genital apparatus. ADH production is rated as (++) for strong, (+) for readily detectable, (rt) for weak or variable, and (m-j for undetectable. Tissues that do not produce ADH in either species or in transformants (e.g., imaginal disks, nervous system, and larval hindgut) have been omitted.

72

DEVELOPMENTALBIOLOGY V0~~~~125,1988

In order to determine the type of transcript in the adult midgut of transformants, RNA was prepared from dissected midguts (with the cardias and crops removed), and the RNA was used as a template for primer extension analysis. Figure 8 clearly shows that the proximal promoter is responsible for most of the Adh transcript from the adult midgut in transformants. Distal transcripts are detected at a level of about 2% of the total in this tissue (Fig. 8B). DISCUSSION

lib L

II, ID FIG. 7. Primer

We have shown that, in most respects, the expression Adh gene is similar or identical of the D. afinidisjuncta and in D. melanogaster. The RNA in D. afinidisjuncta and protein products of this gene are comparable, quantitatively and qualitatively, in the donor and in the host. The developmental switch in transcript type displayed by the endogenous genes in the donor and the host species is displayed also in transformants. By themselves, these observations suggest that transformation of D. melanogaster is a viable approach for further analysis of the evolved regulatory variation described for the Adh genes in the Hawaiian Drosophila. A

extension analysis of adult and larval RNAs. In each case, 100 ng of poly(A)-containing RNA was annealed with a 45-nt primer labeled at its 5’ end with 32P (see Fig. 1 and Materials and Methods). The primer was then extended by reverse transcriptase and the resulting products were resolved on a 5% acrylamide gel containing urea, as described (Maxam and Gilbert, 1980). The lane labeled (M) contains size markers consisting of pBR322 digested with HpaII and end labeled with “P. Marker fragments smaller than 76 nt and unextended primer were electrophoresed off of the gel. The extension products resulting from the various RNAs are labeled according to the D. melunoguster strains from which the RNAs were isolated. The abbreviation (aff.) refers to D. c&nidisjunctu. The bands designated P (223 + 1 nt) and D (249 f 1 nt) correspond to the major products expected for proximal and distal RNA templates, respectively. The smaller labeled fragments seen for the D. u$inidisjuncta larval sample are incomplete extension products that are artifacts of the procedure (Rowan et ah, 1986; Rowan and Dickinson, 1986). These and other incomplete extension products (see also Fig. 8) are not observed reproducibly, but do depend upon the presence of D. ufinidisjunctu Adh RNA. Larger quantities of (nonspecific) RNA and less active preparations of reverse transcriptase enhance the appearance of these products (unpublished observations).

ties that has a substantial level of ADH in the adult is midgut. Second, the adult midgut of D. afinidisjuncta an exceptional adult tissue in that the Adh transcript is made from the proximal promoter (Rowan and Dickinson, 1986). Last, histochemical and electrophoretic analyses on transformed lines (above) suggest that the D. afinidisjuncta Adh gene is active in a cell population in which the endogenous D. melanogaster gene is relatively inactive.

FIG. 8. Primer extension analysis of adult midgut RNA. The primer, hybridization to RNA and gel electrophoresis were the same as in Fig. 7. The designations (P) and (D) are as described in figure 7. In lanes (1) and (2), the extension products are from 100 ng of poly(A)-containing RNA as follows: lane (1) D. a&nidisjunctu adult; lane (2) D. ufinidisjunctu larval. In lanes (3) and (4) extension products are from the total nucleic acids of 90 adult midguts as follows: lane (3) line l-6; lane (4) line 2-35. (A) A 4-hr exposure; (B) an 80-hr exposure of the same gel.

BRENNAN AND DICKINSON

Stronger support for this comes from our general observation that donor and host tissues that are morphologically and embryologically related are also biochemically related. For example, although the tissue-specific adult expression phenotypes are quite similar for the donor and host, ADH production by the anterior portion of the adult midgut in D. afinidisjuncta is a notable exception. ADH production by the adult midgut of transformants is low but, relatively, is high when compared to ADH production by the resident D. melanogaster gene or to ADH production by the D. afinidisjuncta gene in other tissues of transformants. Moreover, the fact that proximal RNA predominates in the adult midguts of both D. afinidisjuncta (Rowan and Dickinson, 1986) and transformants argues for conservation of trans-acting factors in this tissue. The implication is that even for this unusual case, the relevant trans-acting components that are peculiar to these cells have been maintained since the divergence of the two species approximately 40 million years ago (Throckmorton, 1975; Rowan and Dickinson, 1987). Although relative quantitative differences between D. afinidisjuncta and transformants are found for several tissues, general conservation of the trans-acting components seems to apply for the majority of tissues analyzed. By inference, then, the major differences in ADH production between the two species are due to changes in cis-acting regulatory sequences. A similar conclusion was reached by Fischer and Maniatis (1986) following introduction of the duplicate Adh genes from D. mulleri into D. melanogaster. D. mulleri, like D. afjinidisjuncta, is a member of the subgenus Drosophila and is a distant relative of D. melanogaster (Throckmorton, 1975). Indeed, comparison of our results to those of Fischer and Maniatis indicate clearly that donor-specific differences in expression are observed upon introduction of foreign Adh genes into D. melanogaster.

Study of variation in the tissue-specificity of ADH production for the Hawaiian Drosophila has indicated that several tissues show large species-specific differences (Dickinson, 1980a). These tissues are the larval and adult midguts and Malpighian tubules, the larval hypodermis, and the adult hindgut. Preliminary characterization of D. melanogaster transformants carrying the Adh genes of D. grimshawi and D. hawaiiensis indicates that, without exception, ADH is produced in these tissues only by the genes that do so in the donor species (M. Brennan, unpublished observations). Despite remarkable conservation of tissue-specific regulatory factors, the expression of the D. afinidisjuncta Adh gene used in the present study does not follow perfectly the D. afinidisjuncta pattern. The notable case is the larval salivary gland. Possibly, the

Regulation of Drosophila

73

Adh

tissue distribution of trans-acting regulatory components and/or their interaction with &-acting sequences has not been perfectly conserved since the divergence of the donor and host species. If this is the case, the relevant trans-acting factors might evolve independently from one tissue to another, as has been argued for their &s-acting counterparts in the case of Hawaiian Drosophila (Dickinson, 1980a). Alternatively, it is possible that the particular constructions that we used lack an element or elements that normally function to suppress Adh gene expression in the salivary gland. In order to distinguish between these two possibilities, it will be necessary to introduce the same constructions into D. afinidisjuncta or a closely related species. Nucleotide sequence comparisons between the Adh genes of D. melanogaster and D. aflnidisjuncta have revealed only slight sequence conservation in the noncoding regions of the two genes (Rowan and Dickinson, 1987). Given the results of the present study, the functionally important cis-acting regulatory sequences must be small and/or poorly conserved at the nucleotide level. Perhaps this is not surprising since small, but functionally important, regulatory sequences have been described for other eukaryotic genes (e.g., Stuart et al., 1984; Simon et al., 1985). Localization of functionally conserved regulatory sequences is greatly facilitated by preexisting variation in these sequences and an assay for this variation. The Adh genes from the Hawaiian picture-winged Drosophila represent a rich source of regulatory variation. In addition to tissue-specific variation in levels of enzyme production, differences in the tissue- and stage-specific distribution of transcript types (proximal and distal) have been described (Rowan and Dickinson, 1986). Mechanistic understanding of these phenomena requires that they be displayed by the different genes in a homogeneous background. As a host species, D. melanogaster provides such a background and offers the additional advantages of rapid generation time and facile genetic manipulation. Experiments designed to map the regulatory sequences involved in the tissue-specific expression of the D. afinidisjuncta Adh gene (by making and introducing into D. melanogaster Hawaiian Drosophila Adh gene fusions) are in progress. We thank A. Spradling for his helpful advice and support and J. Posakony for supplying D. melanogaster stocks. We also thank T. Tidwell and J. Berry for their technical assistance. This work was supported by NIH grants lROl-GM34961 to M.D.B. and 5ROlHD10723 to W.J.D. REFERENCES BENYAJATI, C., PLACE, A. R., POWERS, D. A., and SOFER, W. (1981). Alcohol dehydrogenase gene of Drosophila melanogaster: Relation-

74

DEVELOPMENTAL BIOLOGY

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