COMPLEX CIS-ACTING REGULATORS AND LOCUS STRUCTURE OF DROSOPHILA TISSUE-SPECIFIC ADH VARIANTS

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Copyright 0 1986 by the Genetics Society of America

COMPLEX CIS-ACTING REGULATORS AND LOCUS

STRUCTURE

OF

DROSOPHILA TISSUE-SPECIFIC ADH

VARIANTS

L E O N A R D R A B I N O W ' AND W. J. D I C K I N S O N

Department of Biology, University of Utah, Salt Lake City, Utah 841 12

Manuscript received September 13, 1985 Revised copy accepted November 8, 1985

ABS TRA C T

Diverse patterns of tissue-specific expression of alcohol dehydrogenase (ADH) among species of the grimshawi subgroup of Hawaiian picture-winged Drosophila suggests control by complex or multiple, independently acting regulatory elements. T h es e elements act by controlling A d h mRNA accumulation in individual tissue types. Restriction mapping of the A d h loci from these species reveals several insertion/deletion differences, on e of which lies just outside t he 5' en d

of t he structural sequences and correlates with differences in larval patterns of

ADH expression. N o tissue-specific rearrangement of A d h sequences was ob-

served.

NE characteristic of eukaryotic development is the genome-encoded pro-

0

gramming of gene activity in specific tissues and at specific times. Nat- urally occurring variation at genetic loci that determine these patterns of expression, termed temporal loci (GANSCHOW 1975), has been described in a number of experimental systems, including maize, mice and Drosophila (for review, see PAIGEN 1979). Both cis and trans-acting temporal loci have been described for a number of enzyme genes. Cis-acting temporal and other regulators have been found to be closely linked to the affected structural gene whenever the experimental system has been amenable to a detailed genetic analysis (CHOVNICK et al. 1976; DICKINSON 1975; SCHWARTZ 1962; WOODMAN and FREELINC 1981).

Many species of the Hawaiian picture-winged Drosophila display dramatic variation in patterns of developmentally programmed tissue and stage-specific expression of a number of enzymes (DICKINSON 1980a). Interspecific hybridization in the laboratory established the existence of both trans- (DICKINSON

1980b) and cis-acting (DICKINSON and CARSON 1979; DICKINSON 1980c) tissue- specific regulatory elements, the properties of which resemble those of the temporal regulatory elzments described within species. Interspecific regulatory variation therefore provides excellent models for the study of developmentally regulated gene function. In addition, studies of this variation may provide clues

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524 L. RABINOW AND W. J. DICKINSON

regarding the nature of events that cause changes in gene regulation during evolution.

Alcohol dehydrogenase (ADH) is one of the enzymes demonstrated to be under the control of cis-acting tissue-specific regulators in the Hawaiian Dro- sophila (DICKINSON and CARSON 1979; DICKINSON 1 9 8 0 ~ ) . In a previous report, a recombinant DNA clone of the corresponding locus (Adh) from

D.

melano- gaster was used as a hybridization probe to demonstrate that the accumulation of ADH-specific mRNA is affected by a cis-acting regulatory locus (RABINOW and DICKINSON 1981). T h e species pair in which this study was undertaken,

D.

grimshawi and

D.

orthofascia, are both members of the grimshawi subgroup of the Hawaiian Drosophila. T h e

D.

melanogaster Adh genomic clone has since been used to isolate genomic clones from one of the members of this subgroup,

D.

uffinidisjuncta (BRENNAN et al. 1984).

We present here several new examples of interspecific variation of ADH tissue-specific expression among four members of the grimshawi species subgroup. Cloned hybridization probes from

D.

afinidisjunctu demonstrate that, in each of the cases examined, the amount of ADH-specific mRNA is affected, although the genomic DNA at Adh is not altered in copy number o r arrangement in any of the tissues examined. T h e existence of discrete regulatory elements is suggested by the ability of ADH expression patterns to vary independently in different tissues. Finally, analysis of genomic sequences in and around the A d h locus in these species reveals insertion/deletion differences, one of which lies at the 5’ end of the structural gene and correlates with the altered patterns of ADH regulation reported.

MATERIALS AND METHODS

Hawaiian Drosophila stocks and culture: The species used in this study (with stock numbers in parentheses) were D. hawaiiensis (J14B8); D. formeEEa (M87G1); D. grimshawi

(Gl); and D. affinidisjuncta (S36G1). The origin and maintenance of these stocks were described previously (DICKINSON 1980a; BRENNAN, ROWAN and DICKINSON 1984).

Tissue preparation, electrophoresis and enzymatic staining for alcohol dehydro- genase: Electrophoresis of ADH in agar noble gels and enzymatic staining were as described elsewhere (DICKINSON and CARSON 1979), with the addition of 0.1 % triton-

X 100 to the staining solution to increase sensitivity approximately two-fold (W. SOFER, personal communication). All tissues for activity analysis, RNA or DNA preparation, were hand dissected in ice-cold Ringer’s solution. To minimize RNA degradation, tissues were immediately frozen on dry ice or in liquid nitrogen following dissection. Adult males > I month old were selected for studies of the adult tissue-specific phenotypes reported. Actively feeding third-instar larvae were used for all experiments in- volving larval phenotypes.

Preparation of Drosophila DNA and genomic library construction: DNA was pre- pared from frozen adult Drosophila as described by BINGHAM, LEVIS and RUBIN (1981) by lysis of isolated nuclei, followed directly by cesium chloride density centrifugation. Larval tissue-specific DNA was prepared from hand-dissected organs, as described above, or by the method of COEN, THODAY and DOVER (1982). Genomic libraries were constructed in the lambda cloning vector L-47.1 (LOENEN and BRAMMAR 1980), as described by BRENNAN et al. (1 984). The numbers of recombinant phage resulting were as follows: D. grzmshawz, 7.8 X lo4; D. hawaiiensis, 2.6 X lo5.

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CIS-ACTING REGULATORS OF ADH 525

juncta subclone pllB-H14 as the probe (see Figure 6). Hybridization was in aqueous solution at 65", using 10% dextran sulfate. Filters were washed to a final salt concen- tration of 0.1 X SSC at 65". At least six phage showing hybridization were recovered from the D. grimshawi and D. hawaiiensis libraries. Only two from each library were

fully characterized.

Subclones were prepared by insertion of phage fragments produced by digestion with HindIII, Sal1 or both enzymes into the corresponding site(s) on plasmid pBR322. Plas- mid DNA was prepared according to CLEWELL and HELINSKI (1969).

Electrophoresis and hybridization of nucleic acids: DNA was size fractionated in gels of from 0.4 to 1.0% agarose. R N A was size fractionated in 1.0% agarose gels containing formaldehyde according to LEHRACH et al. (1977). RNA was prepared from hand-dissected tissues by phenol-SDS extraction as previously described (RABINOW and

DICKINSON 198 1). Transfers of DNA to nitrocellulose were essentially as described by SOUTHERN (1975), and transfers of R N A to nitrocellulose were as described by GOLD-

BERG (1 980). Nick translation was performed according to RICBY et al. (1977). Hybrid- ization was carried out according to the method of WAHL, STERN and STARK'(^^^^) or as described for the isolation of lambda clones. In situ hybridization to polytene chro- mosomes was carried out as previously described (BRENNAN et al. 1984).

RESULTS

Variation in patterns of ADH tissue-specific expression: It has been shown previously that ADH is expressed in the fat body of both larvae and adults in all species examined (DICKINSON 1980a and unpublished results). Expression of ADH in other tissues is highly variable. There are no clear connections among tissues with respect to ADH expression levels. For example, both

D.

affinidisjuncta and

D.

grimshawi express ADH at essentially equal levels in larval carcass and midgut (DICKINSON 1980a). In contrast, although

D.

hawaiiensis

expresses ADH at levels comparable to the former two species in larval carcass, only a trace is found in the midgut, and this is confined to the anterior midgut. As previously reported (DICKINSON 1980a),

D.

formella lacks detectable levels of the enzyme in both larval carcass and midgut, including gastric ceacae. Electrophoretic gels demonstrating these ADH phenotypes are illustrated in Figure 1. T h e qualitatively distinct patterns of ADH activity in midgut were confirmed by histochemical staining of whole dissected digestive tracts. This procedure also reveals that midgut activity in

D.

hawaiiensis is localized in the gastric ceacae (data not shown).

Similar examples may be derived from patterns of ADH expression observed in mature adult midguts and hindguts.

D.

affinidisjuncta demonstrates substan- tial ADH expression in midgut but weak expression in hindgut, whereas

D.

grimshawi displays an essentially reciprocal pattern, with strong expression found in the hindgut and none found in the midgut (Figure

2).

These phenotypes are confirmed by activity gel electrophoresis. Hindgut and midgut from adult males of

D.

formella show essentially the same pattern of ADH expression as that described in

D.

grimshawi, whereas

D.

hawaiiensis has little or no activity in either location. Thus, there is no obvious correlation between larval patterns of expression and those found in adults of the same species. A summary of all the phenotypes presented may be found in Table 1.

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526 L. RARINOW AND W. J. DICKINSON

F I G V R E 1 .--Kcpresent;ttive regulatory differences detected by activity gel electrophoresis. In c.;ich case. dissected parts from five individuals were pooled and homogenized in 15 pl of water,

;itid 5 pl of the extract wits fractionated by electrophoresis and stained for ADH enzymatic activity. I’;uiel A, A D H xtivity i n larval car c~s s (body wall musculature and cuticle). Lanes 1-4: serial five- fold dilutions of D. grimshawi extract: lane 5: undiluted D. formella extract: lanes 6-9: serial five- fold dilutions of D. hawaiiensis extract. In the original, activity is detected in the 25-fold dilution

of D. grimshawi and D. hawaiiensis extracts, but not in undiluted D. formella extract. Panel B. ADH activity in regions of larval gut. Lanes 1-4: D. grimshawi: lanes 5-8: D. hawaiiensis. Within each set. the regions are middle and posterior midgut ( 1 and 5): Malpighian tubules (2 and 6): hindgut (3 and 7): and ;interior midgut (4 and 8).

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CIS-ACTING REGULATORS OF ADH 527

GRIM "m"

GRIM ahq

FIGURE 'L.-Representative regulatory differences detected by histochemistry. Whole organs were hand dissected, fixed and stained for A D H activity. Grin) = D. grimshawi; Affin = D. affini- disjuncta: anig = adult midgut; ahg = adult hindgut.

T A B L E 1

Tissuespecific expression of ADH activity

Stage and tissue

Lirval Adulr

Species Ell Ixxly Gill Carcass Midgiit Hindgut

D. affinidisjuncta

++

+

sf:

D. grimshawi

++

+

D. formdla

++

+

f

-

D. hawaiimsis

++

sf:"

+

Expression of alcohol dehydrogen;tse in selected larval and adult tissues of four members of the grimshawi subgroup of Hawaiian Drosophila.

++

= Strong ADH expression;

+

= readily detectable expression; f = marginal. or very weak, expression;

-

= no detectable expression. See text for more details.

a Confined to gastric ceacae.

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528 L. RARINOW AND W. J. DICKINSON

FIGURE 3.--Cis-action o f an element controlling a difference o f the expression o f ADH in D. hawaiiensis and D. formella. T h e following saniples were electrophoresed and stained as for Figure 1. ( I ) D. formella larval fat body, (2) D. formella larval carcass, (3) D. formella X D. hawaiiensis FI

larval fat body, (4) D. formella X D. hawaiiensis FI larval carcass, (5) D. hawaiiensis larval fat body

and (6) D. hawaiiensis larval carcass.

high ADH levels in the male hindgut of some species is related to, but not inseparable from, a sex-specific morphological difference and should not be attributed to a sex-specific difference in ADH expression per se.

Genetic control of tissue-specific ADH expression in interspecific hybrids:

Cis- or trans-action of a regulatory difference can be distinguished if there are suitable markers for the products of alternative structural loci, such as electrophoretic mobility differences. Among the four stocks considered here, only

D.

hawaiiensis has ADH differentiable from that in other species by activity gel electrophoresis. In

D.

hawaiiensis X

D.

formella hybrids, only the

D.

hawaiiensis form of ADH is present in carcass, even though both are detected in fat body (Figure 3). Thus, parental differences are preserved in hybrids, indicating control of this regulatory difference by a cis-acting element. Examination of the ADH present in larval carcass from hybrids of

D.

formella and

D.

grimshawi yields results consistent with cis-acting regulation of tissue-specific expression of ADH in larval midgut and carcass,

i.

e., levels of expression are intermediate in these tissues of hybrids.

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CIS-ACTING REGULATORS OF ADH 529

FIGURE 4.-Tissue-specific RNA. '1.otaI R N A froin e;rc.li tissue was electrophoresed, blotted to nitrocellulose and probed with D. affinidisjunrfa subclone 1 I R-S8. Panel A, Larval tissue-specific RNAs derived from four species and three tissues. Lanes 1-3: D. afinidisjuncta; lanes 4-6: D. grimshawi; lanes 7-9: D. formella; lanes 10- 12: D. hawaiiensis. Tissues represented from each species itre fat body (1, 4, 7 and 10); gut (2, 5 . 8 and I I); and carcass (3, 6, 9 and 12). Panel B, Adult tissue-specific RNAs. l an es 1 and 2: D. affinidisjuncta; lanes 3 and 4: D. grimshawi. Hindgut-specific RNA is shown in lanes 1 and 3; midgut-specific RNA is in lanes 2 and 4.

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530 L. RABINOW AND W. J. DICKINSON

mologous probe from

D.

afjnidisjuncta also allowed detection of trace amounts of ADH inRNA in the guts of D. hawaiiensis, where enzymatic activity is restricted to the gastric ceacae and is barely detected. This quantitative difference in enzyme activity is thus reflected at the RNA level.

Distribution of ADH-specific sequences in RNA prepared from hindguts and midguts of

D.

afjnidisjuncta and

D.

grimshawi adult males > I month old is shown in Figure 4B. Tissue-specific expression of ADH is again paralleled by the presence o r absence of the mRNA. An additional example of quantitative control of ADH activity and mRNA is found in D. afinidisjuncta adult male hindgut, which demonstrates a low but reproducible level of both.

Chromosomal location: As a first step toward identifying structural correlates of altered regulation, w e examined the possibility that gross chromosomal rearrangements have brought the A d h locus into a new context. I n situ hybridization procedures were used to visualize the A d h locus on polytene chro- mosomes of the four species considered here, as well as species representing all other major subgroups of picture-winged Drosophila. In every case, hybridization is confined to the same position relative to clearly homologous banding patterns. I t is clear that altered regulation is not a consequence of gross rearrangement. D. afinidisjuncta,

D.

grimshawi and D. formella are homosequential

in banding pattern throughout chromosome I l l , on which A d h is located, and there are no visible structural alterations in the bands around the A d h gene (Figure 5). Even when an inversion has moved the A d h locus away from the base of chromosome ZZZ, as in

D.

hawaiiensis, the pattern of bands immediately around the locus is unchanged (see Figure 5).

Genomic Adh sequences of D. afinidisjuncta, grimshawi, formella and ha- waiiensis: Restriction maps of the A d h loci from the four species are compared i n Figure 6 . These maps are based on data obtained from genomic Southern transfers probed with

D.

afinidisjuncta subclones and on isolated recombinant phage from D. afjnidisjuncta, grimshawi and hawaiiensis.

A high degree of sequence conservation at the A d h locus among the four species is indicated by the extensive cross hybridization, as well as by the colinearity and spacing of restriction sites. Although several small fragment- length differences and apparent point changes resulting in gain or loss of some restriction sites are present, no ambiguities in the relationships of comparable restriction sites are found. Repetitive elements localized to the 3’ end of the

D.

afjnidisjuncta A d h locus are present in roughly equal numbers in the ge- nomes of the other species (not shown).

Adh structural sequences are almost entirely contained on a 1.65-kb EcoRI fragment that is conserved in all four species (Figure 7A). Only two insertion/ deletion differences greater than approximately 100 base pairs (bp) were found

i n mapping of between 10 and 20 kb from each of the species. Both of these differences result in fragments shorter than the corresponding

D.

afinidisjuncta

fragments. One difference lies at the 3’ end of the A d h gene and results in a Hind111 fragment of 1.9 kb in

D.

afinidisjuncta, formella and hawaiiensis being shortened to 1.67 kb in

D.

grimshawi. T h e other difference is found at the 5’

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CIS-ACTING REGULATORS OF ADH 53 1

FIGURE 5.--Chromoson:al context of the Adh gene. Top: In situ hybridization of plasniid 32RE1 (HRENNAN ef al. 1984) to the third chronlosonie of D. grimshawi. Below: aceto-orcein stained cliromosoiiies of D. grimshawi ( G ) , D. formella (F) and D. hawaiiensis (H). In each case, the arrow indicates the band in or near which the Adh gene is located. Lines connect homologus bands useful ;IS landmiirks. In D. grimshawi and D. formella, which both have the “standard” sequence of

kinds on chronlosonie / I / . the heavy bands at the right are adjacent to centric heterochromatin. I n D. hawaiiensis, inversion Sg (CARSON and STALKER 1968), which has one breakpoint in the centric lieterochrom;itin, has moved the corresponding bands (and the nearby Adh locus) to a position near the center of chromosonie I / / .

formella) or a 180-bp difference

(D.

hawaiiensis), relative to either

D.

grimshawi o r affinidisjuncta (Figure 7B).

DNA of the Adh locus from specific tissues: To examine the possibility of tissue-specific alteration of Adh genomic sequences, DNA was prepared from 100 hand-dissected larval fat bodies, midguts and carcasses of

D.

grimshawi,

D.

formella and

D.

hawaiiensis. Aliquots were digested with either Hind111 or

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532 L. RABINOW AND W. J. DICKINSON

D aftinidispncta

I l E - S H l 3

I I t € - S E I

t

-

I l l - H I 4

-

I

D hawaiiensis

I +

E

..

. . . .

i

I I I I I I 1 I I

t

-6 -4 -2 -0 + +2 +4

FIGURE 6.--Coniparison of Adh genomic regions by restriction mapping. T h e approximate ex- tent of the transcribed region in indicated by cross-hatching. D. affinidisjuncta subclones (thin lines) and gel-isolated fragments (heavy bars) used as hybridization probes are indicated. T h e maps are aligned at the Hind111 site arbitrarily designated 0.0. Restriction enzyme recognition sites are A = AuaI; B = BglI; E = BcoRI; H = HindIII; P = PstI; R = EcoRV; S = Sall; V = PuuII. An (x) indicates a missing restriction site, and an asterisk (*) indicates a new site or some of the different fragment lengths, all relative to D. afinidisjuncta. Because cloned sequences were not available from D. formella and the presence of repetitive elements near the 3' end of the gene in all species complicated mapping by hybridization to Southern transfers of genomic restriction fragments, some restriction sites present in other species were not mapped in D. formella. Hence, absence of a site on this map does not necessarily mean that the site is missing. T h e ( E ) indicates a possible polymorphism i n D. formella.

have been detected. Additionally, changes in the context of the Adh gene among these tissues would be revealed if any restriction fragments detected by this probe were affected. Only the band(s) expected from mapping of isolated phage o r from adult genomic Southern transfers were observed, thus effec- tively ruling out rearrangement of the gene within 3 kb 5' to the structural gene in these tissues (not shown). T h e intensity of detected bands from different tissue samples was equal, indicating no gross under o r over replication of

Adh genomic sequences on a tissue-specific basis.

DISCUSSION

T h e new regulatory patterns reported here, when considered with previously reported examples (DICKINSON 1980a, 1980b, 1983; RABINOW and DICKINSON

198 l ) , show that evolutionary changes in patterns of gene expression can affect different tissues and stages independently. This suggests the existence of multiple and/or complex regulatory sites that can be altered independently or in

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CISACTING REGULATORS OF ADH 533

2.8

1.01 1.00 0.61

FIGURE 7.-lnterspecific comparisons of restriction fragment lengths. Genomic DNA digested as specified below was electrophoresed, blotted and hybridized to cloned sequences from D. a& idisjuncta as indicated. Panel A. EcoRl digested DNA from D. afinidljuncta (lane l), D. gtimshawi

(lane 2), D. formella (lane 3) and D. hawaiiensl (lane 4) hybridized to t w o gel-purified fragments from 11B-HI4 (Figure 6). T h e first fragment extends from the Hind111 site (0.0) near the middle of the coding region to the EcoRl site (-0.65) near the 5’ end and detects the conserved 1.65-kb fragment that carries most of the coding sequences in all species. T h e second probe is an EcoRl fragment extending from -1.65 to -2.3 in D. affinidljuncta. I t detects the corresponding 0.65- kb fragment in that species and in D. grimshawi, but detects a 2.8-kb fragment in the other species, documenting the absence of the -2.3 site (Figure 6). Panel B HindlII, Sal1 doubledigested DNA from D. grimsham. (lane 1). D. formella (lane 2 ) and D. hawaiiensl (lane 3 ) hybridized to 11B- SH13 (Figure 6). This documents length differences from the central Hindlll site to the Sal1 site 5’ to the coding region. Conservation of the 1.65-kb EcoRI fragment (above) confines the observed length differences to the EcoRISalI fragment adjacent to the 5‘ end of the coding region. T h e length of this fragment in D. afFnidisjuncta (not shown) is almost identical to that in D. grimshawi.

T h e apparent separation of cis-acting sites responsible for expression of Dro- sophila yolk proteins in t w o different tissues recently reported by GARABEDIAN, HUNG and WENSINK (1 985) supports a similar conclusion.

T h e one case for which an appropriate test was possible clearly showed cis-

acting control of the regulatory difference. All other cases gave a result (intermediate expression) consistent with cis-acting control. We believe that the multiple o r complex regulatory sites implicated by the diversity of regulatory patterns are most likely to be located in a cis-acting regulatory region adjacent to the structural gene. However, w e cannot exclude the formal possibility that multiple truns-acting regulators interact with a single cis-acting site in a species- specific or allele-specific manner (i.e., pattern differences are due to different truns-acting regulators, but they appear as cis-acting controls in hybrids, be-

U

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534 L. RABINOW AND W. J. DICKINSON

cause each species’ trans-acting factors interact only with a cis-acting component from the same species).

Results at the RNA level extend the conclusion that the regulatory sites under consideration act at or near transcription to several new cases. T h e low but distinctly detectable presence of both ADH enzyme activity and mRNA in anterior midgut of

D.

hawaiiensis larvae and hindgut of

D.

affinidisjuncta adult males indicates that similar mechanisms apply to quantitative as well as quali- tative (on/off) regulation. We find no evidence for loss, amplification or rearrangement of the Adh locus in any of the tissues affected by regulatory variants. We also find no evidence that differences between expression patterns result from gross chromosomal rearrangements.

Analysis of the structural and flanking sequences of the Adh locus in D.

affinidisjuncta, grimshawi, formella and hawaiiensis reveals a high degree of colinearity at the level of restriction sites and hybridization to subclones. Com- parison of the A d h loci of the four species does not define sequences responsible for the tissue-specific control of Adh expression, but certain regions are circumstantially implicated and bear further study.

D. afinidisjuncta and

D.

grimshawi express ADH reciprocally in adult midguts and hindguts. Comparison of the Adh locus restriction maps of these two species reveals no major differences between them at the 5’ end or within the structural gene. There is a relatively large insertion/deletion difference at the 3’ end (a Hind111 fragment of 1.9 kb in

D.

afinidisjuncta, compared to a 1.67- kb fragment in D. grimshawi), but there is no obvious correlation with pheno- type, because

D.

formella and

D.

hawaiiensis, which resemble D. grimshawi in lacking ADH in adult midguts, have the 1.9-kb restriction fragment.

Correlation between Adh restriction maps and patterns of expression is found with respect to larval phenotypes. Comparison of the A d h restriction maps of either

D.

formella or

D.

hawaiiensis with those of

D.

affinidisjuncta or grimshawi

reveals a large insertion/deletion difference at the 5’ end of the structural gene. Recall that

D.

affinidisjuncta and

D.

grimshawi are identical in their larval patterns of ADH expression, whereas

D.

hawaiiensis expresses low levels of ADH in the larval gastric ceacae, shows none in midgut and shows levels comparable to the former two species in larval carcass. T h e restriction map of the

D.

hawaiiensis A d h locus reveals a deletion relative to the former two species of approximately 180 bp, immediately 5’ to the A d h coding sequences.

D.

formella lacks all ADH expression in larval gut and carcass, and it reveals a 230-bp deletion relative to

D.

affinidisjuncta or

D.

grimshawi at the 5‘ end of

the Adh coding sequences. T h e organization of the

D.

affinidisjuncta Adh gene is similar to that seen in

D.

melanogaster (BENYAJATI et al. 1983), including transcription from two promoters (BRENNAN et al. 1984; R . ROWAN and W. J. DICKINSON, unpublished results). In both cases, the proximal promoter is used primarily in larvae, and the distal promoter is used primarily in adults. T h e length differences in

D.

formella and

D.

hawaiiensis relative to

D.

afjnidisjuncta

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CISACTING REGULATORS OF ADH 535

both

D.

formella and

D.

hawaiiensis. T h e additional 50 bp in the hawaiiensis Adh allele relative to D. formella might then contain information required for ADH expression in larval carcass and gastric ceacae. This is consistent with the presence of multiple, cis-acting elements tightly linked to the structural gene, as suggested above.

One further correlation can be made concerning levels of ADH in larvae of D. formella relative to

D.

grimshawi. We noted previously that D. formella larval fat body expresses only about one-fourth the ADH found in the same tissue of D. grimshawi (RABINOW and DICKINSON 1981). It is interesting that

D.

for- mella bears the larger deletion relative to D. grimshawi o r aflanidisjuncta. There- fore, information determining levels of transcription may also be contained in the fragment missing in D. formella, o r such information may be inseparable from that required for expression in multiple tissues.

These hypotheses are, of course, tentative. Indeed, other studies of Droso- phila Adh locus structure, using strains of D. melanogaster as well as those of several other species, have described DNA polymorphisms without attendant dramatic effects on ADH expression (LANGLEY, MONTGOMERY and QUATTLE- BAUM 1982; BODMER and ASHBURNER 1984; KREITMAN 1 9 8 3 ) . However, tissue- and stage-specific ADH levels were not examined in these studies; thus, the variation in expression patterns described in this report would probably have gone undetected.

Although correlations between ADH expression patterns and Adh locus restriction maps of the grimshawi subgroup variants provide preliminary and tentative evidence concerning the location of tissue-specific regulatory information, unambiguous results await a direct functional demonstration that can only be provided by DNA-mediated transformation.

D.

melanogaster P-element germline transformation is effective in the Hawaiian Drosophila, although no

Adh transformants have so far been recovered (BRENNAN, ROWAN and DICK- INSON 1 9 8 4 , and unpublished results). Transient expression assays following injection of DNA into embryos as described by W. SOFER (unpublished results) have, however, recently begun to provide encouraging results (P. THORPE, unpublished results).

This work was supported by National Institutes of Health grant HD10723 to W.J.D. and National Institutes of Health Predoctoral Fellowship GM07464 to L.J.R. PAULA FEY provided technical assistance in some of these experiments. We would also like to acknowledge the critical comments and continual encouragement of RAY GESTELAND.

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T h e messenger RNA for alcohol dehydrogenase in Drosophila melanogaster differs in its 5’ end in different develop- mental stages. Cell 33: 125-133.

Cloning o f DNA sequences from the white BENYAJATI, C., N. SPOEREL, H . HAYMERIE and M. ASHBURNER, 1983

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536

BODMER, M. and M. ASHBURNER, 1984

L. RABINOW AND W. J. DICKINSON

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Isolation and initial characterization of the alcohol dehydrogenase gene from Drosophila aflnidisjuncta. J. Mol. Appl. Genet. 2: 436-446.

BRENNAN, M., R. ROWAN and W. J. DICKINSON, 1984

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