Mechanisms of Development, 44 (1993) 123-138 123 © 1993 Elsevier Scientific Publishers Ireland, Ltd. 0925-4773/93/$06.00

MOD 00195

In vivo functional characterization of an ecdysone response enhancer in the proximal upstream region of the Fbpl gene

of D. melanogaster

Monique Laval, Francine Pourrain, Jean Deutsch and Jean-Antoine Lepesant *

lnstitut Jacques Monod, CNRS et Universit~ Paris 7; 2, place Jussieu; 75251 Paris Cedex 05, France

Received 3 March 1993; revision received 29 July 1993; accepted 5 August 1993)

Transcription of the D. melanogaster Fat-body-protein-1 (Fbpl) gene is induced by the steroid hormone 20-hydroxyecdysone and is restricted to the fat body tissue at the end of the third larval instar. The location and functional properties of the Fbpl cis-acting regulatory sequences contained in the region from -1386 to + 80 relative to the transcription start were examined by transformation using hybrid constructs with the Adh or lacZ genes as reporters. Regulatory element(s) required for the full level of transcription of the Fbpl gene were located between positions -1386 and -138. Sequences between -138 and -68 were able to drive transcription from a heterologous minimal promoter in the fat body of late third instar larvae. Remarkably, these sequences also conferred 20-hydroxyecdysone inducibility and behaved as an enhancer-like element. These results provide the first functional characterization, at the level of the whole organism, using a direct in vivo ecdysone induction assay, of a discrete ecdysone response element.

20-Hydroxyecdysone; Ecdysone response element; D. melanogaster; Larval fat body

Introduction

The steroid hormone ecdysone (ecdysone is used here as a generic term for all ecdysteroids with hor- monal activity) plays a prominent role in insect devel- opment and modulates the expression of a large frac- tion of the Drosophila genome. Ecdysteroids are pre- sent at all developmental stages, including embryonic ones, but their titer varies considerably throughout the life cycle of the fly (Richards, 1981). There are clear differential gene responses to the hormonal signal with unique patterns of tissue and stage specificities of transcription (see Andres and Thummel, 1992; Andres et al., 1993; Segraves and Richards, 1990, for recent reviews). The molecular basis of this tissue- and stage-

* Corresponding author. Tel: + 33 1 44 27 69 50. Fax: + 33 1 44 27 52 65.

specific restriction of gene activity is still unclear but according to the current model of action of the steroid hormones it can be modulated at two levels: the ecdysone receptor and the ecdysone response elements (EcRE) which bind the hormone-receptor complex.

The biochemical purification of a protein that exhib- ited the activities of an ecdysone receptor and a tran- scription factor was reported by Luo et al. (1991) and Ozyhar et al. (1991). Independently, Koelle et al. (1991) reported the cloning of a gene (Ecdysone Receptor or EcR) coding for three isoforms (Talbot et al., 1993) of a protein with the DNA and ecdysteroid binding prop- erties expected for an ecdysone receptor. More re- cently, Yao et al. (1992), Thomas et al. (1993) and Koelle, Arbeitman and Hogness (personal communica- tion) demonstrated that the formation of an het- erodimer between the EcR protein and the product of the ultraspiracle (usp) gene is actually a prerequisite for hormone and DNA binding activities of the ecdysone receptor. This finding led to the hypothesis that combinatorial interactions of members of the re- ceptor superfamily play a direct role in the tissue and stage specificities of the ecdysone response (Richards, 1992).

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Transfection and DNase I footprinting assays of the 20-hydroxyecdysone responsive sequences of the hsp27 gene of D. melanogaster led Riddihough and Pelham (1986; 1987) to identify a prototype for an ecdysone response element (EcRE) whose activity was confirmed by several studies (Amin et al., 1991; Cherbas et al., 1991; Dobens et al., 1991; Martinez et al., 1991). The further investigation of the ecdysteroid regulation of the Eip28/29 (Cherbas et al., 1991) and hsp23 (Luo et al., 1991) genes using transfection assays of Drosophila cells in culture completed this functional definition of an EcRE. A structural definition was established in the course of in vitro DNA binding assays with crude nuclear extracts from larval fat body (Antoniewski et al., 1993), partially purified EcR (Ozyhar and Pongs, 1991), EcR produced from the cloned gene (Koelle et al., 1991) or radioactive hormone-labelled EcR (Cher- bas et al., 1991) and in vitro transcription experiments with a purified ecdysone receptor preparation (Luo et al., 1991). The consensus sequence of the EcRE, taking into account a large flexibility, was defined by in vitro mutagenesis using transfection and in vitro binding assays (Antoniewski et al., 1993; Cherbas et al., 1991; Dobens et al., 1991; Martinez et al., 1991; Ozyhar and Pongs, 1993).

In this context the Fat-body-protein-1 (Fbpl) gene provides a very valuable model gene for studying the cis-acting elements involved in the ecdysone response in vivo. Its structure and organisation are simple, with a single 59 bp intron in the 5' proximal region of the coding sequence and a unique 3.5 kb poly A+transcript (Maschat et al., 1990). It is expressed in a very re- stricted pattern prior to the onset of metamorphosis. Trancription takes place in the fat body exclusively (Lepesant et al., 1986). The Fbpl transcript starts to accumulate before the onset of wandering and remains at a high level throughout the prepupal period before it decreases sharply at the beginning of pupal develop- ment (Andres et al., 1993; Lepesant et al., 1986). The transcript has not been detected at any other stage or in any other tissue using Northern blot analysis and in situ hybridization on tissue sections (Andres et al., 1993; Lepesant et al., 1986; Paco-Larson et al., 1986). The Fbpl protein sequence does not present any simi- larity with any known protein and its function has not been determined.

Fbpl transcription starts when the global ecdys- teroid titer is still low and then parallels the large increase in the levels of ecdysteroids (Richards, 1981) and EcR transcript (Andres et al., 1993; Karim and Thummel, 1992) detected at the end of the third instar. The Fbpl gene was identified as the first invertebrate gene in which experimental evidence supported the hypothesis that hormonal control acts at the level of gene transcription (Lepesant et al., 1978). The accumu- lation of the Fbpl transcript is severely reduced or

abolished in ecdysteroid-deficient larvae homo- or hemi-zygous for the ecd ~ts or dor 22 mutations, which impair the synthesis or release of ecdysteroids. 20-hy- droxyecdysone restores transcription of the Fbpl gene in these ecdysteroid-deficient larvae (Lepesant et al., 1982; Lepesant et al., 1986). Other ecdysteroids, in- cluding ecdysone and 3-dehydroecdysteroids, induce a similar effect (SommE-Martin et al., 1990). The re- sponse to 20-hydroxyecdysone is not inhibited by cyclo- heximide (Nakanishi and Garen, 1983). Altogether, these results strongly support the idea of a direct effect of ecdysteroids at the transcriptional level and suggest that there are EcRE(s) within the c/s-acting regulatory sequences of the Fbpl gene.

Our previous studies were aimed at the mapping of these regulatory regions. Fbpl sequences of various length were fused to an Ecogpt-SV40 reporter con- struct and expression of the hybrid genes was assayed in transgenic lines by Northern blot. Results showed that correct developmental regulation, including ecdysone inducibility, was conferred on the reporter gene by sequences between positions -1 3 8 6 to + 270 relative to the mRNA start site. Although limited by the low level of expression of the transgenes, these data provided a first indication that essential regula- tory sequences are localized in a - 1 3 8 to - 6 8 seg- ment in the 5' proximal region of the Fbpl promoter (Maschat et al., 1991, 1986).

In the present work, we have confirmed and ex- tended these studies by using a similar gene fusion approach but with the D. melanogaster Adh or E. co# lacZ coding sequences as reporter genes in order to monitor the expression of the hybrid constructs by histochemical staining in addition to Northern blotting.

Our aim was to determine the requirements for Fbpl expression in sequences included in our earlier constructs downstream from the transcription start site and, in particular, the 59 bp intron. A second aim was to determine whether the temporal, tissue and hor- monal specificities of expression of the Fbpl gene are mediated by discrete regulatory elements which retain their functional capacity when tested separately with an heterologous promoter. A third objective was to restrict ecdysteroid inducibility to a short sequence as a step towards a better in vivo functional definition of an EcRE.

Our results demonstrate that regulatory sequences presenting the orientation- and distance-independent functional properties of an enhancer element are lo- cated in the proximal - 1 3 8 to - 6 8 upstream region. Tissue and developmental specificity determinants were not separable from ecdysone regulation but distinct regulatory sequences required to establish the proper level of transcription of the Fbpl gene were located further upstream from the enhancer in the -1 3 86 to

- 138 region.

125

-1386 -256 -138 - 6 8 +80 H M A S +44Hp

j s t

t__ v Fig. 1. Simplified restriction map of the F b p l gene and 5' upstream sequences. The figure is not not drawn to scale. Hatched box: non-transcribed upstream sequences. Double-hatched boxes: transcribed sequences. Empty triangle: intron. The bent arrow indicates the start and direction of transcription. The coordinates of the restriction endonucleases sites (H = H i n d l I I - 1386; M = M s p I ( - 256); A = A l u I ( - 138);

S = S t u l ( - 68); St = S s t I ( + 44); Hp = H p a I ( + 80)) are numbered from the start of transcription as + 1.

Results

Deletion mapping with fusion constructs preserving the Fbp l promoter

Faithful expression of an Fbpl:Adh gene comprising ca. 1.5 kb of Fbpl sequences

This transcriptional fusion (HHp:Adh construct) contained Fbpl sequences from - 1386 to + 80 (Fig. 1) linked to the D. melanogaster Adh coding sequence as a reporter gene (Fig. 2A). The construct retained the Fbpl promoter but downstream Fbpl sequences, in-

cluding the intron, were deleted. (The term 'promoter' is taken sensu stricto, i.e., as the sequences located in the vicinity of the start of transcription, including the TATA box, and required for the proper formation of a transcriptional initiation complex.) Histochemical de- tection of ADH activity in 13 independent transgenic lines showed that the expression of the HHp:Adh construct was restricted to the larval fat body (Fig. 5F) as is the case for the Fbpl endogenous gene (Lepesant et al., 1986; Paco-Larson et al., 1986). Northern blot analysis, using a probe hybridising to the 80 Fbpl nt common to the endogenous Fbpl and hybrid Fbpl-Adh

® HHp:Adh

H Hp E

( ~ 1 2 3 4 5 6 7 8 9 10 11

Fbpl

Fbpl :Adh

Fbp2

Fig. 2. Relative levels of expression of the Fbpl:Adh and Fbpl endogenous transcripts in HHp:Adh transgenic lines. (A) Schematic structure of the HHp:Adh construct. Hatched box: non-transcribed F b p l upstream sequences. Double-hatched box: transcribed F b p l sequences. F b p l

sequences were fused with the D. m e l a n o g a s t e r Adh gene (position + 13 of the coding sequence) and 3' downstream sequences up to the E c o R I

(E) site (stippled boxes). A d h introns are drawn as blank triangles. (B) Northern analysis. The 11 lines presented here are homozygous viable and carry two copies of a single insert of the H H p : A d h transgene per genome. Total RNA was prepared from dissected fat body of staged late third instar larvae as described in Materials and Methods. Approximately 5/~g of RNA were loaded per lane. The level of the endogenous Fbpl (3.5 kb) and hybrid Fbpl :Adh (1.1 kb) RNAs can be directly compared since hybridization was performed using a single-stranded DNA probe hybridizing with the same Fbpl 80 nt sequence common to both RNAs (see text and Materials and Methods). The Fbp2 (formerly P6) RNA (1

kb) (Rat et al., 1991) was probed as a reference for the amount of fat body RNA loaded per lane (lower panel).

126

transcripts, indicated that the level of accumulation of the hybrid transcript is similar to that of the endoge- nous transcript. Some variations from one transgenic line to the other, possibly due to genomic position effects, were observed (Fig. 2B).

The gross developmental profile of the expression of the H H p : A d h transgene was first investigated on pooled individuals collected at various stages from early embryos to adults using an histochemical (2 trans- genic lines) or Northern blot (1 line) assay. In all three cases, expression was not detected at any stage other than the end of the third instar and prepupal period, similar to the pattern of expression of the endogenous Fbpl gene (data not shown). Larvae from one trans- genic line were then precisely staged during the third instar and total RNA was analyzed by Northern blot- ting using the same probe as described previously. The endogenous Fbpl message appeared as early as 90 h after egg eclosion at 20 °, whereas the fusion transcript was not yet detectable at 101 h. At later times, prior to the onset of pupariation, both transcripts reached the same level of accumulation (Fig. 3).

Ecdysone inducibility of the hybrid transgene could be easily tested because the recipient strain for trans- genesis carried the conditional ecd its mutation leading

Fbpl~

F b p l : A d h ~

rp49--~

66 74 90 I01 112 116 I80hrs

a b c d e f g

W Fbp2---~

Fig. 3. Kinetics of the expression of the H H p : A d h construct during the third instar. Northern blot analysis was performed on total fat body R N A extracted from dissected third instar larvae of the H H p : A d h # 9 transgenic line (20 to 25 ~zg per lane, 4 p.g only in the 180 h lane). The amount of total R N A per lane is est imated through hybridization with a probe for the ribosomal protein gene rp49 (Vaslet et al., 1980). Hours after egg eclosion at 20 ° are indicated, with 64 h corresponding to the beginning of the third instar, and 192 h to the onset of pupariation. The same blot was hybridized with a probe for the fat body-specific gene Fbp2. This gene which has been shown to be expressed earlier than the Fbpl gene provides a devel-

opmental marker within the third instar (Lepesant et al., 1982).

to a drastic decrease of the ecdysone titer at non-per- missive temperature (Belinski-Deutsch et al., 1983; Berreur et al., 1984; Garen et al., 1977). The HHp:Adh construct retained full inducibility by 20-hydroxyecdy- sone, with the levels of accumulation of the fusion transcript after 5 h of induction being similar to or even greater than that of the endogenous Fbpl tran- script (Fig. 4A). In this test the accumulation of the hybrid transcript was delayed as indicated by the ratio of hybrid to endogenous transcript after 3 h of hor- mone treatment (Fig. 4B). This delay is similar to the delay in accumulation of the same transcript observed (Fig. 3) in larvae raised at the permissive temperature in the physiological in vivo conditions.

Decrease of the let~el of expression of 5' shorter Fbpl:Adh fusions

A series of shorter Fbpl :Adh fusions were obtained by deleting the HHp:Adh hybrid gene to -256 , - 1 3 8 and - 6 8 . The resulting constructs were tested using the somatic transformation assay of Martin et al. (1986). Control experiments showed first that in this test the level of expression of the HHp:Adh construct in the larval fat body (Fig. 5B) was similar to that of a control plasmid (Posakony et al., 1986) carrying the Adh regu- latory and coding sequences (Fig. 5A). By contrast the level of expression of the reporter gene was drastically decreased and to a similar extent with the constructs retaining sequences to - 2 5 6 ( M H p : A d h ) or to - 1 3 8 (AHp:Adh) . On the other hand, shortening of Fbpl sequences on the 3' side from + 80 t o + 48 in the M H p : A d h or A H p : A d h constructs did not affect the expression of the reporter gene. An example of the level of expression of these constructs is shown in Fig. 5C. Deletion to - 6 8 in the S H p : A d h construct led to a further reduction of the level of expression as com- pared to the M H p : A d h and A H p : A d h constructs (Fig. 5D).

Because the removal of 5' sequences to - 2 5 6 or - 1 3 8 gave similar results in the somatic transforma- tion test, further analysis of the expression of only the A H p : A d h construct was tested in transgenic lines. Northern blot analysis showed that in these lines the level of accumulation of the hybrid transcript was at least 20 fold lower than that of the endogenous Fbpl transcript (Fig. 6A). An $1 protection assay confirmed this result and showed that the Fbpl -Adh RNA was of the expected size for an initiation of transcription from the Fbpl start included in the construct.

Together these results led us to two conclusions. Sequences located between - 1 3 8 6 and - 1 3 8 are re- quired for adjusting the expression of the Fbpl gene to a high level. When these sequences are absent, se- quences between positions - 1 3 8 and - 6 8 are still able to drive a correct temporal and spatial expression of a reporter gene from the Fbpl promoter, but at a

very reduced level. As a further test of the role of the - 138 to - 68 segment it was deleted from the - 1386 to + 80 Fbpl fragment in the HAHp:Adh construct. As i n d i c a t e d by the somatic assay (Fig. 5E) and Northern blot analysis of transgenic lines (Fig. 6A) the expres- sion of this construct was drastically reduced to a very low level as compared to the HHp:Adh construct. It could be then concluded that the - 1 3 8 to - 6 8 se- quences play an essential role in the expression of the Fbpl gene and that more upstream sequences were not able to compensate for their removal.

Autonomous regulatory properties of the Fbpl upstream sequences fused to a heterologous promoter

The regulatory capacity of the - 1 3 8 to - 6 8 seg- ment and in particular its capacity to drive transcrip- tion in an autonomous manner was tested by fusing it to a minimal heterologous promoter. For this purpose, the pHZ50PL vector was selected because its contains

127

a minimal 50 bp hsp70 promoter linked to the lacZ g e n e ( H i r o m i a n d Gehring, 1987) which has been shown in numerous studies to be fully responsive to various regulatory sequences placed in front of it. This strategy also enabled us to use a sensitive histochemical detec- tion procedure.

The expression of four constructs was investigated. The first construct, HS : hsp : lacZ, containing Fbpl sequences from -1 3 8 6 to - 6 8 , was used to test the effect of the replacement of the Fbpl endogenous promoter by the heterologous hsp70 promoter in the context of the long upstream sequences, which were shown in the previous section to drive, faithfully, the transcription of the Fbpl :Adh hybrid gene at a high level. The second construct, AS:hsp : l acZ , contained the - 1 3 8 to - 6 8 sequences only. The two other constructs, SH : hsp : lacZ and SA : hsp : lacZ, contained the same Fbpl fragments in a reverse orientation. The four constructs were injected into the ecd ~ts recipient strain and a total of 40 transgenic lines i.e. 8 to 12 lines per construct, were established.

20 ° 29 ° 29 °

3 hrs 5 hrs 3 hrs

C T -E +E -E +E -E +E

a b c d e f c' d'

Fbp I

Fbp I :Adh

A

Fbp2---I~ C

Fig. 4. 20-Hydroxyecdysone inducibility of the transcription of the HHp:Adh construct. Larvae from the HHp:Adh 5 transgenic line were reared at 20°C for 74 h after egg eclosion, transferred to 29°C and maintained at this temperature for 40 h. Larvae were then transferred to a thick paste of baker's yeast not supplemented ( - E ) (lanes c, e and c') or supplemented with 10 mg/ml 20-hydroxyecdysone (+ E) (lanes d, f and d'). Samples were taken after 3 h (lanes c and d, c' and d') and after 5 h (lanes e and f) of treatment. Lanes c' and d' are a longer exposure of lanes c and d, in order to show that an induced Fbpl:Adh transcript is already present after 3 h of treatment, but at a lower amount. As a control of normal transcription at the end of the third instar, larvae from the same transgenic line (T, lane b) and from the original ecd ~ts, ry 5°6 recipient strain (C, lane a) were kept at 20°C. RNA extraction from dissected fat body and Northern blot analysis of 10/zg total RNA samples were as described in Materials and Methods. A Fbpl-specific probe (see Materials and Methods) was used to detect simultaneously the Fbpl endogenous transcript and the Fbpl-Adh hybrid transcript. As a control for the amount of fat body RNA present in each sample, the same blot was probed with a Fbp2 probe, as it has been shown that the level of expression of this gene is not affected by the transfer to non permissive

temperature and the subsequent addition of 20-hydroxyecdysone under these conditions (Lepesant et al., 1982).

,,.)

)0

[ 11

Q. "I" "I"

i

• "I" " r

<C <:: 3: Z u

129

probe 437 bp

protected fragment

<1---181 bp

n o r t h e r n $1 nuclease p r o t e c t i o n

©

HHp:Adh

Hp

AHp:Adh

A Hp X

H /US Hp E / / m

H Hp:Adh I =

Fig. 6. Expression of .~Hp :Adh and H,:IHp :Adh constructs in transgenic lines. (A) Northern blot analysis of Fbpl :Adh transgenic lines. Twenty /~g (HHp line) or 25/~g (AHp and HAHp lines) of total RNA extracted from from staged ]ate third instar larvae were analyzed as described in the legend of Fig. 3 and Materials and Methods. (13) $1 protection assay of the hybrid transcripts was carried out as described in Materials and Methods. Control lane: 20 ng of the 5' end-labelled 43"7 bp probe incubated without any Drosophila RNA added. 10/~g (lane HHp) and 20/~g (lane ,ad-]p) of fat body RNA extracted from ]ate third instar larvae of the transgenic line HHp:Adh#5 and ,M-]p:Adh# 1 were hybridized with

the same amount of probe before $1 nuclease treatment. (C) Structure of the HHp:Adh, ,~-]p:Adh and H AHp:Adh constructs.

Tissue-specificity of expression of the gene fusions A strong staining was consistently observed in the

late larval fat body of all 40 transgenic lines (see

examples in Figs. 7 and 8). As judged from the inten- sity of histochemical staining the level of expression was higher in the transgenic lines harboring the long

Fig. 5. Histochemical assay for the expression of the Fbpl :Adh fusion gene. (A-E) Somatic transformation. DNA constructs were injected into Adh nuu embryos and the resulting late third instar larvae were dissected and histochemically stained for alcohol dehydrogenase activity. The panels show typical examples of staining of the fat body tissue. (A) Control pSXA1A plasmid containing the D. melanogasterAdh gene (Posakony et al., 1986); (B) H H p : A d h construct containing Fbpl sequences from - 1386 to + 80 (see Fig. 2A). In this case, the level of expression of the construct was found identical to that of the control Adh gene by all criteria mentioned in the Materials and Methods. (C) ASt :Adh construct (Fbpl sequences from - 138 to + 48). Both the staining intensity and the frequency of stained larvae were reduced as compared to the H H p : A d h construct; (D) SHp:Adh construct (Fbpl sequences from - 6 8 to + 80). Staining was very weak. The number of positive larvae was very reduced and stained cells were infrequent in all larvae tested; (E) H A H p : A d h (Fbpl sequences from -1386 to - 1 3 8 and - 6 8 to+80) construct. The frequency of positive larvae and of stained cells per larva was similar to that observed with the ASt : Adh construct. (F) Germ line transformation. Dissected fat body from a late third instar larva from the transgenic line H H p : A d h # 1 1 stained for ADH activity. Scale bars: 100 #m. g: gonad,

sg: salivary gland.

130

direct construct (HS:hsp:lacZ) than the three others. Typically, the larval fat body was heavily stained within 3 h at 37 ° in the HS:hsp: lacZ series, whereas it took 16 h at 37 ° to reach the same staining intensity in the three other series. A major conclusion from these results is that the -138 to -68 sequences are able to drive transcription from the heterologous hsp70 mini- mal promoter in the larval fat body cells irrespective of their orientation relative to the transcription start.

In contrast with the consistent expression of the constructs in the fat body, /3-galactosidase activity was scarcely detected in other tissues. In a small number of lines (1 or 2 over 40 for each tissue) staining was detected in tracheae, gastric caeca and midgut. In a larger number of lines staining was detected in proven- triculus (13/40), Malpighian tubules (MT) (25/40) and central nervous system (CNS) (40/40) (data not shown). For all four constructs the detailed subtissular pattern of stained cells was very variable from line to line in the three tissues. This suggested strongly that the ex- pression of the constructs in the proventriculus, MT and CNS was due to genomic position effects on the transgenes. By contrast, for all four constructs and in 31/40 lines an identical characteristic pattern of a cluster of a few stained cells was observed in the anterior region of the salivary glands. The same pat- tern was observed in one transgenic line bearing one inserted copy of the HZ50PL vector without any Fbpl sequences added (data not shown). It is noteworthy that a similar observation has been reported by Roark et al. (1990) in both somatic and germ line transforma- tion assays using a hsp:lacZ vector bearing a 194 bp hsp70 promoter. Thus, this pattern could be due to a low basal level of expression of the hsp:lacZ minimal construct. Alternatively, the possibility that it reflects a previously undetected expression of the Fbpl gene in the salivary glands cannot be ruled out.

Subtissular pattern of expression in the fat body An unexpected feature was revealed when larvae

from the HS : hsp : lacZ lines were precisely staged dur- ing the whole third instar, and stained for/3-galactosi- dase activity. A differential expression appeared within the fat body with a gradient along the anteroposterior axis, different parts of the tissue being stained at different times in a reproducible sequence (Fig. 7). lacZ expression was first detected in the anteriormost region of the fat body that forms a distinct lobe near the salivary glands. On the basis of morphological and

metabolic criteria, this lobe has been defined by Rizki (1978) as region 1 of the larval fat body. lacZ expres- sion then extended gradually over the more posterior regions of the fat body tissue, giving rise to a mosaic staining and eventually reached a higher and more homogenous level in all fat body cells. A following step was reached when staining faded in region 1 at the time larvae stopped wandering, just prior to the onset of pupariation. At a later stage corresponding to the onset of dissociation and dispersion of the tissue dur- ing the prepupal period, staining decreased in other parts of the fat body (not shown). This pattern was reproducibly observed in several independent H S : h s p : l a c Z transgenic lines and also in the AS:hsp:lacZ transgenic lines bearing only the 70 bp element in the direct orientation in front of the hsp70 promoter. A similarly evolving spatial pattern of ex- pression was observed in the HHp:Adh transgenic lines (see Fig. 5F for an example of fading of histo- chemical staining in region 1 in a late third instar larva). In these lines however, the expression of the transgene at early stages could not be determined accurately because of the lack of sensitivity of the ADH histochemical assay as compared to the X-gal staining assay for/3-galactosidase activity.

Provided that the expression of the hybrid con- structs matches faithfully that of the Fbpl gene, these results furnish the first indication of the earliest time of expression of the gene during the third instar and reveal an unexpected spatial differentiation for this process.

20-hydroxyecdysone induction 20-hydroxyecdysone inducibility of the Fbpl-driven

expression of the hsp : lacZ reporter was monitored by X-gal histochemical staining for all four constructs. In each case, at least two independent transgenic lines were tested and a clear induction of /3-galactosidase activity was observed upon 20-hydroxyecdysone feeding of the ecdysteroid-deficient larvae for 3 or 5 h. Fig. 8 gives an example of this induction in the case of an AS:hsp: lacZ line. This result was confirmed by a Northern blot analysis of the lacZ transcript in the case of one HS : hsp :lacZ and one AS : hsp :lacZ line (Fig. 9). Ectopic staining in other tissues, when it occurred in the tested lines, appeared to be 20-hydroxyecdysone- independent. Such a constitutive expression is illus- trated in Fig. 8 for Malpighian tubules in the case of the AS : hsp : lacZ line.

Fig. 7. Developmental expression of the HS: hsp: lacZ transgene during the third instar. Histochemical staining for/3-galactosidase activity of fat body tissue of third instar larvae of the HS:hsp:lacZ#1 transgenic line. Times refer to h after egg eclosion at 20 °. Scale bars: 200 p,m; Other tissues not separated from the fat body are salivary glands (sg), gonad (g) and Malpighian tubules (Mt). The anteriormost region of the fat body

defined by Rizki (1978) as region 1 is indicated by an open arrow.

66h

13]

74h

104 h

160 h

180 h

132

- 20 O H - e c d y s o n e + 20 O H - e c d y s o n e Fig. 8. 20-Hydroxyecdysone inducibility of the AS:hsp:lacZ transgene. Larvae (AS:hsp: lacZ#2 transgenic line) were reared in conditions described in Fig. 4, and tested for reporter gene expression by histochemical staining. Right panels without, left panels with 10 mg/ml 20-hydroxyecdysone added for 5 h. Upper panels: fat body including a gonad (g) or salivary glands (sg). Lower panels: gut and Malpighian tubules

(Mt) of the same larvae.

133

Discussion

The hybrid constructs as faithful models for Fbpl expres- sion

The expression of the HHp:Adh construct mimics faithfully the expression of the Fbpl gene in all tests

® It

HS:hsp:lacZ

we used. The developmental profile and the tissue specificity of expression of the endogenous gene are preserved, a full 20-hydroxyecdysone inducibility is maintained, and the level of expression of the hybrid message is similar to that of the endogenous one. This is a strong indication that Fbpl sequences from - 1386

$

I

A $

I

( ~ AS:hsp: lacZ#2

kb

HS:hsp: lacZ#1

2 0 = 29 = 2 0 = 29 =

3 hrs 5 hrs 3 hrs 5 his

C T -E +E -E +E T -E +E -E +E

a b c d e f g h i j k

3 . 5 m

0 ° 9 m

hsp:lacZ

rp49

3 . 5 m Fbpl

Fig. 9. 20-Hydroxyecdysone inducibility of the transcription of the AS :hsp: lacZ and HS:hsp : lacZ constructs. Experimental conditions for rearing of larvae and 20-hydroxyecdysone induction test were as described in the legend of Fig. 4. Larvae from either the AS : hsp : lacZ 2 or the HS:hsp : i acZ 1 transgenic lines were either not supplemented ( - E ) (lanes c, e, h and j) or supplemented with 10 m g / m l 20-hydroxyecdysone ( + E) (lanes d, f, i and k). Samples were taken after 3 h (lanes c, d, h and i) and after 5 h (lanes e, f, j and k) of treatment. As a control of normal transcription at the end of the third instar, larvae from the same transgenic lines (T, lanes b and g) and from the original ecd its, ry 5°6 recipient strain (C, lane a) were kept at 20°C. RNA extraction from dissected fat body and Northern blot analysis of 20/~g total RNA samples were as described in Materials and Methods. A hsp:lacZ specific probe (see Materials and Methods) was used for the detection of the hybrid hsp: lacZ RNA transcript. The same blot was hybridized with a probe for the ribosomal protein gene rp49 (Vaslet et al., 1980) as a control of the amount

of RNA loaded. As a control of 20-hydroxyecdysone induction, the same blot was dehybridized and rehybridized with a Fbpl specific probe.

134

to + 80 include most if not all regulatory elements required for the correct developmental and ecdysteroid regulation of the expression of the Fbpl gene at a high level. The only noticeable difference between the ex- pression of the Fbpl endogenous gene and the H H p : A d h construct is the time lag in the appearance of the transcript either in normal physiological condi- tions or in the 20-hydroxyecdysone induction test. It remains to be determined if this is due to the lack of relevant regulatory Fbpl sequences in the construct or to an intrinsic property of this combination of Fbpl and Adh sequences.

These results imply that Fbpl sequences upstream of - 1386 do not play any important regulatory role. As for sequences downstream from the transcription start, two fusion sites were tested, + 80 and + 48, the + 48 site being tested in the somatic transformation assay only. Interestingly the 3'-deleted sequences include the 59 bp Fbpl intron. On the other hand the two introns of the Adh gene were included within the reporter fused sequences. Since these introns had been shown previously to be devoid of any regulatory function (Shen et al., 1989), the lack of the Fbpl intron could not have been compensated for by the intronic Adh sequences. It can thus be concluded that Fbpl se- quences downstream of + 48, including the intron, are dispensable. Bingham et al. (1988) pointed out that Drosophila intronic cis-acting sequences have always been reported in long introns, whereas short introns are apparently not functional. Some ecdysone-respon- sive genes fit the rule, since regulatory elements have been located in the large first intron of the ~Tub60D (t33-tubulin) gene (Gasch et al., 1989), including 20-hy- droxyecdysone inducibility (Bruhat et al., 1990), whereas the short Fbpl intron like that of the 73 bp intron of the sgs3 gene (Mettling et al., 1987) appears to be dispensable. Short introns of a similar small size, ca. 60 bp (Hawkins, 1988), occur at a 5' proximal location in a number of Drosophila genes, and in particular in ecdysone-inducible ones (see Apple and Fristrom, 1991). The recurrence of this observation is intriguing and raises the question as to whether or not these short introns may have retained an as yet undis- covered function.

The properties of the H H p : A d h construct, i.e. faithful expression regarding to developmental, tissue specific and ecdysteroid regulation, are well preserved in the HS : hsp : lacZ construct, where the Fbpl pro- moter and leader sequences are replaced by those of the heterologous hsp70 gene. This provides further evidence that essential specific regulatory sequences of the Fbpl gene lay upstream of the TATA box.

A subtissular pattern of expression extending gradu- ally from region 1 to more posterior regions of the fat body was revealed by the regionalised expression of the hsp:lacZ constructs. It is interesting to note that a

regional pattern of expression has been also described for the expression of a glue gene in salivary glands of D. simulans (ThiJroff et al., 1992). The molecular basis of such a differential expression within a single tissue remains to be elucidated.

A proximal enhancer comprising an ecdysone response element

The - 1 3 8 to - 6 8 segment when isolated from upstream Fbpl sequences is still able to drive tran- scription from the Fbpl or the heterologous hsp70 promoter. This results in a drastically reduced level of transcript accumulation when compared to that from the endogenous gene or from constructs including longer upstream sequences. It suggests that essential regulatory elements are located in sequences upstream of -138 . A further deletion scanning of these se- quences by a genetical method has led to a better characterization of such elements (Lapie et al., 1993).

The c/s-acting sequences contained in the - 1 3 8 to - 6 8 region are functional in an orientation-independ- ent manner and thus share one characteristic with enhancer elements. These sequences are also probably functional in a distance-independent manner although this is based solely on the inversion of the -1 3 86 to - 6 8 region and not on increasing the distance of the - 1 3 8 to - 6 8 segment from the TATA box by inter- vening DNA of unrelated origin. It cannot thus be formally excluded that in the inverted construct more upstream Fbpl sequences when brought closer to the transcription start compensate for the effect of shifting at a distance the 70 bp element.

The 70 bp segment operates in vivo as a c/s-acting ecdysone response element. Such elements were func- tionally characterized to a comparable resolution level in the case of only three genes, hsp27, hsp23 and Eip28/29. In part because of the complexity of their pattern of expression in the whole animal (Andres and Cherbas, 1992; Cohen and Meselson, 1985; Hoffman and Corces, 1986; Pauli et al., 1986; Pauli et al., 1990), the functional definition of the ecdysone response ele- ments of these genes remains so far based solely on transfection experiments carried out with Drosophila cells in culture (Cherbas et al., 1991; Luo et al., 1991; Mestril et al., 1986; Riddihough and Pelham, 1987). By contrast, the simple pattern of expression of the Fbpl gene and its null or undetectable basal level of tran- scription prior to its induction in the third instar facili- tated this first functional characterization, at the level of the whole organism, of a discrete ecdysone response element using a direct ecdysteroid induction assay. In a previous study (Maschat et al., 1991; Maschat et al., 1990) a 15 bp imperfect palindromic sequence sharing some similarity with the EcRE of the hsp27 gene (Riddihough and Pelham, 1987) and the EcRE consen-

sus derived by Cherbas et al. (1991) had been identi- fied in the -138 to -68 region. We have confirmed recently that a 17 bp oligonucleotide encompassing this 15 bp sequence is a bona lute binding site for the EcR-USP heterodimer in vitro (Antoniewski et al., 1993 and manuscript submitted).

In addition to hormone induction, c/s-acting ele- ments located within the -138 to -68 segment re- strict expression of the Fbpl gene to the third larval stage and specify it to the larval fat body. In this study, these three features were not resolved in different discrete elements. Ecdysteroid inducibility, mediated by the binding of a receptor-hormone complex to a specific site when the ecdysteroid titer has reached a sufficient level, cannot be taken as a simple key to account for the induction of the Fbpl gene at the appropriate time. This proposal fails to account for the restriction of Fbpl expression to the fat body when no expression takes place in any other larval tissue at a developmental time when ecdysteroids are at their highest concentration since the beginning of develop- ment (Richards, 1981). If the specific regulatory traits of the Fbpl gene (stage and tissue specificity, hormonal regulation) are mediated by several distinct transcrip- tion factors, the short length of the 70 bp enhancer leads to speculate that these factors have to interact with each other in some way. In this respect the -138 to -68 segment resembles a composite element in which target sequences for multiple trans-acting factors are very close or overlapping as discussed by Miner and Yamamoto (1991), for mammalian hormonally controlled genes. The identification in a nuclear ex- tract from late third instar larvae (Antoniewski et al., submitted) of several trans-acting factors with well-re- solved specific sites in the -138 to -68 region in addition to the EcR-USP heterodimer provides a first argument supporting this hypothesis.

Materials and Methods

Fly stocks and rearing conditions

Most aspects of the present work were based on the use of the ecd ~ts conditional thermosensitive mutant. Shifting early third instar homozygous larvae at the non-permissive temperature (29°C) drastically reduces the ecdysteroid titer at the end of the third instar, thus preventing them from pupariating (Garen et al., 1977). An ecd its, st, ry 5°6 stock was used as a recipient for transgenesis experiments (see below). Rearing of flies and stock maintenance on a standard corn meal, sugar, brewer's yeast medium supplemented with live yeast was performed at the permissive temperature (20°C), unless otherwise stated for the needs of the experi- ments. When used, supplemented or special media are described below in the appropriate sections.

Hybrid DNA constructs

135

Fbpl :Adh fusion genes Fbpl upstream fragments of various length were

fused with the D. melanogaster Adh coding and 3' flanking sequences. They are designated according to the restriction sites used, taking the transcription start site of the Fbpl gene as + 1 for coordinates (Maschat et al., 1990). HHp: HindlII (-1386) -HpaI (+80); HAHp: same as HHp but with a deletion between AluI (-138) and StuI (-68); MHp: MspI (-256) -HpaI (+80); AHp: AluI (-138) -HpaI (+80); ASt :Alu ( - 138) - Sst I ( + 44); SHp: StuI ( - 68) -Hpa I (+ 80). Adh sequences extended 3' to either the EcoRI site or the XbaI site, since both fragments had been previously proved to be efficient (Benyajati et al., 1987).

pHHp:Adh construct. The pHK10 plasmid was de- rived from the transformation vector pHXPR4 (Maschat et al., 1986) by deleting the bacterial Ecogpt coding sequence, the SV40 polyadenylation and termi- nation sequences and the Drosophila rosy sequences. A HindlII-EcoRI fragment containing the coding se- quence as well as the termination and polyadenylation signals of the Adh gene was isolated from the pHAP plasmid (Bonner et al., 1984). It was rendered blunt- ended by filling it with the Klenow enzyme and in- serted into the HpaI and Asp 718 sites of pHK10 also rendered blunt-ended. This resulted in the pHHpA10 plasmid in which Fbpl sequences from - 1386 to + 80 were fused to the Adh sequence at position + 13 (Martin et al., 1989). The pHHp:Adh plasmid was obtained by cloning the 7.3 kb HindlII fragment that included the rosy gene into the EcoRI site of pHHpA10.

pHAHp:Adh construct. The BgllI (-776) -StuI ( -68) Fbpl fragment of pHHpA10 was replaced by a BgllI (-776) -AluI (-138) fragment and the 7.3 kb HindlII rosy fragment was inserted as described above.

pSHp:Adh construct. The pHHpA10 plasmid was deleted from sequences between the EcoRI site up- stream of the HHp Fbpl sequence and the StuI ( -68) site. Religation resulted in the creation of an EcoRI site into which the 7.3 kb HindlII rosy fragment was inserted.

pMHp :Adh, pAHp :Adh and pASt :Adh constructs. These were obtained by inserting the appropriate Fbpl fragment upstream of the promoterless Adh gene in the pDm29 vector (Mismer and Rubin, 1987) which allows neomycin selection of transformants.

Fbpl : hspTO : lacZ fusion genes The starting point for these constructs was the

pHZ50PL plasmid (Hiromi and Gehring, 1987). The insertion of the Fbpl HS (-1386 to -68) and AS ( -138 to -68) fragments into the blunt-ended Not I

136

site of pHZ50PL resulted in four plasmids, pHS: h s p : l a c Z , p S H : h s p : l a c Z , p A S : h s p : l a c Z and pSA: hsp : lacZ with the Fbpl sequence in the direct or reverse orientation with respect to the hsp70 pro- moter.

Transformation assays

Preparation of DNAs, injection procedure and post-injection handling of embryos and larvae were essentially as described by Spradling (1986).

Somatic transformation assay The assay was carried out essentially as described by

Martin et al. (1986). This procedure allows one to gain information about the biological activity of a relevant construct at the tissular level within one week, instead of the one to two months needed to obtain transgenic lines. The injected DNA is distributed randomly during cell divisions. One limitation of this assay is that not all of the individuals express the injected gene, and in those individuals where there is expression, not all cells of the relevant tissue(s) express the reporter gene leading to a random mosaic pattern (see Deutsch et al., 1989; Martin et al., 1986). The level of expression of the injected constructs was estimated according to three criteria: the proportion of larvae staining positively for A D H activity, the number of stained cells per larva and the intensity of staining (Roark et al., 1990; Todo et al., 1990). Recombinant plasmids to be tested were in- jected into early embryos at a concentration of 400 /xg/ml. In some experiments where the same set of injected embryos was used for the somatic and germ line assay, a P helper plasmid (p~-25.7wc) was injected together with the recombinant plasmid at a concentra- tion of 100 t zg /ml as a source of transposase (Karess and Rubin, 1984) Larvae hatching from the injected embryos were reared at 20°C in individual vials of standard medium supplemented with 0.05% Bro- mophenol Blue (Maroni and Stamey, 1983). The clear- ance of the blue dye from the gut was used as an a posteriori criterion, together with morphological and behavioral criteria such as body shortening, eversion of anterior spiracles, arrest of wandering and sticking to the walls of the tube, to select late third instar larvae for dissection and histochemical staining. We estimate that larvae fitting all these criteria were within 30 rain of the onset of pupariation.

Germ line transformation DNA of the recombinant (250 p .g /ml) and helper

plasmid pTr25.7wc (50 ~ g / m l ) w e r e microinjected in embryos of an ecd its, st, ry 5°6 recipient stock. (ry +) transformants were screened for eye color and neo R transformants were selected by raising larvae on com- mercially available ready-mix Drosophila dried food

(Philip Harris International Ltd) supplemented with 1 m g / m l of the antibiotic G418 (Geneticin Sulfate, Gibco BRL) (Steller and Pirrotta, 1985). The number of transgenes in each line was determined by Southern blotting. For histochemical detection of A D H activity, an Adh nu" allele was introduced by classical crosses and recombina t ion into previously established Adh+transgenic lines. Introduction of the Adh nu" al- lele and selection for recombinants and transformants were facilitated by the construction of a CyO, Adh nB, pr, cn; TM3, e, Sb, Ser, ryRK/T(2;3), ap x~ stock. In the AS:hsp:lacZ series, a low number of transgenic lines were obtained from injection. In order to ascertain their pattern of expression, new transgenic lines were obtained by transposing to autosomal locations a previ- ously X-located transposon, using the chromosomal source of transposase P[ry, A2-3](99B) (Robertson et al., 1988) and appropriate crosses.

Ecdysone induction test

Conditions were essentially as described previously (Maschat et al., 1986). Eggs were collected for 2 h periods from 4-10 day old adult females of the recipi- ent ecd ~ts stock or transgenic derivatives and main- tained at 25°C. The following day, first instar larvae were collected every hour at hatching and reared at 20°C at a maximum density of 50 larvae per vial on standard medium supplemented with bromophenol blue. In these conditions, the third instar starts (second molt) at 64 h after hatching and spans about 192 h before the onset of pupariation. At 10 h after the second molt, a sample of larvae was transferred to 29°C, while a control sample remained at 20°C. After 40 h at 29°C, 10 to 20 larvae were collected, washed and transferred into an Eppendorf tube containing a thick paste of live yeast supplemented or not with 20-hydroxyecdysone at 10 m g / m l final concentration. After 3-5 h of treatment, larvae were harvested, rapidly rinsed with cold phosphate buffer saline (PBS), and dissected at 4°C, for either RNA extraction or histo- chemical staining.

Histochemical staining procedures

Larvae were dissected in PBS (pH 7.5) at 4°C. Histochemical staining of dissected tissues for ADH activity detection was performed according to the pro- cedure described in Martin et al. (1986), with the modifications described in Martin et al. (1989). X-gal staining of /3-galactosidase activity was performed ac- cording to Ashburner (1989). The preparations were mounted in glycerol and stored at 4 ° in the dark. Photographs were taken on a Wild-Leitz Kombistereo photomicroscope equipped with a Wild-Leitz MPS46

Photoautomat on Kodak Ektachrome 160T profes- sional films.

Transcript detectiotzs

Total RNA was prepared as previously described (Maschat et al., 1986) from fat bodies of staged larvae rapidly dissected at 4°C in cold PBS. Electrophoresis in MOPS buffer in 1.5% agarose-MOPS/formaldehyde gels, transfer in 20 X SSC onto nitrocellulose mem- branes (Schleicher & Schuell), and hybridization proce- dures were as described in Sambrook et al. (1989).

A single-stranded Fbpl specific probe was gener- ated from a M13mpl0 recombinant phage containing a Hpa I( + 80) -Msp I( - 256) fragment. The complemen- tary strand was synthesized and labelled by polymeriza- tion with the Klenow enzyme using the M13 hybridiza- tion probe primer (Biolabs), in such a way that the Fbpl fragment remained single-stranded. This proce- dure yielded a high specific activity probe hybridizing with the first 80 nucleotides common to the endoge- nous Fbpl and the hybrid Fbpl :Adh transcripts, rp49 and Fbp2 probes were labelled by nick-translation of appropriate plasmids (Rat et al., 1991; Vaslet et al., 1980). A specific hsp:lacZ probe was generated by preparing the appropriate XbaI-SalI fragment from the HZ50PL plasmid (Hiromi and Gehring, 1987).

S1 nuclease protection assay

Ten to 20 tzg of total fat body RNA (corresponding to approximately 1 to 2 ng of Fbpl RNA, respectively) and 20 ng of the labelled probe were coprecipitated with ethanol. A Fbpl :Adh MspI-MspI fragment puri- fied from the pHHp:Adh plasmid was used as a probe. This probe encompassed 336 bp of Fbpl sequences and 101 bp of Adh ones; it hybrized specifically to the fusion message onto 181 nt. Preparation of the S1 nuclease-resistant RNA-DNA hybrids was carried out essentially according to the procedure of Favaloro et al. (1980) except that digestion was performed at 37°C for 30 min in the presence of 1800 units of S1 nuclease. The Sl-resistant RNA-DNA hybrids were analyzed by electrophoresis on denaturing 6% polyacrylamide gels.

Acknowledgements

We are grateful to C. Benyajati and J. Posakony for Adh plasmids, to Y. Hiromi and W. Gehring for the pHZ50PL plasmid, M. Ashburner, P. Martin and W. Sorer for information and fly stocks and R. Dettman and A. Kropfinger for reading of the manuscript. This work was supported by grants from the Centre Na- tional de la Recherche Scientifique, the Institut Na- tional de la Sant6 et de la Recherche M6dicale (grant

137

88-1011), the Fondation pour la Recherche M6dicale, and the Ligue Nationale contre le Cancer to J.-A.L.

References

Amin, J. Mestril, R. and Voellmy, R. (1991) Mol. Cell. Biol 11, 5937-5944.

Andres, A. and Cherbas, P. (1992) Development 116, 865-876. Andres, A.J. and Thummel, C.S. (1992) Trends Genet 8, 132-138. Andres, J.A., Fletcher, J.C., Karim, F.D. and Thummel, C.S. (1993)

Development, in press. Antoniewski, A., Laval, M. and Lepesant, J.-A. (1993) Insect

Biochem. Molec. Biol. 23, 105-114. Apple, R.T. and Fristrom, J.W. (1991) Dev. Biol. 146, 569-582. Belinski-Deutsch, S., Busson, D., Lamour-Audit, C., Pocheron, P.,

Morini~re, M. and Berreur, P. (1983) J. Insect Physiol. 29, 509- 514.

Benyajati, C., Ayer, S., McKeon, J., Ewel, A. and Huang, J. (1987) Nucl. Acids Res. 15, 7903-7920.

Berreur, P., Porcheron, P., Morini~re, M., Berreur-Bonnenfant, J., Belinski-Deutsch, S., Busson, D. and Audit-Lamour, C. (1984) Gen. Comp. Endocrinol. 54, 76-84.

Bingham, P.M., Chou, T.-B., Mims, I. and Zachar, Z. (1988) Trends Genet. 4, 134-138.

Bonner, J.J., Parks, C., Parker-Thornburg, J., Martin, M.A. and Pelham, H.R.B. (1984) Cel 37, 979-991.

Bruhat, A., Tourmente, S., Chapel, S., Sobrier, M.L., Couderc, J.L. and Dastugue, B. (1990) Nucl. Acids Res. 18, 2861-2865.

Cherbas, L., Lee, K. and Cherbas, P. (1991) Genes Dev. 5, 120-131. Cohen, R.S. and Meselson, M. (1985) Cell 43, 737-746. Deutsch, J., Laval, M., Lepesant, J.A., Maschat, F., Pourrain, F. and

Rat, L. (1989) Dev. Gen. 10, 220-231. Dobens, L., Rudolph, K. and Berger, E. (1991) Mol. Cell. Biol. 11,

1846-1853. Favaloro, J., Treisman, R. and Kamen, R. (1980) In Grossman, L.

and Moldave, K. (eds.), Methods in Enzymology. Academic Press, New York, Vol. 65, pp. 718-749.

Garen, A., Kauvar, L. and Lepesant, J.A. (1977) Proc. Natl. Acad. Sci. USA 74, 5099-5103.

Gasch, A., Hinz, U. and Renkawitz-Pohl, R. (1989) Proc. Natl. Acad. Sci. USA 86, 3215-3218.

Hawkins, J.D. (1988) Nucl. Acids Res. 16, 9893-9908. Hiromi, Y. and Gehring, W.J. (1987) Cell 50, 963-974. Hoffman, E. and Corces, V. (1986) Mol. Cell. Biol. 6, 663-673. Karess, R.E. and Rubin, G.M. (1984) Cell 38, 135-146. Karim, F.D. and Thummel, C.S. (1992) EMBO J. 11, 4083-4093. Koelle, M.R., Talbot, W.S., Segraves, W.A., Bender, M.T., Cherbas,

P. and Hogness, D.S. (1991) Cell 67, 59-77. Lapie, P., Nasr, F., Lepesant, J.A. and Deutsch, J. (1993) Genetics,

in press. Lepesant, J.A., Kejzlarov~-Lepesant, J. and Garen, A. (1978) Proc.

Natl. Acad. Sci. USA 75, 5570-5574. Lepesant, J.A., Levine, M., Garen, A., Lepesant-Kejzlarov,~, J., Rat,

L. and Somme-Martin, G. (1982) J. Mol. Appl. Genet. 1,371-383. Lepesant, J.A., Maschat, F., Kejzlarov~-Lepesant, J., Benes, H. and

Yanicostas, C. (1986)Arch. Insect. Biochem. Physiol., sl, 133-141. Luo, Y., Amin, J. and Voellmy, R. (1991) Mol. Cell Biol. 11,

3660-3675. Maroni, G. and Stamey, S. (1983) Drosophila Inf. Serv. 59, 142-143. Martin, M., Giangrande, A., Ruiz, C. and Richards, G. (1989)

EMBO J. 8, 561-568. Martin, P., Martin, A., Osmani, A. and Sorer, W. (1986) Dev. Biol.

117, 574-580. Martinez, E., Givel, F. and Wahli, W. (1991) EMBO J. 10, 263-268.

138

Maschat, F., Dubertret, M.L. and Lepesant, J.A. (1991) Mol. Cell. Biol. 11, 2913-2917.

Maschat, F., Dubertret, M.L., Th6rond, P., Claverie, J.M. and Lepe- sant, J.A. (1990) J. Mol. Biol. 214, 359-372.

Maschat, F., Roux, J., Benes, H., Pictet, R., Jami, J. and Lepesant, J.A. (1986) EMBO J. 5, 583-588.

Mestril, R., Schiller, P., Amin, J., Klapper, H., Ananthan, J., A. and Voellmy, R. (1986) EMBO J. 5, 1667-1673.

Mettling, C., Giangrande, A. and Richards, G. (1987) J. Mol. Biol. 196, 223-226.

Miner, J.N. and Yamamoto, K.R. (1991) Trends Biochem. Sci. 16, 423-426.

Mismer, D. and Rubin, G.M. (1987) Genetics 116, 565-578. Nakanishi, Y. and Garen, A. (1983) Proc. Natl. Acad. Sci. USA 80,

2971-2975. Ozyhar, A. and Pongs, O. (1993) J. Steroid Biochem. Molec. Biol. 46,

135-146. Ozyhar, A., Strangmann-Diekmann, M., Kiltz, H.H. and Pongs, O.

(1991) Eur. J. Biochem 200, 329-335. Paco-Larson, L., Nakanishi, Y., Levine, M. and Garen, A. (1986)

Dev. Genet. 7, 197-203. Pauli, D., Spierer, A. and Tissi~res, A. (1986) EMBO J. 5, 755-761. Pauli, D., Tonka, C.-H., Tissibres, A. and Arrigo, A.-P. (1990) J. Cell

Biol. 111, 817-828. Posakony, J.W., Fischer, J.A. and Maniatis, T. (1986) Cold Spring

Harbor Symp. Quant. Biol. 50, 515-520. Rat, L., Veuille, M. and Lepesant, J.A. (1991) J. Mol. Evol. 33,

194-203. Richards, G. (1981) Mol. Cell. Endocrinol. 21, 181-197. Richards, G. (1992) Current Biol. 2, 657-659.

Riddihough, G. and Pelham, H.R.B. (1986) EMBO J. 5, 1653-1658. Riddihough, G. and Pelham, H.R.B. (1987) EMBO J. 6, 3729-3734. Rizki, T.M. (1978) In Ashburner, M. and Wright, T.R.F. (eds.), The

Genetics and Biology of Drosophila. Academic Press, London, Vol. 2b, pp. 561-597.

Roark, M., Raghavan, V., Todo, T., Mayeda, C.A. and Meyerowitz, E.M. (1990) Dev. Biol. 139, 121-133.

Robertson, H.M., Preston, C.R., Phillis, R.W., Johnson-Schlitz, D.M., Benz, W.K. and Engels, W.R. (1988) Genetics 118, 461-470.

Sambrook, J., Fristch, F.F. and Maniatis, T. (1989) In: Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory.

Segraves, W.A. and Richards, G. (1990) lnvertebr. Reprod. Dev. 18, 67-76.

Somme-Martin, G., Colardeau, J., Beydon, P., Blais, C., Lepesant, J.A. and Lafont, R. (1990) Arch. Insect Biochem. Physiol. 15, 43-56.

Spradling, A.C. (1986) In Roberts, D.B. (eds.), Drosophila a Practical Approach. IRL Press., Oxford, UK. pp, 175-197.

Steller, H. and Pirrotta, V. (1985) EMBO J. 4, 167-171. Talbot, W.S., Swyryd, E.A. and Hogness, D.S. (1993) Cell 73, 1323-

1337. Thomas, H.E., Stunnenberg, H.G. and Stewart, A.F. (1993) Nature

362, 471-475. Thiiroff, E., St6ven, S. and Kress, H. (1992) Mech. Dev. 37, 81-93. Todo, T., Roark, M., Raghavan, K.V., Mayeda, C. and Meyerowitz,

E. (1990) Mol. Cell. Biol. 10, 5991-6002. Vaslet, C.A., O'Connel, P., Izquierdo, M. and Rosbach, M. (1980)

Nature 285, 674-676. Yao, T.P., Segraves, W.A., Oro, A.E., McKeown, M. and Evans,

R.M. (1992) Cell 71, 63-72.