Oct 2, 1995 - ... for the design of anti-plasmodial constructs at a later stage. ... interactions between the host and the parasite are necessary for the eventual ...
The EMBO Journal vol.15 no.2 pp.344-350, 1996
Conserved function of Anopheles gambiae midgutspecific promoters in the fruitfly
G.Skavdis"4, I.Siden-Kiamosl, H.-M.MuIler2, A.Crisanti2'3 and C.Louis1' 45 'InstitLIte
of Molecular Biology and Biotechnology.
Foundation for
Research and Technology. 711 1) Heraklion. Crete. Greece, 21stituto di Parassitologia, Universit'I LTa Sapienza. Piala.ale Aldo Moro 5. 00 128 Rorna. Italy. Department ot Parasitology. Itiperial College of Science and Technology. L ondon. UK and 4Departimient of Biology. University of Crete. 711 10 Heraklion. Crete. Greece
5Corresponding author
Control of malaria by a methodology that would permit the effective blockage of the Anopheles gambiae midgut wall penetration by Plasmodium parasites requires a detailed understanding of both the physiology of the mosquito's digestion, and of the interactions between the parasite and its host. We have transformed Drosophila melanogaster with several constructs that allow the study of the promoter region of two of the major late trypsin genes of A.gambiae. Using several deletions, we have identified, for both genes, small genomic segments that are sufficient to confer tissue specificity to the promoter in a species that is far away in evolution from the mosquito. This will allow further studies that will enable both the understanding of the blood meal digestion, and may potentially be useful for the design of anti-plasmodial constructs at a later stage.
Ke,ywords:
A nopheles gamibiae/Drosophilai mneilaiogaister/
malaria/midgut/trypsin
genes
Introduction One of the main reasons why malaria still constitutes a central health problem in tropical countries can be found in the failure to control the anopheline vectors effectively. An intensification of the study of the biology of Anopheles gamnbiaei and A nopheles arabien.sis could ultimately lead to a control of malaria, or at least to a reduction of the problem in sub-Saharan Africa where, together with Anopheles fitnestus, they are collectively responsible for hundreds of millions of malaria cases per year, leading to > 1.5 million deaths. The prospects for a better understanding of the mosquito biology have been improved recently since several groups have initiated the use of molecular techniques that have led, among others, to the construction of a genetic map (Zheng et al., 1991, 1993; Collins and Besansky, 1994) and to better and faster diagnostics for the differentiation of meinbers of the A.ganbicae complex (Hill eit Cal., 1992; Scott et al., 1993; Wilkerson et cil., 1993; Favia et cli., 1994). In addition to the development of these new tools, molecular biology, through the study of more basic questions, has opened up ways that could lead ultimately to a better control of malaria through the genetic manipulation of the anopheline vectors (Jamiies,
1992; Collins, 1994: Curtis, 1994). It should be stressed, though, that the usefulness of molecular biology is not globally endorsed (Spielman, 1994). The mosquito midgut is a target tissue for the potential use of anti-parasitic chimaeric constructs, designed to block the transmission of Pla.inodica, ingested with an infected blood meal. Here, mating takes place, and development proceeds to the ookinete stage. After the penetration of the midgut epithelium, the oocyst will form, which will rupture to release sporozoites. Complex interactions between the host and the parasite are necessary for the eventual successful invasion of the salivary gland, and several genetically determined conditions have been identified in A.gambica that can block the process at different stages (Collins et al., 1986; Paskewitz et al., 1989; Vernick et al., 1995). Moreover, it is known that mosquito-encoded serine proteases, secreted into the midgut during the digestion of the blood meal, are important in mediating the journey of the ookinete through the peritrophic membrane (Rosenberg et al., 1984: Shahabuddin et al., 1993). Seven trypsin-encoding genes (although not necessarily the ones involved in the processes mentioned above) have been characterized in A.gambiae (Muller et al., 1993). These genes are located in an -1 1 kb long segment, but they are not coordinately expressed. The cluster includes some genes whose products can be distinguished only several hours after the ingestion of the blood and some that are expressed constitutively before blood feeding. These strictly midgut-specific genes, in addition to their importance in answering fundamental questions concerning the digestion physiology of Anopheles, could be used in designing chimaeric constructs that potentially would be utilized to express anti-parasitic agents, once germline transformation has been achieved for A.gambiaC. We decided to test the functionality of the promoter of two major late trypsin genes, Antrvpl and Anttrvp2, in transgenic Drosophila inielantiogacster, hoping that the fruitfly would have retained the ability to recognize the specific control elements of its distant relative.
Results To test whether the mosquito promoters are, in principle, recognized by Drosophila, we first subcloned the two genes to be studied in an appropriate transformation vector and introduced them in the germline of D.mnelanogastei(Figure IA). In the first construct (Tyl-2c4.8), both genes were present, including flanking sequences encompassing 1.7 kb upstream of the Antrvp2 gene and 0.4 kb downstream of the Ant-ApI gene. A second construct (Tylc5.2) was used to test Antrvypl by itself, the 3'-flanking sequence extending this time to 2.7 kb, while the upstream isequences of - 1.7 kb included a truncated part of Antrvp2. Six transformed lines were established that carried single
344 3C4 Oxford University Press
A.gambiae promoters in the fruitfly Tyl -2c4.8 Ty6
A
A
Ty4
Ty5
Ty2
Ty7 Ty3
-mL-mw
th
ViT
Ty1
I
I Tylc5.2
-1638
-952
3'SV4o Tyl cBam Tyl cXho Tyl cHin -360 B6gal3'AgTyl Tyl cBst 13gal3'AgTyl Tyl cRsa
|
Bga
-68130gal
B
3'AgTyl
3'AgTyl
-360
-360
3Hsp7O
Tyl cEco
3' SV40
Ty2cHin Ty2cNde
-99
-1043 Bga
-418
B-galS3'8V40 -1B683 '
~~SV4
Ty2cFok
Fig. 1. (A) The trypsin gene cluster of A.gambiae. The black boxes labelled TvJ-Tv7 indicate the seven genes and their direction of transcription. The parts of the cluster present in the constructs Tylc5.2 and Tyl-2c4.8 are also indicated. (B) The six constructs used to analyse the Antrypl promoter and the three constructs designed for the analysis of the Antryp2 promoter in transgenic fruitflies. The thin lines show the upstream sequences used (the numbers refer to the distances from the corresponding starts of transcription) and the shaded box indicates the Hsp7O minimal promoter element. The 3' sequences used are indicated for each construct (see text). Only the upstream regions are drawn to scale.
insertions of Tyl -2c4.8, while only one was obtained for Tylc5.2. The expression of the mosquito trypsin genes was monitored subsequently by Northern analysis using oligonucleotides specific for each gene as probes (Muller et al., 1993). The Northern blots revealed that a band of the expected size of -1 kb could be detected in all transgenic lines carrying the mosquito trypsin-encoding genes (not shown), but not in the transformation host (see Figure 2). To obtain a rough assessment of the tissues in which the trypsin gene was expressed, we then dissected adults from both sexes to obtain guts and the remaining carcasses. Figure 2 shows such an experiment performed for the line transformed with Ty1c5.2, as well as one of the lines carrying Tyl-2c4.8. In these preparations, a hybridization signal of the same size is only visible in the lanes containing total RNA isolated from guts of both sexes, while, even if overexposed, no signal is apparent in carcass material. This is true for lines carrying both transposons, and these results suggest that the DNA ranging -1.7 kb upstream and -0.4 kb downstream of the two trypsin genes may contain all sequences that are necessary to confer tissue specificity to their expression. To test in more detail the function of the mosquito promoter in the context of the fruitfly genome, we constructed several lacZ fusions and transformed D.melanogaster hosts. These transposons carried deletions extending towards the 5' end of both Antrypl and Antryp2 (Figure iB), leaving intact the proximal parts of the presumed control regions and the 5'-untranslated part of the mRNA. To evaluate the possible contribution of downstream sequences to the expression of the constructs, most of the Antrypl fusions carried 387 bp from the Antrypl 3'flanking region (starting 5 bp after the stop codon), instead
Fig. 2. Expression of the Antrypl and Antryp2 genes in transgenic fruitflies. Total RNA (10 ,ug per lane) from the transformation host (th; equal number of males and females) as well as from transgenic female adults (wf), dissected guts (mg: male guts, fg: female guts), and the remaining carcasses (mc: male carcasses, fc: female carcasses) were probed with (a) an Antrypl-specific oligonucleotide probe in flies transformed with the construct Tyl-2c4.8, (b) an Antrxp2-specific oligonucleotide probe in flies transformed with the construct Tyl-2c4.8 and (c) an Antrypl-specific oligonucleotide probe in flies transformed with the construct Tylc5.2. Ribosomal RNA was used in the same tracks to quantitate the signals obtained.
of an -850 bp long fragment from SV40 that encompasses polyadenylation sequences, provided by the vector and present in TylcBam, and all three Antryp2 constructs (Thummel et al., 1988). Finally, in TylcEco, a 261 bp long fragment ranging from nucleotide -360 to nucleotide -99 (relative to the mosquito start of transcription; H.-M.Muller and A.Crisanti, in preparation) was fused upstream of a minimal promoter element containing the TATA box of the D.melanogaster Hsp7O gene (Hiromi and Gehring, 1987) and the lacZ reporter gene. This construct also carried the 3' polyadenylation signal containing sequences from the Hsp7O gene of D.melanogaster. Three to six transgenic lines were obtained and analysed for each construct to minimize potential position effects due to the site of integration in the Drosophila genome. With the exception of three lines carrying Antrypl-specific constructs (labelled with an asterisk in Table I), that were homozygous lethal and were kept balanced, all others were homozygosed for the respective insertion. The expression patterns of the lacZ gene driven by the A.gambiae sequences were monitored both by staining with X-gal in hand-dissected flies, and by an enzymatic assay that allows for a quantitative measurement of the ,B-galactosidase activity [chlorophenol red-p-D-galactopyranoside (CPRG) assay], and whose results are not affected by endogenous enzymatic activity from the fruitfly host (Simon and Lis, 1987). The results of this analysis are shown in Figure 3 and Table I. In the case of the constructs testing the Antrypl promoter, while the transformation host used as a control, as well as all of the lines carrying the construct TylcRsa, failed to reveal any stain in either midgut or carcass, all other lines displayed different intensity of histochemical staining, which was detectable exclusively in the midgut and, in a few cases, in the malpighian tubules. The rest of the body did not reveal any ,B-galactosidase activity, even after prolonged staining. It must be pointed out that the intensity of the colour as well as the extent to which the midgut and, in the few exceptional cases, the malpighian tubules were blue varied not only when comparing lines carrying different transposons, but also when comparing individuals from the same line. 345
G.Skavdis et al. Table I. Quantitative assays of (3-gal expression in the transgenic flies
Antrypl promoter constructs
Antryp2 promoter constructs
Transposon
Ty I cBam
Ty I cXho
TylcHin
TylcBst
Line #1 Line #2 Line #3 Line #4 Line #5 Line #6 Mean value
30.0 ± 1.4a 50.5 + 5.7 46.7 + 2.5
60.2 53.0 50.0 54.5 42.5 49.5 51.6
64.6 42.7 40.2 32.9 36.9 43.4
31.2 18.1 44.3 38.6 38.6 47.8 36.4
-
42.4
+ ± 1 + ±
3.1 3.5 4.2 6.9 3.5 6.2
± 3.4 ± 2.3
± 2.6 + 1.6 1 3.1
+ + + ± +
TylcRsa
3.4 1.6 2.0a 0.5 2.8 0.9 3.4 0.6 5.0 0.8 6.6 0.9
± + + 1 ±
0.1 0.0 0.1 0.1 0.1
Ty1cEco
Ty2cHin
149.3 + 6.4 47.7 ± 4.8 83.4 + 2.9a
4.6 4.2 6.4 3.9
-
93.5
+ ± + +
0.3 0.3 0.6 0.3
Ty2cNde
Ty2cFok
7.9 6.1 6.6 6.5 7.0
0.5 0.3 0.1 0.6 2.4 0.8
-
-
4.8
6.85
+ + ± 1 +
1.0 0.8 0.7 0.8 0.6
+ + + ±
0.1 0.2 0.1 0.2
0.4b
The table shows the mean value of the CPRG assay determined for the individual lines carrying the transposons indicated. The standard error was calculated for each line tested. The numbers indicate OD595/mg of total protein. Five samples of 10 flies each (five females and five males) were used for every line assayed. aHomozygous lethal lines. bThe only line expressing (3-gal that carries the transposon Ty2cFok.
In the case of the transformants testing the Antryp2 promoter, lines carrying the constructs Ty2cHin and Ty2cNde stained the midgut in a fashion similar to that which was was observed with the Antrypl constructs, while in the case of the shortest deletion derivative (Ty2cFok), one of five lines displayed very weak blue stain in the midgut, while no blue colour was revealed in the others, even following prolonged staining times. Again, as in the case of the Antrypl constructs, all lines carrying deletion derivatives of the Antryp2 mosquito gene failed to stain any tissue other than the midgut, even after prolonged times, with the occasional exception of some light stain in the malpighian tubules present in some preparations. Table I shows the results of the CPRG assays performed on adults from the transformed lines. Only slight variations were detected in the quantitative assay of 3-galactosidase activity among the lines carrying deletions encompassing the region up to nucleotide -360 of the Antrypl gene, while, on- the other hand, lines carrying the transposon with the shortest upstream element present, Ty 1 cRsa, hardly express the reporter gene. The lines carrying TylcEco, that use a foreign minimal promoter element in addition to the short mosquito segment from -360 to -99, again express 13-galactosidase, the activity reaching levels which are higher than those observed for any other construct. These results suggest that the DNA segment extending from -360 to -99 contains all sequences necessary and sufficient to confer tissue specificity to the Antrypl gene in adult fruitflies. In the case of the Antryp2 promoter constructs, the CPRG assays showed that while moderate ,-gal expression can be seen in all lines carrying the two long fusion constructs (although to a lesser degree than in the case of the Antrypl constructs), the expression level drops considerably in all of the five lines carrying the shortest deletion derivative. In this case, the Ty2cFok line that was weakly stained in the previous experiment also shows somewhat higher ,-gal activity, although considerably less than that which was observed for the longer constructs (line #5, labelled with a double asterisk in Table I). Here again, our results indicate that the Anopheles segment extending from the start site of transcription of Antryp2 (H.-M.Muller and A.Crisanti, in preparation) to nucleotide -418 contains all necessary and sufficient cis-acting elements for the correct spatial expression of the gene.
346
Although the two Anopheles trypsin-encoding genes under study represent major late genes whose expression reaches a maximum value several hours after the ingestion of a blood meal (Muller et al., 1993), the product of Antrypl can first be detected (at a low level) at the pupal stages and also in females before the uptake of the blood meal, while Antryp2 is strictly blood meal inducible. We therefore also tested the developmental expression profile of the transgenic lines to determine whether the fruitfly can also recognize the signals responsible for the temporal expression pattern. These results are shown in Figure 4. Using the CPRG assay, no f3-galactosidase activity at all can be detected in embryos, irrespective of the construct and of the promoter tested. In the case of the Antryplspecific constructs, it is apparent that lines carrying the transposon TylcRsa expressed ,B-galactosidase at about background levels (Figure 4A). For most other lines tested, the activity rises in each successive instar, is then reduced in pupae, and reaches high values again in adults. Interestingly, the lines carrying TylcEco, the construct utilizing a foreign minimal promoter element in addition to the short mosquito segment from -360 to -99, have a very reduced level of expression in all pre-adult stages, while, in contrast, they show the highest levels of expression in the adults. Since A.gambiae does not express any of the two trypsin genes in larval stages, we analysed histochemically third instar larvae of transformants carrying Antrypl constructs to determine the spatial distribution of the ,-gal activity detected by the CPRG assay. We examined at least one line for each construct and no tissue other than the midgut was stained. In contrast to the adults, all activity was found to be restricted strictly to the anterior part of the midgut (Figure 3j). This was also true for the two TylcEco lines examined, which displayed very weak stain in the same area of the gut. As was the case for the adults, no stain was apparent in the TylcRsa lines examined. The developmental pattern observed for the Antryp2 constructs is different from that observed for the Antrypl gene (Figure 4B). Here, hardly any expression is detected before the pupal stages, and the expression levels are reduced even in the adults. The negative result of the CPRG assays was further confirmed by the absence of any visible staining in the histochemical analysis of third instar larvae. The fact that the two short mosquito segments extending
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Fig. 3. Histochemical determination of lacZ expression in midguts of adult transgenic fruitflies, driven by the Antrypl promoter sequences (a) TylcBam, (b) TylcXho, (c) TyIcHin, (d) TylcBst, (e) TylcRsa and (f) TylcEco, the Antryp2 promoter sequences (g) Ty2cHin, (h) Ty2cNde and (i) Ty2cFok and in third instar larvae carrying the construct TylcBst (j). Hand-dissected guts were stained with X-gal for 1-2 h. Blue colour cannot be observed (even when stained overnight) in the midguts of adult flies of lines carrying the transposon TylcRsa and Ty2cFok (with the exception of line #5).
347
-114
C TCRGCRRTTGRTRTCGCTRTCRATTRA fCCGCCGRRCGGGRCAAGT Antrypl
-103
C
RTCRGTRTTTGRTAGTRCTATCRARTRR
1
..............
CTRGT Antryp2
-64 TCACT ..... GCCRCGGRRCCTGGRCTGRCCGCRCTGTRTRTATRGTTGG Antrypl
II
11 11
i11111111111
-67
TARTTCGCTRGTTRCARRRRTRGGRCCG CTGCRTGGTATTTRAGGCGTC Antryp2
-19
CRTCTRCRTTCCCATCGRTCRTflC6R6T6CC66RCRCGRTG +22
Antryp 1
-18
+22
Antryp2
l1l11111 111111111111111111ii liii GRGTTTCCTTCCART RATCRTRC6A6T6CCT6RCRA6
Fig. 5. Comparisons of the sequences upstream of the transcription start sites (arrowheads) of Antrypl and Antryp2. The two sequences were aligned using the BESTFIT programme of the GCG package. The transcribed sequences are indicated by bold characters and the AUG translation start codons are underlined. The two TATA boxes are double-underlined, while the boxed sequences represent the upstream conserved segment. The arrows over the sequences indicate the extent of the palindromic sequences present in this segment (see Results for details).
Fig. 4. Developmental pattern of expression of the transformed lines. The bars show the results of the CPRG assay expressed as OD595/mg total protein. At least 10 whole adult individuals from two lines were used for each developmental stage and for each transposon indicated. (A) Antrypl constructs, (B) Antryp2 constructs.
to about -400 bp upstream of both trypsin genes seem to contain the elements responsible for the gut-specific expression, made us look for sequence similarities that
could potentially pinpoint these signals. Alignments of the two sequences ranging from the AUG to nucleotide -360 (in the case of Antrypl) and to nucleotide -418 (in the case of Antryp2) revealed two areas of high similarity (Figure 5). The one segment with extensive similarities encompasses the entire 5 '-untranslated region of both genes (19/21 identities), extending to two nucleotides before the capping site. The second segments in which substantial similarities can be seen (23/29) are the sequences from -112 to -84 and from -101 to -73 for Antrypl and Antryp2 respectively. This segment is interesting in that it contains palindromic sequences, which, in the case of Antryp2, are perfect. Similar boxes are found in front of all other genes in the A.gambiae trypsin gene cluster (H.-M.Muiller, unpublished observations), but they are absent from the D.melanogaster cluster that contains eight trypsin genes (Wang et al., 1994). Finally, database searches using the fragment from nucleotide -99 to -360 relative to the transcription start site of Antrypl, which was shown to be sufficient to express the reporter gene in the midguts of the transformed Drosophila, failed to detect any similar sequences, especially in DNA segments in the vicinity of genes expressed in insect guts. Negative results were also obtained when the corresponding segments of Antryp2 were used to search the nucleic acid databases.
Discussion The results presented here indicate that we have identified two short genomic segments carrying all sequences sufficient to drive the expression of the A.gambiae Antrypl and Antryp2 genes in a tissue-specific manner. Further-
348
more, these segments also contain cis-acting elements that regulate the expression of genes in a temporally specific fashion. While this is clearly not the first case where an insect promoter is recognized by D.melanogaster across millions of years of evolution (e.g. Mitsialis and Kafatos, 1985), it is remarkable that the specificity of the Antrypl promoter is developmentally 'shifted' towards its new genomic environment (Davis et al., 1985; Yun and Davis, 1989), in contrast to the Antryp2 promoter, whose function in Drosophila parallels, to some extent, the situation in the mosquito. In the Antrypl transgenics, the mosquito trypsin gene promoter is activated immediately after the embryonal stages, at a time where the digestion machinery is set on, and it remains active, though at different levels, throughout the life of the fruitfly, including the pupal stages. This differs from the situation in A.gambiae, where Antrypl activity is first observed at pupal stages. Antrypl is also expressed in adult females at low levels before blood feeding, and a maximum is reached several hours after the blood meal, together with Antryp2, which is not expressed before the blood meal (Muller et al., 1993 and unpublished observations). Whether these differences are due to the absence of specific sequences in the constructs used in this analysis, or the inability of Drosophila to fully understand and comply with the necessary signals cannot be determined, given the dramatically different eating behaviour of the two insects. The nature of the cis-acting elements cannot be determined by this analysis. We have used three different 3' segments in our constructs, but this fact cannot account for any of the differences observed in the expression pattem of our transformed lines. Indeed, the lines carrying the construct TylcBam, which contains downstream sequences derived from SV40, did not show any significant differences in their expression pattern when compared with lines carrying constructs attached to the mosquito downstream segments. Obviously, we cannot eliminate the possibility that cis-acting elements controlling the expression of the two trypsin genes may be found in 5' or 3' segments that were not included in the constructs. For example, we failed to see a 'blood meal-related' induction process for both promoters in Drosophila (our unpublished observations) but, as stated above, there is no way of determining whether this is due to the lack of
A.gambiae promoters in the fruitfly the necessary signals or to the inability of the fruitfly to understand them. Moreover, the remarkably conserved palindromic sequences in front of the two genes cannot be involved in the regulation processes maintained in the transformed flies, since all flies carrying the construct Ty I cEco, in which the sequences containing this box have been partially deleted still express 3-gal exclusively in the midgut. Thus, if a biological role were to be assigned to this sequence, this could not be related directly to the spatial specificity of the promoter, also considering the absence of a similar box from the corresponding areas of the D.inelaniogaster trypsin-encoding genes. Finally, given the fact that flies transformed with TylcEco express the reporter gene at a very reduced level during pre-adult stages and at a much higher level in the adults (when compared with the lines carrying the other Antrypl constructs), the sequences closest to the transcription site may contain signals that specify elements which fine-tune the expression of Antryp]. It is clearly remarkable that, even if the digestive physiology is extremely different in the two insects, Drosophila still recognizes the signals that are responsible for specifying a strict expression of the trypsin genes in the gut. This specificity is also found in third instar larvae of Antrvpl transgenics, a developmental stage at which both promoters studied are silent in the mosquito. There is no obvious explanation for the variability observed in the level of expression as well as in the extent to which the midgut could be stained in the adults. This variability was not only observed when comparing different lines carrying the same transposon. It was even present when comparing adult individuals from the same line, a fact that excludes position effects due to the site of integration of the transposon being the source of the variability. The biggest difference between the situation in A.gamnbiae and that in the transgenic flies is the fact that no males of the former species ever express any of the two genes. Given the similar behaviour of all constructs in both sexes of D.melanogaster, the most acceptable conclusion is that the lack of expression in the male mosquitoes is due to the absence of the necessary induction signal, mediated through the blood meal, rather than the presence or absence of independent sex-specific signals. Finally, we can only offer guesses as to why one of the five lines carrying the transposon Ty2cFok expresses 13-gal. This may represent an enhancer trap, but this would have to have occurred close to a gut-specific enhancer, given the midgut-specific stain of the transformants. Altematively, the deletion may encompass a part of a regulatory sequence that confers 'leakiness' to the reporter gene under its control. The latter possibility could explain the very low level of expression detected biochemically. Clearly, once available, a mosquito transformation system would be the ideal tool for the study of potential anti-plasmodial constructs. On the other hand, the fact that the regulatory elements of the malaria mosquito are grosso modo recognized by D.melanogaster suggests that, until such a system is developed, the fruitfly can be of use for the further understanding of the mosquito's gene
physiology.
Materials and methods Unless otherwise indicated, all molecular techniques were performed as described in Sambrook et a!. (1989).
Construction of plasmids The two DNA fragments used to prepare the Tyl-2c4.8 and Tylc5.2 constructs were isolated from the kEMBL3A clone TY3.3 (Muller et al.. 1993) using appropriate restriction enzymes, and subcloned in pBluescript. They were subsequently re-cloned between the unique Sall and Notl sites of the D.mielanogaster transformation vector pDM30 (Mismer and Rubin, 1987). All DNA fragments from the upstream and downstream regions of Antrypi and Antrvp2 genes tested in the various lacZ constructs were first subcloned in pBluescript. They were then reisolated using appropriate restriction enzymes and cloned in a P-elementbased transformation vector. With the exception of TylcEco, for which the transformation vector was pHZ50PL (Hiromi and Gehring. 1987), all remaining lacZ constructs were made using either the expression vector pCaSpeR-AUG-p-gal (Thummel et al.. 1988) or a derivative of this vector, in which an 850 bp long fragment containing SV40 polyadenylation sequences was replaced by a 387 bp long fragment from the Antrypl 3-flanking region starting 5 bp immediately after the translation stop codon. The integrity of the resulting constructs was checked by detailed restriction analysis.
Germline transformation Germline transformation of Drosophila was performed essentially as described by Spradling (1986). The transposon p7t25.7wc was used as a source of transposase (Karess and Rubin, 1984). Transformed flies were kept at 25°C on standard corn-meal food, supplemented with yeast and tetracycline to suppress bacterial growth and avoid background galactosidase activity.
/3-Galactosidase assays The quantitative assays for the determination of P-galactosidase activity (CPRG assays) were performed essentially as described by Ashburner ( 1989). Measurements were taken every 30 min to ensure that the reaction was linear. The protein concentration in each sample was determined according to Bradford (1976), using bovine serum albumin as a standard. Results are given as OD595/mg of total protein, measured 2 h after the initiation of the reaction. For every line, five samples of ten 2-7-day-old adult flies were analysed. To determine the developmental pattern of 3-galactosidase activity, two transformed lines from each construct were examined. At least two samples containing five individuals were analysed for each pre-adult stage. Individuals from the transformation host strain were used as negative controls in each experiment. The histochemical staining was performed on hand-dissected flies according to Reichardt et al. (1992). At least three adult flies were stained with X-gal for 1-2 h. Three to five third instar larvae from selected lines (at least one transformed line from each construct) and from the transformation host strain (as negative control) were also analysed in an identical way.
DNA sequence analysis Sequence analysis was performed using the GCG software package (Devereux et al.. 1984). Database searches were performed using the BLAST search programmes (Altschul et al.. 1990).
Acknowledgements We thank Dr G.Markakis for his invaluable help with statistical analysis and Y.Livadaras for expert technical assistance with the transformation experiments. This work was supported by grants from the European Union and the John D. and Catherine T.McArthur Foundation.
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