Nucleic Acids Research, 1993, Vol. 21, No. 15 3545-3551

Regulation of tissue-specific expression of the esterase S

gene in Drosophila virilis

Pavel V.Sergeev1, Grigorii N.Yenikolopov1'+, Natalia l.Peunova1l+, Boris A.Kuzin3',Ruben A.Khechumian1§, Leonid l.Korochkin2 and Georgii P.Georgiev1 21Engelhardt Institute of Molecular Biology, Russian Acad. Sci., 32 Vavilov str., 117984 Moscow,2Institute of Gene Biology, Russian Acad. Sci., 34/5 Vavilov str., 117334 Moscow and 31nstitute ofDevelopmental Biology, 26 Vavilov str., 117984 Moscow, Russia

Received March 3, 1993; Revised and Accepted June 14, 1993

ABSTRACT

The esterase S gene (estS) of Drosophila virills isspecifically expressed in the ejaculatory bulbs of males.Its sequencing shows similarities between estS productand other esterases of different origin. Thetranscription of estS in ejaculatory bulbs is at least 2orders of magnitude higher than in other tissues ofmales. Two promoters, P1 (distal) and P2 (proximal),and two different transcripts were identified. Thepromoter P2 Is used much more efficiently, and in astringent, tissue-specific manner. The transcriptionfrom P1 takes place in different tissues and stages ofdevelopment of D. virilis. However, the mRNAtranscribed from P1 seems to be inactive in translationas there are three open-reading frames (ORF) betweenP1 and P2, which may block the translation in P1initiated mRNA. Insertion of sequence containing thethree ORFs into the 5' untranslated region of the CATgene strongly inhibited expression of CAT. Pointmutations destroying the three ORFs completelyeliminate the inhibitory effect. Hence tissue-specificexpression of the estS gene may depend on control atthe level of transcription, promoter selection andtranslation.

INTRODUCTION

The estS gene of Drosophila virilis encoding esterase S is an

attractive model for studies on the control of gene expressionas this gene is switched on at a well defined period of development(the 3rd day after emergence), and in one tissue (the epitheliumof ejaculatory bulbs in males). The enzyme is carboxylic-esterhydrolase. It enzymatic activity is determined with hydrolysingbeta-naphtylacetate. The enzyme is responsible for processes offemales fertilisation. Esterase S is accumulated in ejaculatorybulbs, excreted and transferred into genitals of females uponcopulation (1-5). A sharp start of the estS expression seems toresult from some internal signal and does not depend on

EMBL accession no. X70351

extracellular factors. Its activation occurs even if genital imaginal

disk giving rise to ejaculatory bulbs is transplanted into femaleabdomen. Possibly its on switch is a part of the internaldevelopmental program of the corresponding cells (1,2,6).Several data indicate the estS gene is under control of at leastthree genes located in X, IV and V chromosomes. The internalprogram is modulated by hormonal influences (5,7).The question arises as to which step of gene expression is

critical for the control of tissue specificity. Here, we found thattissue specificity is determined at the level of transcription.Obtained results showed also the possibility of regulation ofesterase S gene expression at the level of translation.

MATERIALS AND METHODSSequencingSequencing method was the dideoxy chain termination method(8,9). Subclones for sequencing were obtained with the aid ofpartial DNAase I digestion in the presence of Mn2+ (10).Plasmid DNA was isolated by alkaline method (11) with minormodifications.

Mapping of 5'-ends in poly(A)+RNATotal and poly(A)+RNA were isolated according to (12,13) withminor modifications. S1 analysis was performed by a standardmethod (14) varying temperature and time of annealing and S1nuclease concentration.

In vitro transcriptionCell-free extracts of HeLa cells (obtained from 'BRL', USA)were used for in vitro btanscription experiments (15). The reactioncontained 6-15 1l of cell extract, 0.5-3 /tg DNA, 0.1 mMEDTA, 1 mM creative phosphate and 0.5 mM all four nucleosidetriphosphates.

Site directed mutagenesis and CAT assay

Following single stranded oligodesoxyribonucleotides weresynthesized: 1).5'-CAACTTAGAAATGGGGAATCATCTAT-

Present addresses: 'Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA, hnstitute of Biochemistry, Yerevan, Armenia and IJeffersonMedical College, Philadelphia, PA 19107, USA

3546 Nucleic Acids Research, 1993, Vol. 21, No. 15

aEST S

No l P1 as MeMs Pi1 sN o e 3ml No

R1 R1 I I H?IRII IIpBR32I I SI 6185i

RI RI elRI 8Xi X

Im pBR322

lkb

b~TGA CTTCAAAGTA AlTTTCAAA

_ATATA&ATTG 33

AGAAGAGGAG TTCTAAGTGA TACGGGGAGA TTTGAGCTCA GCTGATATTT 83CTTOATAAG CTCATTTGCG TTCACTMA CGAAACTCAG CCCCTCATTC 133

ATAATMTTAC AGTTGCTAAG AGCGGAACAA CTTAGAAATG GGGAATCATC 183

TATTAAAGAC CCCAGAAACT AAGCGCTGAA CCCAACTTCC AAACTCTCTC 233

GCCTCTATAA AACAAAIGGC AGAGTCTTAA ATTTTCAAAC TCAGTTTAAC 283

TGCCAATGCC AAGGGGCCAG CCATCGGAAC TAATCAATAGCTCTCTTCT 333

TTTCTCACGT GAACAAAACT TAGACTCAAG TGACC AAGGATTG 383

GCTGTCTCCG IT1TT TTCAACTTTG AWCTCCGICL TCTCCACACC 433T ACT CAA ATA CTG TTG CCG ATC GCG TTG CTC TGT CTG TTT GCA 4T8

Ttr Gln Ile Leu Leu Pro Ile Ala Leu Leu Cys Leu Phe Ala 15

GCA TCA ACC CTC AWC MT CCC CTG CTC GTG CAG TTG CCA MT GGA 523Ala Ser Tir Leu Sor Asn Pro Leu Leu Val Clu Leu Pro Asn Gly 30

GAA TTG CGG GGA CGC GAC MT GGA TTC TAT TAC AGC TAC GAG TCA 588Glu Leu Arg Gly Arg Asp Asn Gly Phe Tyr Tyr 8er Tyr Glu Ser 45ATA CCT TAT GCC GAA CCC CCA ATC GAT GAT CTG TGC TTG GAA GAA 613Ile Pro Tyr Ala Glu Pro Pro Ile Asp Asp Leu Cys Leu Glu Glu 60

CCT CGT CCC TAT ACC GM AGA TGG GM AAT ACC Trr GAC GCG ACT 858Pro Arg Pro Tyr Tbr Gln Arg Trp Glu Ann Ttr Phe Asp Ala Thr T5

CCC CCC CCA GTT GAC TGC CTG CAG TGG AGT CM CTC ATT TCA CAC 703Arg Pro Pro Val Glu Cys Leu Gln Trp Her Gln Leu Ile Bar Gln 90

CCT MT MG CTG ACA GGG AGC GAG GAC TGT CTA ACC CTC ACC ATC 748Pro Asn Lys Leu Tiw Gly Ser Glu Asp Cys Leu Thr Val Ser Ile 105

TAC MG CCA MG MT CTG ACT CGC ATC TCT TmT CCG CTG GTG GCC 793Tyr Lys Pro Lys AIn Lou Thr Are Ile Ser Ph. Pro Val Val Ala 120

CAT ATA TTT GGC GC GC TGG TCA TTT GGT GCT GCG ATC GAT GAC 838His Ile Phe Gly Gly Gly Trp Her Phe Gly Ala Ala Ile Asp Asp 135

GGA GTG AGG CCC TTC AGC AWC AGC GGC MT GTG ATA GTG CTG MC 883Gly V1 Arg Pro Phe 8er Her Her Gly Asn Val Ile Val Val Lys 150

ACA ACC ACA GAG TGG GAG CCC TTG GCT TT ATG AGC ACT GGT GAT 928Thr Tir Thr Glu Trp Glu Arg Leu Gly Phe Met Ser Tiw Gly Asp 165TCT GTG ATT CCG GCC MC TTC GGA CTA MG GAT CAG CGT CTG GCA 973Her Val Ile Pro Gly Asn Phe Gly Leu Lys Asp Gln Arg Leu Ala 180

ATC AAA TGG ATT AGG MC MC ATT GCA CGC TT GCC GGA GAT CCA 1018Ile Lys Trp Ile Arg Asn Ann Ile Ala Arg Phe Cly Cly Asp Pro 195

CAT MT ATA ATT CTT CTC GGT TTC ACT ACA GCC GGC TCC TCG CTG 1063His Asn Ile Ile Leu Leu Gly Phe Her Tnr Gly Gly Her Her Val 210

CAC TTC CAG CTT ATG CAC MG GAA TAT GGA CAG CTG CTi AAG CCG 118

H1i Leu Gln Leu Met S1i Lys Glu Tyr Gly Gln Leu Val Lys Gly 225

GCC ATA TCC ATT ACT GGA ACT WCA ACC CTT CCC TGG GCT OTA CAG 1153Ala Ile Her Ile Ser Gly Tiw Ala Tir Val Pro Trp Ala Val Gln 240

GCC AAT WCA CGT GAT CTC GCA TTC CGA TAT GC AAA CTC TTG GGT 1198Ala Asn Ala Arg Asp Leu Ala Ptw Arg Tyr Gly Lys Leu Leu Gly 255

TGT AAT AAC CCT AAA AAT TCA CGC GAG CTG MA GAT TWC CTG AAA 1243Cys Asn Asn Pro Lys Asn Her Arg Glu Leu Lys Asp Cys Leu Lys 2TO

AAA ACG CAT GCG GAA CAA TTC GTC AGC ACC TTA AGG CAC CTT CAG 1208Lys Thr Asp Ala Glu Clu Ph. Val Her Thr Leu Arg His Leu Gln 285

GTG TlT GAC TAT GTG CCT TTT GGT CCG TTT GGC CCA GTC GTA GAG 1333Val Pile Asp Tyr Val Pro Phe Gly Pro Phe Gly Pro Val Val Glu 300

TCC CCC GM GTG GAA AGC CCC TTC CTC ACC GAG CTG CCC CTC GAC 1378Her Pro Glu Val Glu Her Pro Ptie Leu Ttr Glu Leu Pro Leu Asp 315

ACC ATC AGA AGT GGA MC mT GCT CM GTG CCT TGG TTG WCC AWC 1423Tin Ile Arg Her Gly Asn Ptie Ala Gln Val Pro Trp Leu Ala Her 330

TAC ACA CCC GAG AAT GGT ATC TAT AAC GCC GCT CTT CTT TTA GCT 1408Tyr Thr Pro Glu Asn Gly Ile Tyr Asn Ala Ala Len Leu Leu Ala 345

AAG GAC GCC MT GGT AAA GAG AGG ATT CM GAG TTA MC ACT CGC 1513Lys Asp Ala Asn Gly Lys Glu Arg Ile Glu Glu Leu Ann Tir Arg 360

TGG AAC GM CTG WCT CCA TAC m TTG GCT TAC CCA TAC ACA TTG 1558Trp Asn Glu Leu Ala Pro Tyr Phe Lnu Ala Tyr Pro Tyr Tir Lou 375

AMG AGG TCT GAA ATG AMT GCT CAT TCT CAG AAA CTG AAA TAT CM 1003Lys Arg Her Glu Not Asn Ala His Her Gln Lys Leu Lys Tyr Gln 390

TAT CTA GCA TAT MC AAC TTC AGC GTC OTA AAC TAT TTC GAT GTT 1048Tyr LeGuly lys Tyr A7n Pht nr Val Val A n Tyr Phe Asp Val 405

CAG CC TTG mTT ACA MC GAC TTG TAC AAA AAA GGC ATC GM CTG 1093

Gln Arg Leu Pie Tiw Asn Glu Leu Tyr Lys Lys Gly Ile Glu Leu 420

TCA TTA GAT TCA CAT CGC MG CAC GGA GCC AWC CCC GTC TAT GCG 1738Her Leu Asp Ala His Arg Lys Hls Gly Ala Her Pro Val Tyr Ala 435

TAC GTC TAC CAC AAT CCC GCG GAT AAG TCG CTG WCA CM TTC CTG 1783Tyr Val Tyr Asp Asn Pro Ala Asp Lys Her Leu Ala Gln Phle Leu 450

GCC AAC AGA TCT GAT ATA TCC TTG GGTAAGATMTGCTAATGCTCCAAATT 1034Ala Lys Arg Her Asp Ile 8er Leu G

GTGCAGTMCTMTATCAATGATCTATTCTCAGGC ACC GGA ATG GGC GAC GAT 1087ly Tir Gly Not Gly Asp Asp 465

TAC TAT CTC TTG ATC AAC AAT CCA CTC CGT GAA CCC CTG CGA GCT 1932Tyr Tyr Leu Leu Nit Asn Asn Pro leu Arg Glu Pro Leu Arg Ala 480

GAC GAG AAA ATC GTT TCA TOC AAG CTA GTC AAG ATG GTG GAA CAT 1977Asp Glu Lys Ile Val Ser Trp Lye Leu Val Lys Net Val Glu Asp 495

TTC WCC GCG CAC GAG ACC TTG GTC TAT CAC CAC TGT GTG TTC CCA 2022Phs Ala Ala His Glu Tin Leu Val Tyr Asp Asp Cys Val Phs Pro 510

AAC AAC TTGGCC AAA AAG AAA TTC CAG TTC GTG GTCATA GGACCT 2067Asn Asn Leu Gly Lys Lys Lys Phe Gln Leu Val ValIleGly Arg 525

AAC TAT TGC AAG CAA TTGGAAGTG GAA TCG TTTGCC CGA CACGGT 2112Asn Tyr Cys Lys Gln Leu Glu Val Glu Ser Pte Ala ArgHis Gly 540

CTC CAA TAA TCTACCACCA AATCCAATTG TAATTTACTG AGCCGATCTC 2161Val Gln 542

TATGAAACGA TCAAATCAAT AGTCGATGGT TGTAAATTTC TATAAATCTA 2211

TATGTGGGTA TGTAATCTTC TGCAAATAAA TCATGGCATT ATTTAATAAC 2261

TCAA

Figure 1. The structure of the estS gene. (a) Physical map of the pVE9 plasmidcontaining genomic copy of the estS gene of D. virilis. (A1-XbaI, B1-Bgl,B2-BgllI, Cl-Cla,H3-HindIII, P1-Pstl, Si-Sal, Rl-EcoRI, R5-EcoRV,X1-XhoI). The direction of the EstS gene transcription is designated with anarrow. (b) Nucleotide sequence of regulatory and coding regions of the estS gene.The major transcribed region is framed. Putative TATA-boxes are underlinedwith a waved line. The initiation codons are underlined with solid line. Putativeglycosylated codons are designated with an arrow.

TAAAGACCCCATAAACTAAGCGCTGAACCCAACTTC-CAAACTCTGTGGCCTCTATAAAACAAATG-3' 2).5'-CAG-CTATTCGATTAGTTCCGATGGCTGGCCCCTTGGCATTG-GCAGTTAAACTGCGTTTCAAAATTTAAGACTCTGC-CATTTGTTTTATAGAGG-3' 3).5'-CAACTTAGAACCGG-GGAATCATCTATTAAAGACGCCACAAACTAAGCGCT-GAACCCAACTTCCAAACTCTGTGGCCTCTATAAAACA-ACCG-3'4).5'-CAGCTGTTCGATTAGTTCCGATGGCTGG-CCCCTTGGCGGTGGCAGTTAAACTGCGTTTCAAAATT-TGAGACTCTGCCGGTTGTTTTATAGAGG-3'

17 nucleotides at the 3'-ends of fragments 1) and 2) and of3) and 4) respectively are mutually complementary (underlined).The pairs were taken in equimolar amounts and annealed in 6mM MgCl2, 50 mM tris-HCl, pH 7.8 for 30 min at 370C andfor 30 min at 25°C. Thereafter the mixture of 3 mM dATP,dCTP, dGTP and dTTP was added together with 10 U of KlenowDNA polymerase I. The mixture was incubated for 30 min at370C.

Reaction results were checked by electrophoresis in 1.8%agarose gel. The double stranded fragments were inserted intopSV2CAT plasmid (16) in the region corresponding to the5'-untranslated part of mRNA. Orientation of the fragment wasdetermined by sequencing. For sequencing the synthetic primer5'-GCTCCTGAAAATCTCGCC-3' was used. The transfectionwas performed by DEAE-dextran sulfate method as modified forsuspension culture of chicken fibroblasts HD3 (17). 14C-Chloramphenicol from Pharmacia was used in CAT assay (18).

RESULTSStructure of estS gene and its productEarlier, we cloned a genomic copy of the estS gene from D. virilis(6). The physical map of the pVE9 plasmid containing the estSgene is shown on Fig. la. In addition, an incomplete cDNA copyof estS had previosly been cloned. 5' end of obtained cDNA copyextended towards 5' end of the major transcription start fromthe promoter P2 of the estS gene 212 nucleotides. The cDNAcopy was sequenced completely and comparison both sequencesobtained from genomic copy and cDNA copy of the estS genemade possible it exon-intron structure and amino-acid sequenceof the gene product to determine.One can see that the estS gene consists of two exons separated

by a 59 bp intron, The esterase S protein was deduced to consistof 542 amino acids. Esterase S has previosly been shown to beglycosylated (5). The putative glycosylation sites are shown onFig. lb.

Nucleic Acids Research, 1993, Vol. 21, No. 15 3547

aminoacid sbquences ororigin of esterase active centre

EstS or Drosophila virills N I I L L G F S T G G S S V H L Q

Est6 of Drosophila melanogaster N V L L V C H S A G G A S V It L Q

Acetylcholinesterase of skate T V T I F G E S A G G A S V G M H

Acetylcholinesterase or eel G G E S S E G A A G

Alyesterase of horse F G E S A G A A S

Cholinesterase or human S V T L F C E S A G A A S V S L H

Carboxylesterase or chicken G E S A C G I S

Carboxylesterase of pig G E S A G G E S

Carboxylesterase of sheep G E S A G G E S

Carboxylesterase or bull G E S A G G E S

G E S A G G A S V

EstS AATATAATTCTTCTCGGTTTCAGTACAGGCGGCTCCTCGGTGCACTTGCAGAsnI leI leLeuLeuGlyPheSerThrGlyGlySerSerValHisLeuGln197 213

1 2 3 4 5 6 7

-4.2

-n 2.3-1.8

-0.9

Figure 3. Transcription of the estS gene in D. vinlis ontogenesis as follows fromNorthern blot hybridization. Poly(A)+RNA was prepared from (1)-larvae,(2)-pupae, (3)-imago females (the 3rd day after emergence), (4)-imago males(the 3rd day), (5)-imago females (the 10th day), (6)-imago males withoutejaculatory bulbs, (7)-ejaculatory bulbs of imago males (the 10th day). (1-7)amount of poly A' RNA-10 /g. Molecular size of marker fragments (kb) areindicated.

Figure 2. Comparison of amino acid sequences of active centers inD. rWlis esteraseS and beta-esterases from other species.

A significant homology in the region of the putative activecenter of the enzyme was detected with such evolutionary distantspecies as human and ray (Fig. 2).The analysis of nucleotide sequence of the genomic estS copy

also allows one to detect putative promoter sequences fortranscription initiation. Two typical eukaryotic TATA-boxelements are present 64 and 411 bp upstream to the esterase SAUG translation initiation codon.

In vivo transcription of the esterase S gene and mapping ofmRNA 5'-endsFirst, Northern blot hybridization was performed with totalpoly(A)+RNA obtained at different stages of D. virilisdevelopment (Fig. 3). One can see that estS trascription is tissue-specific. It is almost absent in females at all stages ofdevelopment. In males it is restricted to the stage of imagoappearing at the 3rd day after emergence and reaching maximumat the 10th day. In imago, only ejaculatory bulbs were foundto express the gene. However, the absence of a recognisablesignal on Northern blot does not mean that the transcription doesnot exist at all. In fact, an increase ofRNA concentration allowsone to detect a signal of estS mRNA in other tissues and infemales. However the level of expression was at least 100 timeslower (data not shown).To confirm the specificity of this signal, we performed SI

mapping of mRNA 5'-ends.Si analysis was performed with poly(A)+RNA transcribed

from P2 and P1 promoter taken at different stages of developmentfrom males and females (Fig.4,5). RNA was prepared repeatedlyvery carefully from various tissues of Drosophila virilis withoutcross contamination.The major P2-associated mRNA could not be found in

embryos, larvae or pupae. It appears in males of imago stageon the 3rd day after emergence and reaches the maximum onthe 10th day. All P2 mRNA is concentrated in ejaculatory bulbs.No signal was obtained with RNA from any other organ or fromwhole males after removal of ejaculatory bulbs, even when a largeexcess of poly(A)+RNA (100-200 xfold) was used forhybridization. Thus, the level of transcription from the proximal

P2 promoter in ejaculatory bulbs is at least four orders ofmagnitude higher than in other tissues of males (Fig. 4a,b)

In females, the transcription from the P2 promoter is alsoundetectable before the imago stage. At the same time as in males,the P2 transcripts appear, though at the peak of transcription theircontent in females is 50-100 times lower than in males.Considering the absence of P2 transcripts in male tissues otherthan ejaculatory bulbs, one can suggest that low expression ofthe estS gene in female occurs in a certain sex-specific part ofthe female. This question has not been further analysed. Theprecise positions of the transcription start points were obtainedwith a comparison of the sequencing products of the probes usedin SI mapping with S1 mapping products.(Data not shown) Themultiple bands in fig.4(a,b) sometimes occur in Si mappingbecouse of influence of 5' end cap structure ofmRNA. The dataof sequence analysis of the region of the transcription start pointsdetermined with SI mapping showed that here common sequencesfor ends of introns do not exist. The experiments of transcriptionin vitro supported that SI mapping does not detect introns outside,but transcription starts of the estS gene.

Additionally, another mRNA start site was found downstreamfrom the distal putative P1 promoter TATA box. (Fig. 5a,b).This signal can clearly be observed if larger amounts ofpoly(A)+mRNA are used in hybridization experiments. Theratio of concentration of poly(A)+RNA from the distal andproximal promoters constitutes about 1:100 in ejaculatory bulbsof adult males. In malpighian tubules and ejaculatory bulbs ofmature males we detected also the signals of RNA of unknownorigin. At present we are studying the origin of this RNA.The picture is quite different in the case of transcripts initiated

by the P1 promoter (Pl-transcripts). They are detectable atdifferent stages of development and in different tissues of D.virilis. It is inactive in embryos. P1 is switched on in the thirdinstar larvae. It is switched off in pupae and again switched onin imago on the first day after emergence. Later its activitydecreases. The total poly(A)+RNA isolated from 5 day malesfail to protect corresponding DNA fragment upon the S1mapping, although a weak signal can be obtained withpoly(A)+RNA from ejaculatory bulbs. The P1 promoter isreactivated on the 10th day after emergence and7after that itsactivity does not change.

In larvae, the P1 promoter is used in genital imaginal disks

3548 Nucleic Acids Research, 1993, Vol. 21, No. 15

1 2 3 4 5 6 7 8 9 10 111213 14 151617 18

309- -o

e217- -4201-

160- 40

b1 22:-4 5 7 8

9 10 11 12 1314 1516 17sbaa aea

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1 3 5 7- 9 1 1 1 3 1I5 1 -7 19". '21 "2325)~

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Figure 4. Mapping of transcription initiation site from the proximal promotorP2 in the estS gene. a) Si1 mapping in the region of putative proximnal promoter.Poly A' RNA was preparated from (1-5)-ejaculatory bulbs of imago males:(1)-0,1 pg, (2)-0,3 jig, (3)-i jig, (4)-3 jig, (5)-b0 Mg; (6)-imago malewithout ejaculatory bulbs, (7)-malpigian tubules, (8)-ventriculus), (9)-salivaryglands, (10)-fat body, (1 1)-imaginal disks, (12)- female heads, (13)-maleheads, (14)-ovaries, (15)-female genitals without ovaries, (16)-controlhybridization with poly A+ RNA from mouse, (6-16)-amount of poly A+RNA-10 jig. Figures at the left-size of marker fragments (bp). b) Si mappingin the region of putative proximal promoter. (1,2)-control hybridization withtotal RNA from E.coli and mouse, poly A+ RNA was preparated from (3)-embryous, (4)-larvae, (5)-pupae, (6, 8, 10, 12, 14, 16, 18, 20, 22, 24)-imago females of varios ages: (6)-i day, (8)-2 days, (10)-3 days, (12)-Adays, (14)-S days, (16)-6 days, (18)-7 days, (20)-10 days, (22)-16 days.(24)-20 days; (7, 9, 11, 13, 15, 17, 19, 21, 23, 25)-imago males of variosages: (7)-i day, (9)-2 days, (11)-3 days, (15)-A days, (17)-S days, (19)-10days, (21)-12 days, (23)-16 days, (25)-20 days. Labeled fr-agment used asa probe in (a,b) is fr-om 124 to 590 bp Figib. In all cases for analysis wereused 10 M.g poly A+ RNA.

and in malpighian tubules. In adult males, the only place wherethe P1 promoter was active was in the ejaculatory bulbs, whilein females, its activity is undectable.

It is interesting that in the third instar larvae neither imagirnaldisks nor malpighian tubules contain (3-esterase activity at leastin quantities detectable by the techniques used (2,3,4). Thus, twopromoters are used in estStranscription. We have designated thedistal promoter as P1 and the proximnal one as P2. The distancebetween P1 and P2 is equal to 342 bp.

Figure S. Mapping of transcriptibn initiation site from the distal promoter P1in the estS gene. (a) SI mapping in the region of putative distal promoter. (1)-marker fragments (pBR322 DNA hydrolysed by Hpall endonuclease), (2)- labeledfragment used as a probe (see also Fig. lb, from -90 to 317 bp); (3)-controlhybridization with poly(A)+RNA from mouse, (4)-from embryos, (5)-larvae,(6)-pupae, (7)-the 1st day females (after emergence), (8)-the 1st day males,(9)-the 3rd day females, (10)-the 3rd day males, (11I)-the 5th day females,(12)-the 5th day males, (13)-the 10th day females, (14)-the 10th day males,(15)-the 16th day females, (16)-the 16th day males, (17)-imaginal disks oflarvae (18)-RNA transcribed in vitro from the P1 promoter (see Fig.6). Figuresat the left-size of marker fragments, bp. (b) SI mapping in the region of putativedistal promoter. (1,2)- the same as in (a), (3)-hybridization was perfonnedwith control RNA fr-om E.coli, (4)-RNA of mature females, (5)-mature males,(6)-mature males without ejaculatory bulbs, (7)-salivary glands (8)-imaginaldisks, (9)-fat body (I10)-malpighian tubules, (1 1)-female heads, (12)-maleheads, (13)-ejaculatory bulbs, (14)-ovaries, (15)-female genitals withoutovaries, (16)-ventriculus, (17)-in vitro transcribed RNA.

Mapping of P1 and P2 transcripts in vitroTo further prove that the sites detected by SI analysiscorresponded to the transcription initiation sites, an in vitrotranscription system (15) was employed. The system is verysensitive to such parameters as extract and DNA concentrations,which probably reflects dependence of certain factors on oneanother.

Transcription from P1 promoter was found to be very sensitiveto experimental conditions. The initiation site is located at thedistance of 340 bp from the initiation site of the P2 promoter.A two-fold increase of extract concentration reduced thetranscription three times while a two-fold decrease of extractconcentration completely stopped it. But the transcription fromthe proximal P2 promoter depends to a lesser extend on changesin experimental conditions (Fig.6). The transcription start pointsobtained with Si mapping of the RNA transcribed in vitro arethe same as in the case of the Si1 mapping ofmRNA of estS genetranscribed in vivo. These data supported that with SI mappingofmRNA esterase S gene we had determined 5' ends of mRNA,but not exon-intron boundaries.We conclude that transcription of the estS gene begins from

two promoter sites, the major (proximal) P2 and minor (distal)P1. P2 transcription is highly tissue-specific.The total difference

aI~ ) ~1;,.~ :,. a

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Nucleic Acids Research, 1993, Vol. 21, No. 15 3549

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622- -

527--, __

404- _

309_-

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201-

180-

a

ElEl

160- 0S

rniNA

aORF I ORF 2 ORF 3

pSV2C"A( 1)

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SV'40

pSV92CATr(2)

. ~~ ~ ~~~~~~~

promoter SV40 CAT

AUG codons 3 ORFsare substituted

mRNA translation CATi ." i__-

Figure 6. In vitro transcription of the estS gene. Transcription of RNA fromDNA contained both P1 and P2 promoters of the estS of D.virilis. (1)-markerfragments (pBR322 DNA hydrolysed by Hpall endonuclease), (2)-labeled probe,fig. lb from -60 to 590 bp (3)-hybridization with total mouse RNA, (4)-5td of the cell free extract, (5)-7 1l of the cell free extract, (6)-10 dLl of thecell free extract, (7)-15 1l of the cell free extract.

in content of transcript between ejaculatory bulbs and other tissuesof males is 50-100 x fold less than could be expected from thefact of complete absence of esterase S from any tissue butejaculatory bulbs. Therefore, we suggest that tissue-specificityof estS expression depends not only on transcriptional, but also,translational control.

The role of short open-reading frames in the control of theestS gene expressionComputer analysis of the sequence between the P1 and P2promoters showed the existence in this sequence of three shortopen-reading frames (ORF). They could encode peptides of 9,4, and 11 amino acids long. At the same time, the translationof such ORFs may inhibit further translation of downstreamsequences (21).The two first short ORFs, as well as the estS coding sequence

possess sequences around the initiation AUG which correspondto the consensus sequence for animal initiation codons recentlydetalized for Drosophila genes (22). The nucleotide sequence inneighbourhood of the third ORF is not favorable for initiationof translation. Thus sequence data allow one to suggest that atleast two short ORFs are involved in attenuation of translationfrom mRNA transcribed from the P1 promoter.To check this suggestion, we prepared two constructions based

on the pSV-2CAT one (16). The first contained the region ofestS locus with three ORFs inside the leader sequence of the CATgene. The second contained the same sequence with threesubstitutions which converted initiation AUG codons into neutralones (see Fig. 7a).The chicken HD 3 cells were transfected with both

constructions and analysed for the CAT expression (Fig. 7 b).The insertion of estS sequence containing three ORFs betweenmRNA transcription start and initiation codon of the CAT genedrastically decreased the CAT expression (at least 50-fold). Onthe other hand, the destruction of ORFs by three point mutations

promoter SV-O0 CAT SV40

b

S.0

1 2 3 4

Figure 7. Inhibition of translation in CAT assay by the ORFs from estS leadersequence. (a) Schematic diagram of pSV2CAT(l) and pSV2CAT(2) constructions(1)-the nucleotide sequence used in construction pSV2CAT(l) figlb from 160to 327; (2)-the nucleotide sequence used in construction pSV2CAT(2) figlb from160 to 327, 'A' and 'T' in ATG codons were replaced on 'C' in positions fig. lb:170- 171, 248-249, 288-289. (b) The results ofCAT assay. (1)-non-acetylatedchloramphenicol, (2)-the CAT gene expression after transfection withpSV2CAT(l) construction containing short ORFs of the estS gene (see Fig. 1),(3)-the CAT gene expression after transfection with pSV2CAT(2) constructioncontaining nucleotide substitutions destroying ORFs, (4)-CAT gene expressionafter transfection with pSV2CAT construction.

completely eliminated this effect (Fig. 7b). The transcription ofCAT was not changed upon the point mutations (not shown).Thus, three ORFs located between P1 and P2 promoters canstrongly inhibit translation of mRNA. For example, mRNAtranscribed from the P1 promoter should be very inefficient intranslation comparing to that transcribed from P2.We can conclude that at least three factors determine the high

tissue specificity of estS gene expression: overall level oftranscription; promoter selection and control at the translationlevel. This is schematically presented on Fig. 8.

DISCUSSIONWe have shown that three factors are involved in control of theestS gene expression. First, the overall level of transcription may

3550 Nucleic Acids Research, 1993, Vol. 21, No. 15

EP1 ORF 1 ORF 2 ORF 3 P2 ORF 4 Est S

PL ORF I ORF 2 ORF 3 P2 ORF 4 Est SL - 3

P1 ORF I ORF 2 ORF 3 P2 ORF 4 Est S

Translation Est S blocked

ATGGGGAATCATCTATrTAGACCCCArAA Translation Est SMietGlyAsnHisLeuLeuLysThrPro _5 _ATGGCAGAGTCTTMlAMetAloGluSer//

ATrCCAMGGGGCCAGCCATCGGhCTAATCC^6/MetProAr gGI yGlnPr oSerG luL eul lel /

Fgure 8. Schematic presentation of the control of estS expression. E-in embryos,L-in larvae imaginal disks, B-in mature male ejaculatory bulbs. (blackrectangle-promoter worked, white rectangle-promoter not worked).

be determined by tissue-specific transcription factors. Thisquestion has not yet been fully studied since the material forpreparing transcription factors (nuclei of epithelium cells fromejaculatory bulbs) is not easily available. In general, tissue-specific transcription is well studied at many different systems(23,24,25,26). The existence of two alternative promoters activeat different stages of development has been good deseribed formany genes (21). The second factor is promoter selection. Amongtwo promoters described, one, P2, possesses a very high tissueand time specificity (active only in ejaculatory bulbs epitheliumstarting from the third day after emergence). The role of P2activity in females is not clear. In Drosophila melanogaster,esterase 6 is recovered not only in ejaculatory ducts of males,but also in fat body of females (27,28). Fat body may be theplace of low esterase S expression in Drosophila virilis femalesas well. An interesting problem is to detect the sequencesdetermining such a high specificity of the P2 promoter. It shouldbe pointed out that many sequences similar to other enhancerelements are located in this region.The distal P1 promoter possesses a broader tissue specificity.

Though active in ejaculatory bulbs, it is also used for transcriptionin several other tissues (imaginal disk in the third instar larvae).However this mRNA seems not to be utilized due to translationcontrol (third factor). For example, gene expression is inhibitedat the translation level due to the presence of at least two activeORFs (22,29,30). The combination of such factors may

completely eliminate the estS gene expression from the maletissues other than epithelium of ejaculatory bulbs.The question arises as to why the P1 promoter does exists.

The first possibility is that P1 is only a non-functional promoterwhich has appeared in evolution occasionally, and then, has beenfully inactivated by the formation of short ORFs. The secondpossibility is that it may be connected with general activation ofthe estS domain in ohtogenesis. The low level of estS transcriptiondue to the existence of P1 appears at the earlier stages ofdevelopment. The block of translation may eliminate theunnecessary appearance of esterase S in wrong places. However,

the low level of transcription may change the chromatin structure,and thus, prepare it for the sharp switch on.

Interestingly, the appearance of transcription from the P1promoter coincides with the time of determination of the cellsof genital imaginal disks to a synthesis of the esterase S. It hasbeen shown that transplantation of genital imaginal disks isolatedfrom D. littoralis male larvae 48 hour after the second moultinginto D. virilis female larvae before pupation led to appearanceof esterase S in differentiated grafts from 8-10 days afteremergence. Grafts of genital imaginal disks taken 24 hour afterthe second moulting still expressed esterase S at a rather highlevel while those taken earlier (10-12 hour after the secondmoulting) did not (2,3). The time for determination of thesynthesis of the tissue-specific esterase fits well to the oneestablished previously for the synthesis of the tissue-specificesterase 6 of D.melanogaster ejaculatory ducts (27). Explanationsassigning a functional role to the P1 promoter is attractive:however, there is not evidence for such a role.The third possibility to be considered is that mRNA transcribed

from the P2 promoter is still functional. In this case, translationmay be initiated from the correct internal AUG codon due tothe existence of some sequences in this area. Finding the initiatingAUG codon has been described in few cases (31,32,33). It mightbe, for example, that P1 mRNA translation is blocked for freeribosomes by short ORFs, but the ribosomes bound to membranesor the cellular matrix may translate it.The exceptions may depend on the binding of some protein

factors to a leader sequence changing the mechanism of AUGcodon finding. This model requires some additional assumptionsto explain why esterase S is undetectable in the third instar larva.It may be the result of its different location (i.e. in the complexeswith cytoplasmic membranes). Though unlikely, anotherpossibility is that sensitivity of the method for ,B-esterase activityis not high enough. The control of the estS gene expression isan interesting example of a multistep process, and it deservesfurther investigation to ascertain answers to the questionspresented above. Most important is to determine the function ofthe Pl-initiated mRNA that seems to be inactivated by ORFs inthe 5' leader.

ACKNOWLEDGEMENTThis work was partially supported by grant provided by theRussian State Program 'Frontiers in Genetics'.

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