DEVELOPMENTAL

BIOLOGY

140,105-112

(1990)

The Chorion Genes of the Medfly, Ceratitis capitata II. Characterization of Three Novel cDNA Clones Obtained by Differential Screening of an Ovarian Library PETER P. TOLIAS,* MARY KONSOLAKI,~ KATIA KOMITOPOULOU,* AND FOTIS C. KAFATOS*+’ *Department of Cellular and Developmental Biology, The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138,U.S.A.; TInstitute of Molecular Biology and Biotechnology, Research Center of Crete, P.O. Box 1527,and Department of Biology, University of Crete, Heraklio, 711 10, Crete, Greece; and SDepartment of Biochemistry, Cell and Molecular Biology and Genetics, University of Athens, Panepistimiopolis, Kaupcnia, Athens 15701,Greece Accepted February 1.3,1990 We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library. Their isolation was accomplished by differential screening of the library using as probes 32P-labeled poly(A)+ mRNAs obtained from hand-staged medfly choriogenic versus prechoriogenic follicles. RNA blot hybridization analysis revealed that the genes corresponding to these clones have unique temporal profiles of mRNA accumulation, restricted to specific choriogenic stages. In addition, in vitro translation products encoded by these cDNAs approximately comigrated with polypeptides synthesized de nouo in culture by choriogenic follicles. All three genes are located in regions of the medfly genome that are specifically amplified in female ovaries. DNA sequence analysis has revealed that one of these clones is derived from a homolog of the Drosophila melanogaster ~38chorion gene. It appears that, although D. melanogaster and C capitata are separated by at least 120 million years of evolution, the mechanisms by which chorion genes are expressed and regulated during development have been well maintained. We suggest that the regulatory elements controlling the expression of sex-specific (e.g., chorion) genes may be isolated and used to construct transgenic medfly strains from which females could be eliminated by negative selection; such strains could be used as part of an effort to control this agricultural pest. 0 1990 Academic Press, Inc. INTRODUCTION

However, before such novel approaches can be devised The Mediterranean fruit fly Ceratitis capitata (Dip- and implemented in the field, detailed studies of the tera: Tephritidae), commonly referred to as the medfly, molecular genetics of the medfly must be undertaken, began its migration outside its ancestral sub-Saharan based on the wealth of genetic, biochemical, and molecAfrican home in the 1820s (Back and Pemberton, 1918; ular information already available for the fruit fly, DroSaul, 1986) and today is a major agricultural pest sophila melawgaster. Such studies will also be valuable throughout southern Africa, the Mediterranean, south- in providing a comparative perspective for our rapidly western Australia, Hawaii, and Central and South increasing knowledge of Drosophila. We have initiated a molecular genetic study of the America. It has also been reported sporadically on the structure and regulation of a set of female-specific mainland United States. The medfly has a particularly genes in the medfly: the genes which encode chorion wide host range. Agricultural damage in over 200 vari(eggshell) proteins. In Drosophila, chorion genes are oreties of fruits is initiated when the females deposit their ganized in two tandem clusters located on the Xand 3rd eggs (Christenson and Foote, 1960); the damage is inchromosomes (Spradling, 1981; Griffin-Shea et al., flicted by both the developing larvae (which burrow and 1982). Each gene displays a precise and unique pattern feed on the fruit) and the accompanying growth of miof expression in the ovarian follicles, i.e., sex, tissue, and croorganisms. Conventional chemical as well as sterile insect re- temporal specificity (reviewed in Kafatos et ah, 1987). In addition, both clusters are specifically amplified, exclulease programs have enjoyed limited success in controlsively in the follicular epithelial cells which surround ling the damage and proliferation of this insect. New the oocyte, shortly before transcription of the genes strategies, entailing genetic engineering and exclusive commences (Spradling and Mahowald, 1980). Amplifirelease of sterile males, may increase the effectiveness cation ensures that adequate amounts of chorion proof control programs while minimizing crop damage. teins are synthesized in the short time available. Our initial attempt to clone chorion sequences from i To whom reprint requests should be addressed at Harvard University. the medfly using major Drosophila chorion genes as 105

0012-1606/90 $3.00 Copyright All rights

63 1990 by Academic Press, Inc. of reproduction in any form reserved.

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DEVELOPMENTALBIOLOGY

probes at low stringency of hybridization was successful in recovering a single medfly clone which is a homolog of the Drosophila ~36gene (Konsolaki et al, 1990). In the present study, we have obtained three additional putative medfly chorion cDNA clones, by differential screening of a C. capitata ovarian library using labeled RNA probes obtained from prechoriogenic versus choriogenic-stage follicles. One of these cDNAs encodes a protein which is a homolog of the D. melanogaster ~38 gene product; the other two cDNAs do not cross-hybridize to any of the six known major chorion genes of D. melanogaster. The genes corresponding to these clones are each expressed with a precise and unique temporal pattern, restricted only to choriogenic-stage follicles. Moreover, all three genes are specifically amplified in ovaries but not in any other female or male tissue. These results demonstrate that we have cloned three additional medfly chorion components, and that the expression of these genes in the medfly is regulated by mechanisms similar to those that operate in Drosophila. In light of these findings, we suggest possible molecular genetic approaches which could be used in a scheme to control and possibly eradicate the medfly as a major agricultural pest. MATERIALS

AND

METHODS

Library Construction and Lkflerential Screening The medfly cDNA library was constructed according to the method of Brown and Kafatos (1988), by using total RNA from hand-dissected ovaries (Konsolaki et aL, 1990). Approximately 2500 independent clones from this library were distributed on ten plates and blotted onto nitrocellulose filters in duplicate (Brown and Kafatos, 1988). For differential screening of the duplicate filters, two probes were prepared as follows: Follicles in prechoriogenic stages (comparable to Drosophila stages l-10; King, 1970) and choriogenic stages (comparable to Drosophila stages 11-14) were pooled separately and total RNA was extracted according to Mariani et al. (1988). Both RNA pools were poly(A)+ selected with Pharmacia Poly(U)-Sepharose (Shapiro and Schimke, 1975), hydrolyzed to a mass average of approximately 150 nucleotides (by the carbonate method of Cox et aL, 1984), and 32P-end-labeled with T4 kinase (New England BioLabs) and [T-~~P]ATP (6000 Ci/mmol, New England Nuclear) according to Maniatis et al. (1982). One filter from each duplicate pair was screened with the choriogenie probe, and the other with the prechoriogenic probe, by hybridization at 65°C according to Brown and Kafatos (1988) but with yeast RNA (200 pg/ml) added to the hybridization buffers. Single clones which showed differential hybridization were selected and used to prepare DNA by the alkaline lysis mini-prep

VOLUME 140,199O

procedure (Maniatis et aL, 1982); the DNAs were digested with EcoRI and Hind111 (New England BioLabs), and analyzed by the alkaline Southern blot technique (Delidakis and Kafatos, 1987) using both prechoriogenic and choriogenic RNA probes. Used blots were striped of probe by three 30-min washes at 80°C with 2 mM TrisHCl, pH 8,0.1% SDS, 1 mM EDTA, prior to reusing. Analysis of Developmental Expression and Gene Amplification RNA blot analysis was performed as described in Mariani et aL (1988) with some minor modifications. Briefly, RNA was prepared from staged follicles, electrophoresed on 1.4% agarose gels and blotted onto Zeta-Probe (Bio-Rad) according to Delidakis and Kafatos (1987), except that the gels were not acid-treated prior to blotting and the [NaOH] was reduced to 25 mM. CsCl-purified cDNA probes were nick-translated with Escherichia coli DNA polymerase I, [a-32P]dNTPs (800 Ci/mmol, NEN) (Maniatis et aL, 1982) and purified on 2 ml Bio-gel P60 (Bio-Rad) columns. Chorion proteins synthesized in vivo at distinct stages of oogenesis were labeled in culture, by dissecting and staging individual follicles in cold Drosophila Ringer’s, and incubating for 2 hr as described previously (Thireos et al., 1979) with L-[3,5-3H]-tyrosine (58.8 Ci/mmol, NEN) and 0.5 mM phluoroglucinol (to prevent crosslinking of the chorion) (Mindrinos et aL, 1980). In vitro transcription-translation of cDNA clones was performed as described in Brown and Kafatos (1988). All protein samples were solubilized and electrophoresed on 20% SDS-polyacrylamide gels (Laemmli, 1970) and visualized by autofluorography with EN3HANCE (NEN). Genomic DNA was prepared (Delidakis and Kafatos, 1987) from males, ovariectomized females, and handdissected ovarian follicles. Following Hind111 digestion and agarose gel electrophoresis, the DNAs were blotted onto nitrocellulose filters and hybridized at 65°C (Church and Gilbert, 1984) with medfly chorion and control probes, labeled by nick-translation, and purified as above. The control probe was an &I-Hind111 restriction fragment derived from the single copy, unamplified medfly PS2a gene (Brown et aL, 1989), isolated by M. Frisardi (Harvard University) from a genomic EMBL4 library prepared by M. Rina and C. Savakis (IMBB, Crete). Sequence Analysis of the MedJy C5 cDNA and Alignments The C5 clone corresponding to the C. capitata Ccs38 gene was mapped with various restriction enzymes and subcloned into M13mp18 and M13mp19. Overlapping re-

TOLIAS ET AL.

Churion Genes of the Me&‘ly, Ceratitis capitata

107

vector. The inserts of 23 cDNA clones gave signals of similar relative intensity with both probes and were thus considered false positives. Two additional false positives gave stronger signals with the prechoriogenic probe and were not considered further (Fig. 1, lanes 10-11). However, nine cDNAs gave strong signals with the choriogenic probe and weak or no hybridization with the prechoriogenic probe; these positives were further analyzed as potential chorion sequences (Fig. 1, lanes l-9). A cDNA clone corresponding to Ccs36, the RESULTS medfly 36 chorion gene (Konsolaki et al, 1990), was included in these blots as a positive control. An ovarian cDNA library of C. capitata was conRestriction enzyme mapping of the nine positive structed in the pNB40 vector (Brown and Kafatos, clones showed that some were represented more than 1988), and was screened differentially as described once; the unique clones totaled five. One of these (C2) under Materials and Methods. A total of 34 clones, displayed a restriction enzyme mapping pattern identiwhich gave strong signals with a choriogenic-stage cal to that of the previously isolated medfly a36 cDNA RNA probe and weak or no signals with a prechorioclone (Konsolaki et ak, 1990). Southern blot analysis of genie-stage probe, were picked and analyzed as potenC2 probed with the medfly Cc&‘6 or the D. melanogaster tial chorion sequences. A secondary screen was perDms36 gene confirmed its identity (data not shown). formed by Southern blot analysis. The 34 cloned DNAs Low stringency (50°C) Southern blot analysis of the were digested with Hind111 and EcoRI (which cleave remaining unique clones, using as probes the six major near the cloning vector-cDNA insert junctions) and chorion genes from D. melanogaster (~36, ~38, ~15, ~16, blot hybridized with the same choriogenic RNA probe ~18, and s19) showed that only one clone (C5) cross-hythat was used in the initial screening. The blots were bridized with the ~38 probe (data not shown). As docuthen stripped of probe and hybridized with the premented later, this clone (C5) corresponds to Ccs38, the choriogenic probe (Fig. 1). medfly ~38 chorion gene homolog. In the secondary screen, all 34 clones were shown to To confirm the choriogenic developmental specificity contain cDNA inserts that hybridized with one or both of the five unique positives, RNA blot hybridization probes; these probes did not hybridize with the cloning analysis was performed as described under Materials and Methods. One of the sequences was found to be expressed abundantly in all choriogenic follicles (stages choriogenic probe ll-14), but was also represented, to a minor extent, cc cc within prechoriogenic follicles (stages l-10) and in the CC 12 3 4 5 6 7 6 9iOiis36 carcasses of males and ovariectomized females (data not shown). The RNA accumulation profile suggested that this clone does not represent a chorion gene. However, both the Ccs36clone (C2; Konsolaki et aZ.,1990) and the remaining three clones (C5 [Ccs38], Cl and C4) each displayed developmental expression profiles restricted to choriogenic stage follicles (Fig. 2). Expression was prechoriogenic probe not detected in males nor in the carcasses of ovariectomized females (not shown). FIG. 1. Southern blot analysis of cDNA clones selected by differential screening of a medfly ovarian cDNA library. Lanes 1-11 of the top Clones constructed in the pNB40 vector are often panel contain cDNA inserts excised from the vector as described full-length cDNA copies, and can be conveniently tranunder Materials and Methods and probed with the choriogenic =P-lascribed and translated in vitro because of an SP6 probeled RNA probe. The lane marked Ccs.% contains a sequenced cDNA moter located at the 5’ end of the cloned insert (Brown clone corresponding to the previously identified medfly ~36 gene and Kafatos, 1988). The polypeptides encoded by the (Konsolaki et al., 1990) which was included as positive control. The blot was then stripped of label and reprobed with the prechoriogenic four unique clones that showed chorion-specific RNA probe (bottom panel). Note two false positives (lanes 10 and 11) that expression patterns were synthesized by in vitro tranhybridize more intensely with the prechoriogenic probe. Lanes scription-translation and visualized following SDSmarked Cl, C2, C4, and C5 represent the four unique cDNA clones polyacrylamide gel electrophoresis as described under identified as putative chorion sequences. C2 and C5 represent the and Methods. The apparent molecular medfly homologs Co&‘6 and Ccs38of the D. melonogaster ~36 and ~38 Materials chorion genes, respectively. weights of the proteins encoded by the C2 (Ccs36), C5

striction fragments were sequenced in both directions using the chain termination procedure (Sanger et aZ., 197’7) with [%I thio-dATP (NEN; Biggen et aZ., 1983) and Sequenase (U.S. Biochemicals) (Tabor and Richardson, 1987) and were electrophoresed on 40-cm gradient gels (Biggen et al., 1983). Sequence data and alignments were analyzed using the computer programs of Pustell and Kafatos (1982, 1984) and Staden (1982, 1984).

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FIG. 2. Temporal accumulation of medfly chorion gene transcripts during oogenesis. RNA blot hybridization analysis was performed with staged ovarian follicles as described under Materials and Methods. Total RNAs (8 pg each) from the indicated stages of oogenesis were electrophoresed, blotted, and probed with the =P-labeled C2 cDNA (Ccs86; top panel). The blot was then stripped of label and reprobed sequentially for an additional three cycles using 32P-labeled C5 (Ccs38), Cl and C4, respectively.

in vitro cc

cc

~36 ~38 Cl

VOLUME 140.1990

(Ccs38), Cl, and C4 clones (determined by their relative gel mobility) were approximately 43,32,68, and 59 kDa, respectively. These products approximately comigrated with major polypeptides synthesized by staged choriogenie follicles in culture (Fig. 3): taking into account the possibilities of post-translational modification (signal peptide removal, glycosylation, etc.) these results suggest that the clones may indeed encode full-length medfly chorion proteins. Recently, our cloning of the medfly ~36 gene has revealed the first example of chorion gene-specific amplification occurring outside of the genus Drosophila (Konsolaki et aZ., 1990). Genomic blot analysis with clones C5 (CCSZ?),Cl and C4 demonstrated that all three represent genes that are also amplified at least lo-fold (relative to the single-copy, unamplified PSfi?aintegrin gene) in choriogenic ovaries (Fig. 4). Amplification was not observed in prechoriogenic follicles nor in any other male or female tissue of the medfly. In their totality, the results of the present study suggest that, in addition to Cc.s36,we have identified cDNA clones corresponding to three new medfly chorion genes, and that one of these (C5; Ccs38) is derived from a homolog of the D. melanogaster ~38 gene. The latter was

in viva

C4

10~ 1% I\ 43 kDa

- Ccs38 - PSZCT

clones

staged follicles

FIG. 3. Labeling of medfly chorion proteins in vitro and in culture. The four unique chorion cDNA clones (Ccs36, Ccs38, Cl, and C4) were transcribed and translated in vitro, and the products were electrophoresed on a 20% SDS-polyacrylamide gel (left panel). Polypeptides synthesized in culture by individual ovarian follicles from oogenic stages lo-13 were labeled with [3H]tyrosine and electrophoresed as above (right panel: e, early; 1, late). Polypeptides synthesized in culture which approximately comigrate with proteins produced in vitro from various cDNA clones are marked with corresponding symbols. The in viva component corresponding to the Cl product may be the minor 6%kDa band shown (. ), or a more prominent, ca., ‘75-kDa band. Molecular weight markers are included in kilodaltons (kDa).

FIG. 4. Amplification of medfly chorion genes in choriogenic ovaries. High molecular weight genomic DNAs were isolated from males (a), females without ovaries (&arc.), prechoriogenic ovaries (prech. ov.) and choriogenic ovaries (char. ov.), digested with Hind111 and blot hybridized as described in the text. Probing with the 3zP-labeled cDNA clones Ccs88, Cl, and C4 showed that their corresponding genomic equivalents are amplified specifically in choriogenic ovaries, at least lo-fold relative to the single copy PSZa integrin gene.

TOLIAS ET AL.

Genes of the &fed&,

Chorim

109

Ceratitis capitata

1

GACAGTCAGCTAACGGTGCTCTTGCCTTGAGAACACACAT~CAT~CTT~GAGG~CA~CACACGCCGACGGTGTATTAGC~GTTCCGC~CCCAGGCAGCAG 109

CAACAACAACAGTTTGCGTCAAA ATG AAT CGT TTT CAA CAC CTG CTT TAC ATC TGC GCT ATC GCA ATG ATT GCG TGC AGC ACA ACA Met Asn Arg Phe Gln His Leu Leu Tyr Ile Cys Ala Ile Ala Met Ile Ala Cys Ser Thr Thr 195

GCC CAT GCT TCG Ala His Ala Ser 176 _._ GGT GAT GCG CTC Gly Asp Ala Leu

TAC GGC TCG CAG GGA TCG TCA GGT GGC TAT GGT AGC GGT GGC GCT GGT GCT GGC GCT GGT GCT ATT GGT Tyr Gly Ser Gln Gly Ser Ser Gly Gly Tyr Gly Ser Gly Gly Ala Gly Ala Gly Ala Gly Ala Ile Gly AGT GGC ATC ATC CAG GGC ATC GGT TGC GGC TCG ATT CAA GGG CAT GGC AAT GGT GTA CCC AAC CCT TGC Ser Gly Ile Ile Gln Gly Ile Gly Cys Gly Ser Ile Gln Gly His Gly Asn Gly Val Pro Asn Pro Cys

357

GGT AAA ATC TTG CCT GCT CAA AGC ATC CCC TAC TCG TTG GGT CAA CGT GCC AAC ACA GTG AGC TCT TCC ATC ACT TAT CCA Gly Lys Ile Leu Pro Ala Gln Ser Ile Pro Tyr Ser Leu Gly Gln Arg Ala Asn Thr Val Ser Ser Ser Ile Thr Tyr Pro

439

CAA AAC AAA GGT GAA ATT CTT ATC CAT CGT CCG GCT GCT ATC ATT GTT AAG CGT CCA CCC ACC AAA GTG TTG GTG AAC CAT Gln Asn Lys Gly Glu Ile Leu Ile His Arg Pro Ala Ala Ile Ile Val Lys Arg Pro Pro Thr Lys Val Leu Val Asn His 519

CCT Pro 600 CAT His

CCA CTA GTT GTG AAA CCC GCA CCA GTT GTC TTG CAC AAA CCC CCA GCA GTT GTA TTG CGC AAG GTC TAT GTT AAG CAT Pro Leu Val Val Lys Pro Ala Pro Val Val Leu His Lys Pro Pro Ala Val Val Leu Arg Lys Val Tyr Val Lys His CCA CGT CCA GTT AAA GTA GAA CCC GTG TAC GTT AAT GTT GTC AAG CCA CCA GCA GAG AAG TAC TTT GTT AAC GAG AAG Pro Arg Pro Val Lys Val Glu Pro Val Tyr Val Asn Val Val Lys Pro Pro Ala Glu Lys Tyr Phe Val Asn Glu Lys

691

CAA CAA CAG GCC TAT AGC AGC GCT TAT GGC AAT AGC GGT TAT GGT GCT GGT GCT GGA GCT GGT GGT AAT GCT GGC AGT GCA Gln Gln Gln Ala Tyr Ser Ser Ala Tyr Gly Asn Ser Gly Tyr Gly Ala Gly Ala Gly Ala Gly Gly Asn Ala Gly Ser Ala 162

GCA AGT GCT GGC AAC GCC GAA GCT GCT GGA TAC CAG CTA TTA CAA GGT AGC CAA GGA TTA GCT GCT TTG GCC AAT ATC GCC Ala Ser Ala Gly Asn Ala Glu Ala Ala Gly Tyr Gln Leu Leu Gln Gly Ser Gln Gly Leu Ala Ala Leu Ala Asn Ile Ala 643

AAT GGT GGC CAA GAT GCT TAT CAT GGT GGT AAT GCC GGT CAG GGT GGT TAT GCC GCG CCA ACA GCA CAA CCT GGT TAT AAT Am Gly Gly Gln Asp Ala Tyr His Gly Gly Asn Ala Gly Gln Gly Gly Tyr Ala Ala Pro Thr Ala Gln Pro Gly Tyr Asn

921

GCT GGC AGC TCG TCA CAA GGC AGC TAC GCC TTC CCA GCA GCT CCA CAA TAC TAA AGACTACTATTTGAT ACATGGGTAG GATAGGAAT Ala Gly Ser Ser Ser Gln Gly Ser Tyr Ala Phe Pro Ala Ala Pro Gln Tyr End 1012 TAAGTCACCGAAAATATGTTCTGTTTACGGATAGAAAATGGTTATTTTAT~CCT~T~TTTTTTTACTGCACC~GGTGCC~T~TGCTTTTTT ****** FIG. 5.DNA sequence of the medfly Ccs38cDNA clone and the deduced protein sequence which itencodes.The putative polyadenylation signal(AATAAA) is highlighted. The sequence shown is immediately followed by a poly(A) track of approximately 90 A residues.

confirmed by DNA sequence analysis of the C5 clone, D. melanogaster, the medfly protein is almost perfectly which is presented in Fig. 5 along with the deduced conserved in a central domain of approximately 86 protein sequence. A protein-sequence comparison be- amino acids (positions 97 to 182 in Cc&%;95% identity tween the D. melanogaster ~38 (Dms38) and the C5 and no deletions). In contrast, regions flanking this cDNA confirmed that the latter corresponds to the central domain are largely not conserved and show mulmedfly a38 homolog (Ccs38;Fig. 6). Note that relative to tiple deletions/insertions. CCS38 Dms38

1 1

Dms38

60 5-l

CCS38 Dms38

106 117

Ccs38 Dms38

166 111

Ccs38 Dms38

219 231

Ccs38 Dms38

250 297

Ccs38

MnRfqhlly~cAiam~~s~tAhAs~G-..~~ssgg~~~gaga~gAiggdAlsgiiqG~ c' * . MtRstyiwalaAcl-%&--As?+&% ~ygpes~~~asdgd~dAasaaAaaaggaGg gcGsiqGhgnGvpnpcgkiipA-------------Qs~~llslgqra-~~vsS~~~~~~~ agGeygGanaGagalesgadaAgvaqagqssygsdQn~~~kpvntkg~~lt~~~~~

. 9 @&

182

-~qqaysSayg~sGyGaGsgs~GnaGsssss~nAea-----~ nEtPgygqgSqshghGhGhGghghGhsGhghgghgAgphgPgph

deduced D. melanogaster Dms38and C. cupitataCcs38 proteins.Exactmatches are designated by FIG. 6. Sequence alignmentbetweenthe uppercaseletters.Shadingindicatesblocksofatleasttwoconservedamino acids.DotsarepositionedateverylOalignedaminoacidpositions. Numberstotheleftofthesequencesindicateactualaminoacidpositions.Thehighlyconservedcentraldomainisdelimitedbynumbers(97and 182)thatindicate its coordinates in the medfly.

110

DEVELOPMENTALBIOLOGYV0~~~~140,1990 DISCUSSION

These results exemplify the potential of differentially screening cDNA libraries for the recovery of DNA sequences that are differentially expressed at certain developmental stages. Our low density differential screening of 2500 clones from an ovarian cDNA library resulted in the identification of four unique medfly genes which are transcriptionally regulated and specifically amplified, in a manner characteristic of Drosophila chorion genes. One of these cDNAs (C2) represents the previously characterized medfly ~36 gene homolog, Ccs36 (Konsolaki et aZ., 1990). The remaining three cDNAs appear to represent new C. capitata chorion genes. Low stringency Southern blot analysis first suggested that the C5 clone may be homologous to the D. melanogaster ~38 gene. This supposition was confirmed by DNA sequence analysis and comparisons of the encoded protein. Conservation between the D. melanogaster and the medfly protein sequences is sharply limited to a central region of approximately 86 amino acids. That region is nearly coterminous with the “central domain” of the Drosophila ~38 protein, as proposed by Hamodrakas et al. (1989). That domain is rich in valine, proline, and lysine, and is predicted to consist largely of P-sheet strands alternating regularly with P-turns. The structural predictions and the remarkable evolutionary conservation suggest that this central domain may be critical for s38 protein function. Similarly, a central domain of the s36 protein is extensively conserved between medfly and Drosophila (Konsolaki et al, 1990), and is flanked by more variable “arms”; the variable arms of s36 and s38 share certain compositional similarities (e.g., rich in alanine and serine). Silkmoth chorion proteins are also characterized by highly conserved central domains, flanked by variable arms that frequently show repetitive peptides and compositional bias (Lecanidou et aZ., 1986). Interestingly, both the amino and the carboxyl terminal arms of s38 are slightly truncated in the medfly relative to Drosophila, whereas the carboxyl terminal arm of the medfly s36 protein is extended (Konsolaki et ah, 1990). This results in an unexpected size reversal of the ~36 and ~38protein products: according to their deduced amino acid sequences, they have molecular weights of 30.1 and 30.4 kDa in D. melanogaster, as opposed to 32.3 and 28.2 kDa, respectively, in C. capitata (not corrected for removal of the signal peptide) (Spradling et al, 1987; Konsolaki et al., 1990). It should be noted that the chorion components migrate on SDS gels more slowly than expected from their actual molecular weight, both in Drosophila and in Ceratitis.

RNA blot hybridization analysis displayed the accumulation profile of Ccs38mRNA during oogenesis (Fig. 2). The transcripts accumulated mostly at stage 12, with some minor expression detected at stage 13. This profile is virtually identical to that previously observed for the medfly Ccs36 gene (Konsolaki et aL, 1990), and fairly similar to the Drosophila ~36 and ~38 genes whose expression is initiated slightly earlier, at stage 11, but terminated as above (Orr et al, 1984; Parks et a& 1986). The apparent difference in temporal expression may be due to the criteria used for staging, which have been defined independently for each insect species. Alternatively, the difference may be real, corresponding to the morphological differences between the medfly and Drosophda chorions. Interestingly, the earliest expression of the D. melanogaster ~36 gene (as evidenced in transformation studies using s36/ZacZfusions) is localized at the anterior end of the follicle, in cells that produce the dorsal appendages (Tolias and Kafatos, 1990); these appendages are absent from the medfly chorion (Margaritis, 1985). In any case, the expression period for Ccs36 and Ccs38mRNAs is relatively early, as is the case for their Drosophila homologs (Parks et ab, 1986). This pattern of RNA accumulation is in excellent agreement with the period of synthesis, in cultured stage 12 medfly follicles (especially late 12), of two major polypeptides which migrate on SDS-polyacrylamide gels with relative mobilities similar to the mobilities of the in vitro products of the Ccs36 and Ccs38clones. Moreover, both clones correspond to genes that were shown to undergo tissue-specific amplification in the follicles (Fig. 4) (Konsolaki et ab, 1990). Thus, the developmental regulatory mechanisms of ~36 and ~38,including both amplification and temporally specific expression in ovarian follicles, are remarkably conserved between Drosophila and Ceratitis. Other similarities between Drosophila and medfly ~36 and ~38 genes may include their physical organization in the genome. Recently, in situ hybridization studies of medfly polytene chromosomes have revealed that the ~36 and ~38homologs (C2 and C5) map to identical positions within the medfly 5th chromosome (A. Zacharopoulou, unpublished results). This suggests that these early chorion genes might be arranged in a single chromosomal cluster in the medfly, just as they are found on the Drosophila X-chromosome, in a tandem arrangement (Spradling, 1981). The medfly 5th chromosome appears to be equivalent to the Drosophila X, as it also contains nonchorion genes homologous to several other Drosophila X-linked genes (A. Zacharopoulou and M. Frisardi, unpublished results). Similar experiments have shown that the Cl and C4 cDNAs map to a single location on the medfly 6th chromosome (A. Zacharo-

TOLIAS ET AL.

Chtion

Genes of the MedfEy, Ceratitis

poulou, unpublished results), suggesting that they may represent two clustered medfly late chorion genes. The isolation and mapping of phage from a medfly genomic library should confirm the clustered arrangement of chorion genes and may lead to the identification of additional genes within each cluster, as in Drosophila (Spradling, 1981; Griffin-Shea et aZ.,1982). Though the accumulation profiles of Cl and C4 mRNAs are unique, both appear relatively late in oogenesis compared to the transcripts of early medfly chorion genes, such as Ccs36 and Ccs38 (Fig. 2). These late expression patterns correlate with the appearance of labeled polypeptides of appropriate molecular weight in cultured follicles (Fig. 3). Interestingly, Cl and C4 correspond to relatively high molecular weight components (68 and 59 kDa, respectively). Thus, they may not be homologous to the Drosophila low molecular weight late chorion components, ~15, ~16, ~18, and s19, which are encoded by an amplified autosomal gene cluster (Griffin-Shea et ab, 1982), although it is also possible that these genes have undergone major changes in size during evolution. If Cl and C4 are novel genes, they would be of special interest, since the only autosomal amplified cluster that has been identified thus far in Drosophila is the sl5/sl6/sl8/s19 cluster. Beyond their intrinsic interest for comparative developmental analysis, the present studies are potentially useful for medfly control (Louis et al, 1987). This major agricultural pest is susceptible to control by the sterile male technique which involves sterilization by radiation (Anwar et al., 1971) and subsequent release of mixed, male plus female medflies. However, the sterile females are highly undesirable. They can inflict serious crop damage while reducing the efficacy of pest control, because of preferential mating with coreleased sterile males. Thus, a genetic sexing strain permitting production and release of only sterile males would be most valuable. One potential approach for prerelease elimination of females would be to construct, by genetic engineering procedures, a strain bearing a dominant lethal gene under the control of a female-specific promoter, such as a chorion gene promoter. In Drosophila, it is possible to select for or against females on the basis of alcohol dehyrogenase expression in the follicles, under the control of a chorion promoter, in a construct that amplifies during choriogenesis (A. Spradling, personal communication). An alternative approach would be positive selection of males bearing a gene that confers resistance to a toxic agent, under the control of a malespecific promoter. Although genetic transformation procedures are not currently available for the medfly, recent progress in other systems, including the demonstration that the simple FLP site-specific recombinase

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