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Characterization and sequence of follicle cell genes selectively expressed during vitelline membrane formation in Drosophila
DEVELOPMENTAL
BIOLOGY
124,441-450
(1987)
Characterization and Sequence of Follicle Cell Genes Selectively Expressed during Vitelline Membrane Formation in Drosophila THOMAS BURKE, GAIL L. WARING, ELLEN POPODI, AND PARVIZ MINOO Biology Department,
Marquette
University,
Milwaukee,
Wisconsin 53235
Received March 2.4 1987; accepted in revised form August 3,
1987
To isolate genes involved in vitelline membrane production, an ovarian cDNA library was screened with eggchamber RNAs labeled in vivo. Two cDNA clones encoding RNAs that are selectively expressed in follicle cells during the period of vitelline membrane formation were isolated. Following isolation of homologous genomic clones from a Drosophila library, one gene was localized by in situ hybridization to chromosomal region 26A, and the other to 3C. Developmental Northern blots demonstrated that both genes produce 700-800 nucleotide transcripts that accumulate during the stages of vitelline membrane synthesis. In vitro translation products from hybrid selected RNAs and DNA sequence analysis both indicate that the 26A region gene encodes a major protein component of the vitelline membrane. The structural properties of the 3C region follicle cell gene seem more compatible with an intracellular function. o 1987 Academic PESS, IIIC. INTRODUCTION
During the later stages of oogenesis in Drosophila, ovarian follicle cells become actively engaged in the production of the eggshell layers, Vitelline membrane proteins are synthesized during the vitellogenic stages (8-10) (Petri et a& 1976; Fargnoli and Waring, 1982) along with the yolk polypeptides (Brennen et aZ.,1982). The follicle cells then switch to production of the chorionic sublayers (Petri et al., 1976; Waring and Mahowald, 1979). The insect eggshell provides a favorable model system for studying gene regulation during development since several proteins are synthesized in large amounts in a defined temporal order over a short time period (reviewed in Kafatos et al., 1985, 1986). Recent studies on chorion genes have revealed tissue-specific amplification prior to their expression (Spradling and Mahowald, 1980), individual regulation of genes within tight gene clusters (Griffin-Shea et al, 1982; Kalfayan et al., 1985), and a close proximity of tissue and temporal control elements to the 5’ ends of individual structural genes (Kalfayan et al., 1985; Kafatos et ah, 1985). An understanding of how follicle cells execute this developmental program as a whole will require comparable knowledge of vitelline membrane gene expression. In previous studies we have identified vitelline membrane structural proteins (Fargnoli and Waring, 1982) and vitelline membrane mRNAs in stage 10 follicle cells (Fargnoli and Waring, 1984). In this paper we describe the isolation, expression, and DNA sequence of two genes active in follicle cells during the stages of vitelline membrane formation. The 26A region gene encodes a vitelline membrane protein, while properties of the 3C 441
region gene are more consistent with an intracellular function. MATERIALS
AND METHODS
Library construction and screen. Ovarian (poly)A+ RNA was used for construction of plasmid and X-phage recombinant cDNA libraries. For the plasmid library, double-stranded cDNAs were synthesized with reverse transcriptase (Land et ab, 1981), treated with Sl (Efstradiatis et al., 1976), and inserted into the Pat1 site of pBR322 using the G/C homopolymer tailing method (Rowekamp and Firtel, 1980). Transformed HBlOl colonies were picked individually and transferred to microtiter wells. Recombinant plasmid DNAs (200-400 ng) were spotted onto nitrocellulose (Kafatos et ah, 1979) using a 96-well manifold and screened with either endlabeled (Maniatis et al, 1982) embryonic (poly)A+ or in viva labeled ovarian (poly)A+ RNA. For in viva labeling flies were injected with [32P]orthophosphate at approximately 50 &i/fly. Hybridizations with the RNA probes were at 42°C for 24 hr in 50% formamide, 1X SSPE, 5X Denhardt’s, 100 pg/ml denatured salmon sperm DNA, 100 pg/ml (poly)A, 50 gg/ml yeast tRNA, and 10% dextran sulfate. Recombinant phage libraries were constructed essentially as described by Watson and Jackson (1985). Subcloned genomic restriction fragments labeled by nick translation were used to select the appropriate cDNA clones by plaque hybridization (Benton and Davis, 1977). A Drosophila genomic X-phage library (Maniatis et ak, 1978) was screened in a similar manner with nick-translated inserts from the recombinant cDNA plasmids. 0012-1606/87 $3.00 Copyright All rights
0 1987 by Academic Press. Inc. of reproduction in any form reserved.
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DEVELOPMENTAL BIOLOGY VOLUME124,1987
RNA analyses. Cytoplasmic RNA was isolated from eggchambers and spotted onto nitrocellulose essentially as described by White and Bancroft (1982). For Northern analyses, eggchamber RNA isolated and purified as described by Spradling and Mahowald (1979) was denatured and electrophoresed in a 1.2% agarose gel containing 1.1 M formaldehyde. RNA was transferred to nitrocellulose by capillary blotting using 20X SSC buffer. Blots were hybridized with nick-translated DNA probes for 24 hr at 42°C in solutions containing 50% formamide. Following three washes in 0.2~ SSC at 65”C, blots were dried and exposed to Kodak XAR X-ray film with intensifier screens at -70°C. For transcription mapping, test RNA was incubated with 32P-antisense RNA (104-lo5 cpm) synthesized in vitro using the Riboprobe system (Promega, Madison, WI). Following hybridization overnight at 50°C (Melton et ah, 1984), the mixture was diluted lo-fold with Sl buffer (Maniatis et al., 1982) and Sl nuclease was added to 168 units/ml. After incubation at 37°C for 30 min, the Sl-resistant hybrids were heat denatured and analyzed on denaturing polyacrylamide-urea gels. To prepare templates for antisense RNA production, gel-purified restriction fragments were subcloned into pGemTM-1 or pGEMTM-2 transcription vectors. Following linearization of the template, RNA was synthesized in vitro with SP6 or T7 polymerase. All test RNAs were incubated separately with both probes to determine the sense and antisense strands. Test RNAs from follicle cells and nurse cells were prepared by cutting frozen stage 10 eggchambers at their midline with a razor blade and immediately separating the resultant nurse cell and oocyte-follicle cell halves (Fargnoli and Waring, 1982). A lo-p1 pipet partially filled with RNA extraction buffer was used to recover the nurse cell fraction. After allowing the oocyte contents to leak, the follicle cell fraction was recovered in a similar manner. Hybrid selection. Hybrid selections were done essentially as described by Riccardi et al. (1979). Recombinant plasmid DNAs were denatured by boiling in 0.1 N NaOH for 5 min. Following neutralization with l/lOth vol of 1 M Tris-HCl, pH 7.8, and l/lOth vol of 1 N HCl, NaCl was added to 1.5-3.0 M Using a Millipore filtering apparatus and mild suction, the DNA was spotted onto nitrocellulose filters equilibrated in 20x SSC. Air-dried filters were then baked at 80°C under vacuum. Small pieces of filter containing approximately 5 pg of DNA were hybridized overnight at 50°C with ovarian (poly)A+ RNA (30 pg/lOO ~1) in buffer containing 65% deionized formamide, 10 mM Pipes, pH 6.4, and 0.4 M NaCl. Filters were washed as described by Riccardi et al. (1979). To elute the RNA, filters were boiled in 150 ~1 water for 1 min. After quick freezing in dry ice-ethanol, the eluted RNA was ethanol precipitated in the pres-
ence of 250 mM ammonium acetate and carrier tRNA. RNA was translated in a nuclease-treated rabbit reticulocyte system (Promega) supplemented with high specific activity tritiated amino acids (TRK 550, Amersham). In vitro translation products were analyzed by SDS-PAGE in 15% gels as described by Laemmli (1970). In situ hybridization. Polytene chromosome squashes were prepared as described by Bonner and Pardue (1976). Biotinylated probes were prepared by nick translation with Bio-11-dUTP. Hybridization was carried out according to Enzo Biochemical protocols and signals were detected with a streptavidin biotinylated peroxidase complex. For in situ hybridization to RNA in tissue sections, ovaries were embedded in methacrylate, sectioned (1 pm), transferred to subbed slides, and baked overnight at 65°C. Following removal of the methacrylate, slides were acetylated and hybridized with RNA probes synthesized in vitro at 5 X lo4 cpm/pl. RNA probes were subjected to limited alkaline hydrolysis prior to use (Cox et al., 1984). Sections were hybridized overnight in buffer containing 40% formamide and 10% dextran sulfate at 50°C. Slides were washed with 0.2~ SSC at 55-60°C for 1 hr. DNA sequencing. Drosophila DNA fragments cloned into Gemini transcription vectors were sequenced directly according to the manufacturer’s protocol. Briefly, alkaline-denatured DNA was reannealed in the presence of primers and sequenced with DNA polymerase I (Klenow fragment) or reverse transcriptase by the dideoxy chain termination method (Sanger et ah, 1977). Reactions were run on 0.4 mm X 40-cm urea gels at 1500 V (4 and 8% polyacrylamide gels). DNA and protein sequences were analyzed by programs in the BioNet Resource. RESULTS Cmstruction of an Ovarian cDNA Library and Selection of Stage-Spec$ic Follicle Cell Clones
Yolk and vitelline membrane polypeptides are the major synthetic products of stage 10 follicle cells (Fargnoli and Waring, 1982). The concomitant synthesis of abundant follicle cell RNAs encoding the small vitelline membrane proteins (Fargnoli and Waring, 1984) suggests that vitelline membrane genes are regulated at the transcriptional level. To isolate follicle cell genes selectively transcribed during the period of vitelline membrane formation, we screened an ovarian cDNA library with RNA probes labeled in vivo. By enriching for sequences transcribed in stage 10 eggchambers, we hoped to isolate less abundant as well as abundant follicle cell sequences involved in vitelline
BURKEETAL.
Vitelline
membrane formation. A cDNA library was constructed using (poly)A-containing RNA from whole ovaries. DNA from 669 clones was spotted onto nitrocellulose filters and screened with end-labeled RNA from preblastoderm embryos to eliminate clones containing mitochondrial or housekeeping sequences from further consideration. Plasmid DNA from 384 remaining clones was spotted in duplicate onto nitrocellulose filters and hybridized with in viva labeled (poly)A containing RNA from either previtellogenic ovaries (stages O-7) or whole stage 10 eggchambers. Forty clones preferentially hybridized with the stage 10 probe (data not shown). To ensure the isolation of genes active in follicle cells and to eliminate chorion genes, these cloned sequences were rescreened with in vivo labeled RNA from choriogenic eggchambers (stages 12-14) and stage 10 follicle cells (data not shown). The results indicated three clones contained follicle cell sequences transcribed during the time of vitelline membrane formation. Subsequent cross-hybridization analyses showed that two independent sequences had been isolated. Cytogenetic Localization
of the Cloned Follicle
Cell Genes
To facilitate cytogenetic localization of the cloned follicle cell sequences we isolated homologous genomic clones from the Maniatis X-bacteriophage library of D. melanogaster genomic DNA. Recombinant phage DNA was nick translated in the presence of biotinylated dUTP and hybridized in situ to salivary gland chromo-
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FIG. 2. Localization of the 26A-1 and 3C-1 follicle cell genes. (a) The top line shows BamHl restriction sites in genomic DNA from the 26A region. An expanded map of approximately 1 kb containing sequences complementary to pDmo26A-1 (hatched box) is shown below. The polarity and approximate boundaries of the transcription unit are denoted by the arrow. Lettered lines indicate restriction fragments subcloned into plasmid transcription vectors. (b) EcoRI restriction sites are shown for the 3C region. The expanded map shows two adjacent EcoRI fragments which contain the pDmo3C-1 transcription unit (arrow). As above, the hatched box indicates the position of the pDmo3C-1 cloned cDNA while the lettered lines show the boundaries of subcloned restriction fragments. The break in the arrow indicates the approximate position of a small intron within the 3C-1 gene.
somes. As shown in Fig. 1, both genomic clones hybridized to single chromosomal sites, at regions 26A of the second chromosome and 3C of the X chromosome, respectively. Figure 2a shows a BamHI restriction map of 3 overlapping clones from the 26A region; an EcoRI map of the 3C region clone is shown below (Fig. 2b). The 26A region cDNA clone (pDmo26A-1) hybridized to a single 0.9-kb BamHI fragment (H). Hybridization with the 3C region cDNA clone (pDmo3C-1) was restricted to a single 2.0-kb EcoRI fragment (F). The directions of transcription indicated by the arrows below the expanded restriction maps were determined by hybridizations with single-stranded RNA probes and by DNA sequencing (see below). FIG. 1. Cytogenetic localization of genomic clones complementary to the cloned follicle cell cDNAs. Biotinylated recombinant phage DNAs were hybridized in situ to salivary gland chromosomes. (a) Peroxidase reaction signals localized at 26A of the second chromosome; (b) reaction product near the tip of the X chromosome at 3C.
Changes in the Concentration of pDm26A-1 and pDM.YC-1 RNAs during Oogenesis
The differential screen of our cDNA library with in vivo labeled RNA probes indicated that synthesis of the
444
DEVELOPMENTAL
BIOLOGY
3C and 26A region transcripts was maximal during the period of vitelline membrane formation. To determine if the accumulation patterns were similar to the synthesis patterns, cytoplasmic RNA was isolated from previtellogenic ovaries and from stage 10 and stages 12-14 eggchambers. The RNA was spotted in triplicate on nitrocellulose filters and each filter was hybridized to nick-translated insert DNA from one of three clones: pDmo26A-1, pDmo3G1, or pDmo7F (a chorion cDNA (~38) reisolated in our screen). Figure 3a shows that both the 3C and the 26A region cDNAs hybridize to stage 10 eggchamber RNA but not to RNA from previtellogenic or choriogenic stages (12-14). As expected, the ~38 chorion cDNA probe showed intense hybridization to RNA from stages 12 to 14. To more precisely define the stages of RNA accumulation and to determine the sizes of the RNAs complementary to pDmo26A-1 and pDmo3C-1, total RNA was isolated from equal numbers of the eggchamber stages shown in Fig. 3, electrophoresed through a 1.2% agaPv
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VOLUME124, 1987 3C
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FIG. 4. Coding capacity of 26A-1 and 3C-1 genes. Ovarian poly(A)+ RNA was hybrid selected with recombinant plasmid DNA containing either fragment H (Fig. 2) from the 26A region or fragment F from the 3C region. Translation products were fractionated on a 15% SDS-polyacrylamide gel and fluorographed. In viwo labeled vitelline membrane proteins (VM) were run in parallel; their approximate sizes (kDa) are shown on the left. The arrows indicate RNA-dependent translation products not seen in the endogenous lane (EN).
12-14
rose-formaldehyde gel, and transferred to nitrocellulose. The developmental Northern blots were then probed with genomic restriction fragments A and E shown in Fig. 2. Fragment A includes pDmo26A-1 sequences while fragment E includes pDmo3C-1 sequences. As shown in Fig. 3c, RNA complementary to pDmo26A-1 accumulates throughout the period of vitelline membrane formation (stages 8-11) and falls off precipitously in stage 12 eggchambers. RNA complementary to the 3C region sequences (Fig. 3b) is present in stage 9, reaches maximal expression in stages lOa-11, and is undetectable in stage 12 eggchambers. Both the 26A and 3C region RNAs are in the 700- to 800-nt size range. Coding Capacity and Gene Structure
The sizes and stages of expression of the RNAs complementary to the 26A and 3C region sequences are consistent with those expected to encode the small vitelline membrane proteins. To establish their translatability, genomic subclones containing sequences comFIG. 3. Stage specific accumulation of 26A-1 and 3C-1 RNA during plementary to the cDNAs were immobilized on nitrooogenesis. Cytoplasmic RNA isolated from 10 previtellogenic ovaries cellulose and hybridized to (poly)A+ RNA from (PV), stage 10, and stage 12-14 eggchambers was spotted in triplicate approximately 1000 ovaries. Messages selected by the onto nitrocellulose (a). Blots were hybridized with nick-translated 0.9-kb BamHI fragment (Fig. 2, H) from the 26A region insert DNA from either pDmo26-Al, pDmo3C1, or pDm7F, a ~38 chorion gene probe. (b, c) RNAs isolated from equal numbers of egg- and the 2.0-kb EcoRI fragment (Fig. 2, F) from the 3C chambers in the stages indicated (b, 30; c, 50) were electrophoresed, region were translated in vitro in a rabbit reticulocvte transferred to nitrocellulose, and hybridized to nick-translated DNAs lyiate. One-dimensional SDS gel electrophoresis of ihe at 4 X lo5 cpm/ml; (c) 950-bp HindIII-SmaI fragment A (Fig. 2) conproducts showed both messages encoded proteins of aptaining pDmo26-Al sequences; (b) 1.3-kb EcoRI-PvuII fragment E proximately 17 kDa (Fig. 4). The intensity of the 3C (Fig. 2) containing pDmo3C-1 sequences. The sizes of single-stranded RNA markers run in parallel are indicated at the left. translation product was considerably less than its 26A
BURKEETAL.
Vitelline Membrane Fmatim
counterpart. This may reflect differences in message abundance, efficiency of translation in vitro, or both. Both products migrate slightly more slowly than the major 17-kDa vitelline membrane protein. Hybridization of the 26A and 3C region cDNAs to Southern blots of recombinant phage DNA localized each on single restriction fragments: 0.9 kb BamHI and 1.3 kb EcoRI-PvuII, respectively. After subcloning these fragments into Gemini transcription vectors, the polarity of transcription was determined by making single-stranded RNA probes of opposite orientations and finding which strand was complementary to the mRNA by hybridization. Once the directions of transcription (Fig. 2) were established the 5’ ends of the genes were localized by Sl mapping.32P-labeled, singlestranded RNA probes made from the subcloned fragments illustrated in Fig. 2 were hybridized in excess to RNA extracted from stage 10 or 14 eggchambers. Double-stranded RNAs resistant to Sl nuclease were then denatured and sized on a sequencing type polyacrylamide gel (Fig. 5). Lane 1 shows that a single fragment of approximately 700 nt was protected when RNA from stage 10 eggchambers was hybridized with a 950-nt HindIII-SmaI RNA probe (fragment A) from the 26A region. As expected, RNA from stage 14 eggchambers failed to confer protection (lane 2). By using a smaller RNA probe, the 5’ end of the gene was placed near the 5’ end of the 170-bp HaeIII fragment (B) shown in Fig. 2. Probe B protected a stage 10 specific band in the 150- to T
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FIG. 5. Sl nuclease mapping of 26A-1 and 3C-1 5’ ends. “P-labeled, single-stranded RNA probes made from restriction fragments encompassing most of the 26A-1 gene (Fig. 3a) or its 5’end (Fig. 3b) were hybridized with RNA extracted from 30 eggchambers (stage 10 or 14). Following digestion with Sl nuclease, the resistant fragments were fractionated on a 4% denaturing polyacrylamide gel (lanes 1, 2, 4). The RNA transcript (T) used to localize the 5’end of 26A-1 is shown in lane 3. For 3C-1, a 700-nt transcript (lane 5) made from fragment C (Fig. 3~) was used for positioning the 5’ end (lane 6). The intron was localized (lane 7) with a 500-nt RNA made from fragment D (Fig. 3d). 3C region probes were hybridized with RNA extracted from 40 stage 10 eggchambers. Autoradiograms of the gels are shown with RNA (697 and 1400 nt) and DNA size markers (<622 nt).
445
160~nt size range (lane 4) whereas probe G failed to yield protected fragments (data not shown). Unlike 26A-1, two fragments of approximately 500 and 200 nt were protected by a RNA probe that spanned the 3C-1 gene (2.2-kb PvuII fragment, Fig. 2) (data not shown). More refined analyses (lane 6) indicated that the 5’ end of the gene was localized in fragment C (Fig. 2) approximately 300 bp upstream from the BamHI site. The protection of two small fragments with probe D (approximately 250 and 100 nt, respectively) indicated a small intron was located within this region. These results agree with subsequent DNA sequencing. Follicle
Cell-Specific Expression
Although the results from the screen of our cDNA library indicated both 26A-1 and 3C-1 RNAs were synthesized by follicle cells, concomitant synthesis by the metabolically active nurse cells remained a possibility. To demonstrate follicle cell-specific expression, ovarian tissue sections were hybridized with single-stranded RNA probes complementary to the 26A-1 and 3C-1 RNAs. When restriction fragment H in Fig. 2 (26A region) was used as template, grains were selectively localized over the follicle cells of stages 8, 9, and 10 eggchambers (Fig. 6a). As expected, no hybridization was detected with previtellogenic (O-7) or choriogenic (12-14) staged eggchambers. 3C-1 region probes failed to show grains over background levels. As an alternative means to demonstrate the follicle cell specificity of the 3C region transcript, we cut stage 10 eggchambers at their midline (arrow, Fig. 6) to generate follicle cell and nurse cell fractions. RNA was extracted from each fraction, hybridized to 32P-labeled single-stranded RNA probes in excess, and assayed for Sl protected fragments of the predicted size. The results with 26A-1 shown in Fig. 6b confirm the reliability of the method. As expected, when fragment A (Fig. 2) was used as template a 700-nt fragment was protected with follicle cell but not nurse cell RNA. The comparable amount of probe protected by follicle cell and stage 10 eggchamber RNA is also consistent with its follicle cell-specific expression. Similar results were obtained with 3C-1 (Fig. 6~). A protected fragment of the expected size (250 nt) was obtained with whole eggchamber and follicle cell RNA extracts. The absence of signal in the nurse cell fraction coupled with the similar signal intensities in the stage 10 and follicle cell fractions indicate that, like 26A-1, the expression of 3C-1 is limited to the follicle cells. DNA Sequence Analysis
To map the transcripts more precisely, determine the structures of the genes in detail, and gain insights into
DEVELOPMENTAL BIOLOGY
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VOLUME 124, 1987
Analysis of the DNA sequence predicts a message size of 629 bp before (poly)A addition. A single long open-reading frame is found that begins at a methionine codon 82 bp from the 5’ end and extends to within 124 bp of the 3’ end. Conceptual translation predicts a 14,322-Da protein product with properties expected for Drosophila vitelline membrane proteins. The amino terminal segment possesses features typical of secreted proteins with known signal peptides. A hydrophobic core of 18 amino acids is followed within four to six residues by predicted cleavage sites (arrows). The high
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FIG. 6. Follicle cell-specific expression of 26A-1 and 3C-1. (a) Ovarian tissue sections were hybridized with alkaline-digested RNA probe complementary to the 26A-1 transcript (fragment H, Fig. 3). Eggchambers at different developmental stages (PV, previtellogenic) are shown; cell types are marked for a stage 10 eggchamber (fc, follicle cell; nc, nurse cell; 00, oocyte). Stage 10 eggchambers were cut at the arrow to give nurse cell and follicle cell fractions for Sl analysis (b, c). RNA from 50 stage 10 eggchambers or the follicle cell and nurse cell fractions from an equivalent number were hybridized with “P-labeled RNA probes made from restriction fragments A and D (Fig. 3). The results are shown in (b) and (c), respectively. Unlike the nurse cell lanes, protected fragments are apparent in the stage 10 eggchamber and follicle cell lanes.
functional roles, we sequenced both cDNAs and genomic DNAs by the dideoxy chain termination method. The sequencing strategy and complete genomic sequence of 26A-1 is shown in Fig. 7. The 3’ end of the gene was positioned by the (poly)A tract found at the end of the cDNA. The putative 5’ terminus of the mRNA was specified by consideration of the genomic sequence. As expected from our Sl analyses, a mRNA consensus start site, CATCAGT, was found near the 5’ end of the 170-bp HaeIII fragment. The sequence TATAAAA appears 35 bp upstream from this postulated transcription initiation point. This is consistent with the spacing and sequence of typical eukaryotic promotors.
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FIG. 7. DNA and predicted amino acid sequence of 26A-1. A restriction map of the 950-bp HindIII-SmaI fragment from region 26A is shown with arrows that indicate the extent and direction of the sequence analysis. The solid arrows represent sequences read from genomic subclones, while the dashed arrows indicate cDNA sequences read. Sequences determined from the cDNA clone are underlined. The proposed 5’ end is shown by a rightward arrow. Nucleotide 1 is the putative cap site. The coding region with the predicted amino acid sequence is set apart. The downward arrows mark potential signal peptide cleavage sites. The box indicates a potential TATA sequence at -35. The open arrowhead on the restriction map indicates the proposed transcription start site.
BURKE ET AL.
Vitelline Membrane Fm-rnaticm
alanine, glycine, proline, and serine contents of the mature protein (9.5,14.7,11.2, and 12.9%, respectively) are consistent with the average amino acid composition of the total vitelline membrane measured by Petri et al.
447
(1976). The predicted molecular weight of the mature protein is 12,080 Da. The 3C-1 cDNA and genomic sequences are shown in Fig. 8. Sl analysis placed the 5’ end of the gene (open
FIG. 8. DNA and predicted amino acid sequence of 3C-1. A restriction map of the 3C region that encompasses the 3C-1 transcription unit is shown. The arrows indicate the extent and direction of the sequences read from the subcloned regions: solid arrows, genomic sequences; dashed arrows, cDNA sequences. Nucleotides in lower case were derived from genomic subclones while those in upper case are from both cDNA and genomic readings. Potential 5’ ends are shown by the rightward arrows; the 3’ end predicted by the plasmid cDNA clone (minus l-4As?) is marked by a leftward arrow. Numbering in the coding region is based on the cDNA sequence. The boxes indicate the potential TATA sequence at -32 and the potential (poly)A addition site at 768. The open arrowhead on the restriction map indicates the approximate position of the proposed site of transcription initiation.
448
DEVELOPMENTALBIOLOGY
arrowhead) approximately 300 bp upstream from the BamHI site shown in Fig. 8. Within that range are two sequences, CATCCAT at position 1 and CATCCAG at position 16, that show a reasonable match to the proposed cap site consensus sequence for Drosophila (Snyder et al, 1982). Although the 5’ end of a 3C cDNA clone isolated from a X ovarian cDNA library (see Materials and Methods) terminated at position 17, the position at a potential TATA box at -32 is more compatible with the +l cap site. Sl analyses using subclone E in Fig. 2 indicates this 3C region mRNA terminates approximately 200 bp downstream from the EcoRI site (data not shown). The short plasmid cDNA clone, pDmo3C-1, ended in a 3’ (poly)A tract at position 789, 199 bp downstream from the EcoRI site shown in Fig. 8. The presence of a consensus (poly)A addition site (AATAAA) 1’7 bp upstream from the 3’ terminus of this cDNA is consistent with this interpretation of the 3’ end. The cDNA and genomic sequences were identical over most of their lengths except for a short 73-bp intron located at position 568. The sequence of the intron borders conform to the GT . . . AG rule of 5’-donor 3’-acceptor splice sites and the position of the intron is in excellent agreement with that predicted by our Sl data. Sequence analyses predicted a single long openreading frame starting with an ATG codon at nt 113 and ending with a TGA termination codon at nt 743. While in the general size range, the size of the predicted protein product is slightly larger than was expected based on the mobility of the 3C-1 hybrid selected translation product. Conceptual translation of the openreading frame yields a protein product of 22,346 Da. Unlike 26A-1, the predicted 3C polypeptide does not have an amino terminal peptide that resembles a leader sequence. The predicted amino acid composition indicates the protein is rich in serine and threonine (both 11.4%). DISCUSSION
Using in viva labeled RNA probes we have isolated two genes transcribed in follicle cells during the period of vitelline membrane formation. While the function of the 3C-1 gene product remains speculative, as indicated earlier, several properties of the 26A-1 gene are consistent with a vitelline membrane structural gene. The size of the primary translation product and the relative abundance of the 26A-1 transcript are most compatible with Sv17.5, a major vitelline membrane protein reported previously (Fargnoli and Waring, 1982). A precursor comparable in size to the 26A-1 product was noted in earlier translation studies of T2 follicle cell RNA from stage 10 eggchambers (Fargnoli and Waring, 1984). When translated in vitro with microsomal mem-
VOLUME124,1987
branes, T2 yielded a processed product that comigrated with Sv17.5. Preliminary partial peptide data provide additional support for 26A-1 encoding Sv17.5. When Sv17.5 and the primary translation product of 26A-1 were cleaved with Vs protease, both proteins produced the same large proteolytic fragment (P. Minoo and G. Waring, unpublished data). The predicted size of the 26A-1 translation product from DNA sequence analysis, 14,322, is smaller than that estimated by its mobility on SDS gels. A similar discrepancy has been reported for the small chorion proteins (Levine and Spradling, 1985; Wong et aZ.,1985). The predicted molecular weights from sequence analysis are smaller than the size estimates of Waring and Mahowald (1979) but agree with those of Petri et al. (1976). Despite discrepancies in actual size, the relative migration of ~18, Sv17.5, and s15 in our gel system (unpublished) is consistent with the relative sizes of the mature proteins predicted by DNA sequence analysis: 15,027, 12,080, and 9467 Da, respectively. The 26A region has been previously implicated as a vitelline membrane structural gene site based on complementarity of cloned sequences to mRNAs that are selectively expressed in vitellogenic eggchambers (Higgins et aZ., 1984; Mindrinos et al, 1985). Based on map location and transcript size, 26A-1 appears to be comparable to the cr-1 transcription unit described by Higgins et al. (1984) and Mindrinos et al. (1985). Like the chorion genes, vitelline membrane genes appear to be clustered. Within an S-kb region, we have identified four genes, including 26A-1, that are selectively expressed in follicle cells during the period of vitelline membrane formation (Minoo et ab, 1986). We have also mapped a female sterile mutation affecting the synthesis of one of the major vitelline membrane proteins (Sv23) to the 26A region (S. Savant and G. Waring, unpublished results). 26A-1, like the chorion genes (Levine and Spradling, 1985; Wong et aZ.,1985), share structural features typical of most eukaryotic genes. A TATA box is found 30 bp upstream from the transcription start site and typical consensus sequences are found at the mRNA and translation start sites. As with the chorion genes, 26A-1 has short 5’ and 3’ untranslated regions and hydropathicity plots of the predicted amino acid sequence (not shown) indicate multiple hydrophilic and hydrophobic segments in an alternating arrangement (Wong et aZ., 1985). Whereas all chorion genes analyzed to date show a short 5’ intron separating one small and one large exon, the 26A-1 gene lacks introns. While the evidence that 26A-1 encodes a vitelline membrane structural protein is reasonably compelling, the function of the 3C region follicle cell gene remains speculative. Although the timing of its expression is
BURKEET AL.
Vitelline
consistent with a role in vitelline membrane formation, several of its properties do not conform to those expected of the typical eggshell protein gene. The predicted amino acid composition is not characterized by a high alanine, proline, or glycine content and a potential signal peptide was not revealed by DNA sequence analysis. Based on Kyte and Doolittle’s hydrophobicity requirements (1982), 3C-1 does not appear to have lipophilic spikes of sufficient length and magnitude to participate in membrane binding (data not shown). Like 26A-1, 3C-1 appears to be part of a gene cluster. Another low abundance transcript with a similar temporal profile has been localized within 2 kb of the 3’ end of 3C-1 (E. Popodi and G. Waring, unpublished). The 3C region has previously been implicated in vitelline membrane formation by genetic studies. Komitopoulou et al. (1983) described a 3C region female sterile mutation (K93) whose expression was dependent on the somatic line (Perrimon and Gans, 1983). Mutant flies laid flaccid eggs that showed strong uptake of dye, characteristics that might be expected of a vitelline membrane defect. Of the 24 female-sterile complementation groups characterized, permeability changes were only reported in K93 eggs. In this regard, it is interesting to note that eggs from the 26A vitelline membrane mutant mentioned previously also display altered permeability properties (S. Savant, unpublished). In summary, our data suggest that 26A-1 encodes a major early eggshell protein. While a definitive assignment is not possible in the absence of protein sequence data, we feel it is most likely that 26A-1 encodes Sv17.5 (~14.5) (Mindrinos et ab, 1985). Unlike 26A-1, 3C-1 encodes a follicle cell protein of unknown function. No significant homologies were detected with any proteins in the NBRF library. Although the amino acid sequence of the predicted protein appears to be more compatible with an intracellular function (no signal peptide), immunocytochemical localization will be required to resolve this question. We thank Ms. B. DeNoyer for help in the preparation of the manuscript and J. Pollack for expert technical assistance. This work was supported by National Science Foundation Grants PCM-8203171 and DCB-8502633 to G.L.W., who was the recipient of Research Career Development Award HD00524 and by Marquette University and Scholl Foundation predoctoral fellowships to T.B. REFERENCES BENTON,W. D., and DAVIS, R. W. (1977). Screening Xgt recombinant clones by hybridization to single plaques in situ. Science 196, 180-182.
BONNER,J. J., and PARDUE, M. L. (1976). Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster. Chrome sonw, 58,87-99. BRENNEN, M. D., WEINER, A. J., GORALSKI, T. J., and MAHOWALD, A. P. (1982). The follicle cells are a major site of vitellogenin synthesis in Drosophila melanogaster. Dev. Biol 83,225-236.
Membrane
449
Formation
Cox, K. H., DELEON, D. V., ANGERER, L. M., and ANGERER, R. C. (1984). Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101,485-502. EFSTRATIADIS,A., KAFATOS, F. C., MAXAM, A. M., and MANIATIS, T. (1976). Enzymatic in vitro synthesis of globin genes. Cell 7,279-288. FARGNOLI,J., and WARING, G. (1982). Identification of vitelline membrane proteins in Drosophila melanogaster. Dev. Biol. g&306-314. FARGNOLI,J., and WARING, G. L. (1984). Identification and genetic localization of vitelline membrane mRNAs of Drosophila melanogaster. Dev. Biol. 105,41-47.
GRIFFIN-SHEA, R., THIREOS,G., and KAFATOS,F. C. (1982). Organization of a cluster of four chorion genes in Drosophila and its relationship to developmental expression and amplification. Den Biol. 91,325-339.
HIGGINS,M. J., WALKER, V. K., HOLDEN,J. J., and WHITE, B. N. (1984). Isolation of two Drosophila melanogaster genes abundantly expressed in the ovary during vitelline membrane synthesis. Dev. Biol
105,155-165.
KAFATOS,F. C., JONES,C. W., and EFSTRADIATIS,A. (1979). Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucl. Acids Res. 7, 1541-1552. KAFATOS,F. C., MITSIALIS, S. A., SPOEREL,N., MARIANI, B., LINGAPPA, J. R., and DELIDAKIS, C. (1985). Studies on the developmentally regulated expression and amplification of insect chorion genes. Cold Spring Harb. Symp. Quark Biol. 1,537-547.
KAFATOS,F. C., DELIDAKIS, C., ORR,W., THIREOS,G., KOMITOPOULOU, K., and WONG, Y.-C. (1986). Studies on the developmentally regulated amplification and expression of Drosophila chorion genes. Symp. Sot. Dev. Biol. 43,85-101.
KALFAYAN, L., LEVINE, J., ORR-WEAVER,T., PARKS,S., WAKIMOTO,B., DECICCO,D., and SPRADLING,A. C. (1985). Localization of sequences regulating Drosophila chorion gene amplification and expression. Cold Spring Harb. Symp. @ant.
Biol. 1.527-535.
KOMITOPOULOU, K., GANS,M., MARGARITIS,L. H., KAFATOS,F. C., and MASSON,M. (1983). Isolation and characterization of sex-linked female-sterile mutants in Drosophila melanogaster with special attention to eggshell mutants. Genetics 105, 897-908. KYTE, J., and DOOLITTLE,R. (1982). A simple method for displaying the hydropathic character of a protein. J. Mel Biol 157,105-132. LAEMMLI,U. (1970). Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature (London) 227,680-685. LAND, H., GREZ, M., HAUSER, R., WINDENMAIER,W., and SCHUTZ,G. (1981). 5’ terminal sequences of eucaryotic mRNA can be cloned with high efficiency. Nucl. Acids Res. 9,2251-2266. LEVINE, J., and SPRADLING,A. (1985). DNA sequence of a 3.8 kilobase pair controlling Drosophila chorion amplification. Chrorrwsoma 92, 136-142. MANIATIS, T., FRITSCH, E. F., and SAMBROOK,J. (1982). “Molecular Cloning.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MANIATIS, T., HARDISON, R. C., LACY, E., LAUER, J., O’CONNELL,C., QUON,D., SIM, G. K., and EFSTRATIADIS,A. (1978). The isolation of structural genes from libraries of eucaryotic DNA. Cell 15,687-701. MELTON,D. A., KRIEG, P. A., ROBAGLIATTE,M. R., MANIATIS, T., ZINN, K., and GREEN, M. R. (1984). Efficient in vitro synthesis of biologically active RNA and DNA hybridization probes from plasmids containing a bacteriophage sp6 promotor. Nucl. Acids Res. 12, 7035-7036. MINDRINOS,M. N., SCHERER,L. J., GARCINI, F. J., KWAN, H., JACOBS, K. A., and PETRI, W. H. (1985). Isolation and chromosomal location of putative vitelline membrane genes in Drosophila melanogaster. EMBO J. 4,147-153. MINOO, P., POPODI, E., BURKE, T., and WARING,G. (1986). Genetics 113,
552. PERRIMON,N., and GANS, M. (1983). Clonal analysis of the tissue
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DEVELOPMENTALBIOLOGY
specificity of recessive female-sterile mutations of Drosophila melanogaster using a dominant female-sterile mutation Fs(l)K1237. Dev. Biol. 100,365-373. PETRI, W. H., WYMAN, A. R., and KAFATOS, F. C. (1976). Specific protein synthesis in cellular differentiation. III. The eggshell proteins of Drosophila melanogaster and their program of synthesis. Dev. Biol 49, X35-199. RICCIARDI,R. P., MILLER, J. S., and ROBERTS,B. E. (1979). Purification and mapping of specific mRNAs by hybridization selection and cell-free translation. Proc. Natl. Acad Sci. USA 76(10), 4927-4931. ROWENCAMP,W., and FIRTEL, R. A. (1980). Isolation of developmentally regulated genes from Dictyostelzum. Dev. Biol. 79,409-418. SANGER,F., NICKLEN, S., and COULSON,A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad Ski. USA 74, 54635467. SPRADLING,A. C., and MAHOWALD,A. P. (1979). Identification and genetic localization of mRNAs from ovarian follicle cells of DrosoCell 16,589-598. phila mehnogaster.
VOLUME124.1987
SPRADLING, A. C., and MAHOWALD, A. P. (1980). Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proc. Natl. Acad Sci. USA 77,1096-1100. SNYDER,M., HUNKAPILLAR, M., YUEN, D., SILVERT, D., FRISTROM,J., and DAVIDSON, N. (1982). Cuticle protein genes of Drosophila: Structure, organization and evolution of four clustered genes. Cell 29,1027-1040. WARING, G. L., and MAHOWALD,A. P. (1979). Identification and time of synthesis of chorion proteins in Drosophila mdanogastw. Cell 16, 599-607. WATSON,C. J., and JACKSON,J. F. (1985). An alternative procedure for the synthesis of double-stranded cDNA for cloning in phage and plasmid vectors. In “DNA Cloning” (D. M. Glover, Ed.), Vol. 1, pp. 79-88. IRL Press, Oxford. WHITE, B. A., and BANCROFT,F. C. (1982). Cytoplasmic dot hybridization: Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J. Biol. Chem. 257,8569-8572.
BIOLOGY
124,441-450
(1987)
Characterization and Sequence of Follicle Cell Genes Selectively Expressed during Vitelline Membrane Formation in Drosophila THOMAS BURKE, GAIL L. WARING, ELLEN POPODI, AND PARVIZ MINOO Biology Department,
Marquette
University,
Milwaukee,
Wisconsin 53235
Received March 2.4 1987; accepted in revised form August 3,
1987
To isolate genes involved in vitelline membrane production, an ovarian cDNA library was screened with eggchamber RNAs labeled in vivo. Two cDNA clones encoding RNAs that are selectively expressed in follicle cells during the period of vitelline membrane formation were isolated. Following isolation of homologous genomic clones from a Drosophila library, one gene was localized by in situ hybridization to chromosomal region 26A, and the other to 3C. Developmental Northern blots demonstrated that both genes produce 700-800 nucleotide transcripts that accumulate during the stages of vitelline membrane synthesis. In vitro translation products from hybrid selected RNAs and DNA sequence analysis both indicate that the 26A region gene encodes a major protein component of the vitelline membrane. The structural properties of the 3C region follicle cell gene seem more compatible with an intracellular function. o 1987 Academic PESS, IIIC. INTRODUCTION
During the later stages of oogenesis in Drosophila, ovarian follicle cells become actively engaged in the production of the eggshell layers, Vitelline membrane proteins are synthesized during the vitellogenic stages (8-10) (Petri et a& 1976; Fargnoli and Waring, 1982) along with the yolk polypeptides (Brennen et aZ.,1982). The follicle cells then switch to production of the chorionic sublayers (Petri et al., 1976; Waring and Mahowald, 1979). The insect eggshell provides a favorable model system for studying gene regulation during development since several proteins are synthesized in large amounts in a defined temporal order over a short time period (reviewed in Kafatos et al., 1985, 1986). Recent studies on chorion genes have revealed tissue-specific amplification prior to their expression (Spradling and Mahowald, 1980), individual regulation of genes within tight gene clusters (Griffin-Shea et al, 1982; Kalfayan et al., 1985), and a close proximity of tissue and temporal control elements to the 5’ ends of individual structural genes (Kalfayan et al., 1985; Kafatos et ah, 1985). An understanding of how follicle cells execute this developmental program as a whole will require comparable knowledge of vitelline membrane gene expression. In previous studies we have identified vitelline membrane structural proteins (Fargnoli and Waring, 1982) and vitelline membrane mRNAs in stage 10 follicle cells (Fargnoli and Waring, 1984). In this paper we describe the isolation, expression, and DNA sequence of two genes active in follicle cells during the stages of vitelline membrane formation. The 26A region gene encodes a vitelline membrane protein, while properties of the 3C 441
region gene are more consistent with an intracellular function. MATERIALS
AND METHODS
Library construction and screen. Ovarian (poly)A+ RNA was used for construction of plasmid and X-phage recombinant cDNA libraries. For the plasmid library, double-stranded cDNAs were synthesized with reverse transcriptase (Land et ab, 1981), treated with Sl (Efstradiatis et al., 1976), and inserted into the Pat1 site of pBR322 using the G/C homopolymer tailing method (Rowekamp and Firtel, 1980). Transformed HBlOl colonies were picked individually and transferred to microtiter wells. Recombinant plasmid DNAs (200-400 ng) were spotted onto nitrocellulose (Kafatos et ah, 1979) using a 96-well manifold and screened with either endlabeled (Maniatis et al, 1982) embryonic (poly)A+ or in viva labeled ovarian (poly)A+ RNA. For in viva labeling flies were injected with [32P]orthophosphate at approximately 50 &i/fly. Hybridizations with the RNA probes were at 42°C for 24 hr in 50% formamide, 1X SSPE, 5X Denhardt’s, 100 pg/ml denatured salmon sperm DNA, 100 pg/ml (poly)A, 50 gg/ml yeast tRNA, and 10% dextran sulfate. Recombinant phage libraries were constructed essentially as described by Watson and Jackson (1985). Subcloned genomic restriction fragments labeled by nick translation were used to select the appropriate cDNA clones by plaque hybridization (Benton and Davis, 1977). A Drosophila genomic X-phage library (Maniatis et ak, 1978) was screened in a similar manner with nick-translated inserts from the recombinant cDNA plasmids. 0012-1606/87 $3.00 Copyright All rights
0 1987 by Academic Press. Inc. of reproduction in any form reserved.
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DEVELOPMENTAL BIOLOGY VOLUME124,1987
RNA analyses. Cytoplasmic RNA was isolated from eggchambers and spotted onto nitrocellulose essentially as described by White and Bancroft (1982). For Northern analyses, eggchamber RNA isolated and purified as described by Spradling and Mahowald (1979) was denatured and electrophoresed in a 1.2% agarose gel containing 1.1 M formaldehyde. RNA was transferred to nitrocellulose by capillary blotting using 20X SSC buffer. Blots were hybridized with nick-translated DNA probes for 24 hr at 42°C in solutions containing 50% formamide. Following three washes in 0.2~ SSC at 65”C, blots were dried and exposed to Kodak XAR X-ray film with intensifier screens at -70°C. For transcription mapping, test RNA was incubated with 32P-antisense RNA (104-lo5 cpm) synthesized in vitro using the Riboprobe system (Promega, Madison, WI). Following hybridization overnight at 50°C (Melton et ah, 1984), the mixture was diluted lo-fold with Sl buffer (Maniatis et al., 1982) and Sl nuclease was added to 168 units/ml. After incubation at 37°C for 30 min, the Sl-resistant hybrids were heat denatured and analyzed on denaturing polyacrylamide-urea gels. To prepare templates for antisense RNA production, gel-purified restriction fragments were subcloned into pGemTM-1 or pGEMTM-2 transcription vectors. Following linearization of the template, RNA was synthesized in vitro with SP6 or T7 polymerase. All test RNAs were incubated separately with both probes to determine the sense and antisense strands. Test RNAs from follicle cells and nurse cells were prepared by cutting frozen stage 10 eggchambers at their midline with a razor blade and immediately separating the resultant nurse cell and oocyte-follicle cell halves (Fargnoli and Waring, 1982). A lo-p1 pipet partially filled with RNA extraction buffer was used to recover the nurse cell fraction. After allowing the oocyte contents to leak, the follicle cell fraction was recovered in a similar manner. Hybrid selection. Hybrid selections were done essentially as described by Riccardi et al. (1979). Recombinant plasmid DNAs were denatured by boiling in 0.1 N NaOH for 5 min. Following neutralization with l/lOth vol of 1 M Tris-HCl, pH 7.8, and l/lOth vol of 1 N HCl, NaCl was added to 1.5-3.0 M Using a Millipore filtering apparatus and mild suction, the DNA was spotted onto nitrocellulose filters equilibrated in 20x SSC. Air-dried filters were then baked at 80°C under vacuum. Small pieces of filter containing approximately 5 pg of DNA were hybridized overnight at 50°C with ovarian (poly)A+ RNA (30 pg/lOO ~1) in buffer containing 65% deionized formamide, 10 mM Pipes, pH 6.4, and 0.4 M NaCl. Filters were washed as described by Riccardi et al. (1979). To elute the RNA, filters were boiled in 150 ~1 water for 1 min. After quick freezing in dry ice-ethanol, the eluted RNA was ethanol precipitated in the pres-
ence of 250 mM ammonium acetate and carrier tRNA. RNA was translated in a nuclease-treated rabbit reticulocyte system (Promega) supplemented with high specific activity tritiated amino acids (TRK 550, Amersham). In vitro translation products were analyzed by SDS-PAGE in 15% gels as described by Laemmli (1970). In situ hybridization. Polytene chromosome squashes were prepared as described by Bonner and Pardue (1976). Biotinylated probes were prepared by nick translation with Bio-11-dUTP. Hybridization was carried out according to Enzo Biochemical protocols and signals were detected with a streptavidin biotinylated peroxidase complex. For in situ hybridization to RNA in tissue sections, ovaries were embedded in methacrylate, sectioned (1 pm), transferred to subbed slides, and baked overnight at 65°C. Following removal of the methacrylate, slides were acetylated and hybridized with RNA probes synthesized in vitro at 5 X lo4 cpm/pl. RNA probes were subjected to limited alkaline hydrolysis prior to use (Cox et al., 1984). Sections were hybridized overnight in buffer containing 40% formamide and 10% dextran sulfate at 50°C. Slides were washed with 0.2~ SSC at 55-60°C for 1 hr. DNA sequencing. Drosophila DNA fragments cloned into Gemini transcription vectors were sequenced directly according to the manufacturer’s protocol. Briefly, alkaline-denatured DNA was reannealed in the presence of primers and sequenced with DNA polymerase I (Klenow fragment) or reverse transcriptase by the dideoxy chain termination method (Sanger et ah, 1977). Reactions were run on 0.4 mm X 40-cm urea gels at 1500 V (4 and 8% polyacrylamide gels). DNA and protein sequences were analyzed by programs in the BioNet Resource. RESULTS Cmstruction of an Ovarian cDNA Library and Selection of Stage-Spec$ic Follicle Cell Clones
Yolk and vitelline membrane polypeptides are the major synthetic products of stage 10 follicle cells (Fargnoli and Waring, 1982). The concomitant synthesis of abundant follicle cell RNAs encoding the small vitelline membrane proteins (Fargnoli and Waring, 1984) suggests that vitelline membrane genes are regulated at the transcriptional level. To isolate follicle cell genes selectively transcribed during the period of vitelline membrane formation, we screened an ovarian cDNA library with RNA probes labeled in vivo. By enriching for sequences transcribed in stage 10 eggchambers, we hoped to isolate less abundant as well as abundant follicle cell sequences involved in vitelline
BURKEETAL.
Vitelline
membrane formation. A cDNA library was constructed using (poly)A-containing RNA from whole ovaries. DNA from 669 clones was spotted onto nitrocellulose filters and screened with end-labeled RNA from preblastoderm embryos to eliminate clones containing mitochondrial or housekeeping sequences from further consideration. Plasmid DNA from 384 remaining clones was spotted in duplicate onto nitrocellulose filters and hybridized with in viva labeled (poly)A containing RNA from either previtellogenic ovaries (stages O-7) or whole stage 10 eggchambers. Forty clones preferentially hybridized with the stage 10 probe (data not shown). To ensure the isolation of genes active in follicle cells and to eliminate chorion genes, these cloned sequences were rescreened with in vivo labeled RNA from choriogenic eggchambers (stages 12-14) and stage 10 follicle cells (data not shown). The results indicated three clones contained follicle cell sequences transcribed during the time of vitelline membrane formation. Subsequent cross-hybridization analyses showed that two independent sequences had been isolated. Cytogenetic Localization
of the Cloned Follicle
Cell Genes
To facilitate cytogenetic localization of the cloned follicle cell sequences we isolated homologous genomic clones from the Maniatis X-bacteriophage library of D. melanogaster genomic DNA. Recombinant phage DNA was nick translated in the presence of biotinylated dUTP and hybridized in situ to salivary gland chromo-
Me&ane
443
Formation
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FIG. 2. Localization of the 26A-1 and 3C-1 follicle cell genes. (a) The top line shows BamHl restriction sites in genomic DNA from the 26A region. An expanded map of approximately 1 kb containing sequences complementary to pDmo26A-1 (hatched box) is shown below. The polarity and approximate boundaries of the transcription unit are denoted by the arrow. Lettered lines indicate restriction fragments subcloned into plasmid transcription vectors. (b) EcoRI restriction sites are shown for the 3C region. The expanded map shows two adjacent EcoRI fragments which contain the pDmo3C-1 transcription unit (arrow). As above, the hatched box indicates the position of the pDmo3C-1 cloned cDNA while the lettered lines show the boundaries of subcloned restriction fragments. The break in the arrow indicates the approximate position of a small intron within the 3C-1 gene.
somes. As shown in Fig. 1, both genomic clones hybridized to single chromosomal sites, at regions 26A of the second chromosome and 3C of the X chromosome, respectively. Figure 2a shows a BamHI restriction map of 3 overlapping clones from the 26A region; an EcoRI map of the 3C region clone is shown below (Fig. 2b). The 26A region cDNA clone (pDmo26A-1) hybridized to a single 0.9-kb BamHI fragment (H). Hybridization with the 3C region cDNA clone (pDmo3C-1) was restricted to a single 2.0-kb EcoRI fragment (F). The directions of transcription indicated by the arrows below the expanded restriction maps were determined by hybridizations with single-stranded RNA probes and by DNA sequencing (see below). FIG. 1. Cytogenetic localization of genomic clones complementary to the cloned follicle cell cDNAs. Biotinylated recombinant phage DNAs were hybridized in situ to salivary gland chromosomes. (a) Peroxidase reaction signals localized at 26A of the second chromosome; (b) reaction product near the tip of the X chromosome at 3C.
Changes in the Concentration of pDm26A-1 and pDM.YC-1 RNAs during Oogenesis
The differential screen of our cDNA library with in vivo labeled RNA probes indicated that synthesis of the
444
DEVELOPMENTAL
BIOLOGY
3C and 26A region transcripts was maximal during the period of vitelline membrane formation. To determine if the accumulation patterns were similar to the synthesis patterns, cytoplasmic RNA was isolated from previtellogenic ovaries and from stage 10 and stages 12-14 eggchambers. The RNA was spotted in triplicate on nitrocellulose filters and each filter was hybridized to nick-translated insert DNA from one of three clones: pDmo26A-1, pDmo3G1, or pDmo7F (a chorion cDNA (~38) reisolated in our screen). Figure 3a shows that both the 3C and the 26A region cDNAs hybridize to stage 10 eggchamber RNA but not to RNA from previtellogenic or choriogenic stages (12-14). As expected, the ~38 chorion cDNA probe showed intense hybridization to RNA from stages 12 to 14. To more precisely define the stages of RNA accumulation and to determine the sizes of the RNAs complementary to pDmo26A-1 and pDmo3C-1, total RNA was isolated from equal numbers of the eggchamber stages shown in Fig. 3, electrophoresed through a 1.2% agaPv
a
26.4-l 3c-I 7F
b kb 1.38-0.55-
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VOLUME124, 1987 3C
EN
VM
26A
130?O-
23-24-f
FIG. 4. Coding capacity of 26A-1 and 3C-1 genes. Ovarian poly(A)+ RNA was hybrid selected with recombinant plasmid DNA containing either fragment H (Fig. 2) from the 26A region or fragment F from the 3C region. Translation products were fractionated on a 15% SDS-polyacrylamide gel and fluorographed. In viwo labeled vitelline membrane proteins (VM) were run in parallel; their approximate sizes (kDa) are shown on the left. The arrows indicate RNA-dependent translation products not seen in the endogenous lane (EN).
12-14
rose-formaldehyde gel, and transferred to nitrocellulose. The developmental Northern blots were then probed with genomic restriction fragments A and E shown in Fig. 2. Fragment A includes pDmo26A-1 sequences while fragment E includes pDmo3C-1 sequences. As shown in Fig. 3c, RNA complementary to pDmo26A-1 accumulates throughout the period of vitelline membrane formation (stages 8-11) and falls off precipitously in stage 12 eggchambers. RNA complementary to the 3C region sequences (Fig. 3b) is present in stage 9, reaches maximal expression in stages lOa-11, and is undetectable in stage 12 eggchambers. Both the 26A and 3C region RNAs are in the 700- to 800-nt size range. Coding Capacity and Gene Structure
The sizes and stages of expression of the RNAs complementary to the 26A and 3C region sequences are consistent with those expected to encode the small vitelline membrane proteins. To establish their translatability, genomic subclones containing sequences comFIG. 3. Stage specific accumulation of 26A-1 and 3C-1 RNA during plementary to the cDNAs were immobilized on nitrooogenesis. Cytoplasmic RNA isolated from 10 previtellogenic ovaries cellulose and hybridized to (poly)A+ RNA from (PV), stage 10, and stage 12-14 eggchambers was spotted in triplicate approximately 1000 ovaries. Messages selected by the onto nitrocellulose (a). Blots were hybridized with nick-translated 0.9-kb BamHI fragment (Fig. 2, H) from the 26A region insert DNA from either pDmo26-Al, pDmo3C1, or pDm7F, a ~38 chorion gene probe. (b, c) RNAs isolated from equal numbers of egg- and the 2.0-kb EcoRI fragment (Fig. 2, F) from the 3C chambers in the stages indicated (b, 30; c, 50) were electrophoresed, region were translated in vitro in a rabbit reticulocvte transferred to nitrocellulose, and hybridized to nick-translated DNAs lyiate. One-dimensional SDS gel electrophoresis of ihe at 4 X lo5 cpm/ml; (c) 950-bp HindIII-SmaI fragment A (Fig. 2) conproducts showed both messages encoded proteins of aptaining pDmo26-Al sequences; (b) 1.3-kb EcoRI-PvuII fragment E proximately 17 kDa (Fig. 4). The intensity of the 3C (Fig. 2) containing pDmo3C-1 sequences. The sizes of single-stranded RNA markers run in parallel are indicated at the left. translation product was considerably less than its 26A
BURKEETAL.
Vitelline Membrane Fmatim
counterpart. This may reflect differences in message abundance, efficiency of translation in vitro, or both. Both products migrate slightly more slowly than the major 17-kDa vitelline membrane protein. Hybridization of the 26A and 3C region cDNAs to Southern blots of recombinant phage DNA localized each on single restriction fragments: 0.9 kb BamHI and 1.3 kb EcoRI-PvuII, respectively. After subcloning these fragments into Gemini transcription vectors, the polarity of transcription was determined by making single-stranded RNA probes of opposite orientations and finding which strand was complementary to the mRNA by hybridization. Once the directions of transcription (Fig. 2) were established the 5’ ends of the genes were localized by Sl mapping.32P-labeled, singlestranded RNA probes made from the subcloned fragments illustrated in Fig. 2 were hybridized in excess to RNA extracted from stage 10 or 14 eggchambers. Double-stranded RNAs resistant to Sl nuclease were then denatured and sized on a sequencing type polyacrylamide gel (Fig. 5). Lane 1 shows that a single fragment of approximately 700 nt was protected when RNA from stage 10 eggchambers was hybridized with a 950-nt HindIII-SmaI RNA probe (fragment A) from the 26A region. As expected, RNA from stage 14 eggchambers failed to confer protection (lane 2). By using a smaller RNA probe, the 5’ end of the gene was placed near the 5’ end of the 170-bp HaeIII fragment (B) shown in Fig. 2. Probe B protected a stage 10 specific band in the 150- to T
10
IO 3)
-26L
622-
309-
-IL7
II)
+ 264-
12
a
5
C
6
d7
FIG. 5. Sl nuclease mapping of 26A-1 and 3C-1 5’ ends. “P-labeled, single-stranded RNA probes made from restriction fragments encompassing most of the 26A-1 gene (Fig. 3a) or its 5’end (Fig. 3b) were hybridized with RNA extracted from 30 eggchambers (stage 10 or 14). Following digestion with Sl nuclease, the resistant fragments were fractionated on a 4% denaturing polyacrylamide gel (lanes 1, 2, 4). The RNA transcript (T) used to localize the 5’end of 26A-1 is shown in lane 3. For 3C-1, a 700-nt transcript (lane 5) made from fragment C (Fig. 3~) was used for positioning the 5’ end (lane 6). The intron was localized (lane 7) with a 500-nt RNA made from fragment D (Fig. 3d). 3C region probes were hybridized with RNA extracted from 40 stage 10 eggchambers. Autoradiograms of the gels are shown with RNA (697 and 1400 nt) and DNA size markers (<622 nt).
445
160~nt size range (lane 4) whereas probe G failed to yield protected fragments (data not shown). Unlike 26A-1, two fragments of approximately 500 and 200 nt were protected by a RNA probe that spanned the 3C-1 gene (2.2-kb PvuII fragment, Fig. 2) (data not shown). More refined analyses (lane 6) indicated that the 5’ end of the gene was localized in fragment C (Fig. 2) approximately 300 bp upstream from the BamHI site. The protection of two small fragments with probe D (approximately 250 and 100 nt, respectively) indicated a small intron was located within this region. These results agree with subsequent DNA sequencing. Follicle
Cell-Specific Expression
Although the results from the screen of our cDNA library indicated both 26A-1 and 3C-1 RNAs were synthesized by follicle cells, concomitant synthesis by the metabolically active nurse cells remained a possibility. To demonstrate follicle cell-specific expression, ovarian tissue sections were hybridized with single-stranded RNA probes complementary to the 26A-1 and 3C-1 RNAs. When restriction fragment H in Fig. 2 (26A region) was used as template, grains were selectively localized over the follicle cells of stages 8, 9, and 10 eggchambers (Fig. 6a). As expected, no hybridization was detected with previtellogenic (O-7) or choriogenic (12-14) staged eggchambers. 3C-1 region probes failed to show grains over background levels. As an alternative means to demonstrate the follicle cell specificity of the 3C region transcript, we cut stage 10 eggchambers at their midline (arrow, Fig. 6) to generate follicle cell and nurse cell fractions. RNA was extracted from each fraction, hybridized to 32P-labeled single-stranded RNA probes in excess, and assayed for Sl protected fragments of the predicted size. The results with 26A-1 shown in Fig. 6b confirm the reliability of the method. As expected, when fragment A (Fig. 2) was used as template a 700-nt fragment was protected with follicle cell but not nurse cell RNA. The comparable amount of probe protected by follicle cell and stage 10 eggchamber RNA is also consistent with its follicle cell-specific expression. Similar results were obtained with 3C-1 (Fig. 6~). A protected fragment of the expected size (250 nt) was obtained with whole eggchamber and follicle cell RNA extracts. The absence of signal in the nurse cell fraction coupled with the similar signal intensities in the stage 10 and follicle cell fractions indicate that, like 26A-1, the expression of 3C-1 is limited to the follicle cells. DNA Sequence Analysis
To map the transcripts more precisely, determine the structures of the genes in detail, and gain insights into
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Analysis of the DNA sequence predicts a message size of 629 bp before (poly)A addition. A single long open-reading frame is found that begins at a methionine codon 82 bp from the 5’ end and extends to within 124 bp of the 3’ end. Conceptual translation predicts a 14,322-Da protein product with properties expected for Drosophila vitelline membrane proteins. The amino terminal segment possesses features typical of secreted proteins with known signal peptides. A hydrophobic core of 18 amino acids is followed within four to six residues by predicted cleavage sites (arrows). The high
a
IOObP
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NC
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FIG. 6. Follicle cell-specific expression of 26A-1 and 3C-1. (a) Ovarian tissue sections were hybridized with alkaline-digested RNA probe complementary to the 26A-1 transcript (fragment H, Fig. 3). Eggchambers at different developmental stages (PV, previtellogenic) are shown; cell types are marked for a stage 10 eggchamber (fc, follicle cell; nc, nurse cell; 00, oocyte). Stage 10 eggchambers were cut at the arrow to give nurse cell and follicle cell fractions for Sl analysis (b, c). RNA from 50 stage 10 eggchambers or the follicle cell and nurse cell fractions from an equivalent number were hybridized with “P-labeled RNA probes made from restriction fragments A and D (Fig. 3). The results are shown in (b) and (c), respectively. Unlike the nurse cell lanes, protected fragments are apparent in the stage 10 eggchamber and follicle cell lanes.
functional roles, we sequenced both cDNAs and genomic DNAs by the dideoxy chain termination method. The sequencing strategy and complete genomic sequence of 26A-1 is shown in Fig. 7. The 3’ end of the gene was positioned by the (poly)A tract found at the end of the cDNA. The putative 5’ terminus of the mRNA was specified by consideration of the genomic sequence. As expected from our Sl analyses, a mRNA consensus start site, CATCAGT, was found near the 5’ end of the 170-bp HaeIII fragment. The sequence TATAAAA appears 35 bp upstream from this postulated transcription initiation point. This is consistent with the spacing and sequence of typical eukaryotic promotors.
C -
6io
FIG. 7. DNA and predicted amino acid sequence of 26A-1. A restriction map of the 950-bp HindIII-SmaI fragment from region 26A is shown with arrows that indicate the extent and direction of the sequence analysis. The solid arrows represent sequences read from genomic subclones, while the dashed arrows indicate cDNA sequences read. Sequences determined from the cDNA clone are underlined. The proposed 5’ end is shown by a rightward arrow. Nucleotide 1 is the putative cap site. The coding region with the predicted amino acid sequence is set apart. The downward arrows mark potential signal peptide cleavage sites. The box indicates a potential TATA sequence at -35. The open arrowhead on the restriction map indicates the proposed transcription start site.
BURKE ET AL.
Vitelline Membrane Fm-rnaticm
alanine, glycine, proline, and serine contents of the mature protein (9.5,14.7,11.2, and 12.9%, respectively) are consistent with the average amino acid composition of the total vitelline membrane measured by Petri et al.
447
(1976). The predicted molecular weight of the mature protein is 12,080 Da. The 3C-1 cDNA and genomic sequences are shown in Fig. 8. Sl analysis placed the 5’ end of the gene (open
FIG. 8. DNA and predicted amino acid sequence of 3C-1. A restriction map of the 3C region that encompasses the 3C-1 transcription unit is shown. The arrows indicate the extent and direction of the sequences read from the subcloned regions: solid arrows, genomic sequences; dashed arrows, cDNA sequences. Nucleotides in lower case were derived from genomic subclones while those in upper case are from both cDNA and genomic readings. Potential 5’ ends are shown by the rightward arrows; the 3’ end predicted by the plasmid cDNA clone (minus l-4As?) is marked by a leftward arrow. Numbering in the coding region is based on the cDNA sequence. The boxes indicate the potential TATA sequence at -32 and the potential (poly)A addition site at 768. The open arrowhead on the restriction map indicates the approximate position of the proposed site of transcription initiation.
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arrowhead) approximately 300 bp upstream from the BamHI site shown in Fig. 8. Within that range are two sequences, CATCCAT at position 1 and CATCCAG at position 16, that show a reasonable match to the proposed cap site consensus sequence for Drosophila (Snyder et al, 1982). Although the 5’ end of a 3C cDNA clone isolated from a X ovarian cDNA library (see Materials and Methods) terminated at position 17, the position at a potential TATA box at -32 is more compatible with the +l cap site. Sl analyses using subclone E in Fig. 2 indicates this 3C region mRNA terminates approximately 200 bp downstream from the EcoRI site (data not shown). The short plasmid cDNA clone, pDmo3C-1, ended in a 3’ (poly)A tract at position 789, 199 bp downstream from the EcoRI site shown in Fig. 8. The presence of a consensus (poly)A addition site (AATAAA) 1’7 bp upstream from the 3’ terminus of this cDNA is consistent with this interpretation of the 3’ end. The cDNA and genomic sequences were identical over most of their lengths except for a short 73-bp intron located at position 568. The sequence of the intron borders conform to the GT . . . AG rule of 5’-donor 3’-acceptor splice sites and the position of the intron is in excellent agreement with that predicted by our Sl data. Sequence analyses predicted a single long openreading frame starting with an ATG codon at nt 113 and ending with a TGA termination codon at nt 743. While in the general size range, the size of the predicted protein product is slightly larger than was expected based on the mobility of the 3C-1 hybrid selected translation product. Conceptual translation of the openreading frame yields a protein product of 22,346 Da. Unlike 26A-1, the predicted 3C polypeptide does not have an amino terminal peptide that resembles a leader sequence. The predicted amino acid composition indicates the protein is rich in serine and threonine (both 11.4%). DISCUSSION
Using in viva labeled RNA probes we have isolated two genes transcribed in follicle cells during the period of vitelline membrane formation. While the function of the 3C-1 gene product remains speculative, as indicated earlier, several properties of the 26A-1 gene are consistent with a vitelline membrane structural gene. The size of the primary translation product and the relative abundance of the 26A-1 transcript are most compatible with Sv17.5, a major vitelline membrane protein reported previously (Fargnoli and Waring, 1982). A precursor comparable in size to the 26A-1 product was noted in earlier translation studies of T2 follicle cell RNA from stage 10 eggchambers (Fargnoli and Waring, 1984). When translated in vitro with microsomal mem-
VOLUME124,1987
branes, T2 yielded a processed product that comigrated with Sv17.5. Preliminary partial peptide data provide additional support for 26A-1 encoding Sv17.5. When Sv17.5 and the primary translation product of 26A-1 were cleaved with Vs protease, both proteins produced the same large proteolytic fragment (P. Minoo and G. Waring, unpublished data). The predicted size of the 26A-1 translation product from DNA sequence analysis, 14,322, is smaller than that estimated by its mobility on SDS gels. A similar discrepancy has been reported for the small chorion proteins (Levine and Spradling, 1985; Wong et aZ.,1985). The predicted molecular weights from sequence analysis are smaller than the size estimates of Waring and Mahowald (1979) but agree with those of Petri et al. (1976). Despite discrepancies in actual size, the relative migration of ~18, Sv17.5, and s15 in our gel system (unpublished) is consistent with the relative sizes of the mature proteins predicted by DNA sequence analysis: 15,027, 12,080, and 9467 Da, respectively. The 26A region has been previously implicated as a vitelline membrane structural gene site based on complementarity of cloned sequences to mRNAs that are selectively expressed in vitellogenic eggchambers (Higgins et aZ., 1984; Mindrinos et al, 1985). Based on map location and transcript size, 26A-1 appears to be comparable to the cr-1 transcription unit described by Higgins et al. (1984) and Mindrinos et al. (1985). Like the chorion genes, vitelline membrane genes appear to be clustered. Within an S-kb region, we have identified four genes, including 26A-1, that are selectively expressed in follicle cells during the period of vitelline membrane formation (Minoo et ab, 1986). We have also mapped a female sterile mutation affecting the synthesis of one of the major vitelline membrane proteins (Sv23) to the 26A region (S. Savant and G. Waring, unpublished results). 26A-1, like the chorion genes (Levine and Spradling, 1985; Wong et aZ.,1985), share structural features typical of most eukaryotic genes. A TATA box is found 30 bp upstream from the transcription start site and typical consensus sequences are found at the mRNA and translation start sites. As with the chorion genes, 26A-1 has short 5’ and 3’ untranslated regions and hydropathicity plots of the predicted amino acid sequence (not shown) indicate multiple hydrophilic and hydrophobic segments in an alternating arrangement (Wong et aZ., 1985). Whereas all chorion genes analyzed to date show a short 5’ intron separating one small and one large exon, the 26A-1 gene lacks introns. While the evidence that 26A-1 encodes a vitelline membrane structural protein is reasonably compelling, the function of the 3C region follicle cell gene remains speculative. Although the timing of its expression is
BURKEET AL.
Vitelline
consistent with a role in vitelline membrane formation, several of its properties do not conform to those expected of the typical eggshell protein gene. The predicted amino acid composition is not characterized by a high alanine, proline, or glycine content and a potential signal peptide was not revealed by DNA sequence analysis. Based on Kyte and Doolittle’s hydrophobicity requirements (1982), 3C-1 does not appear to have lipophilic spikes of sufficient length and magnitude to participate in membrane binding (data not shown). Like 26A-1, 3C-1 appears to be part of a gene cluster. Another low abundance transcript with a similar temporal profile has been localized within 2 kb of the 3’ end of 3C-1 (E. Popodi and G. Waring, unpublished). The 3C region has previously been implicated in vitelline membrane formation by genetic studies. Komitopoulou et al. (1983) described a 3C region female sterile mutation (K93) whose expression was dependent on the somatic line (Perrimon and Gans, 1983). Mutant flies laid flaccid eggs that showed strong uptake of dye, characteristics that might be expected of a vitelline membrane defect. Of the 24 female-sterile complementation groups characterized, permeability changes were only reported in K93 eggs. In this regard, it is interesting to note that eggs from the 26A vitelline membrane mutant mentioned previously also display altered permeability properties (S. Savant, unpublished). In summary, our data suggest that 26A-1 encodes a major early eggshell protein. While a definitive assignment is not possible in the absence of protein sequence data, we feel it is most likely that 26A-1 encodes Sv17.5 (~14.5) (Mindrinos et ab, 1985). Unlike 26A-1, 3C-1 encodes a follicle cell protein of unknown function. No significant homologies were detected with any proteins in the NBRF library. Although the amino acid sequence of the predicted protein appears to be more compatible with an intracellular function (no signal peptide), immunocytochemical localization will be required to resolve this question. We thank Ms. B. DeNoyer for help in the preparation of the manuscript and J. Pollack for expert technical assistance. This work was supported by National Science Foundation Grants PCM-8203171 and DCB-8502633 to G.L.W., who was the recipient of Research Career Development Award HD00524 and by Marquette University and Scholl Foundation predoctoral fellowships to T.B. REFERENCES BENTON,W. D., and DAVIS, R. W. (1977). Screening Xgt recombinant clones by hybridization to single plaques in situ. Science 196, 180-182.
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KOMITOPOULOU, K., GANS,M., MARGARITIS,L. H., KAFATOS,F. C., and MASSON,M. (1983). Isolation and characterization of sex-linked female-sterile mutants in Drosophila melanogaster with special attention to eggshell mutants. Genetics 105, 897-908. KYTE, J., and DOOLITTLE,R. (1982). A simple method for displaying the hydropathic character of a protein. J. Mel Biol 157,105-132. LAEMMLI,U. (1970). Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature (London) 227,680-685. LAND, H., GREZ, M., HAUSER, R., WINDENMAIER,W., and SCHUTZ,G. (1981). 5’ terminal sequences of eucaryotic mRNA can be cloned with high efficiency. Nucl. Acids Res. 9,2251-2266. LEVINE, J., and SPRADLING,A. (1985). DNA sequence of a 3.8 kilobase pair controlling Drosophila chorion amplification. Chrorrwsoma 92, 136-142. MANIATIS, T., FRITSCH, E. F., and SAMBROOK,J. (1982). “Molecular Cloning.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MANIATIS, T., HARDISON, R. C., LACY, E., LAUER, J., O’CONNELL,C., QUON,D., SIM, G. K., and EFSTRATIADIS,A. (1978). The isolation of structural genes from libraries of eucaryotic DNA. Cell 15,687-701. MELTON,D. A., KRIEG, P. A., ROBAGLIATTE,M. R., MANIATIS, T., ZINN, K., and GREEN, M. R. (1984). Efficient in vitro synthesis of biologically active RNA and DNA hybridization probes from plasmids containing a bacteriophage sp6 promotor. Nucl. Acids Res. 12, 7035-7036. MINDRINOS,M. N., SCHERER,L. J., GARCINI, F. J., KWAN, H., JACOBS, K. A., and PETRI, W. H. (1985). Isolation and chromosomal location of putative vitelline membrane genes in Drosophila melanogaster. EMBO J. 4,147-153. MINOO, P., POPODI, E., BURKE, T., and WARING,G. (1986). Genetics 113,
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SPRADLING, A. C., and MAHOWALD, A. P. (1980). Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proc. Natl. Acad Sci. USA 77,1096-1100. SNYDER,M., HUNKAPILLAR, M., YUEN, D., SILVERT, D., FRISTROM,J., and DAVIDSON, N. (1982). Cuticle protein genes of Drosophila: Structure, organization and evolution of four clustered genes. Cell 29,1027-1040. WARING, G. L., and MAHOWALD,A. P. (1979). Identification and time of synthesis of chorion proteins in Drosophila mdanogastw. Cell 16, 599-607. WATSON,C. J., and JACKSON,J. F. (1985). An alternative procedure for the synthesis of double-stranded cDNA for cloning in phage and plasmid vectors. In “DNA Cloning” (D. M. Glover, Ed.), Vol. 1, pp. 79-88. IRL Press, Oxford. WHITE, B. A., and BANCROFT,F. C. (1982). Cytoplasmic dot hybridization: Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J. Biol. Chem. 257,8569-8572.