Organization of a cluster of four chorion genes in Drosophila and its relationship to developmental expression and amplification

DEVELOPMENTAL

BIOLOGY

91,325-336

(1982)

Organization of a Cluster of Four Chorion Genes in Drosophila and Its Relationship to Developmental Expression and Amplification Cellular

and Developmental

RUTH

GRIFFIN-SHEA,~

Biology,

The Biological Received

GEORGE THIREOS, Laboratories,

December

Harvard

16, 1981; accepted

AND

University, in revised

form

FOTIS

C.

KAFATOS

16 Divinity

Avenue,

February

8, 1982

Cambridge,

Massachusetts

02138

Four chorion genes, coding for low-molecular-weight proteins produced chiefly toward the end of choriogenesis, are clustered within 6 kb of DNA in region 66D. All four genes amplify to the same extent during development, but are not equally expressed into RNA. The quantitative as well as temporal differences in RNA accumulation indicate that, in addition to coordinate amplification, choriogenesis entails the regulated expression of individual genes. If this regulation operat.es through differential transcription, it may be related to the organization of genes in individual, directly oriented transcription units, as opposed to the divergent, coordinately expressed gene pairs characteristic of the silkmoth ehorion locus. DNase I hypersensitive sites flank all three chorion genes examined. Only one gene, s151, shows a bimodal temporal profile of RNA accumulation, the first peak of which may possibly indicate a link between transcription and the onset of amplification.

structural genes, and explore the possible relationships between their organization, amplification, and expression. This cluster corresponds to the third chromosome chorion locus (region 66DlO-15), which was first identified by in situ hybridization of two cDNA clones (Spradling et al., 1980; Griffin-Shea et al., 1980).

INTRODUCTION

Formation of the chorion (eggshell) by the ovarian follicular cells in Drosophila melanogaster is a favorable model system for the molecular analysis of cell differentiation. Choriogenesis entails the production of several structural proteins in quick succession according to a precise temporal and quantitative program (Petri et al., 1976; Waring and Mahowald, 1979). Such programs of differential :gene expression are of general importance in differentiation, and may be regulated at many levels of information flow. The amount of specific mRNA produced per cell in each period of time could be controlled post-transcriptionally, transcriptionally, or through cell-specific genomic alterations. One such genomic alteration, gene amplification, is thought to be associated with developmental needs for accumulation of a gene product in massive amounts in a relatively short period of time (Brown and Dawid, 1968). Gene amplification has been shown to be an important aspect of choriogenesis in Drosophila (Spradling and Mahowald, 1980; Spradling, 1982). However, the details of the protein synthetic and RNA accumulation programs, as compared to the time course of DNA amplification (Thireos et al., 1980), suggest that amplification is not the only important regulatory step in choriogenesis. The arrangement of genes in the chromosome may have important consequences for their developmental regulation, including both amplification and transcription. Here we describe a tight cluster of four chorion ‘Present address: Centre de Genetique

Centre National de la Recherche Moleculaire, 91190 Gif-sur-Yvette,

MATERIALS

AND

METHODS

Materials [a-32P]dNTPs (600 Ci/mmole) and [3H]Pro (110 Ci/ mmole) were from New England Nuclear; restriction endonucleases, DNA polymerase I, and T4 ligase from New England Biolabs; and RNase A, RNase Tl, DNase I, proteinase K, and Sl nuclease from Boeringer-Mannheim. Low-temperature-melting agarose (Sea Plaque) was obtained from Marine Colloids, Inc., and DBM and nitrocellulose filters from Schleicher and Schuell. The D. melanogaster stock was Oregon R, homozygosed for the third chromosome by M. Meselson. Flies were grown at 23°C in bottles for 10 days and then conditioned for 10 days before dissections by daily transfer to fresh bottles containing powdered yeast. At the day of dissection the representation of individual follicle stages in the ovary reflected their relative duration (David and Merle, 1968). Genomic Ckmes and Subclones A partial EcoRI genomic library was constructed by K. Jacobs from embryonic Drosophila DNA, using X Charon 4 as vector, as described by Maniatis et al.,

Scientifique, France. 325

0012-1606/82/060325-12$02,00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form resewed.

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(1978). Phages were propagated in Escherichia coli CSH 18. This library, and a random-shear library from the Canton S strain (provided by T. Maniatis) were screened (Benton and Davis, 1977) for sequences homologous to the DmcAl-1 cDNA clone. One clone from each library was used for detailed analysis. The Oregon R clone (named XAB) was digested with EcoRI or Hind111 and subcloned in pBR322 as described (Jones and Kafatos, 1980b). Subclones were recovered using the appropriate EcoRI or Hind111 fragments of XAB as probes in colony hybridization (Grunstein and Hogness, 1975).

bridizations were for 12 hr in the same mixture but without glycine and with the addition of 10% dextran sulfate. Nick-translated 32P-labeled probes were used for the hybridizations at 3 X lo5 cpm/ml at lo* cpm/ pg. Following hybridization the filters were washed in 2X SSC, 0.1% SDS at 25°C for 30 min and in 0.1X SSC, 0.1% SDS at 50°C for 60 min, and subjected to autoradiography. Filters were reused after complete dehybridization, accomplished by incubation at 70°C for 8 hr with 90% formamide, 2X SSC, 0.5% SDS, 25 mM phosphate buffer (pH 6.0).

Nucleic Acid Preparations

DNA

Plasmid DNAs were purified according to the method of Clewell (1972); phage DNA was purified by the method of Blattner et al. (1977). DNA fragments were generated by restriction endonucleases and electrophoresed on low-temperature-melting agarose gels (usually 0.8%). Fragments were identified by ethidium bromide staining and appropriate gel pieces were excised, melted at 65°C for 15 min with an equal volume of electrophoresis running buffer, and phenol-extracted twice. Following ether extraction the DNA was precipitated with ethanol and could be used as a probe after nick translation (Maniatis et al., 1975), or for further mapping experiments (single, double, or partial enzyme digestions of cold or 32P-end-labeled fragments). Cytoplasmic RNA from ovaries and other nucleic acids were prepared as described (Thireos et al., 1979). Total nucleic acids were prepared from ovaries or dissected follicles of conditioned female flies, by brief homogenization in 7 M urea/2% SDS/10 mM Tris-HCl, pH 7.5/0.135 M NaCl/l mM NazEDTA, followed by phenol-Sevag extractions and ethanol precipitation. Chromosomal DNA from embryos or ovaries was prepared by brief homogenization in 10 mM Tris-HCl, pH 7.5/0.5% SDS/l mM EDTA/50 mM NaCl, folowed by incubation at 37°C for 10 min with 50 pg/ml proteinase K and phenol-Sevag extractions. The aqueous phase was treated with 50 pg/ml RNase A and 1 pg/ml of Tl RNase for 1 hr at 4O”C, and reextracted with phenolSevag.

Following electrophoresis, DNA fragments were transferred onto nitrocellulose filters as described (Southern, 1975) with the addition of 1 N ammonium acetate in the transfer buffer. For efficient transfer of large fragments the partial depurination procedure of Wahl et aE. (1979) was used. Following transfer the filter was washed for 30 min with 1X SSC, 1 N ammonium acetate, air-dried, baked at 80°C for 2 hr, and prehybridized with 5X SSC, 0.1 M sodium phosphate buffer, pH 7.0, 5~ Denhardt’s solution, and 0.5 mg/ml sheared calf thymus DNA at 65°C for 3 hr. Hybridizations were for 12 hr in the same buffer with 10% dextran sulfate and 3 X lo5 cpm/ml of 32P-labeled DNA probe. The filters were washed at 65°C with 1X SSC, 0.1% SDS for 1 hr and at 50°C with 0.1X SSC, 0.1% SDS for 1 hr before autoradiography. The filters were reused after dehybridization with 0.5 NNaOH, 1 MNaCl at 25°C for 15 min, followed by neutralization in 5X SSC, 0.1 M phosphate buffer, pH 7.0.

RNA Electrophoresis,

Transfer,

and Hybridizations

RNA was electrophoresed in 1.5% agarose gels containing 10 mM methylmercury hydroxide according to Bailey and Davidson (1976), and transfered onto DBM paper as described by Alwine et al., (1977). The paper was prehybridized for 3 hr at 60°C in 5X SSC, 50% formamide, 50 mM sodium phosphate buffer, pH 6.0, 5X Denhardt’s solution (Denhardt, 1966), 0.5 mg/ml sheared calf thymus DNA, and 1% (w/v) glycine. Hy-

Transfers and Hybridizations

DNA Protection

Experiments

Following the general procedure of Berk and Sharp (1978), a solution containing 1 mg/ml cytoplasmic ovarian RNA, 10 pg/ml of XAB DNA, 80% formamide, 0.6 M NaCl, 1 mM EDTA, and 10 mM Pipes buffer (pH 6.8) was kept at 70°C for 10 min and then at 54°C for 2 hr. Aliquots were added to an equal volume of 0.3 M NaCl, 6 mM ZnCla, 100 mM Na-acetate (pH 4.0), 50,000 u/ml Sl nuclease, incubated for 5 min at 37”C, phenol-extracted, and precipitated with ethanol. For ExoVII digestions, aliquots of the hybridization mixture were chromatographed in a Sephadex G-50 column in 8 mM EDTA, 10 mM ,&mercaptoethanol, 67 mM K-phosphate (pH 7.8). ExoVII was added to 10 u/ml, and the solution was kept at 37°C for 30 min followed by phenol extraction and ethanol precipitation. The DNA was electrophoresed on 1.5% agarose gels in 30 mM NaOH, 2 mM EDTA (McDowell et al., 1977), transferred onto nitrocellulose filters, and hybridized as described.

GRIFFIN-SHEA,

THIREOS,

Hybrid-Selected Translations and Immunoprecipitations In vitro translations used the wheat germ system as described (Thireos and Kafatos, 1980). Hybrid selections were performed according to Griffin-Shea et al. (1980), immunoprecipitations and SDS-polyacrylamide gel electrophoresis according to Thireos et al. (1979). Purification

of Nuclei

and DNase I Digestions

Ovaries from 100 flies were homogenized with a Teflon homogenizer (0.1 mm clearance) in 4 ml of 1 M sucrose/3.3 mM CaClz and 0.1% Triton X-100 with five strokes. The homogenate was centrifuged for 10 min at 5000~. The resulting pellet was washed once in 1 M sucrose/3.3 mM CaC&., repelleted, and resuspended in 0.5 ml of 0.25 M sucrose, 60 ImM KCl, 15 mM CaC12, 3.0 mM MgClz (Wu, 1980). Aliquots (0.1 ml) were digested with varying amounts of DNAse I at 25°C for 3 min and the reaction was terminated with the addition of EDTA to 10 mM and SDS to 0.5%. The DNA was isolated as described above and sulbjected to restriction endonuclease digestion and Sou.thern blot hybridization. RESULTS

Characterization

of Genomic Clone AAB

A library of OreR genomic DNA, constructed in the Charon 4 vector by the partial EcoRI procedure, was “3

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Genes in Drosophila

screened with the cDNA clone, DmcAl-1, which encodes the ~15-1 chorion protein (Griffin-Shea et al., 1980). A positive clone, XAB (12.0 kb), was recovered, and a restriction map (Fig. 1) was constructed. A corresponding clone was also isolated from the Lauer-Maniatis Canton S genomic library (Maniatis et al., 19’78), and proved to encompass the entire length of XAB, plus 1.5 and 2.5 kb to the left and right, respectively. In the region of overlap, the OreR and Canton S clones were identical with respect to the EcoRI, HindIII, and XhoI sites mapped. Southern blot-hyridization experiments, and other experiments to be described below, established that four distinct, closely linked genes exist in XAB (Fig. 1) and are expressed in the ovary. The gene second from the right strongly hybridizes with and has an identical restriction map with DmcAl-1, which is a single-copy sequence; thus, that gene must encode the ~15-1 protein. To determine the orientation of transcription, XAB DNA was strand-separated, blotted, and hybridized with total ovarian cDNA. Only one strand detectably hybridized (data not shown). Since the same cDNA preparation showed strong hybridization with various restriction fragments, each containing one of the three right-most genes, we conclude that these genes are transcribed from the same strand, i.e., in the same orientation. The actual orientation was established using the cDNA clone DmcAl-1, as detailed below. The

XDI

H3

OR CS

. . .

-

. . .

-mm Sl6-I

Sl9-I --e--

Sl5-I

518-l

FIG. 1. Restriction map of XAB. Identification and approximate positioning of the four genes were based on the use of specific restriction fragments, A through K. Hind111 fragment D was subjected to more detailed restriction analysis, allowing exact positioning of genes ~15-1 and ~18-1 which correspond to cDNA clones DmcAl-1 (Griffin-Shea et al., 1980) and Dmc5G2 (Spradling et al., 1980). The genes are indicated by black rectangles corresponding in height to the respective RNA abundance in steady-state ovarian RNA (see Fig. 4b). Arrows indicate the direction of transcription. The genomic clone from the Canton S strain (CS) has the same restriction sites for HindIII, %I, and EcoRI in the region of overlap with the Oregon R XAB clone (OR). Rl, EcoRI; H3, HindIII; Xl, XhoI; Sal, SalI; Xbl, XbaI; Al, AM; Hfl, Hi&; Hl, HhaI; 7 , PvuI; P , HindII.

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91, 1982

b B E7 C

C Sl

E7

Dm cAl-1 E7 Sl

Sl

E E7

Sl

D

.70-

.60-

. .70 .67’

,65.65

.75.65-

FIG. 2. Identification of mRNA sizes and intron-exon structure of chorion genes. (a) Total ovarian RNA was electrophoresed in a methylmercury-containing gel, transferred onto DBM paper, and parallel lanes containing equal amounts of RNA were hybridized with nicktranslated fragments from XAB as indicated in Fig. 1 (A-G). Only fragments B-D showed hybridization, with two molecular weight classes of RNA (0.65 and 0.75 kb). (b) XAB DNA was hybridized with total ovarian RNA (see Materials and Methods). Half of the reaction mixture was digested with Sl and half with &oVII. Each digest was split in half, run on parallel lanes of an alkaline agarose gel, transferred onto nitrocellulose paper, and probed with a2P-nick-translated DmcAl-1 or fragment E. The same filters were dehybridized and probed with fragments C and-B, respectively.

orientation of the fourth gene was not established unequivocally, since the intensity with which the cDNA preparation hybridized with restriction fragments containing only that gene was not sufficient to guarantee its detection above background in the strand-separation experiment. The entire XAB clone was used as probe in Northern analysis of RNAs from embryos, larvae, pupae, adult males, and adult females. Only the last preparation detectably hybridized, suggesting that the XAB genes not only are expressed in the ovary (as shown by Southern analysis), but are also specific for it. The ~15-1 mRNA is 0.65 kb long (Griffin-Shea et al., 1980), as is the mRNA encoded by the fourth gene from the right; the first and third genes encode mRNAs 0.75 kb in length (Fig. 2a). Thus, genes of one mRNA size class alternate the genes of a second mRNA size class. Each gene is distinct in sequence, however, as shown by cross-hybridization analysis (see below). Localization

of the Genes in AAB

The locations of the genes within XAB DNA were determined by a combination of Southern, Northern,

and Berk-Sharp analyses. For convenience, subcloned EcoRI and Hind111 fragments were used in most of these experiments (fragments A through G in Fig. 1). Fragments A, F, and G did not detectably hybridize with ovarian RNA or cDNA, and were not studied further. For gene s&l, precise localization and determination of the transcriptional orientation were possibly by aligning its restriction map with that of the corresponding cDNA clone, DmcAl-1; that clone has been sequenced (A. Georgi, unpublished results) and its coding strand has been determined by Northern analysis using singly end-labeled probes specific for each strand. The right-most gene was also precisely localized, by comparison with the restriction map of the cDNA clone Dmc5G2 (Spradling et al., 1980) (see below). The third gene from the right, which is split by a Hind111 site, was localized with reasonable accuracy, from the relative intensity of Southern hyridizations of ovarian cDNA with the short flanking EcoRI-Hind111 and HindIII-XhoI fragments, and from the absence of hybridization with the 0.5-kb XhoI-Hind111 fragment further to the right. The location of the fourth gene is known less accurately (see Discussion). Berk-Sharp analysis revealed the existence of introns

GRIFFIN-SHEA,

THIREOS,

AND KAFATOS

in all four genes (Fig. 21)). Ovarian RNA was hybridized with XAB DNA, and aliquots of the hybridized mixture were digested either with Exonuclease VII or Sl nuclease, electrophoresed on a denaturing gel, transferred to nitrocellulose, and probed with labeled restriction fragments corresponding to individual genes (or with DmcAl-1 DNA for the ~15-1 gene). The ExoVII-digestions yielded protected fragments invariably longer than those of the Sl digestions. In each case, the size of the Sl-resistant fragment, relative to the known mRNA size, indicated that most of the mRNA length corresponds to a sinle large exon, equalling from 64 to 93% of the mRNA plus poly(A) length. The intron(s) in each gene must be small, as well as located near one end of the gene. In the case of ~15-1, the intron(s) must be approximately 120 bp long, since DmcAl-1 DNA is 0.55 kb, and the ExoVII-resistant fragment, 0.67 kb; the intron(s) is presumabl:y located near the 5’ end, since the restriction maps of the genomic and cDNA clones

HST+I B

C

T

Chvrion

Genes

329

in Drosophila

are identical in the 3’ region (Fig. 1). Comparisons of the lengths of mRNAs and ExoVII-resistant fragments indicated that the introns of the remaining genes equal in length the length of the poly(A) plus approximately 100, 50, and 50 bp, from right to left (Fig. l), respectively. Characterization of the Genes in AAB by the HybridSelected Translation The four genes were characterized by hybrid-selected translation, permitting us to identify them as encoding the major chorion proteins ~18-1, ~15-1, s19-1, and s161, from right to left, respectively. DNAs from the cloned subfragments B, C, and CD, containing the fourth, third, and first through third genes from the right, respectively (Fig. l), were spotted on nitrocellulose and hybridized with cytoplasmic ovarian RNA. The bound RNA was eluted and translated

HST

T

CD

CDC

B

,-mm918-l

~15-1

s19-1

916-l

FIG. 3. Identification of the genes on XAB by hybrid-selected translation. Fragments B, C, and C + D were used to select the corresponding mRNAs from ovarian RNA. The hybridized message was eluted and translated in the wheat germ system. One-half of each reaction mixture was immunoprecipitated using the indicated chorion protein-specific sera and analyzed on a lo-15% SDS-polyacrylamide gradient gel, in parallel with the untreated ‘half (left and right panels, respectively). The mobilities of precursors to chorion proteins s19, ~18, ~16, and 915 (Thireos et al., 1979) are indicated in the translation products of total ovarian RNA (lanes T) and in the corresponding immunoprecipitate using mixed antichorion sera (lane M). Longer exposures of the immunoprecipate of C + D-selected products reveal ps18, which is present in amounts not detectable at the exposure used here.

330

DEVELOPMENTAL

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in the wheat germ system. Aliquots of the translation products were characterized by SDS-polyacrylamide gel electrophoresis, either directly or after immuno-

VOLUME

precipitation size classes The results

91, 1982

with antiserum directed against specific of chorion proteins (Thireos et al., 1979). (Fig. 3) clearly identified the proteins en-

a OVARY

EMBRYO

n

“%I

‘kbl

4.22.92.41.2.6-

2 Fragment

4

Kb

Size

b

919-l ~16-1

916-l s15-1

FIG. 4. Extent of amplification and of RNA accumulation for each gene in XAB. (a) Ovarian and embryonic genomic DNA (4 Fg each) were digested with Hind111 + EcoRI (H3/Rl) or XhoI + XbaI (Xl/Xbl), chosen since they generate fragments each containing a single gene (see Fig. 1). The digests were electrophoresed on an agarose gel, transferred to nitrocellulose paper, and hybridized with 32P-nick-translated XAB DNA (left panel). Densitometry showed a linear relationship between extent of hybridization and fragment size (right panel, based on the H3/Rl lane of ovarian DNA). (b) Total RNA from steady-state ovaries was electrophoresed on methylmercury agarose gel, blotted onto DBM paper, and parallel lanes were hybridized with the indicated fragments, each containing two adjacent genes in overlapping combinations (H, K, and D). Each lane was scanned by densitometry and the abundance of each RNA species was expressed relative to ~15-1.

GRIFFIN-SHEA, THIREOS, AND KAFATOS

Chwim

Genes

331

in Drosophila

b

a E 8+9 B ~16-1

10 II+12

13

14

E

+

8+9 IO I-l+1213

14

s19-1 s15-1

818-l s15-1

FIG. 5. Developmental 8 + 9, 10, 11 + 12, 13, sequentially hybridized, same filter as in a was

accumulation of RNAs corresponding to the XAB genes. (a) Total RNA was extracted from 40 follicles each of stages and 14, and from embryos (E), electrophoresed on methylmercury gels, and blotted on DBM paper. The paper was following dehyhridization, with “P-nick-translated fragments, each specific for one gene (B, C, DmcAl-1, E.). (b) The probed sequentially with fragments H, K, and D, each containing two genes in overlapping combinations.

coded by the fourth and third genes (in B and C, respectively) as components of the s16 and s19 chorion size classes, respectively. The RNA hybridizing with the CD subclone encoded proteins of the ~15, ~18, and s19 chorion size classes; since by itself the C fragment encodes an s19 protein, and the adjacent gene is known to encode the ~15-1 protein, by subtraction we infer that the first gene encodes an ~18 protein. In agreement with this interpretation, three restriction sites mapped in the ~18-1 encoding cDNA clone, Dmc5G2 (Spradling et al., 1980), are also found, at the expected distance from each other, in the right-most gene (Fig. 1). Quantitative Expression of Chorion Genes

and Developmental

SpeciZcity

The entire DNA region represented in XAB is amplified in the follicular cells during oogenesis (Spradling and Mahowald, 1980; Spradling, 1982). Although amplification is apparently equal throughout this region (Fig. 4a), the four genes are unequally expressed into RNA over all oogenetic stages combined (Fig. 4b).

Ovarian RNA was prepared from the ovaries of wellconditioned flies (rapidly laying, steady state), in which the various stages of oogenesis are represented at frequencies closely paralleling the relative durations of the stages. The RNA was Northern-blotted and hybridized with overlapping nick-translated probes, each corresponding to two genes (producing one 0.65 and one 0.75kb mRNA in each case). The autoradiograms were quantified by densitometry, using nonsaturating exposures. Because of this experimental design, it was possible to determine the relative RNA abundances for various pairwise combinations of genes, and ultimately to express all of these abundances on an internally consistent relative scale. It was evident that the various RNAs were represented in the following order of decreasing abundance (approximately threefold range): ~15-1, ~18-1, s19-1, and ~16-1. Experiments of similar design were also performed using stage-specific rather than total RNA, in order to determine the developmental specificities of the genes (Fig. 5). RNAs were prepared from embryos and from pools each of 40 staged follicles, corresponding to King’s

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5a) or, as in the previous experiment, to overlapping pairs of genes (Fig. 5b). Probing with pairs of genes permitted densitometric quantification according to an internally consistent relative scale (Fig. 6). The results clearly indicated that, although all four genes are expressed most abundantly toward the end of choriogenesis (stages 13 and 14), they differ in detailed timing. Within the sensitivity of the assay, s161 and ~18-1 mRNAs were first detectable at stage 11 + 12, and showed peak accumulation at stages 13 and 14. The s19-1 mRNA was a little earlier: it was detectable at stage 10 and reached a maximum at stage 13. The ~15-1 mRNA was unique in showing clearly biphasic kinetics of accumulation. “Premature” transcript was present at stages 8 + 9 and 10, when amplification occurs but the ~15-1 protein is not synthesized in viva (Thireos et al., 1980); this transcript essentially disappeared at stage 11 + 12, and was replaced by a properly late transcript at stages 13 and especially 14, when synthesis of the protein is maximal in vivo (Petri et al., 1976; Waring and Mahowald, 1979). The amount of premature ~15-1 mRNA accumulated depended on the physiological condition of the flies: in aged flies (see Materials and Methods) it reached as much as 20% of the stage 14 maximum, whereas in young flies it was barely detectable (data not shown).

~16-1

Cross-Hybridization

Duration (hr) Stage

10.7

8+9

(

5

12.41Oij2

IO

II+12

) I3 14

FIG. 6. Quantitative diagram of developmental accumulation of mRNA corresponding to the four genes in the XAB cluster. The DmcAl-1 autoradiogram of Fig. 5a, as well as the combined autoradiograms of Fig. 5B were scanned densitometrically, and the abundance of each mRNA was expressed relative to the ~15-1 mRNA. The width of each bar corresponds to the duration of that stage, permitting estimation of temporally integrated abundance; these estimates are in good agreement with the combined stage estimates in Fig. 4b.

stages 8 plus 9, 10, 11, plus 12, 13, and 14 (King, 1970). Stages 8 through early 10 are involved in vitellogenesis and vitelline membrane production, and stages late 10 through 14 in chorion formation. To maximize reproducibility, the same filter containing the blotted stagespecific RNAs was hybridized sequentially with all probes, after removal of the previously hyridized RNA. The probes used corresponded to individual genes (Fig.

Analysis of Chorim Genes

The similarities in transcript sizes and intronic lengths and apparent locations (Fig. 2) suggested that some or all of the genes in XAB may be homologous. However, cross-hybridization analysis failed to detect significant sequence homologies, between different genes. For this analysis, XAB DNA was digested into fragments containing individual genes and blot-hybridized with probes also containing individual genes plus surrounding sequences (or with the cDNA clone DmcAl1). Even though the hybridization conditions were modestly permissive (55”C, 5~ SSC), only the fragment(s) corresponding to the probe itself hybridized (data not shown). DNase I Hypersensitive Cloned Genes

Sites in the Vicinity

of the

Hypersensitive sites were revealed, by DNase I digestion, in the vicinity of the three best localized chorion genes ~18-1, ~15-1, and s19-1, which are contained in the large (7.8 kb) EcoRI fragment of XAB. Nuclei were prepared from the ovaries of 100 flies, aliquoted, and digested with varying doses of DNase I. The DNA of each sample was then extracted, redigested with EcoRI to

GRIFFIN-SHEA,

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AND KAFATOS

Chwitm

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333

in Drosophila

I

DNase

I

9.2 46:; 2.1 1.9 .8

R

1 C

Ia I Rl

‘t,,:l~~~i~i H3 4 019-l

I Xl

tv

v

I IH3Xl --

I I H3 Xbl

xbl s15-1

H3

I

1

I Xbl

I Rl

~18-1

FIG. ‘7. DNase I hypersensitive sites. Equal aliquots of nuclei from ovaries were digested for 3 min with various amounts of DNase I 0.2, 0.5, 1, and 2 units/ml). The DNA was extracted, digested with EcoRI, electrophoresed, and blotted onto nitrocellulose. The paper hybridized first with fragment R (see bottom panel), then dehybridized and reprobed with fragment L. Both probings detected complementary bands, bounded by an EcoRI site and a DNase I sensitive site (a-d). In the second probing (L), two additional faint bands were detected relatively high concentrations of DNaseI (asterisks), corresponding to a site too close to the left end for resolution in the probing with From the sizes of the bands, t.he DNase I sensitive sites could be mapped consistently from both the left (L) and the right (R) end of EcoRI 7.8-kb fragments ( j and v , respectively).

completion, Southern-blotted, and probed with a nicktranslated fragment (R in Fig. 7) from the right end of the 7.8-kb region. After autoradiography, the filter was dehybridized and reprobed with a second fragment

(0.1, was at (R). the

(L in Fig. 7), from the left end of the region. Thus, DNase I hypersensitive sites could be detected and mapped by the Wu procedure (Wu, 1980), from both directions, to ensure greater accuracy.

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Four DNase I hyperactive sites were detected from both orientations, and mapped with an accuracy of 50 to 200 bp (a through d in Fig. 7). The sites detected flank each of the three chorion genes in the region assayed. No sites were found within the genes themselves. Only one site (d in Fig. 7) was not correlated with a known gene. DISCUSSION

In this report we have characterized a tight cluster of major chorion genes, and have explored the possible significance of their clustered arrangement for the temporal and quantitative control of gene expression. The data available establish unequivocally the existence of at least four chorion genes within the 6-kb region extending from the Hind11 site of fragment B to the right end of fragment D. Only this region detectably hybridizes with ovarian cDNA. Specifically, four DNA segments hybridize with 0.65- or 0.75-kb mRNAs, in alternating order. Hybridization-translation proves that the two 0.65-kb mRNAs are different from each other, as are the two 0.75-kb mRNA; together with the gene sizes estimated by ExoVII digestion, this eliminates the possibility that only two overlapping genes exist (one corresponding to 0.65- and the other to 0.75-kb mRNA). We have positioned one gene between the Hind11 and the right-end EcoRI site of fragment B, based on the relative intensities of hybridizations of ovarian cDNA with the pertinent HindII-PvuI and PvuI-EcoRI fragments. The data are not adequate, however, to exclude the possibility that a second gene may exist in the same segment. We note that minor, lower molecular weight bands are observed in the Berk-Sharp and Northern analysis of fragment B: these are compatible with the possibilities of discrete RNA degradation products, shorter RNAs from minor polyadenylation sites, or RNA products of a second gene, contained either within fragment B or elsewhere in the genome but cross-hybridizing with fragment B. Further work is necessary to discriminate between these possibilities. The four genes localized have been characterized by hybrid-selected translation as encoding major chorion proteins of the ~15, ~16, ~18, and s19 size classes. In agreement with these results, the ~15-1 and ~18-1 genes have been shown to map to the same cytogenetic locus (66DlO-15) by in situ hybridization of cDNA clones (Spradling et al., 1980; Griffin-Shea et al., 1980), and a variant of the s19-1 protein has been mapped to the same general area by recombination (Yannoni and Petri, 1982). A cDNA clone which encodes an sl6-size protein has been selected by weak cross-hybridization

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with the sl5-l-encoding clone, DmcAl-1, and has been mapped to a different site (54CD, second chromosome; Griffin-Shea et al., 1980). However, Northern analysis has shown that this stage is expressed in embryos, testes, as well as all stages of oogenesis (unpublished observations), and thus encodes a protein which is not unique to chorion (and possibly not even a chorion component). We have named the gene contained in fragment B ~16-1 on the basis of the size and immunoprecipitability of its product with sl6-chorion-specific antiserum, the tissue and developmental specificity of its expression, and its reasonably abundant expression into RNA. Three distinct s16 protein components have been recognized (Waring and Mahowald, 1979; Margaritis et al., 1980; Yannoni and Petri, 1980), and thus it remains to be determined which one corresponds to the ~16-1 gene defined here. All four chorion genes show structural similarities, including small size (0.70 to 0.85 kb according to BerkSharp analysis) and the presence of small intron(s) near one end (probably the 5’ end). These are reminiscent of the similarities between silkmoth chorion genes, which are members of multigene families, i.e., evolutionarily homologous (Jones and Kafatos, 1980b). Surprisingly, no cross-hyridization was detected between the various XAB genes, even though peptide fingerprints clearly suggest the existence of homologies between chorion proteins (e.g., the ~18-1 and s19-1 proteins) (Spradling et al., 1980). The sensitivity of the cross-hybridization analysis should be reduced proportionately by the inclusion of flanking as well as coding sequences in the probes; moreover, if a flanking region includes a simple, internally repetitive sequence, the self-hybridization signal might be emphasized still more. Pending sequence analysis, it appears that, if the XAB genes have homologies, they have diverged to the point that homology is no longer detectable under our conditions. Unlike silkmoth chorion genes, which are organized in divergently and coordinately transcribed pairs (Jones and Kafatos, 1980a), three and possibly all four Dro sophila chorion genes in this locus are transcribed in direct orientation, from the same strand. The absence of divergent gene pairs may be related to the temporal and quantitative differences in the mRNA accumulation profiles: transcriptional control signals immediately linked to individual genes may be responsible for these differences. In this respect, it is interesting that DNase I hypersensitive sites flank all three genes examined; site d (Fig. 7) may reflect the existence of a gene not detected as yet (perhaps expressed in a different tissue). It is also possible, however, that the differences in the RNA accumulation profiles are mediated at the level of RNA processing or stability, rather than

GRIFFIN-SHEA,

THIREOS,

AND

KAFATOS

transcription. Furthermore, we cannot exclude the possibility that the clustered gene arrangement has some coarse regulatory role, somehow ensuring transcription primarily toward the end of oogenesis. The clustered arrangement does seem to result in equal amplification of these genes during choriogenesis (Fig. 4a). Again, however, the clustered arrangement at most has a coarse regulatory role: equal amplification does not result in equal mRNA accumulation. Equal amplification ma,y be a simple consequence of the clustered gene arrangement, as much as a selective advantage for it. The most striking feature of the temporal RNA profiles is the premature transcription and unique bimodal accumulation of ~15-1 mRNA. It is tempting to speculate that the ~15-1 gene is at or very near a unique amplification origin, and that premature transcription is somehow related to the mechanism of amplification. It is notable, however, that the amount of premature ~15-1 transcript accumulated is subject to change according to the physiological state of the fly. That raises the possibility that other genes (e.g., s19-1) may also be transcribed at stage 8 + 9, albeit at a lower rate, or into transcripts which fail to accumulate in amounts detectable under our conditions. Clearly, a prerequisite to further analysis of the significance of chorion gene organization is direct determination of the rates of transcription, as opposed to mRNA accumulation. This could be accomplished by high-sensitivity Southern analysis, using as probes pulse-labeled RNAs from nuclei of the various developmental stages. Recovery and molecular analysis of chorion mutants mapping in this region should also be informative. We thank K. Jacobs for clone XAB and some restriction mapping experiments, A. C. Spradling for exchange of information, and M. Muskavitch for helpful discussions and suggestions. We thank S. Luper-Foy for secretarial assistance, C. Phillips for artwork, and B. Klumpar for photography. The work was supported by grants from the NSF (R.G.S.), and the ACS and the NIH (G.T. and F.C.K.). RE;FERENCES ALWINE, J. C., KEMP, D. J., and STARK, G. R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzylomethyl-paper and hybridization with DNA probes. Proc. Nut. Acad Sci. USA 74, 5350-5354. BAILEY, J. M., and DAVIDSON, N. (1976). Methyl mercury as a reversible denaturing agent for agarose gel electrophoresis. Anal. B&hem. 70,75-85. BENTON, W. D., and DAVIS, R. W. (197’7). Screening Xgt recombinant clones by hybridization to single plaques in situ. Science 196, 180182. BERK, A. J., and SHARP, P. A. (1978). Spliced early mRNAs of SV40. Proc. Nat. Acad. Sci USA 75, 1274-1278. BLATTNER, F. R., WILLIAMS, B. G., DENNISTON-THOMPSON, K., FABER, H. E., FURLONG, L., GRUNWALD, D. J., KIEFER, D. O., MOORE, D. D.,

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