Identification and cytogenetic localization of vitelline membrane messenger RNAs in Drosophila

DEVELOPMENTAL

BIOLOGY

105, 41-47 (1984)

Identification and Cytogenetic Localization of Vitelline Membrane Messenger RNAs in Drosophila JOSEPH FARGNOLIAND GAIL L. WARING' Department

of Biology,

Marquette

University,

Milwaukee,

Wticmzsin 53233

Received December 1, 1983; accepted in revised form April

4, 1984

During stages 9 and 10 of oogenesis in Drosophila the major proteins involved in vitelline membrane (VM) formation are synthesized and secreted by the somatic follicle cells surrounding the oocyte. To identify potential mRNAs involved in VM protein synthesis, newly synthesized poly(A)-containing RNA from egg chambers of different developmental stages was studied. Urea-agarose gel electrophoresis revealed two RNA bands in stage 10 egg chambers in the size range expected for those which encode the smaller VM proteins. These RNA bands, T, and Tz, are specifically enriched in stage 10 follicle cell preparations. In vitro translations in reticulocyte lysates in the absence and presence of microsomal membranes showed both RNA bands code for products that are synthesized in precursor forms which are processed to species that comigrate with VM proteins. Tz directed the synthesis of processed species that comigrated with the 23- to 24-kDa and 17.5-kDa VM proteins (J. Fargnoli and G. L. Waring, 1982, Dev. Biol. 92, 306-314) while the Ti translation product comigrated with the ll-kDa protein. To determine the cytogenetic location of the genes encoding T1 and Tz RNAs, radiolabeled Ti and Tz RNAs were hybridized in situ to salivary gland chromosomes. The results suggest that the structural genes coding for the small vitelline membrane proteins are localized at two sites on the second chromosome: 39DE and 42A.

proteins (Fargnoli and Waring, 1982). In the present study we have electrophoretically resolved two follicle cell-enriched RNA bands whose time of synthesis coincides with vitelline membrane production. In vitro translation of these RNAs in the presence of pancreatic microsomal membranes yielded three processed protein products that comigrated with authentic vitelline membrane proteins on SDS-polyacrylamide gels. In situ hybridization of these RNAs to salivary gland chromosomes has led to the identification of two putative vitelline membrane gene sites on the second chromosome.

INTRODUCTION

Eggshell production in Drosophila involves the sequential expression of a set of functionally related genes. During the vitellogenic stages (g-lo), the ovarian follicle cells produce five major proteins that comprise the first eggshell layer or vitelline membrane (Fargnoli and Waring, 1982). Following vitelline membrane synthesis, the follicle cells switch to production of the chorionic sublayers. During stages 11-14 approximately 20 chorion proteins are synthesized in a defined temporal sequence (Petri et aZ., 1976; Waring and Mahowald, 1979). The structure, organization, and expression of the chorion genes have been the focus of several recent studies. The major chorion genes are organized into two gene clusters (Spradling et al, 1980; Griffin-Shea et ab, 1980) which undergo tissue-specific amplification prior to their expression (Spradling and Mahowald, 1980; Griffin-Shea et ah, 1982). Temporal as well as quantitative differences in RNA accumulation patterns indicate that chorion production entails the regulated expression of individual genes within these clusters (Griffin-Shea et al, 1982). In an effort to understand factors involved in initiating the eggshell synthesis program, we have recently focused our attention on vitelline membrane gene expression. By biochemical and immunological analyses we have identified six major size classes of vitelline membrane i To whom correspondence

MATERIALS

AND

METHODS

Radiolabeling and Isolation of Egg Chambers and Egg Chamber Cell Types Drosophila melanogaster (Oregon R, P2 strain) were obtained from stocks grown in mass culture as previously described (Allis et al., 19’77). For in vivo labeling, flies were injected with radioisotopes according to the methods of Spradling and Mahowald (1979). Egg chambers in stages 10 through 14 and previtellogenic ovaries (egg chambers, stages O-7) were isolated as described previously (Fargnoli and Waring, 1982). To separate egg chamber cell types, stage 10 egg chambers were placed on glass slides and cut in half at the interface between the nurse cells and oocyte. The nurse cells were removed immediately from the oocyte and follicle cell fraction. Following release of the oocyte contents, the remaining

should be addressed. 41

0012-1606/84 Copyright All rights

$3.00

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

42

DEVELOPMENTAL BIOLOGY

egg chamber remnants consisting of follicle cells, vitelline membrane, and some adhering oocyte contents were transferred with forceps to a microcentrifuge tube and processed as an enriched follicle cell fraction. The extruded oocyte contents were then recovered by rinsing the slide with small volumes of extraction buffer. RNA Purification RNA was extracted, purified by oligo(dT)-cellulose chromatography, and recovered by ethanol precipitation according to the methods of Spradling and Mahowald (1979). RNA fractions containing and lacking poly(A) were analyzed on 3% agarose-urea gels as described by Rosen and Monahan (1981). After electrophoresis, the gel was either dried or wrapped in Saran Wrap and exposed to X-ray film at -80°C. When desired, appropriate regions of the wet gel were excised with X-acto knives and electrophoretically eluted into dialysis tubing (Maniatis et aL, 1982) supplemented with tRNA at 200 pg/ml. Eluted RNA was ethanol precipitated and repurified by oligo(dT)-cellulose chromatography. Translation in Vitro Poly(A)-containing RNAs used for translations were ethanol precipitated from solutions containing 0.25 M ammonium acetate, pH 5.0, and 2 pg carrier tRNA. Precipitated RNAs were washed three times with 95% ethanol prior to their use in a mRNA-dependent amino acid-depleted rabbit reticulocyte suspension (Amersham N.150). Translation reactions, performed according to the manufacturer, were allowed to proceed for 1.5 hr at 30°C. Ten microcuries of a high-specific-activity 3H-labeled amino acid pool consisting of phenylalanine, leucine, lysine, tyrosine, and proline (Amersham) was used per assay (0.4 mCi/ml). Amino acids not included in the radioactive pool were added at 50 PM. Where indicated, 0.5 ~1 of dog pancreas microsomal membranes (New England Nuclear) was included in the lysate mixture (25 ~1). Translation products and follicle cell proteins were analyzed electrophoretically on 15% acrylamide gels by fluorography as previously described (Fargnoli and Waring, 1982). In Situ Hybridizations Salivary gland squashes from third-instar larvae bearing the giant mutations go’/&’ (Kaufmann, 1971) were prepared as described by Bonner and Pardue (1976). Heat-treated (Bonner and Pardue, 1976), NaOH-denatured squashes (Pardue and Gall, 1975) were hybridized in a solution containing 40% formamide, phosphate buffer, 4X SSC, pH 6.8, and 5-10 X lo3 cpm of 3H-labeled RNA bands electroeluted from agarose-urea gels. Fol-

VOLUME 105, 1984

lowing hybridization, squashes were exposed to RNase, rinsed, dehydrated, and processed for autoradiography as described by Pardue and Gall (1975). RESULTS

Identiiification of Stage-Speci$c RNAs In previous studies on RNA synthesis patterns during egg chamber development, Spradling and Mahowald (1979) electrophoretically resolved six poly(A)-containing RNA bands whose synthesis was restricted to the choriogenic stages (11-14). Four of these RNA bands (E3-E6) were follicle cell products (Spradling and Mahowald, 1979) that encoded major chorion proteins (Thireos et al, 1979; Spradling et ak, 1980). In addition to choriogenic stage transcripts, Spradling and Mahowald (1979) noted three low-molecular-weight bands which were only labeled in stage 10 egg chambers, the period of maximal vitelline membrane protein synthesis (Fargnoli and Waring, 1982). The abundance, sizes, and temporal profiles of these bands suggested that they might encode some of the smaller vitelline membrane proteins which are major synthetic products of stage 10 egg chambers (Fargnoli and Waring, 1982). To look for putative vitelline membrane RNAs, RNA synthesis was studied in egg chambers at various stages of development following in viva labeling with [““PIorthophosphate for 1 hr. Figure 1 shows the patterns o-7

11-12

10

13-1L

84

E3 E4 E5 E6 .

1

2

3

L

FIG. 1. Electrophoretic analysis of RNA synthesis in egg chambers at different developmental stages. Flies were injected with [32P]orthophosphate (loo-150 &i/&. After 1 hr egg chambers were separated according to their developmental stage. Poly(A)-containing RNA was purified, resolved by urea-agarose gel electrophoresis, and analyzed by autoradiography. Numbers above each lane indicate the developmental stages from which RNA was extracted. Approximately 10K cpm were applied to each lane. The arrowhead indicates stage lo-specific RNA. B4 is the major mitochondrial poly(A)-containing RNA described by Spradling et al. (197’7). The E bands have been tentatively identified as major chorion mRNAs (Spradling and Mahowald, 19’79) based on their abundance, sizes, and production periods.

FARGNOLI AND WARING

Messenger

of poly(A)-containing RNA species resolved by ureaagarose gel electrophoresis. As previously described by Spradling and Mahowald (1979), the majority of labeled RNAs in previtellogenic ovaries (stages O-7) and stage 10 egg chambers are heterogenous (lanes 1 and 2). No unique RNA species were resolved in previtellogenic ovaries. One RNA band whose synthesis was restricted to stage 10 egg chambers was clearly distinguishable in the low-molecular-weight range (arrow, lane 2). Other than the major mitochondrial RNA species B4 (Spradling et al., 1977), two abundant bands were readily detected in stages 11-12. The sizes, abundance, and temporal profiles of these bands are consistent with the E3 and E4 chorion RNAs. Likewise, the two prominent lowmolecular-weight RNAs in stages 13-14 are characteristic of E5 and E6 chorion RNAs (Spradling and Mahowald, 1979). Stage 10 Follicle

Cell RNAs

If the stage 10 specific RNA noted in Fig. 1 encodes vitelline membrane protein, it should be a follicle cell product since it has previously been demonstrated that follicle cells are the site of synthesis of the vitelline membrane proteins (Fargnoli and Waring, 1982). To determine if this RNA was synthesized by follicle cells, flies were injected with [32P]orthophosphate. After a 1-hr labeling period, enriched follicle cell and oocyte fractions were prepared and RNA was extracted and purified by oligo(dT)-chromatography as described under Materials and Methods. Analysis by urea-agarose gel electrophoresis showed two abundant follicle cell RNA bands (T, and T,) in the low-molecular-weight range. In independent gel runs (data not shown) we have established that T1 migrates slightly faster than E6 RNA, in a position comparable to the stage lo-specific RNA band shown in Fig. 1. The T1 and Tz RNAs are not detected in the oocyte fraction and their relative labeling intensities in follicle cells are greater than in whole stage 10 egg chambers (lane 3). In addition they are not apparent in the poly(A)-lacking RNA fraction from stage 10 egg chambers (lane 4). It should be noted that the detection of radiolabeled Tz RNA in extracts from whole stage 10 egg chambers has been variable. In five independent labeling experiments, we have seen equivalent labeling intensities of T1 and T2 RNA in two experiments (e.g., lane 3, Fig. 2), T1 labeling greater than T2 in one experiment, and Tz labeling obliterated by background heterogeneous RNA synthesis twice (e.g., lane 2, Fig. 1). This variability could reflect differences in the relative contributions of nurse cells and follicle cells to the total RNA synthetic profile at different times during stage 10 egg chamber development. The proportion of early-, mid-, and late-stage 10 egg chambers can vary significantly in different ovaries. These experiments

RNAs

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in Drosophila

1

2

3

FIG. 2. Analysis of follicle cell RNA synthesis in stage 10 egg chambers labeled in viva. Stage 10 egg chambers were isolated from flies labeled in wivo for 1 hr with [“Plorthophosphate. Oocyte and follicle cell fractions were prepared as described under Materials and Methods. RNA containing (lanes l-3) and lacking (lane 4) poly(A) was analyzed by urea-agarose gel electrophoresis. Lane 1, A+ RNA oocyte fraction; lane 2, A+ RNA follicle cell fraction; lane 3, A+ RNA whole stage 10 egg chambers; lane 4, A- RNA from whole stage 10 egg chambers. T1 is the stage lo-specific RNAs noted in Fig. 1. The positions of the BlO and D RNA species previously described by Spradling and Mahowald (1979) are indicated in lane 4. Lanes l-3 are equivalent in terms of counts (20,000) and exposure time (20 hr).

represent random collections of stage 10 egg chambers, hence some variability in labeling patterns might be expected. In contrast to the whole stage 10 labeling patterns, T1 and T2 RNAs have been readily apparent in every stage 10 follicle cell preparation analyzed to date (approximately 10). The appearance and enrichment of the T1 and T2 RNA bands in the follicle cell fraction coupled with their small sizes make them likely candidates for mRNAs encoding the abundant low-molecular-weight (l424kDa) vitelline membrane proteins. In Vitro Translation

of T1 and T2 RNA

To establish a relationship between T1 and T2 RNAs and specific vitelline membrane proteins, these RNAs were introduced into a rabbit reticulocyte lysate cellfree translation system. Poly(A)-containing RNA was purified from 3000 stage 10 egg chambers and fractionated on urea-agarose gels. Excised T1 and T2 RNA bands were electrophoretically eluted and translated in an amino acid-depleted rabbit reticulocyte lysate. Use of the amino acid-depleted lysate increased the amount of radiolabel incorporated into the translation products approximately twofold (data not shown). Two translation products (2, y) of similar but slightly larger sizes than authentic vitelline membrane proteins were obtained from translation of Tz RNA (Fig. 3, lane 2). A single translation product (x) that migrated at a position

44

DEVELOPMENTALBIOLOGY fc

T2

1

2

TI

E

3

4

T2

+ b.

-

5

TI

t 6

FIG. 3. Electrophoretic analysis of in vitro translation products. Poly(A)-containing RNA from stage 10 egg chambers was electrophoretically resolved on urea-agarose gels. To determine the positions of the T1 and Ta RNA bands, [“P]RNA extracted from stage 10 follicle cells (Fig. 2, lane 2) was run in an adjacent lane. In vitro translation products of TZRNA (x. y) and T1 RNA (z) in the absence of microsomal membranes are shown in lanes 2 and 3, respectively. Lane 4 shows endogenous protein synthesis in the absence of added RNA. Lanes 5 and 6 show the translation products of T2 and T1 RNA in the presence of microsomal membranes. To relate the positions of the translation products to authentic vitelline membrane proteins, in viva-labeled stage 10 follicle cell proteins were routinely run in adjacent lanes. Follicle cells were prepared from stage 10 egg chambers labeled in viva with a mixture of %-labeled amino acids. The positions of the small vitelline membrane proteins (Sv14, 16, 17.5, and 23-24) are indicated by the arrows and bracket in lane 1. Yolk protein (yp) is also an abundant synthetic product of stage 10 follicle cells. The arrows and bracket to the right of lane 6 indicate the positions of these same proteins run in parallel on the gel from which lanes 5 and 6 were taken. Exposure times: lane 2-l week; lanes 3, 4-2 weeks; lane 52 weeks: lane 6-l month.

similar to vitelline membrane protein Sv16 was obtained from translation of T1 RNA (lane 3). Nearly all secretory proteins have been shown to be synthesized as larger-molecular-weight precursors containing hydrophobic NH2 terminal signal peptides that are removed upon entry into the endoplasmic reticulum (Blobel and Dobberstein, 19’75a,b);Blobel, 19’7’7;Shields and Blobel, 1978). Thireos et al. (1979) as well as Spradling et al. (1980) have demonstrated presecretory chorion proteins for all chorion messenger RNAs tested to date. In all cases chorion protein precursors were 1000-3000 Da larger than their authentic chorion protein counterparts. Since the Tz translation products migrated at positions approximately 1000-2000 Da larger than the in v&o-labeled vitelline membrane proteins, Sv23-24 and Sv1’7.5 (lane l), it seemed probable that the Tz, and perhaps Ti, translation products were vitelline membrane protein precursors. To test if the translation products of T1 and Tz RNAs were precursor forms, dog pancreas microsomal membranes were added to the reticulocyte lysates prior to

VOLUME105.1984

translation. Lane 5 of Fig. 3 shows that when the Tz RNA band is translated in the presence of microsomal membranes to allow processing of signal peptides, two new bands of lower molecular weight appear. The smaller band comigrates with Sv1’7.5while the highermolecular-weight band migrates at the leading edge of the diffuse Sv23-24 band. The sharpness of the processed 23-kDa translation product suggests that the diffuse nature of the in vivo-labeled Sv23-24 band reflects posttranslational modifications of a single species rather than a series of closely related but discrete polypeptide chains. The persistence of a small amount of the X protein in lane 5 may be due to incomplete processing, or may represent a third translation product of Tz that does not undergo processing. This question could not be resolved since higher concentrations of microsomal membranes completely inhibited translation. Translation of T1 RNA with microsomal membranes (Fig. 3, lane 6) resulted in the appearance of a prominent new band identical in size to Sv14. The failure to detect a processed translation product that comigrated with Sv16 is consistent with our previous hypothesis that Sv16 is a modified primary vitelline membrane protein. The 16-kDa protein has been a consistent component of purified vitelline membranes and long-term labeled follicle cells (1 hr). However, unlike the other vitelline membrane proteins, it has not been detected in short-term labeled follicle cells (10 min) (Fargnoli and Waring, 1982). Cytogenetic Localization and T2 RNAs

of the Genes Coding for the Tl

Production of T1 and T2 RNAs by follicle cells during the period of vitelline membrane deposition coupled with the sizes of their processed translation products indicate T1 and Tz RNAs encode the small vitelline membrane proteins Sv14, 17.5, and 23. DNA sequences complementary to abundant radiolabeled RNA species can be detected by in situ hybridization to salivary gland chromosomes (Spradling et ab, 1975; Bonner and Pardue, 1976; Spradling and Mahowald, 1979). To identify the cytogenetic location of these vitelline membrane genes, T1 and T2 RNAs were purified from 1200 stage 10 egg chambers labeled in vivo for 1 hr with high-specificactivity ribonucleosides. T1 and Tz RNA bands electroeluted from urea-agarose gels were hybridized in situ with polytene chromosomes. The hybridization of Tz RNA resulted in grains at one site over the right arm of the second chromosome at band 42A (Figs. 4B and C). Hybridization of the T1 RNA encoding Sv14 was detected on the left arm of the second chromosome site 39DE (Figs. 4A and D). As indicated in Table 1, hybridization sites were not randomly distributed. With

FARGNOLI

AND

WARING

Messenger

RNAs

in Drosophila

A

D

370

FIG. 4. In situ hybridization of T1 and Tz RNA to salivary gland chromosomes. Flies were injected and labeled for 1 hr with an equimolar mixture of tritiated adenosine, uridine, cytidine, and guanosine (approximately 5 &i/fly). Poly(A)-containing RNA from 1200 stage 10 egg chambers was resolved on urea-agarose gels. Ti and Tz RNAs were located and recovered as in Fig. 3 and hybridized individually to salivary gland chromosomes. There were 8 X 10’ cpm of T1 hybridized (A, D) and 5 X lo3 cpm of T2 (B, C). Slides were exposed for 63 days. (A) Hybridization of T, RNA to the 2L arm near the chromocenter (C). (D) Stretched and magnified region of the 2L arm to illustrate the 39DE hybridization site. (B) Hybridization of T2 RNA to the 2R arm near the chromocenter. The inset (b) shows the tip of the same chromosome. (C) Stretched and magnified region of 2R arm to illustrate 42A site.

T1, 100% of the hybridized sites were restricted to 39DE with a mean number of 10.8 grains. With Tz, 92% of the sites showing three or more grains were 42A. The mean number of grains observed was 5.2. The occurrence of grains in only two cases at sites other than 42A suggests these are randomly located background grains. DISCUSSION

Vitelline Membrane RNAs The T1 and T2 RNAs characterized in this study are believed to be responsible for the synthesis of three

major vitelline membrane proteins based on four major criteria: (1) Stage 10 follicle cell populations, known to be the site of synthesis of the vitelline membrane proteins, showed selective enrichment of the T1 and Tz RNAs. These same RNA species were not observed in either the oocyte nor poly(A)-lacking RNA fractions from the same in viva-radiolabeled egg chambers. (2) The sizes of these RNAs were in the expected range for those messenger RNAs encoding the smaller molecular weight vitelline membrane proteins. (3) Synthesis of T1 RNA was detected only in stage 10 egg chambers, the stage of maximal vitelline membrane protein synthesis.

46

DEVELOPMENTALBIOLOGY TABLE 1 IN SITU HYBRIDIZATION

RNA band

Nuclei scored

Nuclei hybridized

T, T*

21

13

44

25

Percentage nuclei hybridized 62 57

Vitelline Membrane Genes

Percentage hybridization 39DE 100

VOLUME105, 1984

42A 92

Mean no. grains per site 10.8 5.2

Note. Sevenor more grains at 39DE and three or mwe grains at 42A were scoredas sites of hybridization. In the case of Tz (42A). a site having three grains and another having five grains were observedat two different sites other than 42A.

(4) In vitro translation of these RNAs in the presence of microsomal membranes resulted in processed protein species identical in size to three major vitelline membrane proteins. The labeling properties of both putative vitelline membrane RNA bands are consistent with what is known about protein synthesis during Drosophila oogenesis. The analysis of protein synthesis patterns throughout oogenesis has indicated only a few changes that are related to developmental stage. Loyd et al. (1981) have described a 9’7-kDa germ line component (nurse cell) whose synthesis is restricted to early egg chamber stages. The most striking examples of stage specific synthesis occur in the follicle cells. During late oogenesis (stages 11-14) the major synthetic activity of the egg chambers is chorion protein production by the follicle cells (Petri et a& 1976; Waring and Mahowald, 1979). During stage 10 the follicle cells synthesize yolk (Brennan et aL, 1982) and vitelline membrane proteins (Fargnoli and Waring, 1982). Both yolk and major vitelline membrane protein synthesis are readily detected in whole egg chamber extracts. Based on these data the appearance of abundant stage-specific RNA species would be expected to be of follicle cell origin. While most of the experiments described in this paper focus on newly synthesized RNA species, the analysis of the in vitro translation products depended on RNA mass. It might be argued that abundant germ line (oocyte-nurse cell) products, which comigrate with the follicle cell RNAs, accumulate during early oogenesis and are preferentially translated in vitro. We feel this is unlikely since the synthesis of abundant poly(A)-containing RNA species in this size range has not been detected in stages l-9 (Spradling and Mahowald, 1979; Fig. 1 this paper). Furthermore, the primary translation products of both T1 and Tz RNA bands are larger precursor molecules that are processed in the presence of microsomal membranes. These properties are consistent with products of secretory cells such as the follicle cells, not germ line nurse cells and oocytes.

In situ hybridization of T1 and Tz RNA to polytene chromosomes revealed two major sites of hybridization: 39DE and 42A, respectively. It has previously been shown by in situ hybridization that region 39DE includes the histone gene locus (Pardue et al., 1977). Histone mRNA would be expected to migrate in a size range similar to T1 RNA (Spradling and Mahowald, 1979). If histone mRNA is actively transcribed in stage 10 egg chambers and if it is present in significant quantities in the poly(A)-containing RNA fraction, then grains at 39DE may reflect hybridization to the histone gene cluster rather than a vitelline membrane structural gene. Although polyadenylated histone messenger RNA has been reported in amphibian oocytes (Ruderman and Pardue, 1978), the bulk of the histone message from stage 10 egg chambers appears to be nonadenylated (Spradling and Mahowald, 1979). Spradling and Mahowald showed that in vivo-labeled RNA from the nonadenylated RNA fraction hybridized heavily in situ to the histone gene locus. As shown in lane 4 of Fig. 2, no prominently labeled RNA species in the T1 size range are evident in the poly(A)-lacking fraction. Therefore it is reasonable to assume that the proportion of radiolabeled histone mRNA in the polyadenylated T1 fraction is insignificant. The paucity of histone mRNA in this fraction is also supported by the in vitro translation results shown in lanes 3 and 6 of Fig. 3. Hybridization of T1 RNA to the 39DE site has been observed using two independent RNA preparations. Furthermore the average number of grains observed at the 39DE versus the 42A site was proportional to the input counts hybridized. These results would not be expected if the 39DE signal reflected hybridization of contaminating sequences to the repeated histone gene cluster. While histone RNA hybridization cannot be definitively ruled out at this time, taken together, our data indicate otherwise. Whereas the in vitro translation data indicated the T1 RNA band was a homogeneous species, at least two distinct RNAs comprise the T2 band. Despite this species heterogeneity only one site was observed in our in situ hybridization experiments. This may indicate that the Sv17.5 and Sv23-24 genes are closely linked. Clustering the vitelline membrane genes would not be unexpected since clustering of chorion eggshell genes is well established (Spradling, 1981; Griffin-Shea et ah, 1982). Alternately, only one RNA species in the Tz band may be abundant enough to yield a hybridization signal in these experiments. The relative intensities of the processed in vitro translation products shown in lane 5 of Fig. 3 indicate Sv17.5 RNA is the most abundant Tz species. This is also consistent with Sv17.5 being the most abundant protein in vitelline membrane preparations (Farg-

FARGNOLI AND WARING

Messenger

noli and Waring, 1982). Whether the 42A hybridization site contains genes coding for one or both of the Tz RNA species cannot be distinguished by the present data. Analysis of genomic clones from this region should resolve this question. In conclusion, our results indicate genes coding for some of the major vitelline membrane proteins are localized on the proximal portions of the left and right arms of the second chromosome. It is hoped that subsequent analysis of female sterile mutations that map to these regions will provide insight into sequences that play a role in initiating the eggshell gene program. It is of interest to note that a female sterile mutation, fs(2)E’7, tentatively described as a vitelline membrane defect has been mapped in the 42A region (Lindsley and Grell, 1968). The eggs produced by mutant females are collapsed and leak when dechorionated. It has been suggested that this phenotype is a consequence of a weakened vitelline membrane. We thank Ms. B. DeNoyer for help in preparation of the manuscript and Dr. S. Hennen for her helpful comments. This work was supported by Schmitt and Scholl Foundation predoctoral fellowships to J.F. and NSF Grants PCM7823385 and PCM8203171 to G.W. REFERENCES ALLIS, C. D., WARING, G. L., and MAHOWALD, A. P. (1977). Mass isolation of pole cells from Drosophila melanogaster. Dev. Biol. 56, 373-381. BLOBEL, G. (1977). Synthesis and secretion of secretory proteins: The signal hypothesis. In “International Cell Biology 1976-1977” (B. R. Brinkley and K. R. Porter, eds.), pp. 318-325. Rockefeller Univ. Press, New York. BLOBEL, G., and DOBBERSTEIN, B. (1975a). Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin chains on membrane-bound ribosomes of murine myeloma. J. Cell Biol. 67, 835-851. BLOBEL, G., and DOBBERSTEIN, B. (1975b). Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J. Cell Biol. 67, 852-862. BONNER, J. J., and PARDUE, M. L. (1976). Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster. Assay by in situ hybridization. Chromosmna 58, 87-99. BRENNAN, 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. Den Biol 89, 225-236. FARGNOLI, J., and WARING, G. L. (1982). Identification of vitelline membrane proteins in Drosophila melanogaster. Dew. Biol. 92, 306314. GRIFFIN-SHEA, R., THIREOS, G., KAFATOS, F. C., PETRI, W. H., and VILLAKOMAROFF, L. (1980). Chorion cDNA clones of D. melanogaster and their use in studies of sequence homology and chromosomal location of chorion genes, Cell 19, 915-922. GRIFFIN-SHEA, R., THIREOS, G., and KAFATOS, F. C. (1982). Organization

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of a cluster of four chorion genes in Drosophila and its relationship to developmental expression and amplification. Dez. Biol. 91,325336. KAUFMAN, T. C. (1971). Characterization of three new alleles of the giant locus of Drosophila melanogaster. Genetics 71, s28-s29. LINDSLEY, D. L., and GRELL, E. H. (1968). “Genetic Variations of Drosup&la melanogaster” (I. L. Norton, ed.). Carnegie Institution of Washington, Baltimore. LOUD, J. E., RAFF, E. C., and RAFF, R. A. (1981). Site and timing of synthesis of tubulin and other proteins during oogenesis in Dro sophila melavwgaster. Dev. Biol. 86, 272-284. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular Cloning (A Laboratory Manual).” Cold Spring Harbor Laboratory, New York. PARDUE, M. L., and GALL, J. (1975). Nucleic acid hybridization to the DNA of cytological preparations. In “Methods in Cell Biology” (D. Prescott, ed.), Vol. 10, pp. l-34. Academic Press, New York. PARDUE, M. L., KEDES, L. H., WEINBERG, E. S., and BIRNSTIEL, M. L. (1977). Localization of sequences coding for histone messenger RNA in the chromosomes of Drosophila melanogaster. Chrmnosoma 63, 135-151. 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. Deu. Biol. 49, 185-199. ROSEN, J. M., and MONAHAN, J. (1981). In “Laboratory Methods Manual for Hormone Action and Molecular Endocrinology” (W. T. Schrader and B. W. O’Malley, eds.), Chap. 4. Houston Biological Assoc., Houston. RUDERMAN, J. V., and PARDUE, M. L. (1978). A portion of all major classes of histone messenger RNA in amphibian oocytes is polyadenylated. J. Biol Chem. 253, 2018-2025. SHIELDS, D., and BLOBEL, G. (1978). Efficient cleavage and segregation of nascent presecretory proteins in a reticulocyte lysate supplemented with microsomal membranes. J. Biol Chem 253,3753-3756. SPRADLING, A. C. (1981). The organization and amplification of two chromosomal domains containing Drosophila chorion genes. Cell 27, 193-201. SPRADLING, A. C., DIGAN, M. E., MAHOWALD, A. P., SCOTT, M., and CRAIG, E. A. (1980). Two clusters of genes for major chorion proteins of Drosophila melanogaster. Cell 19, 905-914. SPRADLING, A. C., and MAHOWALD, A. P. (1979). Identification and genetic localization of mRNAs from ovarian follicle cells of Drosophila melanogaster. Cell 16, 589-598. SPRADLING, A. C., and MAHOWALD, A. P. (1980). Amplification of genes for chorion proteins during oogenesis in Drosophila melunogaster. Proc. Nat1 Acad Sci. 77, 1096-1100. SPRADLING, A., PARDUE, M. L., and PENMAN, S. (1977). mRNA in heat shocked Drosophila cells. J. Mol. Biol 109, 559-587. SPRADLING, A., PENMAN, S., and PARDUE, M. L. (1975). Analysis of Drosophila mRNA by in situ hybridizations: Sequences transcribed in normal and heat shocked cultured cells. Cell 4, 395-404. THIREOS, G., GRIFFIN-SHEA, R., and KAFATOS, F. C. (1979). Identification of chorion precursors and the mRNAs that encode them in Drosophila melanogaster. Proc. Natl. Acd Sci. USA 76, 6279-6283. WARING, G. L., and MAHOWALD, A. P. (1979). Identification and time of synthesis of chorion proteins in Drosophila melanogaster. Cell 16, 599-607.