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Identification and time of synthesis of chorion proteins in Drosophila melanogaster
Cell,
Vol. 16.599-607.
March
1979,
Copyright
0 1979 by MIT
Identification and Time of Synthesis Proteins in Drosophila melanogaster
correlation between the abundance, sizes and times of synthesis of the chorion proteins and the putative chorion mRNAs was observed.
Gail L. Waring* and Anthony P. Mahowald Program in Molecular, Cellular and Developmental Biology Department of Biology Indiana University Bloomington, Indiana 47401
Results
Summary The chorion of Drosophila melanogaster consists of proteins secreted by the follicular epithelium during late oogenesis. Petri, Wyman and Kafatos (1976) have described six major protein components of the Drosophila chorion and reported the synthesis of these proteins in vitro by mass-isolated egg chambers. We have used two-dimensional gel electrophoresis to identify approximately twenty components in highly purified chorion preparations. The synthesis patterns of these proteins in vivo were determined by isolating egg chambers of different developmental stages from proflies injected with 14C amino acids. Chorion teins constitute a large fraction of the protein synthesized by ovarian egg chambers in stages 12-14. The sizes and times of synthesis of the chorion proteins correlate closely with the production of poly(A)-containing RNAs by the follicle cells (Spradling and Mahowald, 1979). Introduction During development, functionally related structural genes are expressed in a coordinate fashion. The genes coding for the chorion proteins in Drosophila are an example of such a gene set that is particularly amenable to biochemical analysis (Spradling and Mahowald, 1979). Furthermore, numerous putative chorion mutants have been described (King and Mohler, 1975) which may provide information on mechanisms which regulate the activity of these genes. Understanding the role of these genes in the production of the chorion will require detailed knowledge of the chorion proteins themselves. Petri et al. (1976) identified six major Drosophila chorion proteins and studied their synthesis in vitro in mass-isolated egg chambers. We have studied the synthesis in vivo of approximately twenty chorion proteins, including the six previously described. In our study, all the chorion proteins were synthesized during stages 11-14, and at the peak of production (stages 12-13), they were the major products synthesized by the egg chambers. A close * Present address: Department Milwaukee, Wisconsin 53233.
of Biology,
Marquette
of Chorion
University,
Isolation of Soluble Chorion Fragments The egg shell of Drosophila consists of three major layers: the vitelline membrane, the endochorion and the exochorion (Figure 1 B). The vitelline membrane opposes the oocyte surface, while the exochorion is adjacent to the follicle cell layer. Between the vitelline membrane and the exochorion is the endochorion, made up of a thin floor, pillars and a thick roof. A thin electron-dense layer lies between the vitelline membrane and the floor of the endochorion. A procedure was developed for the extensive purification of these structures. Chorions were typically obtained from 500 early stage 14 egg chambers isolated by hand dissection from the ovaries of 25-30 rapidly laying flies. Fragments of the chorion were pelleted by low speed centrifugation following disruption of the egg chambers by sonication. Detergent (Triton X-100) was included in the sonication buffer to prevent yolk and other cytoplasmic proteins from pelleting with the chorion fragments. Morphological analysis of chorions isolated by these procedures showed that the preparations consisted of exochorion and the entire endochorion (Figures 1A and 1C). Scanning electron microscopy of the isolated chorion preparations (Figure 1A) showed pieces of fragmented chorions. The outer surface of the fragments resembles the outer surface of the endochorion in situ (Margaritis, Petri and Kafatos, 1976). The ridges, built up by an external reticulum that projects from the roof of the endochorion (Figure lB), mark the borders between adjacent follicle cells. The inner surface of the fragments resembles the floor of the endochorion. The majority of the exochorion separates from the endochorion during the isolation procedure and appears to pellet in mass as a separate entity (Figure 1C). There is no evidence of either vitelline membrane or the thin electron-dense layer in the preparation. Light microscope analysis indicated that the chorion preparations were morphologically pure. Another indication of the cleanliness of this fraction is the lack of contamination by the major nonchorion proteins. The majority of egg chamber protein consists of three polypeptides that comprise the yolk protein (Warren and Mahowald, 1979) (Figure 2A). No yolk protein is detected following solubilization of microscopically pure chorion fragments (Figure ZB).
Cell 600
Figure
1. Scanning
Electron
Micrograph
and Transmission
Elect1 ron Micrographs
of Chorions
of chorions isolated from early stage 14 oocytes (900x). (A) Scanning electron micrograph showing inner (I) and outer (0) surfaces (13,000~). (VM) vitelline membrane: (EN) endochorion; (8) Transmission electron micrograph of eggshell layers in stage 14 oocytes and endochoric exochorion; (FC) follicle cell. The arrow indicates a thin electron- dense layer that lies between the vitelline membrane and remnants of the particulate exochc (C) Transmission electron micrograph of isolated chorions sb lowing the endochorion (7600x). (25,000x). The insert shows a field of the particulate exochorion iat lower magnification
Identification of Chorion Proteins Eight to ten proteins are detected by staining acrylamide gels of solubilized chorion preparations with Coomassie blue (Figure 2B). To determine whether additional chorion components could be identified, chorions were analyzed following labeling in vivo with radioactive amino acids. The chorion is formed during stages 11-14 of oogenesis (King and Koch, 1963), a period encompassing about 5’hr (David and Merle, 1968). To maximize the number of radioactive species in our preparations, chorions near completion were isolated from stage 14 egg chambers 8-10 hr after the flies were injected with radioisotope. Under the conditions of these experiments, stage 14 egg chambers accumulated in the ovaries during the incubation period. Thus stable radioactive species synthesized in stage 1 l-l 3 as well as stage 14 egg chambers were recovered in the chorion fragment preparation. In one-dimensional SDS-polyacrylamide gels, the four major species migrating as 38,000, 36,000, 19,000 and 18,000 dalton bands are apparent, as well as numerous minor species covering the mo-
(EN m. lrion
lecular weight range 15,000-l 50,000 daltons (Figure 38). In a two-dimensional gel electrophoretogram of proteins from a similar chorion fragment preparation, approximately twenty species are resolved (Figure 4). Since this pattern has been repeated in six independent experiments, all of these species are considered putative chorion proteins. To facilitate discussion, the chorion proteins have been assigned numbers. The numerical designation is based on the position of the protein within the two-dimensional gel electrophoretogram. The first number indicates the approximate molecular weight, while the second number indicates relative migration from the acidic to basic end in the first dimension NEPHGE gel. For this purpose, the major protein of 36,000 daltons is arbitrarily given a value of 50. For example, the four major radioactive species in Figure 4 that correspond to the four major stained species in Figure 28 are designated ~38-60, ~36-50, c19-63 and ~18-68. Where no ambiguity would result, the second half of the designation could be omitted.
Chorion 601
Proteins
of
Drosophila
a
b
130K92 I(68 K43 K-
29 K-
16 KFigure Figure 2. Two-Dimensional from Stage 14 Egg Chambers
Gel Electrophoretogram and Isolated Chorion
of Proteins Fragments
Proteins extracted from 100 stage 14 egg chambers (a) and chorion fragments from 250 early stage 14 egg chambers (b) were analyzed by two-dimensional gel electrophoresis. Gels were stained with Coomassie blue. The arrow indicates yolk protein.
Time of Synthesis of the Chorion Proteins Although the putative chorion proteins are greatly enriched in chorion fragment preparations compared with other egg chamber proteins, an additional test of their identity would be to determine when they are synthesized during oogenesis. Because chorion fragments from stage 11-13 egg chambers cannot be isolated, it is not possible to determine the radiolabeled proteins that are present in the chorion at each developmental stage. Thus to determine the developmental period when specific chorion proteins are synthesized, chorion proteins must be identified against a background of nurse cell and oocyte proteins in pulse-labeled egg chambers. We have used the resolving power of two-dimensional gel electrophoresis to evaluate the time of synthesis of most of the chorion proteins. Flies were injected with a mixture of 14C amino acids, and individual egg chambers, stages 10-14, were isolated by hand dissection 1 hr later. Approximately 25-75 labeled egg chambers from each stage were dissolved in toto, and two-dimensional gel electrophoretograms were prepared (Figure 5). Different exposure times were used to compensate
3. Autoradiogram
of Long-Term
Labeled
Chorion
Proteins
Early stage 14 egg chambers were isolated from flies injected with r4C amino acids 9 hr prior to dissection. Proteins extracted from chorion fragments isolated from 50 egg chambers were electrophoresed on one-dimensional SDS-polyacrylamide gels (b). Molecular weight markers run on the same gel were stained with Coomassie blue (a). Proteins used to calibrate the gel included kgalactosidase (130,000); phosphorylase a (92,000); bovine serum albumin (68,000); ovalbumin (43,000); carbonic anhydrase (29,000); and human hemoglobin (16,000).
for the differing amounts of material present. Comparison of the fluorographs in Figure 5 shows that except for the chorion proteins, the patterns of proteins synthesized by stage lo-14 egg chambers are very similar. Figures 5C and 5D also illustrate that the major synthetic activity in stage 12 and 13 egg chambers involves production of the chorion proteins by the follicle cells. It is clear that putative chorion proteins are synthesized sequentially during the period of chorion deposition (stages 1214). Three major chorion proteins are synthesized in stage 12, most are synthesized during stage 13 and at least one (~15-55) is synthesized during stage 14 only. Figure 6 summarizes the times of synthesis of the chorion proteins. Labeling studies in which flies were injected with 35S-methionine failed to reveal synthesis of the major chorion proteins (~36, ~38, cl8 and c19), although labeling of nonchorion proteins was similar to that in Figure 5. Thus the major chorion proteins in Drosophila appear to be methioninepoor.
Cell 602
80 60 40
20
s I
I
I
10
I
I
30
Figure
4. Two-Dimensional with
Autoradiogram chorion
fragments
of Long-Term isolated
from
Distance Labeled
Chorion
200 %-labeled
Discussion The results in this paper demonstrate that the chorion proteins are major products synthesized in stage 12 and 13 egg chambers. The major labeled species in stage 10 egg chambers are the three polypeptides that constitute yolk protein (Warren and Mahowald, 1979). These yolk polypeptides are sequestered from the hemolymph by the oocyte during the vitellogenic phase of oogenesis (stages 8-10) (King, 1970). The presence of only trace amounts of the yolk polypeptides in stage 11 egg chambers indicates that stage overlap was not a major problem in these experiments. The synthetic profile of stage 12 egg chambers shows that the major chorion proteins c36 and c38 are the predominant products. In addition, c60 synthesis is prominent. Most of the chorion proteins are synthesized in stage 13 egg chambers and, as in stage 12, represent the major synthetic products. Stage 14 egg chambers gave a lower level of labeling of both general proteins and chorion proteins, although synthesis of cl 8 and ~15-55 appears to be maximal during this stage. There is no evidence of residual synthesis of ~36, ~38, c55 or c60 in the stage 14 egg chambers. This demonstrates that rapid and dramatic changes occur in the synthetic
I
I
I
70
50
Relative Proteins extracted electrophoresis.
I
of
Migration
Proteins stage
14 egg
chambers
were
analyzed
by two-dimensional
activities of the follicle cells during choriogenesis. In this study, we have used two-dimensional gel electrophoresis to identify approximately twenty chorion proteins. Proteins present in purified chorion preparations that are synthesized only during the period of chorion deposition are considered chorion proteins. Sixteen species in the chorion fragment preparation shown in Figure 4 were synthesized in stage 12-l 4 egg chambers. The time of synthesis of the more minor species could not be determined with certainty because of the difficulties encountered in detecting these species in extracts from whole egg chambers. It is improbable that these minor components are contaminants since we failed to detect any of the major yolk polypeptides in the purified chorion preparation. It is difficult to exclude the possibility that a sonication-resistant, detergent-insoluble cellular structure co-pellets with the chorion fragments at 40 x g, although we have not found such structures in electron microscopic analyses of the fractions. Several considerations indicate that the number of chorion proteins which we have resolved by twodimensional gel electrophoresis represent most of the major proteins in the Drosophila chorion. Morphological analysis of the fragment preparations show that the two major structural components of
Chorion 603
Proteins
of Drosophila
NEPHGE
Figure
5. Time of Synthesis
-
of the Chorion
Proteins
“C-labeled egg chambers at different developmental stages were isolated from flies which had been pulse-labeled for 1 hr. Proteins extracted from egg chambers from each stage (10-14) were analyzed by two-dimensional gel electrophoresis. The arrows on the autoradiogram indicate some of the major chorion proteins during the stage in which their synthesis appears maximal. (a) 92 stage 10 egg chambers; (b) 23 stage 11; (c) 20 stage 12; (d) 59 stage 13; (e) 93 stage 14. The definitions of the stages during oogenesis are those described by Illmensee, Mahowald and Loomis (1976). The exposure times for each developmental stage were normalized such that the product of number of egg chambers/stage and the length of exposure were constant. The positions of the major chorion proteins were located by stain. The minor chorion proteins were identified by superimposing fluorographs of chorion fragment preparations run on parallel gels.
Cell 604
Developmental 11
12
Stage
13
14
~80-20 c70-45 ~60.50 c55-40 ~48-45 c45-55 c40-35 ~38-60
,
~36.50
---
_-_-
- ---_
-_
~28.58 c19-63 c18-68 -_--.
~16.30
. _-
--_-
~16-35 c15-50 c15-55
(
,
,
,
,
,
0
1
2
3
4
5
Hours Figure
6. Time
of Synthesis
at 25 “C
of the Chorion
The period of maximal synthesis detectable synthesis is indicated
Proteins
is indicated by the dashed
by the line.
solid
line;
the chorion (endochorion and exochorion) are present. All of the proteins which we have solubilized from these structures were synthesized by egg chambers in stages 12-14. If chorion proteins were lost during the isolation procedures, we would expect to see proteins synthesized between stages 12 and 14that are not present in our chorion preparation. Inspection of the synthetic profiles of stage 12-14 egg chambers (Figure 5) indicates that only one detectable protein synthesized specifically during this developmental period was not recovered in the isolated chorion. This stage 12 protein (approximately 40,000 daltons) could be a component of the electron-dense layer which forms during stage 11 and early stage 12, and is not retained in our chorion fraction. Some chorion proteins may not have been detected because they became insoluble immediately following synthesis. Solubility studies (Petri, Wyman and Henikoff, 1977; G. L. Waring and A. P. Mahowald, unpublished observations) indicated that whereas stage 14 egg chambers became less soluble as they became more developmentally advanced, stage 12 and 13 egg chambers dissolved completely. Thus major chorion proteins synthesized in stage 12 and 13egg chambers are probably
soluble in purified chorions. However, any chorion proteins synthesized in stage 14 egg chambers which were rendered insoluble immediately by cross-linking might not have been detected. In addition, the existence of chorion proteins in amounts too low to be detected in Figure 4 cannot be excluded. The purification of six major chorion proteins and their synthesis in vitro by mass-isolated egg chambers from Drosophila has been reported previously (Petri et al., 1976). The six major proteins described by Petri and colleagues correspond closely in molecular weight to those of Figure 3. Although they observed a relative synthesis order for these six major chorion proteins generally similar to that of Figure 5, we found a number of striking differences in the labeling patterns of the egg chambers in vivo compared with the patterns in vitro. Using a mixture of labeled amino acids, it is clear from our studies that the major chorion proteins ~36, ~38, cl 8 and cl9 are the predominant products synthesized in choriogenic egg chambers. This prodigious synthesis was not evident in the study by Petri and co-workers, even though they used specific 3H amino acids which would be expected to enhance labeling of the chorion proteins. Furthermore, protein synthesis in their stage 10 egg chambers was significantly depressed relative to the stage 11-14 egg chambers, whereas we found synthesis of the nonchorion proteins comparable, if not greater, in stage 10 chambers relative to the more developmentally advanced stages. These differences in labeling patterns may reflect differences in the health of egg chambers labeled in vitro after mass isolation versus chambers labeled in vivo. A further complication of in vitro labeling of isolated egg chambers is the possibility of the induction of the “heat shock” response (Petri et al., 1977; Spradling and Mahowald, 1979). It is interesting to compare our results with the chorion proteins in Drosophila with the extensive knowledge of chorion proteins in silkmoths (reviewed by Kafatos et al., 1977). The Drosophila chorion may be less complex in terms of protein multiplicity. We have resolved approximately twenty proteins, whereas Regier et al. (1978) estimate approximately 100 chorion proteins in the silkmoth. The chorion proteins in Drosophila have a much wider distribution on SDS gels than the silkmoth proteins. Drosophila chorion proteins range in size from 15,000 to 150,000 daltons, while silkmoth proteins range from 7000 to 30,000 daltons. The distribution of the chorion proteins in the first dimension NEPHGE gels in this study indicates that the Drosophila proteins have a wider distribution of isoelectric points and are generally more basic than the silkmoth proteins. The isoelectric points of 80% of the silkmoth chorion proteins fall
Chorion 605
Proteins
of Drosophila
within the pH range of 4-6. It is interesting, however, that the small Drosophila chorion proteins synthesized during stage 14 appear to resemble the major silkmoth proteins much more closely than those produced earlier. The chorion proteins are secretory products of the follicle cells. In vitro translation of mRNAs coding for a variety of exportable proteins yield products slightly larger than the secreted product (Deveillers-Thiery et al., 1975; Burstein, Kanto and Schechter, 1976; Blobel, 1977). Current hypotheses concerning the segregation of exportable proteins into the cisternal space of the rough endoplasmic reticulum envision the synthesis of many secretory proteins via larger precursors (Blobel and Dobberstein, 1975). Analysis of the products synthesized and accumulated in stage 12-14 egg chambers indicates that c36 may be formed via a larger precursor. A protein exhibiting a slightly higher molecular weight and slightly more acidic charge appears to be synthesized coordinately with c36 in stage 11, 12 and 13 egg chambers (Figure 5). Although this protein is prominent in the synthetic profile of stage 12 and 13 egg chambers, it is barely detectable on Coomassie blue-stained gels (data not shown). Furthermore, wherease c36 is prominent in stained gels of stage 14 egg chamber extracts, the slightly larger protein is not detectable at this stage (Figure 2A). These properties are consistent with the notion of a c36 precursor protein. In the case of c19, a similar putative precursor was detected, but other possible chorion precursors were not observed. In the accompanying paper, Spradling and Mahowald (1979) describe the synthesis of a small number of poly(A)-containing RNAs during choriogenesis. The abundance, sizes and times of synthesis of these putative mRNAs correlate closely with the abundance, sizes and times of synthesis of the major chorion proteins. In particular, the abundance and stage specificity of E3 correspond closely with the abundance and stage specificity of c36 and ~38, suggesting that this RNA band contains mRNAs for one or both of these two chorion proteins. Similarly, the sizes and stage specificities of the smaller RNAs (E5-E6) correlate with the smaller chorion proteins including ~16-30 and c1555. In situ hybridization of the E3 and E4 mRNAs at one site (7Ell) on the Drosophila polytene chromosomes (Spradling and Mahowald, 1979) suggests that this region contains the structural genes coding for one or more chorion proteins, including c38 and/or ~36. Johnson and sing (1974) have described a putative chorion mutant, ocelliless, that maps genetically in this region. In a second accompanying paper (Spradling, Waring and Mahowald, 1979), we show that both c36 and c38 map
in this region (7ElO-8A4) and that the ocelliless mutation reduces the production of these two major chorion proteins. Experimental
Procedures
Isolation of Egg Chambers and in Vlvo Labeling Drosophila melanogaster (Oregon R, P2 strain) were collected from a stock grown in mass culture as described previously (Allis. Waring and Mahowald, 1977). Egg chambers of the appropriate stage were isolated from the ovaries by hand dissection as described by Spradling and Mahowald (1979). To label with radioactive amino acids, flies were injected with isotope, prior to dissection, by the methods of Spradling and Mahowald (1979). Briefly, lo-20 &I of a “C amino acid mixture (100 &i/ml; New England Nuclear) were taken to dryness and resuspended in water at 0.5-I .O &I/$. The pH of the resuspended amino acids was monitored with pH paper prior to injection to insure that HCI from the supplier had been removed. Approximately 0.3-0.4 ~1 of the resuspended isotope were injected into the abdomen of each female fly. lsolatlon of Chorion Fragments Approximately 500 early stage 14 egg chambers isolated by hand dissection from adult ovaries were resuspended 1 ml of Tricine buffer [50 mM Tricine, 75 mM KCI, 25 mM sucrose, 0.2 mM MgCI, (pti 7.3)] containing 5% p-mercaptoethanol and 1% Triton X-100 (CalBiochem). The resuspended egg chambers, in plastic Eppendorf tubes, were disrupted by sonication for 2 min at 4°C using a Branson sonicator operated at maximum output. Pieces of the chorion were pelleted by centrifugation at 40 x g for 5 min. The pellet was resuspended in the sonication buffer by vortexing and repelleted by centrifugation as described above. The entire cycle was then repeated. Chorion fragments were always prepared from stage 14 egg chambers because the mechanical stability of the developing chorion (stages 12 and 13) was not sufficient to withstand our isolation procedures. Ultrastructural Analysis Chorion fragments pelleted in IO x 75 mm Pyrex tubes were fixed in 2% glutaraldehyde for 1 hr at 4°C and post-fixed in 1% osmium tetroxide for 2 hr. After dehydration, pellets were embedded in the test tube in DER 732-322 plastic. Glass was removed from the hardened plastic by immersing the test tube in liquid nitrogen. Thin sections were double-stained with uranyl acetate and lead citrate, and examined with a Phillips 300 electron microscope. Chorion fragments were prepared for scanning electron microscopy by the methods of Turner and Mahowald (1976). Samples were examined with an ETEC Autoscan scanning electron microscope. Egg chambers were fixed by the method of Kalt and Tandler (1971). post-fixed with 0~0, and prepared for transmission electron microscopy (Mahowald. 1972). Chorion and Egg Chamber Solubillxation Chorion fragments from 500-700 egg chambers were dissolved in 100 ~1 of buffer [2 mM Tris. 1 mM MgCI, (pH 7.4)] containing 2% SDS and 5% p-mercaptoethanol. The samples were boiled for 5 min to facilitate solubilization. Approximately 100 egg chambers were suspended in 50 ~1 of sonication buffer [2 mM Tris. 1 mM MgCI, (pH 7.4)] containing RNAase (Worthington) at 100 pg/ml. The resuspended preparations were sonicated for 2 min as previously described. After 45 min of incubation at O’C, SDS and P-mercaptoethanol were added to 2 and 5%. respectively. The samples were then boiled for 5 min. Stage 12 and 13 egg chambers dissolved in toto under these conditions, whereas a small residue usually remained with stage 14 egg chambers. Chorions were isolated from early stage 14 egg chambers since the proportion of stage 14eggs chambers with insoluble chorions increased with developmental advancement. 10% of the early stage 14 egg chambers remained either
Cel I 606
intact or as empty chorion shells under these solubilizing conditions, whereas 40+X1% of the more developmentally advanced chambers remained intact. SDS-Polyacrylamide Gel Electrophoresis Slab gels (0.75 mm thick) containing gradients of acrylamide were poured between 17 x 14 cm plates. The gels were composed of a resolving gel (9 cm high) and a stacking gel (1.5 cm high). The resolving gel, similar to that of Laemmli (1970), contained a 7.5 17% linear gradient of acrylamide. The 4% stacking gel was similar to that of Laemmli but contained 15% glycerol. Aqueous samples were diluted with 4 vol of Laemmli sample buffer and electrophoresed in Laemmli electrode buffer for 4-5 hr at constant voltage (90 V). Gels were fixed for at least 1 hr in 10% TCA, stained with 0.1% Coomassie blue in 50% methanol and 7.5% acetic acid, and destained in a solution containing 5% methanol and 7.5% acetic acid. Two-Dimensional Gel Electrophoresis First dimension nonequilibrium pH gradient electrophoresis gels (NEPHGE) (O’Farrell, Goodman and O’Farrell, 1977) were prepared as previously described (Waring, Allis and Mahowald. 1978). Prior to electrophoresis, ampholines and solid urea were added to SDS-solubilized samples (described above) at final concentrations of 1.5% and 8.6 M. respectively. NP40 (Particle Data Laboratories) was then added such that the ratio of NP40: SDS in the final preparation was 1O:l. Samples (100 ~1) were loaded at the anode onto 11 x 0.24 cm cylindrical gels and overlaid with 20 ~1 of 7.5 M urea containing 5% NP40 and 0.75% ampholines of the desired pH range (3.5-10. 4-6, 5-8, 7-9, mixed in equal parts). Using 0.01 M NaOH and 0.01 M H,PO, as electrode buffers, electrophoresis was carried out for 4.5 hr at 500 V. First dimension gels were equilibrated for 2 hr in buffer [62 mM Tris-HCI, 2.3% SDS, 5% P-mercaptoethanol (pH 6.8)] and sealed to the second dimension slab gel (prepared as described above) with 1% agarose (Biorad) in 125 mM Tris-HCI (pH 6.8) as previously described (Waring et al., 1978). Electrophoresis was carried out at constant voltage for 700 V hr. Gels were fixed and stained as described above. Fluorography Slab gels containing proteins with radioactive amino acids were subjected to fluorography as described by Bonner and Laskey (1974). The RP-Royal film (Kodak) was preexposed following the method of Laskey and Mills (1975) and then exposed for the appropriate length of time at -70°C.
We would like to thank Dr. F. R. Turner for preparing the scanning electron micrograph; Joan Caulton for help in preparing the transmission electron micrographs: and Dr. Allan Spradling for assistance in dissection and many valuable discussions. This work was supported by grants from the NSF and the NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. September
Bonner, W. M. and Laskey, R. A. (1974). for tritium-labeled proteins and nucleic gels. Eur. J. Biochem. 46, 83-88.
A film detection method acids in polyacrylamide
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Acknowledgments
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Chorion 607
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Spradling. A. C., Waring, G. L. and Mahowald. A. P. (1979). Drosophila bearing the ocelliless mutation underproduce two major chorion proteins both of which map near this gene. Cell 76, 609-616. Turner, F. R. and Mahowald, A. P. (1976). Scanning electron microscopy of Drosophila embryogenesis: I. The structure of the egg envelopes and the formation of the cellular blastoderm. Dev. Siol. 50, 95-108. Waring, G. L., Allis, C. D. and Mahowald, A. P. (1978). Isolation polar granules and the identification of polar granule-specific protein. Dev. Biol. 66, 197-206.
of
Warren, T. and Mahowald, A. P. (1979). Isolation and partial chemical characterization of the three major yolk polypeptides from Drosophila melanogaster. Dev. Biol. 68, 130-139.
Vol. 16.599-607.
March
1979,
Copyright
0 1979 by MIT
Identification and Time of Synthesis Proteins in Drosophila melanogaster
correlation between the abundance, sizes and times of synthesis of the chorion proteins and the putative chorion mRNAs was observed.
Gail L. Waring* and Anthony P. Mahowald Program in Molecular, Cellular and Developmental Biology Department of Biology Indiana University Bloomington, Indiana 47401
Results
Summary The chorion of Drosophila melanogaster consists of proteins secreted by the follicular epithelium during late oogenesis. Petri, Wyman and Kafatos (1976) have described six major protein components of the Drosophila chorion and reported the synthesis of these proteins in vitro by mass-isolated egg chambers. We have used two-dimensional gel electrophoresis to identify approximately twenty components in highly purified chorion preparations. The synthesis patterns of these proteins in vivo were determined by isolating egg chambers of different developmental stages from proflies injected with 14C amino acids. Chorion teins constitute a large fraction of the protein synthesized by ovarian egg chambers in stages 12-14. The sizes and times of synthesis of the chorion proteins correlate closely with the production of poly(A)-containing RNAs by the follicle cells (Spradling and Mahowald, 1979). Introduction During development, functionally related structural genes are expressed in a coordinate fashion. The genes coding for the chorion proteins in Drosophila are an example of such a gene set that is particularly amenable to biochemical analysis (Spradling and Mahowald, 1979). Furthermore, numerous putative chorion mutants have been described (King and Mohler, 1975) which may provide information on mechanisms which regulate the activity of these genes. Understanding the role of these genes in the production of the chorion will require detailed knowledge of the chorion proteins themselves. Petri et al. (1976) identified six major Drosophila chorion proteins and studied their synthesis in vitro in mass-isolated egg chambers. We have studied the synthesis in vivo of approximately twenty chorion proteins, including the six previously described. In our study, all the chorion proteins were synthesized during stages 11-14, and at the peak of production (stages 12-13), they were the major products synthesized by the egg chambers. A close * Present address: Department Milwaukee, Wisconsin 53233.
of Biology,
Marquette
of Chorion
University,
Isolation of Soluble Chorion Fragments The egg shell of Drosophila consists of three major layers: the vitelline membrane, the endochorion and the exochorion (Figure 1 B). The vitelline membrane opposes the oocyte surface, while the exochorion is adjacent to the follicle cell layer. Between the vitelline membrane and the exochorion is the endochorion, made up of a thin floor, pillars and a thick roof. A thin electron-dense layer lies between the vitelline membrane and the floor of the endochorion. A procedure was developed for the extensive purification of these structures. Chorions were typically obtained from 500 early stage 14 egg chambers isolated by hand dissection from the ovaries of 25-30 rapidly laying flies. Fragments of the chorion were pelleted by low speed centrifugation following disruption of the egg chambers by sonication. Detergent (Triton X-100) was included in the sonication buffer to prevent yolk and other cytoplasmic proteins from pelleting with the chorion fragments. Morphological analysis of chorions isolated by these procedures showed that the preparations consisted of exochorion and the entire endochorion (Figures 1A and 1C). Scanning electron microscopy of the isolated chorion preparations (Figure 1A) showed pieces of fragmented chorions. The outer surface of the fragments resembles the outer surface of the endochorion in situ (Margaritis, Petri and Kafatos, 1976). The ridges, built up by an external reticulum that projects from the roof of the endochorion (Figure lB), mark the borders between adjacent follicle cells. The inner surface of the fragments resembles the floor of the endochorion. The majority of the exochorion separates from the endochorion during the isolation procedure and appears to pellet in mass as a separate entity (Figure 1C). There is no evidence of either vitelline membrane or the thin electron-dense layer in the preparation. Light microscope analysis indicated that the chorion preparations were morphologically pure. Another indication of the cleanliness of this fraction is the lack of contamination by the major nonchorion proteins. The majority of egg chamber protein consists of three polypeptides that comprise the yolk protein (Warren and Mahowald, 1979) (Figure 2A). No yolk protein is detected following solubilization of microscopically pure chorion fragments (Figure ZB).
Cell 600
Figure
1. Scanning
Electron
Micrograph
and Transmission
Elect1 ron Micrographs
of Chorions
of chorions isolated from early stage 14 oocytes (900x). (A) Scanning electron micrograph showing inner (I) and outer (0) surfaces (13,000~). (VM) vitelline membrane: (EN) endochorion; (8) Transmission electron micrograph of eggshell layers in stage 14 oocytes and endochoric exochorion; (FC) follicle cell. The arrow indicates a thin electron- dense layer that lies between the vitelline membrane and remnants of the particulate exochc (C) Transmission electron micrograph of isolated chorions sb lowing the endochorion (7600x). (25,000x). The insert shows a field of the particulate exochorion iat lower magnification
Identification of Chorion Proteins Eight to ten proteins are detected by staining acrylamide gels of solubilized chorion preparations with Coomassie blue (Figure 2B). To determine whether additional chorion components could be identified, chorions were analyzed following labeling in vivo with radioactive amino acids. The chorion is formed during stages 11-14 of oogenesis (King and Koch, 1963), a period encompassing about 5’hr (David and Merle, 1968). To maximize the number of radioactive species in our preparations, chorions near completion were isolated from stage 14 egg chambers 8-10 hr after the flies were injected with radioisotope. Under the conditions of these experiments, stage 14 egg chambers accumulated in the ovaries during the incubation period. Thus stable radioactive species synthesized in stage 1 l-l 3 as well as stage 14 egg chambers were recovered in the chorion fragment preparation. In one-dimensional SDS-polyacrylamide gels, the four major species migrating as 38,000, 36,000, 19,000 and 18,000 dalton bands are apparent, as well as numerous minor species covering the mo-
(EN m. lrion
lecular weight range 15,000-l 50,000 daltons (Figure 38). In a two-dimensional gel electrophoretogram of proteins from a similar chorion fragment preparation, approximately twenty species are resolved (Figure 4). Since this pattern has been repeated in six independent experiments, all of these species are considered putative chorion proteins. To facilitate discussion, the chorion proteins have been assigned numbers. The numerical designation is based on the position of the protein within the two-dimensional gel electrophoretogram. The first number indicates the approximate molecular weight, while the second number indicates relative migration from the acidic to basic end in the first dimension NEPHGE gel. For this purpose, the major protein of 36,000 daltons is arbitrarily given a value of 50. For example, the four major radioactive species in Figure 4 that correspond to the four major stained species in Figure 28 are designated ~38-60, ~36-50, c19-63 and ~18-68. Where no ambiguity would result, the second half of the designation could be omitted.
Chorion 601
Proteins
of
Drosophila
a
b
130K92 I(68 K43 K-
29 K-
16 KFigure Figure 2. Two-Dimensional from Stage 14 Egg Chambers
Gel Electrophoretogram and Isolated Chorion
of Proteins Fragments
Proteins extracted from 100 stage 14 egg chambers (a) and chorion fragments from 250 early stage 14 egg chambers (b) were analyzed by two-dimensional gel electrophoresis. Gels were stained with Coomassie blue. The arrow indicates yolk protein.
Time of Synthesis of the Chorion Proteins Although the putative chorion proteins are greatly enriched in chorion fragment preparations compared with other egg chamber proteins, an additional test of their identity would be to determine when they are synthesized during oogenesis. Because chorion fragments from stage 11-13 egg chambers cannot be isolated, it is not possible to determine the radiolabeled proteins that are present in the chorion at each developmental stage. Thus to determine the developmental period when specific chorion proteins are synthesized, chorion proteins must be identified against a background of nurse cell and oocyte proteins in pulse-labeled egg chambers. We have used the resolving power of two-dimensional gel electrophoresis to evaluate the time of synthesis of most of the chorion proteins. Flies were injected with a mixture of 14C amino acids, and individual egg chambers, stages 10-14, were isolated by hand dissection 1 hr later. Approximately 25-75 labeled egg chambers from each stage were dissolved in toto, and two-dimensional gel electrophoretograms were prepared (Figure 5). Different exposure times were used to compensate
3. Autoradiogram
of Long-Term
Labeled
Chorion
Proteins
Early stage 14 egg chambers were isolated from flies injected with r4C amino acids 9 hr prior to dissection. Proteins extracted from chorion fragments isolated from 50 egg chambers were electrophoresed on one-dimensional SDS-polyacrylamide gels (b). Molecular weight markers run on the same gel were stained with Coomassie blue (a). Proteins used to calibrate the gel included kgalactosidase (130,000); phosphorylase a (92,000); bovine serum albumin (68,000); ovalbumin (43,000); carbonic anhydrase (29,000); and human hemoglobin (16,000).
for the differing amounts of material present. Comparison of the fluorographs in Figure 5 shows that except for the chorion proteins, the patterns of proteins synthesized by stage lo-14 egg chambers are very similar. Figures 5C and 5D also illustrate that the major synthetic activity in stage 12 and 13 egg chambers involves production of the chorion proteins by the follicle cells. It is clear that putative chorion proteins are synthesized sequentially during the period of chorion deposition (stages 1214). Three major chorion proteins are synthesized in stage 12, most are synthesized during stage 13 and at least one (~15-55) is synthesized during stage 14 only. Figure 6 summarizes the times of synthesis of the chorion proteins. Labeling studies in which flies were injected with 35S-methionine failed to reveal synthesis of the major chorion proteins (~36, ~38, cl8 and c19), although labeling of nonchorion proteins was similar to that in Figure 5. Thus the major chorion proteins in Drosophila appear to be methioninepoor.
Cell 602
80 60 40
20
s I
I
I
10
I
I
30
Figure
4. Two-Dimensional with
Autoradiogram chorion
fragments
of Long-Term isolated
from
Distance Labeled
Chorion
200 %-labeled
Discussion The results in this paper demonstrate that the chorion proteins are major products synthesized in stage 12 and 13 egg chambers. The major labeled species in stage 10 egg chambers are the three polypeptides that constitute yolk protein (Warren and Mahowald, 1979). These yolk polypeptides are sequestered from the hemolymph by the oocyte during the vitellogenic phase of oogenesis (stages 8-10) (King, 1970). The presence of only trace amounts of the yolk polypeptides in stage 11 egg chambers indicates that stage overlap was not a major problem in these experiments. The synthetic profile of stage 12 egg chambers shows that the major chorion proteins c36 and c38 are the predominant products. In addition, c60 synthesis is prominent. Most of the chorion proteins are synthesized in stage 13 egg chambers and, as in stage 12, represent the major synthetic products. Stage 14 egg chambers gave a lower level of labeling of both general proteins and chorion proteins, although synthesis of cl 8 and ~15-55 appears to be maximal during this stage. There is no evidence of residual synthesis of ~36, ~38, c55 or c60 in the stage 14 egg chambers. This demonstrates that rapid and dramatic changes occur in the synthetic
I
I
I
70
50
Relative Proteins extracted electrophoresis.
I
of
Migration
Proteins stage
14 egg
chambers
were
analyzed
by two-dimensional
activities of the follicle cells during choriogenesis. In this study, we have used two-dimensional gel electrophoresis to identify approximately twenty chorion proteins. Proteins present in purified chorion preparations that are synthesized only during the period of chorion deposition are considered chorion proteins. Sixteen species in the chorion fragment preparation shown in Figure 4 were synthesized in stage 12-l 4 egg chambers. The time of synthesis of the more minor species could not be determined with certainty because of the difficulties encountered in detecting these species in extracts from whole egg chambers. It is improbable that these minor components are contaminants since we failed to detect any of the major yolk polypeptides in the purified chorion preparation. It is difficult to exclude the possibility that a sonication-resistant, detergent-insoluble cellular structure co-pellets with the chorion fragments at 40 x g, although we have not found such structures in electron microscopic analyses of the fractions. Several considerations indicate that the number of chorion proteins which we have resolved by twodimensional gel electrophoresis represent most of the major proteins in the Drosophila chorion. Morphological analysis of the fragment preparations show that the two major structural components of
Chorion 603
Proteins
of Drosophila
NEPHGE
Figure
5. Time of Synthesis
-
of the Chorion
Proteins
“C-labeled egg chambers at different developmental stages were isolated from flies which had been pulse-labeled for 1 hr. Proteins extracted from egg chambers from each stage (10-14) were analyzed by two-dimensional gel electrophoresis. The arrows on the autoradiogram indicate some of the major chorion proteins during the stage in which their synthesis appears maximal. (a) 92 stage 10 egg chambers; (b) 23 stage 11; (c) 20 stage 12; (d) 59 stage 13; (e) 93 stage 14. The definitions of the stages during oogenesis are those described by Illmensee, Mahowald and Loomis (1976). The exposure times for each developmental stage were normalized such that the product of number of egg chambers/stage and the length of exposure were constant. The positions of the major chorion proteins were located by stain. The minor chorion proteins were identified by superimposing fluorographs of chorion fragment preparations run on parallel gels.
Cell 604
Developmental 11
12
Stage
13
14
~80-20 c70-45 ~60.50 c55-40 ~48-45 c45-55 c40-35 ~38-60
,
~36.50
---
_-_-
- ---_
-_
~28.58 c19-63 c18-68 -_--.
~16.30
. _-
--_-
~16-35 c15-50 c15-55
(
,
,
,
,
,
0
1
2
3
4
5
Hours Figure
6. Time
of Synthesis
at 25 “C
of the Chorion
The period of maximal synthesis detectable synthesis is indicated
Proteins
is indicated by the dashed
by the line.
solid
line;
the chorion (endochorion and exochorion) are present. All of the proteins which we have solubilized from these structures were synthesized by egg chambers in stages 12-14. If chorion proteins were lost during the isolation procedures, we would expect to see proteins synthesized between stages 12 and 14that are not present in our chorion preparation. Inspection of the synthetic profiles of stage 12-14 egg chambers (Figure 5) indicates that only one detectable protein synthesized specifically during this developmental period was not recovered in the isolated chorion. This stage 12 protein (approximately 40,000 daltons) could be a component of the electron-dense layer which forms during stage 11 and early stage 12, and is not retained in our chorion fraction. Some chorion proteins may not have been detected because they became insoluble immediately following synthesis. Solubility studies (Petri, Wyman and Henikoff, 1977; G. L. Waring and A. P. Mahowald, unpublished observations) indicated that whereas stage 14 egg chambers became less soluble as they became more developmentally advanced, stage 12 and 13 egg chambers dissolved completely. Thus major chorion proteins synthesized in stage 12 and 13egg chambers are probably
soluble in purified chorions. However, any chorion proteins synthesized in stage 14 egg chambers which were rendered insoluble immediately by cross-linking might not have been detected. In addition, the existence of chorion proteins in amounts too low to be detected in Figure 4 cannot be excluded. The purification of six major chorion proteins and their synthesis in vitro by mass-isolated egg chambers from Drosophila has been reported previously (Petri et al., 1976). The six major proteins described by Petri and colleagues correspond closely in molecular weight to those of Figure 3. Although they observed a relative synthesis order for these six major chorion proteins generally similar to that of Figure 5, we found a number of striking differences in the labeling patterns of the egg chambers in vivo compared with the patterns in vitro. Using a mixture of labeled amino acids, it is clear from our studies that the major chorion proteins ~36, ~38, cl 8 and cl9 are the predominant products synthesized in choriogenic egg chambers. This prodigious synthesis was not evident in the study by Petri and co-workers, even though they used specific 3H amino acids which would be expected to enhance labeling of the chorion proteins. Furthermore, protein synthesis in their stage 10 egg chambers was significantly depressed relative to the stage 11-14 egg chambers, whereas we found synthesis of the nonchorion proteins comparable, if not greater, in stage 10 chambers relative to the more developmentally advanced stages. These differences in labeling patterns may reflect differences in the health of egg chambers labeled in vitro after mass isolation versus chambers labeled in vivo. A further complication of in vitro labeling of isolated egg chambers is the possibility of the induction of the “heat shock” response (Petri et al., 1977; Spradling and Mahowald, 1979). It is interesting to compare our results with the chorion proteins in Drosophila with the extensive knowledge of chorion proteins in silkmoths (reviewed by Kafatos et al., 1977). The Drosophila chorion may be less complex in terms of protein multiplicity. We have resolved approximately twenty proteins, whereas Regier et al. (1978) estimate approximately 100 chorion proteins in the silkmoth. The chorion proteins in Drosophila have a much wider distribution on SDS gels than the silkmoth proteins. Drosophila chorion proteins range in size from 15,000 to 150,000 daltons, while silkmoth proteins range from 7000 to 30,000 daltons. The distribution of the chorion proteins in the first dimension NEPHGE gels in this study indicates that the Drosophila proteins have a wider distribution of isoelectric points and are generally more basic than the silkmoth proteins. The isoelectric points of 80% of the silkmoth chorion proteins fall
Chorion 605
Proteins
of Drosophila
within the pH range of 4-6. It is interesting, however, that the small Drosophila chorion proteins synthesized during stage 14 appear to resemble the major silkmoth proteins much more closely than those produced earlier. The chorion proteins are secretory products of the follicle cells. In vitro translation of mRNAs coding for a variety of exportable proteins yield products slightly larger than the secreted product (Deveillers-Thiery et al., 1975; Burstein, Kanto and Schechter, 1976; Blobel, 1977). Current hypotheses concerning the segregation of exportable proteins into the cisternal space of the rough endoplasmic reticulum envision the synthesis of many secretory proteins via larger precursors (Blobel and Dobberstein, 1975). Analysis of the products synthesized and accumulated in stage 12-14 egg chambers indicates that c36 may be formed via a larger precursor. A protein exhibiting a slightly higher molecular weight and slightly more acidic charge appears to be synthesized coordinately with c36 in stage 11, 12 and 13 egg chambers (Figure 5). Although this protein is prominent in the synthetic profile of stage 12 and 13 egg chambers, it is barely detectable on Coomassie blue-stained gels (data not shown). Furthermore, wherease c36 is prominent in stained gels of stage 14 egg chamber extracts, the slightly larger protein is not detectable at this stage (Figure 2A). These properties are consistent with the notion of a c36 precursor protein. In the case of c19, a similar putative precursor was detected, but other possible chorion precursors were not observed. In the accompanying paper, Spradling and Mahowald (1979) describe the synthesis of a small number of poly(A)-containing RNAs during choriogenesis. The abundance, sizes and times of synthesis of these putative mRNAs correlate closely with the abundance, sizes and times of synthesis of the major chorion proteins. In particular, the abundance and stage specificity of E3 correspond closely with the abundance and stage specificity of c36 and ~38, suggesting that this RNA band contains mRNAs for one or both of these two chorion proteins. Similarly, the sizes and stage specificities of the smaller RNAs (E5-E6) correlate with the smaller chorion proteins including ~16-30 and c1555. In situ hybridization of the E3 and E4 mRNAs at one site (7Ell) on the Drosophila polytene chromosomes (Spradling and Mahowald, 1979) suggests that this region contains the structural genes coding for one or more chorion proteins, including c38 and/or ~36. Johnson and sing (1974) have described a putative chorion mutant, ocelliless, that maps genetically in this region. In a second accompanying paper (Spradling, Waring and Mahowald, 1979), we show that both c36 and c38 map
in this region (7ElO-8A4) and that the ocelliless mutation reduces the production of these two major chorion proteins. Experimental
Procedures
Isolation of Egg Chambers and in Vlvo Labeling Drosophila melanogaster (Oregon R, P2 strain) were collected from a stock grown in mass culture as described previously (Allis. Waring and Mahowald, 1977). Egg chambers of the appropriate stage were isolated from the ovaries by hand dissection as described by Spradling and Mahowald (1979). To label with radioactive amino acids, flies were injected with isotope, prior to dissection, by the methods of Spradling and Mahowald (1979). Briefly, lo-20 &I of a “C amino acid mixture (100 &i/ml; New England Nuclear) were taken to dryness and resuspended in water at 0.5-I .O &I/$. The pH of the resuspended amino acids was monitored with pH paper prior to injection to insure that HCI from the supplier had been removed. Approximately 0.3-0.4 ~1 of the resuspended isotope were injected into the abdomen of each female fly. lsolatlon of Chorion Fragments Approximately 500 early stage 14 egg chambers isolated by hand dissection from adult ovaries were resuspended 1 ml of Tricine buffer [50 mM Tricine, 75 mM KCI, 25 mM sucrose, 0.2 mM MgCI, (pti 7.3)] containing 5% p-mercaptoethanol and 1% Triton X-100 (CalBiochem). The resuspended egg chambers, in plastic Eppendorf tubes, were disrupted by sonication for 2 min at 4°C using a Branson sonicator operated at maximum output. Pieces of the chorion were pelleted by centrifugation at 40 x g for 5 min. The pellet was resuspended in the sonication buffer by vortexing and repelleted by centrifugation as described above. The entire cycle was then repeated. Chorion fragments were always prepared from stage 14 egg chambers because the mechanical stability of the developing chorion (stages 12 and 13) was not sufficient to withstand our isolation procedures. Ultrastructural Analysis Chorion fragments pelleted in IO x 75 mm Pyrex tubes were fixed in 2% glutaraldehyde for 1 hr at 4°C and post-fixed in 1% osmium tetroxide for 2 hr. After dehydration, pellets were embedded in the test tube in DER 732-322 plastic. Glass was removed from the hardened plastic by immersing the test tube in liquid nitrogen. Thin sections were double-stained with uranyl acetate and lead citrate, and examined with a Phillips 300 electron microscope. Chorion fragments were prepared for scanning electron microscopy by the methods of Turner and Mahowald (1976). Samples were examined with an ETEC Autoscan scanning electron microscope. Egg chambers were fixed by the method of Kalt and Tandler (1971). post-fixed with 0~0, and prepared for transmission electron microscopy (Mahowald. 1972). Chorion and Egg Chamber Solubillxation Chorion fragments from 500-700 egg chambers were dissolved in 100 ~1 of buffer [2 mM Tris. 1 mM MgCI, (pH 7.4)] containing 2% SDS and 5% p-mercaptoethanol. The samples were boiled for 5 min to facilitate solubilization. Approximately 100 egg chambers were suspended in 50 ~1 of sonication buffer [2 mM Tris. 1 mM MgCI, (pH 7.4)] containing RNAase (Worthington) at 100 pg/ml. The resuspended preparations were sonicated for 2 min as previously described. After 45 min of incubation at O’C, SDS and P-mercaptoethanol were added to 2 and 5%. respectively. The samples were then boiled for 5 min. Stage 12 and 13 egg chambers dissolved in toto under these conditions, whereas a small residue usually remained with stage 14 egg chambers. Chorions were isolated from early stage 14 egg chambers since the proportion of stage 14eggs chambers with insoluble chorions increased with developmental advancement. 10% of the early stage 14 egg chambers remained either
Cel I 606
intact or as empty chorion shells under these solubilizing conditions, whereas 40+X1% of the more developmentally advanced chambers remained intact. SDS-Polyacrylamide Gel Electrophoresis Slab gels (0.75 mm thick) containing gradients of acrylamide were poured between 17 x 14 cm plates. The gels were composed of a resolving gel (9 cm high) and a stacking gel (1.5 cm high). The resolving gel, similar to that of Laemmli (1970), contained a 7.5 17% linear gradient of acrylamide. The 4% stacking gel was similar to that of Laemmli but contained 15% glycerol. Aqueous samples were diluted with 4 vol of Laemmli sample buffer and electrophoresed in Laemmli electrode buffer for 4-5 hr at constant voltage (90 V). Gels were fixed for at least 1 hr in 10% TCA, stained with 0.1% Coomassie blue in 50% methanol and 7.5% acetic acid, and destained in a solution containing 5% methanol and 7.5% acetic acid. Two-Dimensional Gel Electrophoresis First dimension nonequilibrium pH gradient electrophoresis gels (NEPHGE) (O’Farrell, Goodman and O’Farrell, 1977) were prepared as previously described (Waring, Allis and Mahowald. 1978). Prior to electrophoresis, ampholines and solid urea were added to SDS-solubilized samples (described above) at final concentrations of 1.5% and 8.6 M. respectively. NP40 (Particle Data Laboratories) was then added such that the ratio of NP40: SDS in the final preparation was 1O:l. Samples (100 ~1) were loaded at the anode onto 11 x 0.24 cm cylindrical gels and overlaid with 20 ~1 of 7.5 M urea containing 5% NP40 and 0.75% ampholines of the desired pH range (3.5-10. 4-6, 5-8, 7-9, mixed in equal parts). Using 0.01 M NaOH and 0.01 M H,PO, as electrode buffers, electrophoresis was carried out for 4.5 hr at 500 V. First dimension gels were equilibrated for 2 hr in buffer [62 mM Tris-HCI, 2.3% SDS, 5% P-mercaptoethanol (pH 6.8)] and sealed to the second dimension slab gel (prepared as described above) with 1% agarose (Biorad) in 125 mM Tris-HCI (pH 6.8) as previously described (Waring et al., 1978). Electrophoresis was carried out at constant voltage for 700 V hr. Gels were fixed and stained as described above. Fluorography Slab gels containing proteins with radioactive amino acids were subjected to fluorography as described by Bonner and Laskey (1974). The RP-Royal film (Kodak) was preexposed following the method of Laskey and Mills (1975) and then exposed for the appropriate length of time at -70°C.
We would like to thank Dr. F. R. Turner for preparing the scanning electron micrograph; Joan Caulton for help in preparing the transmission electron micrographs: and Dr. Allan Spradling for assistance in dissection and many valuable discussions. This work was supported by grants from the NSF and the NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. September
Bonner, W. M. and Laskey, R. A. (1974). for tritium-labeled proteins and nucleic gels. Eur. J. Biochem. 46, 83-88.
A film detection method acids in polyacrylamide
Burstein, Y.. Kanto. F. and Schechter, I. (1976). Partial amino acid sequence of the precursor of an immunoglobulin light chain containing NH-terminal pyroglutamic acid. Proc. Nat. Acad. Sci. USA 73, 2604-2608. David, J. and Merle, J. (1968). A re-evaluation of the duration of egg chamber stages in oogenesis of Drosophila melanogaster. Drosophila Information Service 43, 122-123. Deveillers-Thiery, A., Kindt, Homology in amino-terminal creatic secretory proteins. 5020. Illmensee. K., Mahowald. ontogeny of germ plasm Biol. 49, 40-65.
T.. Scheele, G. and Blobel. G. (1975). sequence of precursors to panProc. Nat. Acad. Sci. USA 72, 5016A. P. and Loomis, M. R. (1976). during oogenesis in Drosophila.
The Dev.
Johnson, C. C. and King, R. C. (1974). Oogenesis in the ocelliless mutant of Drosophila melanogaster Meigen (Diptera: Drosophilidae). Int. J. Insect Morphol. Embryol. 3, 385-395. Kafatos, F. C., Regier, J., Mazur, G., Nadel. M., Blau. H., Petri, W. H., Wyman, A. R., Gelinas, R., Moore, P., Paul, M.. Efstratiadis, A., Vournakis, J., Goldsmith, M.. Hunsley, J., Baker, B. and Nardi, J. (1977). The eggshell of insects: differentiationspecific proteins and the control of their synthesis and accumulation during development. In Results and Problems in Cell Differentiation, 8. W. Beerman, ed. (Berlin: Springer-Verlag), pp. 45-143. Kalt. M. R. and Tandler. amphibian embryos for Res. 36, 633-645.
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King, R. C. and Koch, E. A. (1963). Studies on the ovarian cells of Drosophila. Quart. J. Microbial. Sci. 104, 297-320.
follicle
King, R. C. and Mohler, D. J. (1975). The genetic analysis of oogenesis in Drosophila melanogaster. In Handbook of Genetics, 3, R. C. King, ed. (New York: Plenum Publishing), pp. 757-791. Laemmli. assembly
W. K. (1970). Cleavage of structural proteins during the of the head of bacteriophage T,. Nature 227, 680-685.
Laskey, R. A. and Mills, A. D. (1975). Quantitative film detection of JH and “C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341.
Acknowledgments
Received
membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. Cell Biol. 67, 835-851.
5.1978;
revised
December
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Chorion 607
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Warren, T. and Mahowald, A. P. (1979). Isolation and partial chemical characterization of the three major yolk polypeptides from Drosophila melanogaster. Dev. Biol. 68, 130-139.