Cell-free translation of silkmoth chorion mRNAs: Identification of protein precursors and characterization of cloned DNAs by hybrid-selected translation

DEVELOPMENTAL

BIOLOGY

78,36-46

(1980)

Cell-Free Translation of Silkmoth Chorion mRNAs: Protein Precursors and Characterization of Cloned Selected Translation GEORGE Cellular

and Developmental

Received

THIREOS Biology, Cambridge,

November

AND

FOTIS

C.

KAFATOS

Biological Laboratories, Massachusetts 02138

13, 1979; accepted

in revised

form

Identification of DNAs by Hybrid-

Harvard

December

University,

27, 1979

The secretory silkmoth chorion proteins are synthesized as precursors bearing signal peptides. Precursors are detected upon cell-free translation of chorion mRNAs in the wheat germ system; they are processed into products identical in size to authentic chorion proteins when translation is performed in the presence of microsomal membranes from dog pancreas. Precursors corresponding to specific protein size classes and subclasses are identified by three approaches: comparison of precursors and products encoded by stage-specific mRNAs, comparison of precursors and products encoded by mRNAs specifically hybridizing to individual chorion cDNA clones, and comparison of relative amino acid compositions of precursors and authentic chorion proteins. Translation of stage-specific mRNA preparations indicates that, in general, the developmental changes of in vivo chorion protein synthesis are based on changes in concentrations of the corresponding mRNAs. Characterization of the precursors makes it possible to identify, for any chorion DNA clone, the protein subclass, a member of which is encoded by the clone sequence.

This RNA mixture was translated in the wheat germ system, and yielded products immunoprecipitable with chorion-specific antiserum and (under conditions of low electrophoretic resolution) showing a size distribution comparable, although not identical, to authentic chorion proteins (Kafatos et al., 1977; Efstratiadis and Kafatos, 1976). Although the available evidence indicated that this mixture contains chorion mRNAs, the large number of related chorion proteins precluded the physical purification and characterization of individual mRNAs. To pursue analysis of the chorion system at the nucleic acid level, we have cloned a mixture of double stranded cDNAs (Sim et al., 1979). Individual clones of this “cDNA library” correspond to individual mRNA species. The types of proteins encoded by several clones have been determined by DNA sequencing (Jones et al., 1979) and comparison to known chorion protein sequences (Regier et al., 1978a; Rodakis, 1978).

INTRODUCTION

The silkmoth chorion is a favorable system for studying specific gene expression during cell differentiation (Kafatos et al., 1977). Moreover, since chorion proteins are encoded by multigene families (Regier et al., 1978a; Rodakis, 1978; Jones et al., 1979), the system is also interesting from the perspective of molecular evolution. In earlier work, we demonstrated that the many electrophoretically separable chorion proteins of Antheraea polyphemus are synthesized sequentially during a 52-hr period of choriogenesis, according to a strict developmental program (Paul et al., 1972; Paul and Kafatos, 1975). During choriogenesis, approximately 95% of total protein synthesis in the follicular epithelial cells is devoted to chorion protein synthesis. From this biologically very specialized tissue we identified a class of poly(A)’ RNAs with the size, developmental specificity, and polysomal location expected for chorion mRNAs (Gelinas and Kafatos, 1973, 1977). 36 0012-1606/80/090036-11$02.00/O Copyright 0 1980 by Academic All

rights

of reproduction

in any

Press, form

Inc. reserved.

THIREOS

AND KAFATOS

Because of the extensive knowledge available concerning the developmental kinetics of particular size subclasses of chorion proteins (Paul and Kafatos, 1975; Sim et al., 1979), as well as their location and function (Regier et al., 1980; Mazur et al., 1980), it would be valuable to have a convenient and general method for relating a specific chorion DNA clone to a specific protein size subclass. Theoretically, hybridselected translation should be such a method: hybridization of total chorion mRNA with an individual DNA clone, purification of the hybrids, melting off and translation of the specifically hybridized mRNA that they contain, and electrophoretie analysis of the products. However, a possible complication is that chorion components, like other secretory proteins, might be synthesized as precursors, bearing a signal peptide. Here we demonstrate the existence of these precursors, and show that they can be processed to products identical to authentic chorion proteins in electrophoretie mobility, using a cell-free translation system supplemented with microsomal membranes. Taking advantage of known differences in developmental specificities and amino acid compositions of authentic proteins, we characterize the precursors corresponding to different protein size classesand subclasses.Finally we show that with this knowledge about precursors, we can, in fact, use hybrid-selected translation for clone characterization. Some translation studies reported here indicate that, in general, the protein synthetic program of choriogenesis is based on progressive accumulation and disappearance of specific chorion mRNAs, rather than sequential translational activation of preexisting mRNAs: when the cell-free products of total cytoplasmic RNA from staged follicles are membrane processed, they show electrophoretic patterns essentially identical to those of proteins synthesized in vivo at the corresponding stages. It also appears that hybrid-selected

Silkmoth

Chorion mRNAs

37

translation can reveal the pattern of homologies between chorion proteins, if performed under a hybridization criterion which permits nucleic acid cross-hybridization between closely related members of the chorion multigene families. In the present study we have resolved chorion proteins and their precursors by SDS-polyacrylamide gel electrophoresis. The higher resolution two-dimensional analysis which we have emphasized in other studies (Regier et al., 1980) could not be used, because many chorion proteins are charge-modified in viuo after removal of the signal peptide (Regier, 1975), apparently by cyclization of an exposed N-terminal glutamine (Regier and Kafatos, in preparation), and thus authentic proteins differ in isoelectric focusing from the in vitro processed products; no difference exists in SDS-electrophoresis. This limitation is not very serious for our purposes, since chorion proteins which are very similar in size tend to be very closely related evolutionarily, and to be synthesized in parallel during development (Regier and Kafatos, in preparation). Thus, for present purposes, we have treated each band resolved by SDS-electrophoresis as a unit, although in fact it consists of multiple components. MATERIALS

AND

METHODS

RNA Preparation Poly(A)+ RNA from choriogenic follicles was prepared using the Mg2+ precipitation technique and oligo(dT) cellulose chromatography as described by Efstratiadis and Kafatos (1976). Total cytoplasmic RNA was prepared as follows: choriogenic follicles were briefly homogenized in lysis buffer (25 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 2% Triton X-100), the resulting homogenate was clarified for 5 min at 27,OOOg, made 0.5% in SDS and 10 mM in Na2 EDTA (pH 8.0), and deproteinized by repeated extraction with phenol and Sevag (chloroform:isoamvl \.~~~~~~~~~~ ~~ LI alcohol., 24:l).I

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In Vitro Translations Translation assays used the wheat germ system as described by Efstratiadis and Kafatos (1976). Typical assays contained 20 pg/rnl poly(A)’ RNA or 200 pg/ml unfractionated RNA. Translations in the presence of dog pancreas microsomal membranes (gift of J. Majzoub) and post-translational proteolysis were carried out as described by Shields and Blobel (1978).

Sample Preparations and SDS-Polyacrylamide Electrophoresis Dissolved and 14C-carboxamidomethylated chorions were used as a source of authentic proteins (Efstratiadis and Kafatos, 1976; Regier et al., 1978b). To generate in viuo pulse-labeled, developmentally specific proteins, follicles were incubated with 100 &i/ml [3H]leucine (40 Ci/mmole) in Grace’s medium (Grace, 1962) for 1 hr. The follicles were then washed free of yolk, solubilized as above, and carboxamidomethylated with nonradioactive iodoacetamide. The translation products were also carboxamidomethylated: they were precipitated with 2 vol of ethanol, resuspended in 8 M urea, 0.36 M Tris-HCl, pH 8.4, and 0.05 M DTT and reacted with 0.5 vol of 1.0 M iodoacetamide in 1.2 M Tris, pH 8.4. The reaction was terminated by the addition of two times concentrated sample buffer (Laemmli, 1970) containing 10% v/v /3-mercaptoethanol. To achieve maximum resolution of the chorion polypeptides on analytical SDS-polyacrylamide slab gels, a modification of Laemmli’s procedure (1970) was used as described by Regier et al. (1978b). Fluorography was according to Bonner and Laskey (1974).

Immunoprecipitations An antiserum raised against solubilized and carboxamidomethylated chorion proteins (gift of Dr. J. R. Hunsley) was used for immunoprecipitations. Staphylococcus aureus was used as immunoadsorbent according to Kessler (1976).

VOLUME 78. 1980

Hybrid-Selected mRNAs

Translation

of Chorion

Chorion cDNA clones (Sim et al., 1979) were linearized with the restriction endonuclease XhoI, which cleaves the vector plasmid PML-21 once, outside the inserted DNA. The reaction was terminated by phenol-Sevag extraction and the DNA was ethanol precipitated. The DNA pellet was then resuspended in 99% formamide at a concentration of 2.5 mg/ml and denatured by heating at 100°C for 1 min. Hybridization with follicular poly(A)+ RNA was carried out under conditions that favor a twostep formation of R-loops (Thomas et al., 1976). In the first step, the denatured plasmid DNA was hybridized in a 1O:l sequence excess (2 mg/ml) with chorion mRNA for 2 hr at 50°C in 80% formamide, 0.4 M NaCl, 0.1 M Pipes buffer, pH 6.4, and 10 mM Nap EDTA. These conditions do not allow any DNA renaturation (Casey and Davidson, 1977). The reaction mixture was then diluted with 1 vol of H20 and incubated for 10 min at 50°C. In this second step the conditions allow complete renaturation of the DNA without displacing the hybridized RNA (Hutton, 1977). The reaction mixture was then loaded on an 1.5 x 16 cm agarose A-150 column, developed with 0.8 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM Naz EDTA. Under these conditions, double stranded DNAs (23 kb), including molecules with R-loops, elute in the excluded volume, whereas nonhybridized RNA is in the included volume (Woolford and Rosbash, 1979). The excluded fractions were pooled, ethanol precipitated, resuspended in water, and cell-free translated after heating for 1 min at 100°C. RESULTS

Size Distribution of Chorion Translation Products

Cell-Free

A follicular RNA preparation highly enriched in chorion mRNA sequences can be obtained from choriogenic follicles, by Mg’+ precipitation, phenol-chloroform extrac-

THIREOS

AND KAFATOS

tion, and oligo(dT) cellulose chromatography (Efstratiadis and Kafatos, 1976). This preparation was translated in vitro in the wheat germ system, and the products were analyzed by electrophoresis on an SDSpolyacrylamide slab gel designed to maximize chorion protein resolution (Regier et al., 1978b). Autofluorography revealed a large number of distinct bands that appeared to be in a slightly higher molecular weight range and not to corn&ate with authentic chorion proteins (Fig. 1, lanes 4 and 1, respectively). Like authentic proteins, the components synthesized by the cell-free system were precipitable with specific antiserum directed against total chorion proteins (Fig. 1, lanes 2 and 3, respectively). The higher molecular weight distribution was also observed with translation of total, rather than Mg2+ precipitable, cytoplasmic poly(A)+ RNA (data not shown). As we demonstrate below, specific cell-free translated components can be recognized as precursors of specific chorion bands (protein subclasses), and are accordingly labeled in Fig. 1. In

Vitro Processing Precursors

of Chorion

Protein

Since chorion proteins are secretory, their messages are expected to encode precursors containing an additional aminoterminal “signal” peptide that serves to segragate nascent chains within the cisternae of the endoplasmic reticulum (Blobel and Dobberstein, 1975). Such signal peptides have already been demonstrated for the chorion proteins of Drosophila melanogaster (Thireos et al., 1979). To investigate this possibility, the foiiicular RNA preparation was translated in vitro in the presence of microsomal membranes derived from dog pancreas (Shields and Blobel, 1978). The resulting cell-free products were shifted to lower molecular weights, relative to the components synthesized in the absence of membranes (Fig. 2a, lanes 2 and 1, respectively), and they comigrated with authentic

Silkmoth

Chorion

mRNAs

39

FIG. 1. Proteins resulting from cell-free translation of follicular poly(A)’ RNA (lane 4; labeled with [“Hlleucine) are compared to authentic chorion proteins, %-labeled by in vitro carboxamidomethylation (lane 1). Both in vitro-translated and authentic proteins are immunoprecipitable with a chorion-specific antiserum (lanes 3 and 2, respectively). The in uitro translated proteins are shifted to a higher molecular weight range; numbered subclasses are precursors of correspondingly identified authentic protein bands (see the text and subsequent figures); additional tickmarks in classes B and C correspond to putative subclass precursors suggested by Fig. 3c. In this and subsequent figures, authentic chorion classes and subclasses are numbered as in Regier et al., 1980. Overlapping lines between A5 and Bl indicate uncertainty and probable overlap between the size ranges of these components. Band B7 is fist defined in the present study.

chorion bands (Fig. 2a, lane 3). The products of membrane-supplemented translation were sequestered within the microsomal membranes, since they were generally resistant to proteolysis (Fig. 2a, lane 4). By contrast, the products synthesized in

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123456

B A

FIG. 2. Processing of chorion protein precursors in the presence of microsomal membranes. (a) The [3H]leucine-labeled precursors, synthesized by the wheat germ cell-free system in response to follicular poly(A)+ RNA (lane 1) are shifted to products of lower molecular weight if translation is performed in the presence of microsomal membranes (lane 2), and comigrate with W-labeled authentic chorion proteins (lane 3). The processed products are sequestered within the microsomal vesicles, as shown by their resistance to proteolysis (lane 4). By contrast, cell-free products synthesized in the absence of membranes, or in the presence of membranes disrupted with Triton X-100, are sensitive to proteolysis (lanes 5 and 6, respectively). Dot indicates a band observed only after proteolytic treatment, which is interpreted as an artifactual product of B6 proteins (see Discussion). (b) Membrane-processed cell-free products are immunoprecipitable with chorion-specific antibodies, as are the precursors and authentic proteins. Lanes l-4 are samples identical to those of the corresponding lanes of (a), except that they have been immunoprecipitated.

the absence of membranes, or in the presence of membranes disrupted with Triton X-100, were fully sensitive to proteolysis (Fig. 2a, lanes 5 and 6, respectively). Like precursor and authentic chorion. proteins, the sequestered and protected products of membrane-supplemented translations were immunoprecipitable with chorion-specific antiserum (Fig. 2b). After limited proteolytic digestion a novel band appeared (dot in lane 4, Fig. Ba), which was absent from the membraneprocessed but undigested products. This band was also immunoprecipitable (Fig. 2b). Although it appeared to corn&ate with some relatively minor A5 authentic chorion bands, we believe that it is an artifact of

proteolysis in the in vitro-reconstituted tem (see Discussion).

Developmental mRNAs

Specificity

of

sys-

Chorion

Since the membrane-supplemented wheat germ system translates chorion mRNAs into products identical in size with in vivo synthesized chorion components, it should be possible to use this translation system for assaying specific subclasses of chorion mRNAs in a mixture. A developmental application of this assay is shown in Fig. 3. Two parallel ovarioles, each containing seven progressively more mature choriogenic follicles, were dissected from a single animal. One ovariole was la-

THIREOS

AND

KAFATOS

Silkmoth

Chorion

mRNAs

41

beled with [3H]leucine for 1 hr in Grace’s tissue culture medium, to determine the changing pattern of in vivo protein synthesis for each of these follicles (Fig. 3a). From each follicle of the second ovariole, total cytoplasmic RNA was extracted and translated in membrane-supplemented wheat germ system, to assay the corresponding mRNA population. Comparison of in vivo and in vitro patterns showed an impressive degree of correspondence, within the limits of developmental asynchrony between

Ib Ie III

VI VIII Xa Xc T

c

DI

,

IflIe

II

IV

VI

Ix

.

Xb

T

FIG. 3. Comparisons of in uiuo-synthesized stagespecific chorion proteins, with in vitro synthesized stage-specific precursors and their membrane-processed products. (a) Autofluorogram of in uiuo-synthesized stage-specific proteins. One ovariole containing seven choriogenic follicles was labeled with r3H]leutine for 1 hr in Grace’s tissue culture medium. Individual follicles were washed free of yolk, solubilized, and displayed on an SDS-polyacrylamide slab gel. The lirst seven lanes (left to right) represent progressively more mature follicles (staged as in Paul and Kafatos, 1975, and Sim et al., 1979). ‘%-Carboxamidomethylated total chorion proteins from another animal are displayed in the last lane (T). (b) Autofluorogram of proteins synthesized and processed in a membranesupplemented wheat germ system, in response to total cytoplasmic RNA purified from individual staged follicles. The follicles were from a parallel ovariole, from the same animal as the ovariole used in (a). Follicles of corresponding position in parallel ovarioles are known to be similar but not identical in developmental stage; the samples of this panel were also staged independently, as in Paul and Kafatos (1975) and Sim et al. (1979). The cell-free synthetic patterns are very similar to those observed in uiuo at the indicated stages (a). Dot indicates a product endogenous to the microsomal membrane preparation. (c) Autofluorogram of precursors synthesized and not processed by an unsupplemented wheat germ system, in response to the same stage-specific RNA preparations used in (b) (lanes Ib’ to Xb). The last lane (T) shows the total unprocessed precursors synthesized using follicular poly(A)+ RNA from mixed developmental stages. Comparison with (b) permits characterization of precursor-product relationships. Al, A4, B2, and B6 precursors are clearly identified by additional evidence (see Figs. 4 and 5), and C3 precursors by their very late component; these well-identified subclass precursors are also numbered in other figures. Putative identification of additional subclass precursors is suggested from the translation patterns corresponding to the early stages Ib’ and Ie; these are numbered here but not in other figures.

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ovarioles (Paul and Kafatos, 1975). Consistent small quantitative differences in relative intensities of certain bands could be easily interpreted as due to unequal translational efficiencies. Within these limits, it was clear that both in uiuo (see also Paul and Kafatos, 1975; Sim et al., 1979) and in vitro, most C proteins were synthesized at early stages; D proteins were also relatively early. Protein subclasses A4 and B2 could be described as middle, Al and B6 as late, and a single C3 band as very late; subclasses A2, 3, 5 and Bl, 3, 4, 5 and 7 appeared to contain members of more than one developmental stage. Identification of Specific Chorion Precursors by Developmental Specificity Once it was ascertained that stage-specific mRNAs can be cell-free translated to yield a limited set of chorion bands (corresponding to those synthesized in uiuo), the opportunity arose to identify specific subclasses of precursors from their developmental specificity. Essentially, developmental specificity was used to simplify the chorion protein complexity. The same RNA preparations used for Fig. 3b were also translated in the absence of microsomal membranes (Fig. 3~). Comparison with Fig. 3b permitted identification of the precursors corresponding to the well-characterized stage-specific protein groups: the early C and D proteins, the middle period subclasses A4 and B2, the late subclasses Al and B6, and the very late C3 subclass. In addition, it appeared that, at least within each class, the mobilities of all major bands were shifted approximately in parallel due to the signal peptides. Note in particular the relative intensity ratios of the four C subclasses in the processed products of Stages Ib’ and Ie (Fig. 3b) and the corresponding intensity ratios of putative precursors indicated in Fig. 3c. Identification of Specific Chorion sors by Amino Acid Content Incorporation experiments amino acid analysis of purified

Precur-

as well as components

VOLUME 78. 1980

have revealed significant differences in the composition of chorion proteins belonging to different size classes or subclasses (Paul et al., 1972; Regier et al., 1978b; Rodakis, 1978; and J. C. Regier, unpublished observations). Thus, high or relatively high levels of glycine, cysteine, and leucine are present in the major chorion components; however, relative to glycine, the A proteins are slightly deficient in leucine and enriched in cysteine, whereas the C proteins are deficient in cysteine. Methionine is absent from nearly all A and the major low molecular weight B proteins (B2), but present in the high molecular weight B proteins and in the C’s. Tryptophan is found in very few A’s but nearly all B’s and C’s. Histidine is very rare and mostly limited to some of the C’s. (We have omitted from this summary D and E proteins, since they are an insignificant fraction of the translation products.) A series of cell-free translations in the absence of membranes were performed, each using for label one of the amino acids listed above. Figure 4 shows the results, which are clearly consistent with expectations: they confirm the identifications of A, B, and C class precursors, as well as those of the precursors of B2 proteins in particular (in this case, by the lack of methionine). Identification of Specific Chorion Precursors Using Chorion cDNA Clones Use of cDNA clones (Sim et al., 1979) simplifies the chorion system considerably. Specific mRNA subclasses can be purified by hybridization with specific clones. When these selected mRNAs are translated in the presence and absence of microsomal membranes, comparison of the translation products permits identification of the precursors corresponding to specific mature chorion bands. At the same time, this experiment identifies the protein band encoded by each cDNA clone. For these experiments we used clones which hybridize with very abundant (~~18, ~~401) or moderately abundant (pc609) mRNA subclasses (Kafatos et al., 1979).

THIREOS

Leu AC

Gly

AND

KAFATOS

His

Met Cys

Trp

Silkmoth

Chorion

mRNAs

43

of related sequences belonging to the same multigene family (see under Discussion). In the case of pc18, precursors and products form doublets, apparently corresponding to two closely homologous Al components. In the case of pc609, only minor cross-reacting components are evident among the precursors. DISCUSSION

FIG. 4. Identification of specific chorion precursors by amino acid content. Follicular poly(A)’ RNA was translated in the absence of membranes, using as the label [3H]glycine, [“Hlleucine, [%]cysteine, [‘“S]methionine, [3H]tryptophan, or [3H]histidine, as indicated. The relative prominence of various precursor bands in each translation, in conjunction with the known compositional differences among authentic chorion protein classes and subclasses (see text), permits identification of precursors corresponding to particular authentic chorion components (AC lane).

Clones were linearized by digestion with appropriate restriction enzymes (which do not cut the chorion DNA sequence), and hybridized with total chorion mRNA under conditions that favor a two-step formation of R-loops (see under Materials and Methods). When the mixture was chromatographed through an agarose column, the hybridized RNA was excluded along with the plasmid DNA, and thus separated from the nonhybridized sequences (Woolford and Rosbash, 1979). The isolated hybrids were then melted and the RNA translated. The results shown in Fig. 5 identify definitively precursors of subclasses Al and A4 encoded by clones pc18 and pc609, respectively. In the case of ~~401, a number of bands are detected, with the strongest corresponding to B6 and weaker ones corresponding to B3, 4, 5. We interpret the weaker bands as due to cross-hybridization

The studies described here made four useful contributions to the continuing analysis of the chorion system. These are identification and characterization of chorion protein precursors; development of a procedure for identification of chorion DNA clones through translation; demonstration that during development the translatable mRNA populations contained within the cells change, in parallel with changes in the in vivo pattern of protein synthesis; and suggestion that hybrid-selected translation can be used to assay sequence homologies between related proteins. The chorion precursors bear signal peptides, as demonstrated by their cotranslational processing by microsomal membranes, and the sequestration of the products within membrane vesicles (Blobel, 1977). The existence of such precursors for the secretory chorion proteins is not unexpected, but its documentation is welcome in that it explains the disparity in size distributions of in vivo and cell-free translated chorion proteins. As is true for other systems, the precursors are not detected in intact cells even by very brief pulse labeling (Paul et al., 1972; Kafatos et al., 1977). By a combination of developmental, compositional, and specific hybridization analyses, we characterized the precursors corresponding to various classes and subclasses of mature chorion proteins. As a result, translations can now be used to characterize specific chorion mRNA sequences, as for example to identify the subclass of protein encoded by a chorion DNA clone (cDNA or genomic). Because of the detailed information available on the developmental timing of synthesis of specific

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FIG. 5. Precursors and mature-size chorion proteins corresponding to cDNA clones. Specific mRNAs were selected by hybridization with cDNA clones pc18, p&09, or pc401 and translated in the wheat germ system in the absence (-) of presence (+) of microsomal membranes. The precursors and processed (mature size) products, respectively, are compared to each other, as well as to total precursors (TP) and authentic chorion proteins (AC). These comparisons identify pc18, pc609, and pc401 as encoding mature-size proteins of subclasses Al, A4, and B6, respectively (right-hand identifications), and unambiguously characterize the corresponding precursors (left-hand identifications). Because of homologies between different chorion sequences, the mRNAs selected by pc18 encode two Al bands, homologous minor components are also selected by pc609 (A2,3; because of the exposures evident only in the precursor sample), and by pc401 (B3,4, 5).

SDS-electrophoretic protein bands, this type of characterization is crucial, and cannot be replaced even by sequencing of the clone. Protease treatment of membrane-sequestered translation products yielded one unexpected, novel band (dot in Fig. 2). Because of a concomitant decrease in the intensity of the B6 band, we believe that the novel band is a processing artifact. Internal signal peptides or stop signals have recently been discovered or postulated in membrane proteins and ovalbumin (Lingappa et al., 1979). It is possible that such internal signals might fortuitously exist in some B6 protein(s), resulting in incomplete translo-

cation during cell-free translation. Subsequent exogenous protease treatment would destroy the portion of the protein(s) exposed to the medium; the portion contained within the membrane or vesicle lumen would be spared and appear as a novel band. Among the A proteins, resolution appears to be greater for the precursors than for the mature proteins; the converse is true for the higher molecular weight classes, especially the C proteins. This is probably due to different resolving capacities of the gel for different molecular weight ranges. Although differences in signal peptide length are another possible explanation, se-

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AND KAFATOS

quence analysis of seven rather divergent chorion genes, corresponding to subclasses Al, A4, B2, and B6, suggests that this explanation is unlikely: four of the clone-encoded signal peptides are 21 residues long, and three are 20 residues long (C. W. Jones, personal communication). In the present study it was not possible to detect precursors for the E proteins, because they are not translated very efficiently in the wheat germ system. We have been able to detect their precursors by using reticulocyte lysates (data not shown). Differences in in vitro translatability are common for various mRNAs; in the wheat germ system these differences appear to be rather limited for chorion mRNAs, other than those corresponding to E proteins, which are unlike the rest in composition (Regier et al., 1980). Translations of stage-specific RNAs strongly argue that the program of choriogenesis is fundamentally based on sequential production and transient accumulation of different chorion mRNAs. This conclusion was suggested earlier by experiments of much lower resolution (Gelinas and Kafatos, 1977) and has recently received support from dot hybridization assays of RNA sequence concentrations during development, using cloned chorion cDNAs as probes (Sim et al., 1979). Unlike the two previous studies, however, the translations shown in Fig. 3b used total cytoplasmic RNA, without selection through Mg’+ precipitation and oligo(dT)-cellulose chromatography, and thus assayed all chorion mRNAs which are present within the cytoplasm at a particular stage (irrespective of location or poly(A) content). Similarly, in Dictyostelium discoideum, translatable mRNAs are not detectable in vitro at stages of development before synthesis of the respective proteins begins in vivo (Alton and Lodish, 1977). The possibility of translational modulation for some chorion proteins cannot be tested adequately without two-dimensional electrophoretic analysis, which was avoided in the present study

Silkmoth

Chorion

mRNAs

45

because of the in viva post-translational charge modification of some chorion proteins, as noted in the Introduction. However, the results presented here certainly argue in favor of concentrating developmental studies of the chorion system at the pretranslational level. An observation potentially of great value is that single cDNA clones select by hybridization a small set of, rather than individual, mRNA species. This is due to sequence similarities between chorion mRNAs, resulting from their evolutionary homologies; it was to be expected from the known crosshybridization between distinct cDNA clones (Sim et al., 1979). For evolutionary studies, it would be very convenient to be able to evaluate sequence homologies between mature proteins by hybrid-selected translation, without having to characterize physically the proteins themselves. From the results presented in Fig. 5, we can already conclude that at least two Al components are more related to each other than to other A proteins; and that B6 proteins are more related to B3, B4, and B5 than to Bl and B2 proteins. This type of analysis should be applicable to interspecies as well as intraspecies comparisons, and could be refined by careful attention to the criterion of hybridization, or by introducing stepwise melting of the hybrids after hybridization and before translation. We thank Dr. J. R. Hunsley for the chorion-specific antiserum; Dr. J. Majzoub for the dog pancreas membranes; Dr. J. C. Regier and C. W. Jones for permission to quote unpublished results; B. Klumpar for help with the figures; and S. Byers for secretarial assistance. This work was supported by grants from NSF and NIH to F.C.K. REFERENCES ALTON, T. H., and LODISH, H. F. (1977). Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum. Develop. Biol. 60, 180-206. BLOBEL, G (1977). Synthesis and segregation of secretory proteins: the signal hypothesis. In “International Cell Biology 1976-1977” (B. R. Brinkley and K. R. Porter, edsl, pp. 318-325. Rockefeller Univ. Press, New York.

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