Specific protein synthesis in cellular differentiation: II. The program of protein synthetic changes during chorion formation by silkmoth follicles,and its implementation in organ culture

During their differentiation, the follicular epithelial cells of the silkmoth, Antheraea polyphemus, produce the extracellular proteinaceous eggshell or chorion. Choriogenesis entails continuous changes in cell-specific protein synthesis; the various chorion proteins are synthesized with distinct kinetics. On the basis of protein synthetic profiles, 17 stages of choriogenesis are defined. The average duration of the stages is 3.0 hr, and thus choriogenesis lasts a total of approximately 51 hr. This program of protein synthetic changes is autonomous; i.e., it is implemented with normal kinetics by follicles cultured in isolation in a defined tissue culture medium.     

Specific protein synthesis in cellular differentiation: V. A secretory defect of chorion formation in the Grcol mutant of Bombyx mori

When homozygous, the Gr^col mutation of Bombyx mori causes the production of an eggshell in which most proteins are underrepresented to varying degrees. Neither the relative rates nor the timing of chorion protein synthesis appear to be affected; instead, the mutant phenotype results from the post-translational loss of normally synthesized proteins. The extent of loss of each protein correlates with its developmental timing, being maximal at early to middle stages. At the same stages, secretion appears to be deficient: chorion proteins overaccumulate within mutant cells, and slowly disappear. A preliminary electron microscopic examination has revealed the presence of mutant-specific cytoplasmic vesicles. The deficient complement of secreted proteins fails to form the highly ordered structure characteristic of normal chorion.     

Specific protein synthesis in cellular differentiation: III. The eggshell proteins of Drosophila melanogaster and their program of synthesis

The eggshell of Drosophila melanogaster is composed of a set of proteins synthesized by the follicular epithelium during the last third of oogenesis and organized into an inner zone (vitelline membrane) and an outer zone (chorion). To study these proteins, the authors developed techniques for mass-isolating follicles of mixed stages, mature (stage 14) follicles, chorion from stage 14 follicles, and chorion and vitelline membrane from laid eggs. The eggshell is composed mainly of protein and is unusually rich in proline and alanine. Six proteins of the chorion have been identified on polyacrylamide gels. The program of synthesis of these proteins was studied by incubating follicles of different developmental stages in culture with ³H-labeled amino acids and displaying the labeled proteins on gels with the aid of autofluorography. The proteins are synthesized in a specific overlapping sequence during stages 10–14, a period when chorion deposition is known to occur. In addition, putative vitelline membrane proteins have been identified by their preferential incorporation of [³H]proline and [³H]alanine during stages of active vitelline membrane synthesis.     

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

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.     

Specific protein synthesis in cellular differentiation: VI. Temporal expression of chorion gene families in Bombyx mori strain C108

Detailed patterns of expression for putative members of 5 major chorion gene families have been obtained by separating labeled proteins using two-dimensional polyacrylamide gel electrophoresis. Proteins fall into 4 temporal cohorts called early, early middle, middle, and late on the basis of when they initiate and terminate synthesis. Proteins synthesized during the early, early middle, and late periods are highly synchronous, exhibiting abrupt onset times and relatively uniform termination times. Middle proteins begin synthesis in small groups at staggered times over a relatively long period, but most cease translation as the late proteins turn on. This data is correlated with a previous follicle staging system based on separation of newly synthesized chorion proteins by isoelectric focusing alone. The absolute timing of choriogenesis was determined in vivo, using trypan blue dye to mark vitellogenic follicles. The relative age difference between chorionating follicles was 2.2–2.6 hr; chorion biosynthesis took 4 days in all. These data are discussed in terms of patterns of activity of chorion gene families, the functions of the temporal cohorts, and regulation of the chorion multigene system.     

Effect of the pleiotropic GrB mutation of Bombyx mori on chorion protein synthesis

The Gr^B mutation has a profound pleiotropic effect, leading in the homozygous state to the absence or extreme reduction of a substantial number of chorion proteins. The effect shows developmental specificity: most of the proteins normally synthesized beginning with stage III of choriogenesis or later, but possibly none of these normally beginning with stage II, are eliminated in the mutant. More subtle quantitative effects on certain other proteins are also observed, including prolongation of synthesis of some proteins which normally terminate at stage VIII. The proteins eliminated in the mutant are present in the heterozygote at intermediate levels, quantitatively close to those in the wild-type. The differences in chorion protein composition result from correspondingly altered protein synthesis rather than from post-translational degradation or modification. The missing proteins also fall to be synthesized in vitro when total RNA from mutant follicles is translated in the wheat germ system. It appears that as a consequence of the mutation, a set of mRNAs fails to be synthesized or accumulated. These results are consistent with the possibility that Gr^B is a regulatory mutation, or a deletion eliminating multiple chorion genes, clustered predominantly according to the developmental period of their expression.     

Identification and time of synthesis of chorion proteins in Drosophila melanogaster.

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 flies injected with 14C amino acids. Chorion proteins 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).     

SPECIFIC PROTEIN SYNTHESIS IN CELLULAR DIFFERENTIATION : Production of Eggshell Proteins by Silkmoth Follicular Cells

Silkmoth follicles, arranged in a precise developmental sequence within the ovariole, yield pure and uniform populations of follicular epithelial cells highly differentiated for synthesis of the proteinaceous eggshell (chorion). These cells can be maintained and labeled efficiently in organ culture; their in vitro (and cell free) protein synthetic activity reflects their activity in vivo. During differentiation the cells undergo dramatic changes in protein synthesis. For 2 days the cells are devoted almost exclusively to production of distinctive chorion proteins of low molecular weight and of unusual amino acid composition. Each protein has its own characteristic developmental kinetics of synthesis. Each is synthesized as a separate polypeptide, apparently on monocistronic messenger RNA (mRNA), and thus reflects the expression of a distinct gene. The rapid changes in this tissue do not result from corresponding changes in translational efficiency. Thus, the peptide chain elongation rate is comparable for chorion and for proteins synthesized at earlier developmental stages (1.3–1.9 amino acids/sec); moreover, the spacing of ribosomes on chorion mRNA (30–37 codons per ribosome) is similar to that encountered in other eukaryotic systems.     

Changes in follicle cell morphology,ovarian protein synthesis and ovarian DNA synthesis during oöcyte maturation in Leucophaea maderae: Role of juvenile hormone

Changes in follicle cell morphology were correlated with changes in rates of protein synthesis and DNA synthesis by the ovary during ovarian maturation in Leucophaea maderae. During the vitellogenic period of oöcyte development, which lasts approx, 15 days, morphological changes in the follicle cells are accompanied by moderate rates of ovarian protein synthesis and rapid rates of ovarian DNA synthesis. At approx. 15 days after mating, the shape of the follicle cells changes from cuboidal to squamous, ovarian DNA synthesis is arrested, and ovarian protein synthesis increases slightly. During the final period of oöcyte development, which lasts approx, two days, the interfollicular channels between the follicle cells have disappeared and the squamous follicle cells, which contain an extensive rough endoplasmic reticulum, deposit a chorion around the mature oöcyte. These morphological changes are accompanied by a radical increase in ovarian protein synthesis, while ovarian DNA synthesis remains arrested. Immediately before ovulation, ovarian protein synthesis starts to decline, reaching a minimal level 24 hr post-ovulation.Ovarian maturation is dependent on the presence of juvenile hormone (JH) only during the vitellogenic stage of oöcyte development. Decapitation of insects at any point during the first 10 days after mating arrests the synthesis of DNA and retards the synthesis of protein by the ovary, resulting in degeneration of the oöcyte. Subsequent injection of JH restores both events to normal levels within 72 hr. Decapitation on or after the tenth day following mating does not alter normal oöcyte development, chorion deposition, ovulation or egg case formation.Primary induction of protein synthesis in ovaries from virgin females can be achieved by either an in vivo or in vitro exposure of the tissue to JH, thus confirming a site of action for JH to be ovarian tissue. Electrophoretic analysis of the soluble proteins from JH-exposed ovaries in vivo reveals that JH stimulates general protein synthesis, rather than the synthesis of a specific major protein such as vitellogenin.     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

The silkmoth chorion: Morphogenesis of surface structures and its relation to synthesis of specific proteins

The morphogenesis of four spatially differentiated surface regions of the silkmoth eggshell (chorion) has been documented and correlated with differing patterns of chorion protein synthesis within the corresponding secretory cells. During the first half of choriogenesis the polygonal pattern of ridges which cover the entire chorion appears. Regional differences in the morphology of developing ridges are not accompanied by significant protein differences, and thus presumably reflect differences in secretory cell behavior and shape. During the second half of choriogenesis expanding domes of the chorion located immediately beneath three-cell junctions of the overlying secretory surface become prominent surface features exclusively in the aeropyle crown region. Domes are composed of a thin lamellar skin and an inner buttressing “filler.” Continued filler deposition appears to cause a ripping of the lamellar skin, transforming the dome into a multiple-pronged crown that overflows with filler. Continued synthesis of lamellar chorion components elongates and strengthens the crowns until they can stand alone without the support of filler. In the aeropyle crown region, synthesis of regionally specific proteins begins in the second half of choriogenesis and accelerates until the final stages, in parallel with dome/crown formation. The more numerous proteins which are common to all regions are synthesized at approximately equal rates within all regions, and their synthesis decelerates toward the end of choriogenesis. Fifteen of the proteins (excluding filler) which are found predominantly in the aeropyle crown region may be necessary but not sufficient for crown formation, since they also occur in the stripe region (1); presumably the secretory cell surfaces mold the same components differently in the two regions. Filler appears to play an important scaffolding role in crown formation. A group of eight aeropyle crown region-specific chorion proteins which compose filler have been identified on two-dimensional gels and shown to be restricted to one of five previously described classes of chorion proteins.     

Changes in protein synthesis induced in tomato by chilling

Impaired chloroplast function is responsible for nearly two-thirds of the inhibition of net photosynthesis caused by dark chilling in tomato (Lycopersicon esculentum Mill.). Yet the plant can eventually recover full photosynthetic capacity if it is rewarmed in darkness at high relative humidity. As a means of identifying potential sites of chilling injury in tomato, we monitored leaf protein synthesis in chilled plants during this rewarming recovery phase, since changes in the synthesis of certain proteins might be indicative of damaged processes in need of repair. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins pulse labeled with [³⁵S]methionine revealed discrete changes in the pattern of protein synthesis as a result of chilling. A protein of M_r = 27 kilodaltons (kD), abundantly synthesized by unchilled plants, declined to undetectable levels in chilled plants. Reillumination restored the synthesis of this protein in plants rewarmed for 8 hours. Peptide mapping analysis showed the 27 kD protein to be the major chlorophyll a/b binding protein of the photosystem II light-harvesting complex (LHCP-II). The identity of this protein was confirmed by its immunoprecipitation from leaf extracts by a monoclonal antibody specific for the major LHCP-II species. While chilling abolished the synthesis of the major LHCP-II species, it also induced the synthesis of an entirely new protein of M_r = 35 kD. The protein was synthesized on cytoplasmic ribosomes, and two-dimensional polyacrylamide gel electrophroesis showed it to exist as a single isoelectric species. This chilling-induced 35 kD protein is structurally distinct from the 27 kD LHCP-II and appears to be synthesized specifically in response to low temperature. While the 35 kD protein was found not to be associated with the chloroplast thylakoid membrane, chilling did cause selective changes in thylakoid membrane protein synthesis. The synthesis of two unidentified proteins, M_r = 14 and 41 kD, and the β-subunit of the chloroplast coupling factor were substantially reduced after chilling. These losses may provide clues as to the causes of the overall reduction in net photosynthesis caused by chilling.     

Ecdysterone-induced protein synthesis in vitro

Ecdysterone added in vitro to wing tissue from diapausing Antheraea polyphemus pupae induced the synthesis of several epidermal cell proteins. This is one of few instances in which any steroid hormone in physiological concentrations has been able to induce specific protein synthesis in target tissue in vitro soon after hormone stimulation. Hormone-treated tissue was incubated with ³H-leucine while control tissue was incubated with ¹⁴C-leucine. Polyacrylamide gel electrophoretic distribution of labelled wing tissue proteins after ecdysterone stimulation in vitro for various periods of time was determined. The ${}^3\text{H}{}^{14}\text{C}$ ratio emphasized the areas of increased protein synthesis due to ecdysterone. These areas of increased protein synthesis were reproducible with several ecdysterone concentrations and with different incubation times. Induction of protein synthesis occurs at an earlier time period when the hormone dosage is higher, i.e. the lower the dosage, the longer it is necessary for exposure of tissue to hormone. α-Ecdysone, known to initiate the moulting process in vitro in some insect species, also induced protein synthesis. Cortisol, a mammalian steroid hormone, produced no hormone specific protein synthesis. Therefore, the results seen with ecdysterone and α-ecdysone are not the result of non-specific steroid stimulation. When no hormone was added to the incubation medium (control), only one area of the polyacrylamide gel demonstrated protein synthesis. Therefore, there are a few proteins being synthesized in vitro in wing tissue, removed from diapausing animals without hormone stimulation, which may be related to the ‘injury phenomenon’. Protein banding patterns were also determined and compared with the radioactivity profile. The study of such early biochemical and physiological responses of target tissue to hormones will aid in our understanding of a hormone's mechanism of action, since the earlier an event occurs, the more likely that it is the primary result of hormone stimulation.     

Tubulin and actin synthesis during brain development

Chick brain proteins from 5- through 13-day embryos were labeled with l-[³⁵S]methionine for 30 min in vitro and analyzed by two-dimensional gel electrophoresis. Autoradiographs of the gels were scanned with a computer-coupled densitometer to measure the relative rates of protein synthesis. The actins and the tubulins were the most abundant proteins and had the highest rates of synthesis. β and γ actin were synthesized at constant rates throughout this period of development, but the rate of tubulin synthesis increased fourfold. Six α tubulins and two β tubulins were distinguished, and they were all synthesized at all times. The relative rates of synthesis of these forms changed with development in a complex pattern, but the stoichiometry of α:β remained 1:1.     

Tissue-specific synthesis of yolk proteins in Caenorhabditis elegans

The primary site of yolk protein synthesis in the nematode, Caenorhabditis elegans, has been determined. In animals containing no gonadal cells (obtained by laser ablation of the gonadal precursor cells early in development), yolk proteins are present in abundance. This demonstrates that yolk proteins are made outside the gonad. An examination of proteins present in tissues isolated by dissection, and a comparison of proteins synthesized by isolated tissues incubated in vitro have identified the intestine as the major site of yolk protein synthesis. We propose that yolk proteins are synthesized in the intestine, secreted from the intestine into the body cavity, and taken up from the body cavity by the gonad to reach oocytes. The site of yolk protein synthesis has also been examined in four mutants that have largely male somatic tissues, but a hermaphrodite germ line. Here again, yolk proteins are produced by intestines in a hermaphrodite-specific manner. This suggests that sex determination is coordinately regulated in intestinal and germ line tissues.     

N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.     

Control of bacteriophage T4 DNA polymerase synthesis

Analysis of sodium dodecyl sulphate/acrylamide gels of ¹⁴C-labelled proteins from phage-infected bacteria suggests the existence of a self-regulatory control mechanism in bacteriophage T4.Infection of Escherichia coli with phage T4 carrying a mutation in gene 43 (which codes for the phage DNA polymerase) results in a greatly increased rate of synthesis of the gene 43 protein. Such overproduction of defective polymerase occurs in restrictive infections with all gene 43 amber and most gene 43 temperature-sensitive mutants tested. Gene 43 protein synthesis in gene 43⁺ infections or increased synthesis in gene 43^? infections appears to require no additional function of other phage proteins essential for DNA synthesis. Functional gene 43 protein is needed continuously to keep its own levels down to normal.     

Heat shock response of Dictyostelium

In response to a shift from 22 to 30°C the relative rate of synthesis of a small number of proteins is dramatically increased in Dictyostelium discoideum. The cells neither grow nor develop at this temperature but die slowly with a half-life of 18 hr. The major protein synthesized in response to a heat shock to 30°C in either growing cells or developing cells has an apparent molecular weight of 70,000 (70K). An increase in the relative rate of synthesis of 70K can be seen as early as 20 min following heat shock. Synthesis of 70K remains high for 4 hr at 30°C and then decreases. Similar kinetics of 70K synthesis occur during recovery at 22°C following a 1-hr heat shock. RNA synthesis during the first half-hour of heat shock is essential for the high rate of 70K measured 2 hr later. By isoelectric focusing the 70K protein can be separated into two spots, one of which overlaps one of the major heat shock proteins of Drosophila melanogaster. The relative rate of synthesis of several other proteins (82K, 60K, 43K) increases less dramatically in Dictyostelium during heat shock at 30°C. A heat shock to 34°C results in rapid synthesis of these proteins but not of 70K. The relative rates of synthesis of most other proteins made at 22°C decreases, most notably that of actin. Synthesis of heat shock proteins at 30°C does not significantly affect viability at 30°C but dramatically prolongs the period of time the cells can survive at 34°C. Thus, 30°C appears to be a stasis condition for Dictyostelium which elicits a response essential for protection from lethal temperatures. The similarity of the heat shock response in Dictyostelium to that in Drosophila and vertebrate cells suggests that certain aspects of the response may be universal in eukaryotes.