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.     

A genetic melanotic neoplasm of Drosophila melanogaster

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.     

Establishment and maintenance of stable spatial patterns in lacZ fusion transformants of Polysphondylium pallidum.

Polysphondylium pallidum cells were transformed with a construct containing the Dictyostelium discoideum ecmA promoter fused to a lacZ reporter gene. Two stably transformed lines, one in which beta-galactosidase (beta-gal) is expressed in apical cells of the fruiting body (p63/2.1), and one in which it is expressed in basal cells (p63/D), have enabled us to infer how cells move during aggregation and culmination. Several types of cell movement proposed to occur during slime mold culmination, such as random cell mixing and global cell circulation, can be ruled out on the basis of our observations. Cells of the two transformant lines express beta-gal very early in development. In both cases, stained cells are randomly scattered in a starving population. By mid to late aggregation, characteristic spatial patterns emerge. Marked cells of p63/2.1 are found predominantly at tips of tight aggregates; those of p63/D accumulate at the periphery. These patterns are conserved throughout culmination, showing that marked cells maintain their relative positions within the multicellular mass following aggregation. Neither the apical nor the basal pattern appears to be regulated within the primary sorogen by de novo gene expression or by cell sorting as whorls are formed. However, marked cells within a whorl re-establish the original pattern in secondary sorogens. This must be achieved by cell migration, since beta-gal is not re-expressed.     

Individual regulation of the accumulation of H1 mRNA and core histone mRNAs in sea urchin embryos.

The relative cytoplasmic accumulation of the individual histone mRNAs in sea urchins was determined by gel analysis of 3H-labeled cytoplasmic RNA isolated from embryos of the early cleavage through the mesenchyme blastula stages. A number of separate determinations showed that H1 mRNA accumulates at a molar ratio of 0.5 or less compared with each of the H2 or H3 core histone mRNAs through approximately the first 12 h of embryonic development. After this time, the accumulation of H1 mRNA increases relative to the core histone mRNAs, and approximately equimolar amounts of the histone mRNAs are produced by about the 14-h stage. The equimolar synthesis of H1 mRNA appears to be transient, returning to 0.5-molar levels several hours later. The increase in H1 mRNA accumulation, relative to the core histone RNAs, is coincident with the transition from expression of the early (alpha) sea urchin histone gene set to the late histone genes. Since all five of the early histone genes occur in a 1:1 ratio within repeating units, the data suggest that the genes within a single repeat, or their immediate products, are individually regulated. Gel analysis of the proteins synthesized in vivo by embryos demonstrates that the pattern of synthesis of the histone proteins reflects the changing ratios of the histone mRNAs.     

Induction of unique mRNAs by human interferons

Treatment of human fibroblast cells with human interferon (INF-alpha, IFN-beta, or IFN-gamma) resulted in the accumulation of at least four newly synthesized mRNAs. The mRNAs code for proteins having molecular weights of 56,000, 57,000, 62,000, and 68,000 when characterized in a wheat germ cell-free translation system. A direct relationship was observed between the amount of IFN used and the degree of both the accumulation of the induced mRNAs and the development of an antiviral state. In the case of IFN-alpha or IFN-beta, time course studies indicated that the induced mRNAs appeared as early as 40 min, accumulated for 2 h, then remained ribosome bound for up to 16 h. The ability of fibroblast cells to develop an antiviral state always coincided directly with both the appearance and the level of accumulation of the induced mRNAs. Further mRNA synthesis beyond 2 h had a minimal effect on the development of an antiviral state. Human IFN-gamma also induced the synthesis of the same four mRNAs but required higher interferon titers and a longer incubation time. In addition, IFN-gamma induced a disproportionate amount of the mRNA coding for the 68,000 molecular weight protein and three new mRNAs not detected in cells treated with IFN-alpha or IFN-beta. Mouse interferon induces the original four mRNAs in human cells but to a far lesser extent. This correlated with the inability of these cells to develop much resistance to viral infection.     

Regulation of the synthesis of two carbohydrate-binding proteins in Dictyostelium discoideum

The relative rate of de novo synthesis of two membrane-associated carbohydrate-binding proteins (CBP) has been examined during Dictyostelium development. The results show that the relative rate of CBP synthesis is minimal during the vegetative stage and increases to represent approximately 3.5 to 5% of newly synthesized protein during the aggregation stage after which the relative rate decreases. Analysis of the relative rates of synthesis of CBP-26 and CBP-24 indicate that at the peak period of synthesis (approximately 5 to 9 h of development) CBP-26 is synthesized at a rate which is approximately eight times greater than CBP-24. In addition, we have examined the relative amount of CBP-26 and CBP-24 mRNA during development as assayed by its ability to direct CBP synthesis in in vitro protein-synthesizing systems. We show that there is no detectable CBP mRNA in vegetative cells and that during the pre-aggregating stages, assayable CBP mRNA appears and accumulates with a maximal level at the period of peak in vivo CBP synthesis. These results suggest that the rate at CBP synthesis in vivo is controlled by the relative amount of functional mRNA.     

Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells

Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.     

Synthesis of spore proteins during development of Dictyostelium discoideum.

The pattern of synthesis of the spore coat proteins during development of Dictyostelium discoideum has been determined by using immunoprecipitation with spore protein antibody. SP170, SP103, 'SP94', SP82, SP76 and SP55 are all first synthesized just prior to the 'Mexican hat' stage of development (16-18h), but the synthesis of SP72 is delayed. This protein is apparently synthesized as a precursor, P66, which is modified during spore maturation to yield SP72. The nature of the modification is unknown. At their peak period of synthesis during early culmination (18-20h), the spore coat proteins account for 5-9% of total protein synthesis. Shortly after synthesis, these proteins are inserted into the spore coat, where all except SP103 become disulphide-cross-linked during the period 24-30h. SP3 does not accumulate until disulphide-cross-linking of the major spore coat proteins occurs and is itself disulphide-cross-linked into the spore coat. Several additional proteins that are accumulated during development have also been identified, namely P31, P25, P21 and P18. P25 first appears at 18-20h and then continues to be made throughout development. P31 synthesis begins at 12-14h and then largely ceases after approx. 20 h of development. The genes for both P21 and P18 are first expressed early in development, starting at 9-12h. P21 synthesis ceases at approx. 14h, but P18 continues to be synthesized throughout the rest of development. The marked differences in the time period of accumulation of these proteins compared with the co-ordinated syntheses of SP170, SP103, 'SP94', SP82, SP76 and SP55 provide a useful system for analysis of the mechanism of temporal gene expression during development.     

Histone gene expression during sea urchin embryogenesis: isolation and characterization of early and late messenger RNAs of Strongylocentrotus purpuratus by gene-specific hybridization and template activity

We have examined the molecular mechanisms responsible for the shifts in histone protein phenotype during embryogenesis in the sea urchinStrongylocentrotus purpuratus. The H1, H2A, and H2B classes of histone synthesized at the earliest stages of cleavage are heterogeneous: These proteins are replaced at late embryogenesis by a different set of histone-like polypeptides, some of which are also heterogeneous. The H3 and H4 histones appear to be homogeneous classes and remain constant. We have isolated from both early and late embryos the individual messenger RNAs coding for each of the multiple protein subtypes. The RNAs were isolated by hybridization to cloned DNA segments coding for a single histone protein or by elution from polyacrylamide gels. Each RNA was then analyzed and identified by its mobility on polyacrylamide gels and by its template activity in the wheat germ cell-free protein synthesizing system. The mRNAs for each of the five early histone protein classes are heterogeneous in size and differ from the late stage templates. The late mRNAs consist of at least 11 separable types coding for the 5 classes of histones. Each of the 11 has been separated and identified. The late stage proteins were shown to be authentic histones since many of their templates hybridize with histone coding DNA. The early and late stage mRNAs are transcribed from different sets of histone genes since (1) late stage H1 and H2A mRNAs fail to hybridize to cloned early stage histone genes under ideal conditions for detecting homologous early stage hybrids, (2) late stage H2B, H3, and H4 RNA/DNA hybrids melt at 14, 11, and 11°C lower, respectively, than do homologous RNA/DNA hybrids, and (3) purified late stage mRNAs direct the synthesis of the variant histone proteins which are synthesized only during later stages. The time course of synthesis of the late stage mRNAs suggests that they appear many hours before the late histone proteins can be detected, possibly as early as fertilization. In addition, early mRNAs are synthesized in small quantities as late as 40 hr after fertilization, during gastrulation. Thus, the major modulations of histone gene expression are neither abrupt nor an absolute on-off switch, and may represent only a gradual and relative repression of early gene expression. Two histones are detected only transiently during early cleavage. The mRNA for one of them, a subtype of H2A, can be detected in the cytoplasm for as long as 40 hr after fertilization. However, template activity for the other, a subtype of H2B, can be detected only at the blastula stage. Thus, the histone genes represent a complex multigene family that is developmentally modulated.     

Developmentally regulated expression of temporally distinct beta-glucosidase isozymes in Dictyostelium discoideum

During development of Dictyostelium discoideum, the cellular specific activity of beta-glucosidase increases before aggregation, declines to low levels during pseudoplasmodium formation, and increases rapidly during culmination. In addition, two electrophoretically distinct isozymes of beta-glucosidase are present at different times of development. Using enzyme-specific monoclonal antibodies, we have shown that changes in the level of enzyme specific activity are closely paralleled by changes in the relative rate of beta-glucosidase synthesis in vivo and by corresponding changes in the relative cellular concentration of functional beta-glucosidase mRNA. Thus, the developmental synthesis of beta-glucosidase is controlled at a pretranslational level. Furthermore, our experiments have demonstrated that both beta-glucosidase isozymes consist of a single subunit of identical molecular weight. This result is consistent with the previous finding that both isozymes are encoded by the same gene and suggests that the isozymes differ solely with respect to post-translational modification.     

Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas Flagella

A dramatic stimulation of synthesis of flagellar proteins occurs in Chlamydomonas following flagellar removal or experimentally induced resorption of the flagella into the cell. In this report we show that this stimulation involves an increase in the levels of mRNAs for tubulin and many other flagellar proteins. Total RNA and poly(A) RNA were isolated from cells after deflagellation or flagellar resorption, and were then translated in the reticulocyte lysate system. Two-dimensional gel analysis of the translation products demonstrates that the RNA-directed in vitro synthesis of α and β tubulins, and a number of other flagellar proteins, increases after deflagellation or flagellar resorption. Surprisingly, the α-tubulin synthesized in vitro does not co-migrate on two-dimensional gels with mature flagellar α-tubulin. Moreover, in vivo labeling experiments show that the major α-tubulin synthesized in the cell after deflagellation co-migrates with the major α-tubulin made in vitro, not with the major α-tubulin present in the flagella. These results suggest that flagellar α-tubulin is synthesized as a precursor, and undergoes post-translational modification before assembly into the flagella. In addition, we report that the synthesis of tubulin and other flagellar proteins can be specifically inhibited, as well as stimulated. Treatment of cells with IBMX, which induces flagellar resorption, causes a marked decrease in the levels of translatable mRNAs for tubulin and other flagellar proteins, without affecting levels of mRNAs for nonflagellar proteins.     

Translational control of protein synthesis during the early stages of differentiation of the slime mold Dictyostelium discoideum

As analyzed by two-dimensional polyacrylamide gel electrophoresis, no new proteins are synthesized during the first 60 min of differentiation of Dictyostelium discoideum. The major change observed is the cessation of synthesis of five polypeptides and the reduction in the relative rates of synthesis of several more. We show here that this specific inhibition of protein synthesis is under translational control; the mRNAs for these proteins persevere in the cell in a translatable form for as long as 4 hr of differentiation, but these proteins are not synthesized by the cells after 2 min of development. As determined by analysis of the subcellular distribution of ribosomes and messenger RNA, there is a precipitous drop in the overall rate of polypeptide chain initiation during the first 5 min of differentiation. To interrelate and explain these phenomena, we show that a recent kinetic analysis of mRNA translation can explain how a reduction in the activity of a component of the initiation machinery required for translation of all mRNAs, such as an initiation factor, could result in a reduction in the overall rate of chain initiation and also a preferential inhibition of translation of certain mRNAs.