Metabolism of polyadenylic acid sequences during germination of blastocladiella emersoni: Zoospores.

The metabolism of the poly(A) sequences isolated from Blastocladiella emersonii was followed during the first hour of germination. Poly (A) sequences synthesized during the first 30 min of germination do not undergo detectable changes in size. During the first 45 min of germination, poly(A) sequences synthesized during zoosporogenesis decrease in size to the extent that there is essentially no size overlap between poly(A) fragments which were present in the zoospore and newly synthesized poly(A) sequences. The results presented indicate that during germination, polyadenylation occurs in RNA molecules which were present in the zoospore but lacked poly(A) sequences. No detectable size differences were observed between poly(A) sequences added to newly synthesized RNA compared to those added to the nonpolyadenylated RNA present in the zoospore during germination. Cycloheximide did not prevent the observed decrease in size of the poly(A) sequences during germination.     

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.     

The Changes of Protein Content and Synthetic Activity in Squash Pollen During Germination

The total protein content of squash (Cucurbita moschata Duch.) pollen decreased gradually during in vitro germination. It was caused by the release of wall proteins and part of the cytoplasmic proteins. The release of the pollen wall proteins was not dependent on germination, it was a passive diffusion process. However, the cytoplasmic proteins did not release until the pollen germinated, a fraction of them was synthesized de novo during germination. The RNA and protein synthetic activities initiated soon after in vitro pollen germination. The RNA synthesis decreased during germination. As about half the activity was inhibited by α-amanitin, mRNA might be the major RNA synthesized de novo. The total protein synthesis increased during germination, almost all of this synthesis was inhibited by cycloheximide, and partially by α-amanitin, but it was not affected significantly by actinomycin D. These results indicated that both stored and de novo synthesized mRNA might play a role in the protein synthesis. The content of stored mRNA of squash pollen was about 11-3 pg/grain as measured by UV absorption after its purification from total RNA (2440 pg/grain) by oligo (dT)-cellulose affinity chromatagraphy. Both cycloheximide and α-amanitin inhibited pollen tube growth in vitro. Actinomycin D and tunicamycin inhibited pollen germination in the first hour, however, no reduction ,of the tube length was observed later. Cyclohex,nide inhibited the pollen germination and tube elongation in vivo, that fitted well with the in vitro results. According to these results, it was suggested that the de novo syntheses of mRNA and protein were neccessary for the maintenance of pollen tube growth.     

Changes in Protein Synthesis upon Cytokinin-Mediated Adventitious Bud Induction and during Seedling Development in Norway Spruce, Picea abies

A pulse-treatment of embryos of Picea abies (L.) Karst with cytokinin efficiently and reproducibly induces the coordinate de novo formation of bud primordia from subepidermal cells. The cytokinin treatment also affects the germinative development of the embryo; chloroplast maturation is delayed, and cell elongation is completely suppressed. We have analyzed the protein patterns in developing spruce embryos with the aim of identifying proteins which are differentially synthesized during early bud-differentiation and germination. In addition to a set of major seed storage proteins and a large set of constitutively synthesized proteins, we distinguish two sets of proteins that showed different patterns of synthesis in relation to germination. One was synthesized at high rates during germination, and the second set during post-germinative seedling development. Twenty-two proteins were differentially synthesized in the bud-induced versus the germinating embryos. Interestingly, all 22 belonged to either the germination phase-abundant or the seedling protein sets, whereas the constitutively synthesized proteins were unaffected by the treatment. Proteins synthesized exclusively in bud-induced embryos were not found. In total, the bud-induction treatment caused a maintenance of a protein synthesis pattern typical for the germination phase in the nontreated embryos, and the de novo formation of buds was not preceded by a major change in gene expression in the tissue.     

Decreased activity of Blastocladiella emersonii zoospore ribosomes: correlation with developmental changes in ribosome-associated proteins

Ribosomal proteins isolated from dormant zoospores were compared to the ribosomal proteins found in the active growth phase by two-dimensional polyacrylamide gel electrophoresis. Zoospore ribosomes were found to contain a set of five proteins, designated Z1 to Z5, which were not present in growth phase ribosomes. The Z1-Z5 proteins were not removed by high-salt washes using either 1 M KCl or 1 M NH4 Cl. The Z1 protein is found associated with zoospore 60 S subunits while Z2-Z5 are bound to 40 S subunits. Zoospore monoribosomes and polyribosomes contain comparable levels of each of the five proteins. Approximately 60 min. after sporulation is induced, the Z1-Z5 proteins begin to accumulate on the ribosomes with the highest levels of these proteins found associated with ribosomes at the zoospore stage. During germination, the proteins gradually disappear and are not detectable on the ribosomes after 4 hr of germination. The presence of the Z1-Z5 proteins correlates with a decrease in in vitro protein synthetic activity of the fungal ribosomes. The data are consistent with the hypothesis that the proteins regulate translation by completely blocking protein synthesis on a subset of ribosomes while the remainder of the ribosomes function at normal rates.     

Comparative study of protein synthesis and heat-shock puffing activity in Drosophila salivary glands treated with chloramphenicol

The effects of chloramphenicol (CAP) on puffing activity and incorporation of tritiated amino acids in proteins synthesized by cultured larval salivary glands of Drosophila melanogaster were examined. CAP concentrations exceeding 1 mM were found to inhibit cellular protein synthesis and to induce the special group of heat-shock puffs in the polytene chromosomes. Recovery from a transient treatment with 5 mM CAP for 120 min led to rapid regression of the puffs and resumption of protein synthesis giving a pattern of labelled polypeptides similar to that produced by cells submitted to a temperature shift from 25 to 37 degrees C. Only slight inhibition of protein synthesis was found with thiamphenicol, the methylsulphonyl analogue of CAP, which induced a single puff in the 93D region, but did not alter the pattern of polypeptides. In contrast to the results obtained with CAP, recovery from a transient inhibition of protein synthesis by cycloheximide led to the synthesis of normal proteins as produced by control cells at 25 degrees C. Different effects of CAP which may interfere with protein synthesis and puffing activity are discussed.     

Protein synthesis in enuleated fertilized and unfertilized mouse eggs

Summary Fertilized and unfertilized C57BL/6J eggs were microsurgically enucleated and then analyzed for their capacity to synthesize proteins using 2-dimensional polyacrylamide gel electrophoresis. In both types of enucleated eggs (cytoplasts), protein synthesis continued and was still detected up to three days in culture. Shortly after enucleation, the pattern of polypeptides remained similar to the respective non-operated control eggs but it later became gradually reduced in intensity and complexity. After two days of culture the appearance of some new proteins typical for 2-cell embryos was observed in enucleated fertilized eggs only. Our findings suggest that maternal mRNA stored during oogenesis is utilized during the preimplantation period.     

Protein synthesis and differentiation in a clonal line of teratocarcinoma and in preimplantation mouse embryo.

Undifferentiated cells of a clonal line of teratocarcinoma can differentiate in vitro into embryoid bodies with morphological and biochemical features of early mouse embryo. During the first step of differentiation protein synthesis has been analysed by 2 dimensional gel electrophoresis. While new proteins are synthesized, the synthesis of others turned off with the appearance of endodermal cells in embryoid bodies. We have compared protein synthesis during teratocarcinoma differentiation and during early mouse embryogenesis at three stages of mouse preimplantation embryo. The results demonstrate that only the late blastocyst protein synthesis pattern shows most of the polypeptides identified in the differentiated protein synthesis pattern of teratocarcinoma. In contrast, protein synthesis during the early stages of mouse embryonic development is very different from protein synthesis in undifferentiated teratocarcinoma.     

Two-dimensional gel analysis of proteins in cell lines from the central nervous system of larvalDrosophila

Summary High resolution two-dimensional gel electrophoresis was used to quantitatively analyze the patterns of protein synthesis in three different clones of a nerve cell line (ML-DmBG2) ofDrosophila melanogaster. When patterns of pulse-labeled proteins of the three different clones were compared, I observed quantitative variations affecting the rate of synthesis by twofold or more in 25–30% of the polypeptides and qualitative differences, always affecting less than 2% of the polypeptides. Patterns of protein synthesis were analyzed during the 24 d of culture, revealing both quantitative (increase or decrease; 40%) and qualitative (presence or absence; 3%) differences. More than 70 proteins synthesized in these cultures were secreted into the medium. Among them were two major groups of acidic proteins which disappeared with culture time. When cell lines and intact central nervous systems were compared, large differences in protein synthesis were observed. In fact, only 20% of the synthesized proteins were common to both isolated cells grownin vitro and the original nervous systemin vivo.     

Developmental regulation of protein synthesis during Polysphondylium pallidum cyst germination: A two-dimensional gel electrophoresis study

Microcyst germination in Polysphondylium pallidum can be used as a model for studying gene expression because temporally regulated modulations in protein synthesis occur in this developmental pathway. Germinating cysts were labeled with [³⁵S]methionine for half-hourly periods during the synchronous germination sequence, and the proteins labeled in each period were resolved by two-dimensional polyacrylamide gel electrophoresis. Three major classes of proteins observed were distinguished by the time of onset and duration of their synthesis: (a) proteins made throughout germination; (b) proteins synthesized only during a portion of the germination pathway; and (c) polypeptides whose synthesis started at 1 or 1.5 h and then continued throughout germination.     

Envelope synthesis during the cell cycle in Escherichia coli B/r

The rate of synthesis of envelope proteins and phospholipids during the cell cycle of Escherichia coli B/r has been studied using both synchronous cultures and random cultures, first labelled and then subsequently fractionated on an age basis by the membrane elution technique. The rate of total protein synthesis and of phospholipid synthesis, measured by incorporation of [2-³H]glycerol into whole cells, was found to increase exponentially throughout the cell cycle. Total envelope protein was also synthesized continuously throughout the cycle, but the rate of synthesis showed a stepwise pattern with a discrete doubling in rate in the first half of the cycle. Analysis of the pattern of synthesis of about 29 individual envelope polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography revealed that the great majority followed the pattern of the bulk measurements, with a discrete increase in rate of synthesis early in the cycle. One envelope polypeptide, molecular weight 76,000, was, however, only synthesized during a brief period, near the time of division of the bacteria. Pulse-chase studies of envelope polypeptide synthesis in synchronous cultures demonstrated that (1) synthesis and insertion of polypeptide into the envelope was always completed within the pulse period; (2) no post-synthetic modification of polypeptides was detected; (3) one group of polypeptides, including a major outer membrane protein, maintained a stable association with the envelope, whilst a second group displayed considerable “turnover”; (4) about 70% of newly synthesized 76,000 molecular weight protein was lost from the envelope during the succeeding generation.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Lipid turnover during morphogenesis in the water mold Blastocladiella emersonii

The lipid content of $\text{Blastocladiella emersonii}$ zoospores is 5 pg/cell or about 13% of dry weight. Within the first few minutes of germination 60–70% of total zoospore lipid is lost, with neutral lipid, glycolipid and phospholipid fractions decreasing to about the same extent. These changes in lipid content precede the breakdown during germination of the complex and extensive membrane system of zoospores. During growth, which immediately follows germination, net phospholipid synthesis resumes so that total lipid is maintained at 6% of dry weight, but net synthesis of neutral and glycolipid does not begin until induction of sporulation. During sporulation the phospholipid level decreases so that the distribution of lipid among the three fractions approaches that found in zoospores. These changes in lipid content suggest that zoospore membranes containing neutral and glycolipids are synthesized $\text{de novo}$ during spore formation.     

Ribosomal Heterogeneity of Maize Tissues: Insights of Biological Relevance

In recent years, the selective role of ribosomes in the translational process of eukaryotes has been suggested. Evidence indicates that ribosomal heterogeneity at the level of protein stoichiometry and phosphorylation status differs among organisms, suggesting ribosomal specialization according to the state of development and the surrounding environment. During germination, protein synthesis is an active process that begins with the translation of the mRNAs stored in quiescent seeds and continues with the newly synthesized mRNAs. In this study, we identified differences in the abundance of ribosomal proteins (RPs) in maize embryos at different developmental stages. The relative quantification of RPs during germination revealed changes in six small subunit proteins, S3 (uS3), S5 (uS7), S7 (eS7), two isoforms of S17 (eS17), and S18 (uS13), and nine large subunit proteins, L1 (uL1), L5 (uL18), two isoforms of P0 (uL10), L11 (uL5), L14 (eL14), L15 (eL15), L19 (eL19), and L27 (eL27). Further analysis of ribosomal protein phosphorylation during germination revealed that the phosphorylation of PRP0 (uL10) and P1 increased and that of PRS3 (uS3) decreased in germinated versus quiescent embryos. The addition of insulin during germination increased the phosphorylation of the P1 protein, suggesting that its phosphorylation is controlled by the TOR pathway. Our results indicate that a heterogeneous ribosomal population provides to maize ribosomes during germination a different ability to translate mRNAs, suggesting another level of regulation by the ribosomes.