Cytochemical and Biochemical Analysis of Development of Urechis Oocytes

The echiuroid marine worm Urechis caupo is uniquely suited forthe study of oogenesis. A relatively large quantity of oocytesat various developmental stages can be obtained and subjectedto coordinated cytochemical and biochemical analysis Oocytesat the cluster, early diplotene, mid-diplotene, and diffusediplotene or lampbrush stages are active in the synthesis andaccumulation of ribosomal RNA, several proteins, carbohydrates,lipids, and also, perhaps, yolk constituents. Only corticalgranule formation, which occurs during later stages of oogenesis,appears to be stage specific. Ribosomal RNA genes are also transcribedin the nucleolus of the mature oocytes or unfertilized eggs.However, the rate of production in these eggs appears to beregulated at the level of maturation of rRNA precursor molecules.     

Nucleic Acid, Protein, and Starch Synthesis in Developing Cotyledons of Pisum arvense L.

During the cell-division period of cotyledon development inPisum arvense L. cell volume increases slightly but nuclearvolume shows little variation and the DNA content remains atthe 2C to 4C level. During the main period of cell expansionthere is a close correlation between cell volume, nuclear volume,and nuclear DNA content, the nuclei of the largest storage cellsfinally attaining the 64C level. The rate of RNA synthesis increasesseveral days after the increase in DNA has begun and at thesame time accumulation of reserve protein and starch begins.RNA and starch synthesis apparently cease some time before maturationbut protein synthesis continues until the seeds are ripe. Cotyledondevelopment was found to comprise two distinct phases: an initialphase of cell division and differentiation during which DNA,RNA, and protein per unit volume of cell decline; and a phaseof reserve accumulation in which DNA per unit volume of cellremains constant but RNA and protein per unit volume increase,starch synthesis is initiated, and all the cotyledon cells assumethe properties of storage cells.     

The Concept of Messenger RNA and Cytodifferentiation

The messenger RNA hypothesis serves as a concrete model of genefunction which may be of real use to the developmental biologist.The hypothesis posits that the product of a gene is an RNA moleculewith a base sequence complementary to the sequence of nucleotidebases in the DNA. The operations and restrictions which definea messenger are discussed. Animal and embryonic cells, whileconforming in general to the predictions, display certain variationsfrom the original hypothesis, such as difficulties in obtainingdirected synthesis of protein in vitro, greater stabilityofthe messenger RNA, association of active ribosomes with lipoproteinmembranes, and a possible heterogeneity of ribosomes. Some recent studies on messenger RNA in sea urchins and frogembryos are discussed, with special reference to the natureand importance of the RNA synthesized during cleavage, the significanceof new ribosome synthesis, and the potential importance of controlmechanisms at the 'translational level for regulation of newprotein synthesis The latter point is illustrated further by a discussion of theinitiation of synthesis of hemoglobin in the chick embryo. Theapplication of drugs and antimetabolites which derange and inhibitRNA synthesis (actinomycin, 8-azaguanine, 5-fluorouracil, and5-bromodeoxyuridine) shows that 8 hours prior to its onset,the synthesis of hemoglobin is independent of the synthesisof RNA of high molecular weight. The initiation of hemoglobinsynthesis is probably regulated at the translational level.Preliminary experiments using the heme precursor, delta-amino-levulinicacid, show that heme may be limiting for the onset of hemoglobinsynthesis.     

RNA and Protein Synthesis in Germinating Pine Pollen

The results of autoradiographic experiments demonstrate that,as with the pollen of most other species, both the generativeand vegetative nuclei of Loblolly Pine (Pinus taeda) activelyengage in RNA synthesis from the very early stages of pollengermination. Unlike most other species, however, this newlysynthesized RNA includes rRNA. Evidence is provided for theimportance of this newly synthesized RNA in the process of continuedpollen tube growth. One and two-dimensional gel electrophoretic analysis revealsa number of both qualitative and quantitative differences amongthe proteins synthesized during the early stages of germinationand the later stages of pollen tube growth. One of the mostnotable of these is a 36 kD protein, the synthesis of whichpredominates during the later stages of pollen germination.A similar pattern of 36 kD protein synthesis is observed whenmRNA extracted from pollen at each of these stages is translatedin vitro. Key words: Pinus, pollen tube growth     

RNA Synthesis in the Shoot Apex of Polytrichum formosum Hedw.

RNA metabolism in the cells of the gametophyte shoot apex ofPolytrichum formosum was investigated using both microspectrophotometricand autoradiographic methods along with an accurate measurementof surfaces and volumes of nuclei, nucleoli, free cytoplasmand vacuolar systems. On a per cell basis, the amount of RNAand the rate of RNA synthesis were shown to be highest in theapical cell. On the other hand, both RNA concentration and rateof synthesis for a unit quantity of cytoplasm were found tobe higher in leaf initials and in the cells of young leavesthan in theapical cell, the segments and the segmental derivatives.For the various types of cells in the shoot apex it was establishedthat the more voluminous the nucleolus, the higher the RNA syntheticrate per cell. These results were correlated with the data previouslyobtained on the mitotic cycles in Polytrichum. It is concludedthat in the apical cell, notwithstanding its differentiatedfeatures, RNA metabolism must be considered on the whole tobe very active. These various data are compared with those obtainedon angiosperm shoot apices.     

Macromolecular changes and commitment to sporulation in the fission yeast Schizosaccharomyces pombe

Homothallic cultures of Schizosaccharomyces pombe in stationaryphase may be induced to flocculate by aeration. Flocculationis followed by copulation, conjugation, zygote formation, meiosisand sporulation. This developmental sequence was monitored forrespiratory activity, changes in protein, RNA and DNA, cataboliterepression, and commitment to sporulation. Respiratory activity,apparently a prerequisite to induction, increased 5-fold priorto maximum flocculation and remained at that level up to theend of the sequence. Protein and RNA content increased priorto conjugation but gradually decreased shortly thereafter. Around of premeiotic DNA synthesis occurred after copulation,presumably during conjugation. The developmental sequence wasrepressible by glucose. Cyclic AMP at low concentrations stimulatedsporulation somewhat, but the stimulatory effect was not sufficientto offset repression due to glucose. Commitment to sporulationwas determined by adding glucose at various times during thedevelopmental sequence and then observing refractoriness ofthe events to catabolite repression. Cells not committed wererepressed by glucose and reverted to mitotic cell cycles. Committedcells proceeded to sporulate in the presence of exogenous glucose.Commitment to sporulation appears to occur soon after premeioticDNA synthesis. ¹NRCC No.: 18664. (Received February 22, 1980; )     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA     

Macromolecular Changes Associated with the Growth of Crustacean Tissues

Tissue mass, rate of protein synthesis, content of ribosomalRNA and rates of synthesis of ribosomal RNA have been studiedthroughout the molting cycle in the midgut gland, epithelium,and somatic muscle in the land crab, Gecarcinns lateralis. Inall tissues there is an increase in ribosomal RNA followed byan increase in the rate of synthesis of protein in the premoltperiod. Subsequently, the three tissues differed in that (a)in the midgut gland the level of ribosomal RNA and protein synthesisreturned to the intermolt rates before ecdysis whether or notthe mass of the tissue was increasing or decreasing; (b) ribosomalRNA and protein synthesis in epithelium reached a maximum ata time when epithelial cells reached a maximal size; subsequently,all three parameters decreased toward intermolt levels beforeecdysis; (c) ribosomal RNA and protein synthesis reached a maximumin the premolt period in somatic muscle while the muscle wasin fact decreasing in mass. Muscle ribosomes are very stableand appear to be conserved for weeks or months to be reusedafter ecdysis in a second burst of protein synthetic activityat the time when there is replacement and growth of new musculartissue. The relation of these events with hormonal control ofgrowth is discussed.     

DNA Synthesis and Cell Division During Spore Germination in Lygodium japonicum

This paper describes the ontogenetic sequence of cell divisionsand associated DNA synthetic patterns observed in sectionedspores of Lygodium japonicum (Thunb.) Sw., collected at differentstages of germination. Following exposure to a saturating doseof red light, the spore undergoes an asymmetric division toform a basal cell, which retains nearly all of the storage inclusions,and an apical cell which expands and protrudes from the rupturedsporoderm. Division of the apical cell results in formationof a protonemal cell and an intermediate cell. Subsequently,the latter cell divides to form the primary rhizoid and a wedgecell adjacent to the protonemal cell. Secondary rhizoids mayarise from later divisions of either the basal cell or the wedgecell. In addition, the wedge cell appears to have the capacityto form a secondary prothal-lial filament. Histochemical localizationof cell constituents indicates an increasing concentration ofcytoplasmic RNA and protein in the presumptive protonemal regionof the spore cell prior to division. Autoradiography of ³H–thymidineincorporation has shown that synthesis of nuclear DNA precedeseach cell division. Although strictly nuclear DNA synthesisoccurs during early stages of germination, extra-nuclear DNAsynthesis increases greatly following division of the sporecell. The results are discussed in relation to earlier studieson cell division patterns seen in whole mount preparations ofgerminating spores of different species of Lygodium. Lygodium japonicum, spore germination, cell division, DNA synthesis     

Synthesis of the small molecular weight monodisperse nuclear RNA's in contact-inhibited cell cultures

The small molecular weight monodisperse nuclear RNA's are synthesized in contact-inhibited cultures of 3T3 cells. The level of synthesis of these RNA's, and of ribosomal and transfer RNA's, appears to be only 8–20% of that observed in growing cultures. The synthesis of all of these relatively stable RNA species may thus be coordinately controlled.     

Protein and RNA Content and Synthesis in Embryos and Endosperms from Developing Triticum duram Seeds

Protein and RNA contents and synthesis were evaluated in the course of wheat grain (T. durum cv. Cappelli) development. Embryos and endosperms were considered separately during five phases from the 20th day after anthests until full ripenes was reached. No clean-cut changes were observed in the pattern of soluble proteins of the embryos. In the endosperms protein synthesis continues till the later phases and appears to be due to the albumin + globulin component. Screening of bands of endosperm proteins from electrophoresis indicates that the gliadins are synthesized early, with the exception of a Ω - gliadin. Glutelins with high relative molecular mass also appear to be synthesized when the grain approaches full ripeness. The RNA content of the embryo and the endosperm is high in the early stages, when high cell proliferation occurs, and declines later on. The synthesis of RNA during in vitro imbibition is, however, higher in the later phases of ripening. Most of RNA synthesized in the embryos was ribosomal. To whom correspondence should be sent.     

In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA.

Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.     

Structure and Function of the Golgi Complex in Rice Cells: Characterization of Golgi Membrane Glycoproteins

The structure and synthesis of the saccharide chains of Golgimembrane glycoproteins in suspension-cultured rice (Oryza sativaL.) cells were studied. Peanut lectin (PNA) and Ulex europaeuslectin-I (UEA-I) have high affinity for typical O-linked saccharidechains and both recognized the saccharide chains of rice Golgimembrane glycoproteins. These glycoproteins were also sensitiveto alkali and to O-glycanase. These results indicate that theGolgi membrane glycoproteins have O-linked saccharide chains.Brefeldin A, a specific inhibitor of Golgi-mediated secretion,induced morphological changes in Golgi complexes and preventedthe synthesis of the saccharide chains of the membrane glycoproteinsthat could be recognized by PNA and UEA-I. These glycoproteinswere typically localized in all compartments of the Golgi complex.Monensin can arrest the transport of secretory proteins frommedial to trans Golgi compartments but did not affect the formationand localization of the Golgi membrane glycoproteins. Tunicamycin,an inhibitor of the synthesis of N-linked saccharide chains,did not inhibit the synthesis of the saccharide chains of theseGolgi membrane glycoproteins. These results strongly suggestthat the synthesis of O-linked saccharide chains of Golgi membraneglycoproteins is initiated in the cis Golgi compartment. (Received September 24, 1992; Accepted June 4, 1993)     

Effect of amino acid and serum deprivation on the regulation of RNA synthesis in cultured Chinese hamster ovary cells

In a proline-requiring Chinese hamster ovary cell line, if both proline and serum are removed from the culture medium, net RNA synthesis is reduced to about 12 % of the unstarved control. This reduction in RNA synthesis is comparable to the stringent regulation of RNA in bacteria. A beta-globulin carbohydrate containing (3.5 % $\text{w}\text{w}$) protein factor was isolated and partially purified from fetal calf serum. The isolated serum factor is able to replace whole serum in stimulating cellular RNA synthesis and has an RNAase inhibitory effect in vitro. The effect of proline starvation and serum factor deprivation on RNA synthesis are independent and additive; each regulates about half of the total RNA synthesized. The regulation appears to affect the synthesis of all species of cytoplasmic RNA.     

The Formation of Polyploid Cells in Ripening Cotyledons of Pisum sativum L. in Relation to Ribosome and Protein Synthesis

The present paper deals with the formation of polyploid nucleiand the synthesis of RNA and protein in the parenchyma cellsof developing cotyledons of Pisum sativum L. During seed formationthese cells synthesize large amounts of reserve proteins andstarch, which later on are used up by the embryo during seedgermination. The changes of the amount of DNA per cell in the ripening cotyledontissue have been estimated by Feulgen histophotometry. The amountsof DNA, RNA, and protein in the whole cotyledons have been estimatedby chemical methods. In this way it was possible to correlatethe fluctuations of the amount of DNA, RNA, and protein withchanges at the cellular level. During a preparatory phase, preceding the phase of real cellexpansion and intensive accumulation of seed globulins and starch,the storage cells attain a high level of polyploidy: nucleiwith up to a 64 C DNA content are formed. The results indicate a correlation between the high degree ofpolyploidy in the parenchyma cells of the cotyledon and thehigh rate of RNA and protein synthesis (seed globulins) in thisstorage organ (gene dosage effect).     

Changes in the Synthesis of RNA and Protein during Tracheary Element Differentiation in Single Cells Isolated from the Mesophyll of Zinnia elegans

Cycloheximide (CH) prevented tracheary element (TE) differentiationand cell division in a culture of single cells isolated fromthe mesophyll of Zinnia elegans at the concentrations whichinhibited incorporation of [¹⁴C]-leucine into protein. Whenthe cells were pulse-treated with this inhibitor for 12 h atvarious times of culture, TE formation was inhibited most stronglyby the treatments made between 24 and 60 h of culture. Incorporationof [¹⁴C]-leucine into protein showed a high level during thisperiod. The inhibitory effect of actinomycin D (Act-D) on TEdifferentiation was also marked when it was administered from24 to 60 h of culture when incorporation of [¹⁴C]-uridine intonucleic acid was at a high level. These results indicate thatRNA and protein syntheses are prerequisites for cytodifferentiationto TE and that the syntheses between 24 and 60 h of cultureare closely associated with cytodifferentiation. Studies of qualitative changes in proteins using two-dimensionalelectrophoresis revealed that approximately 400 polypeptidesextracted from [³⁵S]-methionine-labeled cells could be reproduciblyresolved and that most of them were synthesized in both differentiatingand non-differentiating cells. During TE differentiation, however,the synthesis of two polypeptides was shut off and two otherpolypeptides were newly synthesized between 48 and 60 h of culture,preceding the morphological changes. The relationship betweenTE differentiation and the synthesis of RNA and protein is discussed. (Received November 20, 1982; Accepted February 18, 1983)