RHIZOID CELL DIFFERENTIATION IN THE FERN GAMETOPHYTE OF PTERIS VITTATA

A series of events in the differentiation of rhizoid cells in the gametophyte of Pteris vittata L. is described. Differential in vivo staining reactions make it possible to trace a sequence of stages from pre-mitotic nucleocytoplasmic staining (corona stage), through the events of mitosis, to formation of an internally segmented rhizoid cell which then grows out from the parent thallus cell. When the standard ribonuclease test is employed, basophilic protoplasmic staining due to pyronin is prevented in developmental stages up through early formation of the rhizoid cell. Staining in these stages is therefore assumed to be due to ribonucleic acid associated with protein synthesis. A new developmental phase starts just prior to the protrusion growth of this cell when an intensely pyroninophilic material appears at the membrane of the rhizoid cell nucleus and along cytoplasmic strands radiating to the peripheral regions of the cell. The staining of this newly synthesized material cannot be prevented by ribonuclease treatment, and it shows strong positive tests for protein and polysaccharide. As long as the rhizoid continues to grow, this material remains concentrated in the growing tip region. Material showing similar staining reactions is also found in the cells of the meristematic notch area. Although thallus cell walls do not stain, the rhizoid cell wall adsorbs basic stain, in some cases, metachromatically. Finally, it is suggested on the basis of observations reported here that the sometimes neglected role of the nucleus in theories of unequal cell division-differentiation should be re-examined.     

Lack of correlation between overall protein synthesis and the onset of cell elongation in germinating embryos of Haplopappus gracilis

10⁻⁵M abscisic acid (ABA) completely inhibits germination or (if seeds deprived of integuments are used) embryo elongation in Haplopappus gracilis (Nutt.) Gray. Nevertheless, considerable rates of protein and RNA synthesis were found in embryos grown in abscisic acid, at least during the early hours after sowing. On the contrary, seeds grown in cycloheximide + fusicoccin (a powerful promoter of cell expansion), where protein synthesis is almost completely inhibited, show full protrusion of radicle, thus simulating a "germination" process. These results suggest that some of the most important events involved in seed germination, i.e. protein and RNA synthesis, and cell elongation which leads to radicle protrusion, may not necessarily be linked together and are possibly regulated by different control mechanisms. Moreover, when seeds or embryos are grown in abscisic acid + fusicoccin, protein synthesis is considerable, cell elongation is greater than in water controls at least for 12 h, and germination in its early stages appears to be normal; but DNA synthesis and cell division are not resumed, possibly since some other factor is required. All these findings propose a reevaluation of criteria for defining successful germination.     

SPORE GERMINATION AND DEVELOPMENT OF THE YOUNG GAMETOPHYTE OF THE OSTRICH FERN (MATTEUCCIA STRUTHIOPTERIS)

Light is required for the germination of spores of Matteuccia struthiopteris. Histochemical studies show that dormant spores contain no starch, but have an abundance of storage protein granules. Starch accumulates in the numerous chloroplasts of the spore on exposure to light and becomes gradually more extensive. Protein granules disappear as germination progresses. Following this, the centrally located nucleus migrates toward the proximal spore face. Concomitant with the nuclear migration, an increase of cytoplasmic RNA surrounding the nucleus occurs. An equal nuclear division and unequal cell division give rise to a 2-celled gametophyte consisting of a large prothallial cell and smaller rhizoidal cell. A new peripheral wall forms around the entire protoplast at the time of nuclear migration, while a transverse wall forms after nuclear division. The rhizoid emerges through the split raphe along the proximal spore face; it is rich in cytoplasmic RNA but contains very few chloroplasts and little starch. Electron microscopy of the 2-celled stage revealed a greater concentration of mitochondria, Golgi bodies, and a more extensive endoplasmic reticulum in the rhizoid than was found in the prothallial cell, which, however, was far richer in chloroplasts and lipid bodies. As the rhizoid elongates and becomes more vacuolated, cytoplasmic RNA decreases as cytoplasmic protein increases. The rhizoid undergoes no cell divisions, while the prothallial cell retains the potential for further cell division. The possible significance of the distribution of storage products, cell organelles, and other cell components were considered in relation to the non-equational cell division and differentiation of the 2 cells.     

REFORMATION OF NUCLEOLUS-LIKE BODIES IN THE ABSENCE OF POSTMITOTIC RNA SYNTHESIS

The dependence of nucleolar reformation on RNA synthesis that resumes in late anaphase or early telophase has been investigated in synchronously dividing Amoeba proteus. RNA synthesis was completely inhibited throughout all stages of mitosis and the early hours of interphase with high concentrations of actinomycin D. In such cells, nucleolus-like bodies that bind azure B and pyronin were apparent in the reformed nuclei. The bodies appear as dense, fibrous masses with loosely associated, finely fibrillar material. There are no characteristic granular regions in the reformed structures. It is suggested that the bodies probably represent mainly nucleolar protein and residual RNA which can bring about the reorganization of nucleoli in the absence of postmitotic RNA synthesis.     

Intracellular transport of nuclear ribosomal RNA in Acetabularia mediterranea]

The ribosomal RNA transport from a nucleus to a perinuclear cytoplasm and its following distribution in the cytoplasm of Acetabularia mediterranea cells were studied using transplantation of RNA-labeled rhizoid into unlabeled stalk. In addition rifamycin treatment was used for inhibition of cytoplasmic RNA synthesis. Acetabularia nuclei contain the stable RNA fractions similar to those present in some other eukaryotes. Nuclear 25S and 17S ribosomal RNA rapidly enter the rhizoid cytoplasm whereas the following trasfer of them to other regions of the cell is a very slow process. Within two days only an insignificant part of 25S and 17S ribosomal RNA is transferred from the rhizoid to the stalk and is distributed there over the base-apical gradient. No preferential transfer of the nuclear ribosomal RNA to the apical region was observed.     

RIBONUCLEIC ACID SYNTHESIS DURING MITOSIS AND MEIOSIS IN THE MOUSE TESTIS

The pattern of ribonucleic acid synthesis during germ cell development, from the stem cell to the mature spermatid, was studied in the mouse testis, by using uridine-H³ or cytidine-H³ labeling and autoradiography. Incorporation of tritiated precursors into the RNA occurs in spermatogonia, resting primary spermatocytes (RPS), throughout the second half of pachytene stage up to early diplotene, and in the Sertoli cells. Cells in leptotene, zygotene, and in the first half of pachytene stage do not synthesize RNA. No RNA synthesis was detected in meiotic stages later than diplotene, with the exception of a very low rate of incorporation in a fraction of secondary spermatocytes and very early spermatids. At long intervals after administration of the tracer, as labeled cells develop to more mature stages, late stages of spermatogenesis also become labeled. The last structures to become labeled are the residual bodies of Regaud. Thus, the RNA synthesized during the active meiotic stages is partially retained within the cell during further development. The rate of RNA synthesis declines gradually with the maturation from type A to intermediate to type B spermatogonia and to resting primary spermatocytes. "Dormant" type A spermatogonia synthesize little or no RNA. The incorporation of RNA precursors occurs exclusively within the nucleus: at later postinjection intervals the cytoplasm also becomes labeled. In spermatogonia all mitotic stages, except metaphase and anaphase, were shown to incorporate uridine-H³. RNA synthesis is then a continuous process throughout the cell division cycle in spermatogonia (generation time about 30 hours), and stops only for a very short interval (1 hour) during metaphase and anaphase.     

The Effects of Centrifugation on Asymmetric Cell Division and Differentiation of Fern Spores

We have investigated the effects of centrifugation on sporepolarity, asymmetric cell division, and rhizoid differentiationin the sensitive fern Onoclea sensibilis L. Centrifugation at10000 g for 30 min produces a random orientation of spores withstratification of the cell contents. After centrifugation atmost early stages of development, the nucleus retains its normalpattern of migration from the centre of the ellipsoidal sporeto the proximal face and then to an end of the spore, withoutregard to the orientation of stratification. This indicatesthat the polarity of the spore is stable to centrifugation.As long as the nucleus migrates to an end of the spore and asymmetriccell division occurs, the small cell differentiates into a rhizoid.The arrangement of large cytoplasmic organelles appears to haveno influence on nuclear migration, asymmetric cell division,or rhizoid differentiation. The only period during developmentwhen centrifugation blocks asymmetric cell division is immediatelypreceding and during mitosis and cytokinesis. Spores centrifugedat this stage do not complete nuclear migration, and symmetriccell division results, with neither cell differentiating intoa rhizoid. The source of the stable polarity of the spore isdiscussed. cell polarity, rhizoid differentiation, centrifugation, Onoclea sensibilis L., sensitive fern, fern spores     

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     

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.     

Reactivation of Ribonucleic Acid and Protein Synthesis During Germination of Blastocladiella Zoospores and the Role of the Ribosomal Nuclear Cap

Freshly harvested zoospores of Blastocladiella emersonii begin to germinate about 15 min after inoculation into a defined growth medium at a density of 10(6) zoospores per ml. Flagellum retraction accompanies encystment, and dispersal of the ribosomal nuclear cap takes place shortly thereafter. The primary rhizoid begins to emerge at 25 to 30 min and starts to branch at ca. 60 min. The first nuclear division occurs between 120 and 190 min. The dry weight per cell increases linearly after 60 min, whereas the deoxyribonucleic acid per cell doubles between 120 and 240 min. A linear increase in total ribonucleic acid (RNA) is detectable beginning at 40 to 45 min, and in total protein beginning at 80 min; neither process is interrupted during nuclear division. Encystment and nuclear cap disorganization are associated with a sharp rise in the rates of precursor incorporation into RNA and protein. Cycloheximide at 20 mug/ml prevents leucine incorporation at all stages and inhibits development beyond the earliest encystment stage. Actinomycin D at 25 mug to 50 mug/ml prevents uracil incorporation, but it has no effect on leucine incorporation or development until 40 to 45 min. At the latter stage, actinomycin D causes a sharp developmental arrest and begins to inhibit leucine incorporation. It is concluded that early protein synthesis must occur on the ribosomes formed during the prior growth phase and conserved through the zoospore stage in the nuclear cap. The results further indicate that this synthesis is dependent upon messenger RNA already present in the zoospore before germination.     

Messenger RNA synthesis during early ascidian development.

RNA synthesis during early embryogenesis of the ascidian Ciona intestinalis was studied. Embryonic polyribosomes labeled with uridine from 5 to 7 hr after fertilization were isolated and the labeled RNA species were characterized by oligo(dT)-cellulose chromatography and sucrose gradient sedimentation analysis. Since at least 50% of the labeled RNA was polyadenylated and all of it sedimented heterogeneously, it was concluded that mRNA was synthesized during the labeling period. Further, the synthesis of heterogeneously sedimenting, polyadenylated RNA at various stages of development from midcleavage to metamorphosis indicated that gene activity and perhaps mRNA synthesis occurred at earlier and later stages of development as well. Autoradiographic studies showed that the embryonic genome was the site of this activity, since uridine incorporation was localized in embryonic cells and not in accessory cells. Finally, under the labeling conditions employed (2-hr pulses), rRNA synthesis was not detected until larvae hatched.     

Temporal and spatial immunolocalization of glucomannans in differentiating earlywood tracheid cell walls of Cryptomeria japonica

We investigated the deposition of glucomannans (GMs) in differentiating earlywood tracheids of Cryptomeria japonica using immunocytochemical methods. GMs began to deposit at the corner of the cell wall at the early stages of S₁ formation and showed uneven distribution in the cell wall during S₁ formation. At the early stages of S₂ formation, limited GM labeling was observed in the S₂ layer, and then the labeling increased gradually. In mature tracheids, the boundary between the S₁ and S₂ layers and the innermost part of the cell wall showed stronger labeling than other parts of the cell wall. Deacetylation of GMs with mild alkali treatment led to a significant increase in GM labeling and a more uniform distribution of GMs in the cell wall than that observed before deacetylation, indicating that some GM epitopes may be masked by acetylation. However, the changes in GM labeling after deacetylation were not very pronounced until early stages of S₂ formation, indicating that GMs deposited in the cell wall at early stages of cell-wall formation may contain fewer acetyl groups than those deposited at later stages. Additionally, the density of GM labeling increased in the cell wall in both specimens before and after GM deacetylation, even after cell-wall formation was complete. This finding suggests that some acetyl groups may be removed from GMs after cell-wall formation is complete as part one of the tracheid cell aging processes.     

Expression of the mitochondrial genome in HeLa cells. V. Transcription of mitochondrial DNA in relationship to the cell cycle

An investigation of the rate of incorporation of [5-³H]ur dine into mitochondrial RNA in synchronized HeLa cells in different phases of the cell cycle has revealed a considerable acceleration of this incorporation in cells in S and especially in G₂ phase. An analysis of the labeling of the intramitochondrial UTP pool has shown that this acceleration reflects a true increase in the rate of synthesis of mitochondrial RNA: this increase is considerably greater than can be accounted for by the expected doubling of mit-DNA^‡ templates during the S and G₂ phases.     

The cell cycle-apoptosis connection revisited in the adult brain

Adult neurogenesis is studied in vivo using thymidine analogues such as bromodeoxyuridine (BrdU) to label DNA synthesis during the S phase of the cell cycle. However, BrdU may also label DNA synthesis events not directly related to cell proliferation, such as DNA repair and/or abortive reentry into the cell cycle, which can occur as part of an apoptotic process in postmitotic neurons. In this study, we used three well-characterized models of injury-induced neuronal apoptosis and the combined visualization of cell birth (BrdU labeling) and death (Tdt-mediated dUTP-biotin nick end labeling) to investigate the specificity of BrdU incorporation in the adult mouse brain in vivo. We present evidence that BrdU is not significantly incorporated during DNA repair and that labeling is not detected in vulnerable or dying postmitotic neurons, even when a high dose of BrdU is directly infused into the brain. These findings have important implications for a controversy surrounding adult neurogenesis: the connection between cell cycle reactivation and apoptosis of terminally differentiated neurons.     

MACROMOLECULAR EVENTS LEADING TO CELL DIVISION IN TETRAHYMENA PYRIFORMIS AFTER REMOVAL AND REPLACEMENT OF REQUIRED PYRIMIDINES

Tetrahymena pyriformis were brought to a non-growing state by removal of pyrimidines from their growth medium. During pyrimidine deprivation cell number increased 3- to 4 fold, and this increase was accompanied by one or more complete cycles of macronuclear DNA replication. Autoradiographic studies show that endogenous protein and RNA were turning over throughout starvation and that RNA breakdown products were used to support the DNA synthesis that occurred during the early period of starvation. However, after 72 hours of starvation all DNA synthesis and cell division had ceased. Feulgen microspectrophotometry shows the macronuclei of these cells to have been stopped at a point prior to DNA replication (G1 stage). After pyrimidine replacement the incorporation of H³-uridine, H³-adenosine, and H³-leucine was measured by the autoradiographic grain counting method. The results indicate that RNA synthesis began to increase almost immediately, but that there was a lag of almost an hour before an increase in protein synthesis. In agreement with the autoradiographic data, chemical data also show that cellular content of RNA began to increase shortly after pyrimidine replacement but that cellular protein content did not increase until about one hour later. Pulse labeling of the cells with H³-thymidine at intervals after pyrimidine replacement shows that labeled macronuclei first began to appear at 150 minutes; that 98 per cent of the macronuclei were in DNA synthesis at 240 to 270 minutes; and that the percentage then began to decrease from 300 to 390 minutes, at which time only 25 per cent of the macronuclei were labeled. Cellular content of DNA did not increase for at least 135 minutes after pyrimidine replacement; however, just before the first cells divided (360 minutes) the DNA content had doubled. After pyrimidine replacement the cells first began to divide at 360 minutes, and 50 per cent had divided at 420 minutes; however, all cells had not divided until 573 minutes. This technique of chemical synchronization of cells in mass cultures makes feasible detailed biochemical analysis of events leading to nuclear DNA replication and cell division.     

THE RELATIONSHIP BETWEEN RNA SYNTHESIS AND HEMOGLOBIN SYNTHESIS IN AMPHIBIAN ERYTHROPOIESIS : Cytochemical Evidence

A cytochemical study of the relationship between RNA synthesis and hemoglobin synthesis has been performed on splenectomized newts, Triturus viridescens. Employing radioautography, labeled cytidine was incorporated into the RNA of the early developmental stages but was not incorporated in the later stages. Labeled leucine was incorporated into the cellular protein of all stages except mature erythrocytes but was incorporated at a higher level in the later stages. Microphotometric measurements of azure B binding to cytoplasmic RNA revealed a sharp initial increase between the stem cell and proerythroblast followed by a rapid decrease between the basophilic and polychromatophilic stages. The loss of cytoplasmic RNA became more gradual in the late stages and, in the mature erythrocyte, little or no cytoplasmic RNA could be detected. Measurements of cytoplasmic total protein, using fast green staining at pH 2.0, and of heme showed that both curves increased similarly with development, indicating net hemoglobin synthesis. The results are compatible with the hypothesis that, as the stem cell differentiates along erythrocytic lines, a stable "messenger" RNA specifying the production of a given type or types of hemoglobin is formed. This complex probably becomes associated with ribosomal RNA and is retained throughout the process of RBC differentiation.     

NUCLEOLAR AND NUCLEAR RNA SYNTHESIS DURING THE CELL LIFE CYCLE IN MONKEY AND PIG KIDNEY CELLS IN VITRO

The incorporation of 5-³H-uridine and 5-³H-cytidine into nucleolar and nonnucleolar RNA in the nucleus of monkey and pig kidney cells was measured in vitro during the cell life cycle. Time-lapse cinematographic records were made of cells during asynchronous exponential proliferation, in order to identify the temporal position of individual cells in relation to the preceding mitosis. Immediately following cinematography, cells were labeled with uridine-³H and cytidine-³H for a short period, fixed, and analyzed by radioautography. Since the data permit correlation of the rate of RNA labeling with the position of a cell within the cycle, curves could be constructed describing the rate of RNA synthesis over the average cell cycle. RNA synthesis was absent in early telophase, and rose very abruptly in rate in late telophase and in very early G₁ in both the nucleus and the reconstituting nucleolus. Thereafter, through the G₁ and S periods the rate of nuclear RNA synthesis rose gradually. When we used a 10-min pulse, there was no detectable change in the rate for nucleolar RNA labeling in monkey kidney cells during G₁ or S. When we used a 30-min labeling time, the rate of nucleolar RNA labeling rose gradually in pig kidney cells. With increasing time after mitosis, the data became more variable, which may, in part, be related to the variation in generation times for individual cells.