DEGRADATION OF RIBONUCLEIC ACID IN MOUSE FIBROBLASTS TREATED WITH ACTINOMYCIN

The cellular RNA content of mouse fibroblasts incubated with actinomycin decreases at a rate of about 1 to 1.5 per cent per hour, while DNA and protein content remain unchanged. This degradation affects nuclear and cytoplasmic RNA, ribosomal and soluble RNA. The breakdown products appear quantitatively in the acid-soluble fraction of the cells and the medium. Polynucleotides synthesized a short period (120 minutes) prior to exposure to actinomycin are degraded before those synthesized 8 to 12 hours previously.     

METABOLISM OF RIBOSOMAL PRECURSOR RIBONUCLEIC ACID IN KIDNEY

The labile precursors of ribosomal RNA in mouse kidney are preserved when nuclei rapidly isolated after sieving through multiple screens are swollen and cleansed in the presence of an RNase inhibitor before digestion with DNase and phenol extraction. The kinetics of nucleolar labeling analyzed on polyacrylamide gels show that 36S RNA is the major intermediate product in the catabolism of the original 45S RNA precursor to 32S RNA, from which 28S RNA is derived. Each kidney nucleus contains about 200–600 molecules of 45S RNA; the turnover time of the 45S pool is about 3 ± 2 min. Compared with HeLa cells, kidney nuclei have a different major intermediate product and a much smaller and more rapidly turning-over pool of ribosomal precursor RNA.     

RIBONUCLEIC ACID METABOLISM AND CELL EXPANSION IN OAT COLEOPTILE

RNA metabolism in oat coleoptiles was studied using physiologicalresponses to 5-FU and actinomycin D; autoradiographic detectionof RNA and protein synthesis; and estimation of ribosomal concentrationby analytical ultracentrifugation. 5-FU failed to inhibit growthof either intact coleoptiles or isolated coleoptile segmentsbut completely blocked cell division in roots. Actinomycin Dmarkedly inhibited auxin-induced expansion of coleoptile segments.When supplied to isolated segments from coleoptiles of variouslengths the RNA precursors cytidine, adenine and adenosine allshowed weak incorporation into RNA of nuclei and in some cases,to a lesser extent, RNA of cytoplasm. IAA did not affect thisRNA synthesis but it was considerably reduced by actinomycinD. A proportion of the label incorporated from RNA precursorswas not removable with either RNase, PCA or hot TCA but wasextracted by trypsin. The amount of this spurious incorporationincreased with coleoptile age, as did the ability to incorporatelabelled amino acids. The concentration of both free and boundribosomes does not increase in growing coleoptiles and may evendecline. Free ribosomes decline markedly in fully grown coleoptileswhile the proportion of bound ribosomes increases. It is concludedthat young coleoptiles contain a full complement of ribosomesnecessary for subsequent growth but normal growth is dependenton continued production of an actinomycin D-sensitive messenger-typeRNA. No evidence for auxin mediation of RNA synthesis was found. ¹Present address: Laboratory of Cell Biology, Faculty of Science,Osaka City University, Sugimoto-cho, Sumiyoshi-ku, Osaka, Japan.     

THE CONTROL OF THE ORIENTATION OF CELL DIVISIONS IN FERN GAMETOPHYTES

Time-lapse observations of filamentous fern gametophytes were used to evaluate whether the plane of cell division is referable to the plane of minimal surface area before and during the transition to two-dimensional growth. Cell dimensions of the apical cell were related to the length/width ratios associated with minimal area in the transverse plane vs. longitudinal plane, by modeling the apical cell as a hemisphere subtended by a cylinder. Our working hypothesis predicts that filamentous growth is perpetuated by an apical cell geometry that makes the transverse division plane the orientation of minimal surface area, whereas the transition to two-dimensional growth (longitudinal division of the apical cell) occurs once the longitudinal plane becomes the position of minimal surface area. The predictions of this hypothesis are fulfilled regardless of variations in light intensity and light quality, the presence of regulators of metabolism, or whether the experimental perturbation causes a corresponding selective inhibition of the transition to two-dimensional growth. Thus, the control of the plane of cell division in this system seems to depend on thermodynamic considerations of surface area. Furthermore, we favor the conclusion that the role of the genome in the transition to two-dimensional growth involves its influence on apical cell dimensions rather than the induction of specific genes for specific morphogenetic mRNAs.     

THE TRANSITION FROM FILAMENTOUS TO TWO-DIMENSIONAL GROWTH IN FERN GAMETOPHYTES. I. THE REQUIREMENT FOR PROTEIN SYNTHESIS IN GAMETOPHYTES OF PTERIDIUM AQUILINUM

Two-dimensional (2-D) development of gametophytes of Pteridium aquilinum was estimated by the percentage of plants with at least one cell wall at an oblique angle, relative to the long axis. Inhibition of cell division per se was assayed by counting the cell number per filamentous (1-D) gametophyte. Specific concentrations of 8-azaguanine, 5-fluorouracil, p-fluorophenylalanine, actinomycin D, streptomycin, and cycloheximide were found to selectively inhibit 2-D development. A reduction in the percentage of 2-D gametophytes was accompanied by a reduction in the relative protein content (protein per dry weight). A comparable association was found only during the initial stages of 2-D development; after 5 days the relative protein content decreased as the percentage of 2-D gametophytes increased. Different intensities of white light resulted in a 10-fold difference in the percentage of 2-D plants with no alteration of the relative protein content. These results demonstrate that no strict relationship between 2-D development and relative protein content occurred in these gametophytes.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. V. ETHYLENE AND THE EARLY EVENTS OF SPORE GERMINATION

Early events during the germination of spores of the fern Onoclea sensibilis were studied to determine the time during germination when ethylene had its greatest inhibiting effect. Water imbibition by dry spores was rapid and did not appear to be inhibited by ethylene. During normal germination DNA synthesis occurred about four hours before the nucleus moved from a central position to the spore periphery. Following nuclear movement, mitosis and cell division occurred, partitioning the spore into a small rhizoid cell and a large protonemal cell. Cell division was complete approximately six hours after nuclear movement. Ethylene treatment of the spores blocked DNA synthesis, nuclear movement, and cell division. The earliest DNA replication in uninhibited spores was observed after 14 hours of germination, and the maximal rate of spore labeling with ³H-thymidine was between 16 and 20 hours. Spores were most sensitive to ethylene, however, during the stages of germination prior to DNA synthesis, and it was concluded that ethylene did not directly inhibit DNA replication but blocked germination at some earlier fundamental step. The effects of ethylene were reversible. since complete recovery from inhibition of germination was possible if ethylene was released and the spores were kept in light. Recovery was much slower in darkness. It was hypothesized that light acted photosynthetically to overcome the ethylene inhibition of germination. Consistent with this, it was shown that spores exhibit net photosynthesis after only two hours of germination.     

EFFECTS OF NERVE STIMULATION ON THE METABOLISM OF RIBONUCLEIC ACID IN A MOLLUSCAN GIANT NEURONE

—In vitro experiments were performed in order to determine whether nerve stimulation would affect the RNA metabolism of an identified giant neurone (R2) in the abdominal ganglion of Aplysia californica. The electrophysiological activity of the neurone was continuously monitored with an intra- or extracellular microelectrode. The mere presence of an intracellular microelectrode inside the neurone had no significant effect on the incorporation of tritiated nucleosides into the RNA of the giant neurone. Prolonged electrical stimulation of ganglionic nerves, strong enough to elicit post-synaptic spikes in the giant neurone, produced a marked increase in the amount of labelled RNA in the nucleus as well as in the cytoplasm. Electrophoresis studies suggested that this increase in labelling might concern RNA with molecular weights corresponding to ribosomal as well as to non-ribosomal RNA.     

INCOMPATIBILITY OF MERISTEMATIC AND FILAMENTOUS GROWTH IN THE FERN GAMETOPHYTE

Gametophytes of Pteridium aquilinum can be maintained in red light as either 1- or 2-dimensional structures. The mode of growth realized in red light is dependent upon the activity of the meristem. An active meristem in a 2-dimensional structure will permit a continued development of that structure. A breakdown in meristematic activity results in filament formation. It is suggested that a group of actively dividing cells in some manner inhibits cell elongation and thus prevents filament formation in red light.     

BLUE LIGHT IN THE DEVELOPMENT OF FERN GAMETOPHYTES AND ITS INTERACTION WITH FAR-RED AND RED LIGHT

Two effects of blue light on the development of Onoclea sensibilis spores are demonstrated. Brief irradiation promotes the protonemal or filamentous type of growth, and the rate of filament elongation is greater than in darkness. Longer periods of irradiation induce the formation of 2-dimensional prothallia. Blue-light treatments which promote filament elongation interact with elongation-promoting far-red light. Far-red irradiation alone promotes filament elongation to a greater extent than blue light, but a blue-light irradiation, either following or preceding far-red treatment, strongly inhibits the far-red promotion. In darkness, a slow recovery from the blue-light-induced loss of sensitivity to far-red takes place. The recovery may be greatly accelerated by interposing a red-light treatment between blue and far-red irradiation.     

CHANGES IN PROTEIN BIOSYNTHESIS DURING GIBBERELLIC ACID INDUCED INDUCTION AND FORMATION OF ANTHERIDIUM IN THE FERN ANEMIA PHYLLITIDIS

Changes in protein biosynthesis have been studied during the induction and formation of antheridium in Anemia phyllitidis .
Based on incorporation of ¹⁴C-amino acid mixture into TCA precipitable material two distinct phases of accelerated protein biosynthesis were observed. First phase initiates at 4th day, while the second at 8th day of development. The first phase is likely associated with antheridium induction and second with spermatogenesis. From electrophoretic pattern of proteins on stained acrylamid gels and from radioactivity profiles of labelled proteins distinct quantitative differences between vegetative and reproductive prothalli were observed at different stages of antheridial development. Radioactivity profiles reveal characteristic pattern of each stage of antheridial differentiation.     

ORIENTATION OF THE PLANE OF CELL DIVISION IN FERN GAMETOPHYTES: THE ROLES OF CELL SHAPE AND STRESS

Apical cells of Onoclea sensibilis L. protonemata were measured to determine areas of new walls which were formed during both transverse and longitudinal cell division. Actual wall areas were compared with calculated areas of hypothetical walls oriented in the opposite sense (i.e., an actual transverse wall compared with a hypothetical longitudinal wall, and the reverse). Among 87 out of 90 cells which were analyzed the actual walls had the least area. Thus, the minimal area hypothesis of cell partitioning accurately predicts wall orientation in this instance, although it appears, on other grounds, that the hypothesis does not furnish a plausible mechanism for wall orientation. The application of Lintilhac's concept of the orientation of cell walls in response to anisotropic stresses in the cell was explored. Photographs of apical cells during deplasmolysis indicated that unequal stresses might be generated in apical cells as a result of the osmotic distension of the elastic protoplast. It is concluded that the primary factor which determines the plane of cell division in the apical cell, and the transition from one- to two-dimensional growth, is the local pattern of stress which exists at the position of the nucleus at the time of onset of cell division and wall formation. Calculations of some geometrical properties of idealized model cells are interpreted to mean that the accuracy of the minimal area hypothesis results from a coincidence of its predictions with predictions of Lintilhac's hypothesis, and no causal significance is attributed to wall areas.     

INTERACTION OF LIGHT QUALITY AND NUCLEASES IN THE GROWTH OF THE GAMETOPHYTES OF ASPLENIUM NIDUS

Appropriate concentrations of ribonuclease A and B selectively inhibited initiation of two-dimensional morphology in the gametophytes of Asplenium nidus, grown under a photoperiod of 5½ hr white light. Filamentous growth was promoted in such sporelings, the individual cells of which were significantly longer than corresponding cells of the control. Higher concentrations of enzymes were required to inhibit two-dimensional growth in the gametophytes grown in blue light. Concentrations of ribonuclease A or B which inhibited two-dimensional growth in white light promoted growth in length of the protonema in red light. Growth modifications in the sporelings induced by deoxyribonuclease in different light conditions were similar to those induced by the ribonucleases. The results lend further support to the postulated role of RNA in the regulation of two-dimensional growth in fern gametophytes.     

CHANGE IN ENZYME ACTIVITY AND RIBONUCLEIC ACID CONCENTRATION WITHIN THE EPIDERMAL CELL OF THE RAT DURING THE GROWTH STAGE OF THE HAIR CYCLE

The rat hair cycle produces rapid anatomical and physiological changes in the entire skin. By the 4th day of the cycle, the epidermis has doubled in thickness. The present study shows that, during this proliferative period, there is a correlation between the biological state (growth) and the molecular composition (enzyme concentrations) of the epidermal cell. In proliferated 4th day epidermis there has occurred not only an increase in the number of viable cells but also an increase in succinic dehydrogenase and cytochrome oxidase activity and ribonucleic acid (RNA) content in the viable epidermal cell. Moreover, RNA content and enzyme activities appear to be correlated. The significance of these findings is discussed in the light of recent evidence supporting the relationship between RNA and enzyme synthesis and the role of hormones in the induction of enzyme adaptation.     

THE TRANSITION FROM FILAMENTOUS TO TWO-DIMENSIONAL GROWTH IN FERN GAMETOPHYTES. IV. INITIAL EVENTS

Time-lapse photomicrography and autoradiography were used to study the initial events leading to the first cell division following transfer from red to white light. The first responses to white light, an apical swelling and a reduced rate of elongation, were often observed within the first 1–2 hr after the transfer; these two changes were not always initiated at the same time. Only the dome region of the cell participated in the elongation in red, as demonstrated by the displacement of anion exchange resin markers. The dome region as well as the region proximal to the dome was associated with the apical swelling, including regions formed in red. The average duration of the G1, S, G2, and M phases was found to be approximately 4, 3, 3.5, and 1 hr, respectively.