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.     

ACTION OF PURINE AND PYRIMIDINE ANALOGS ON THE GROWTH AND DIFFERENTIATION OF THE GAMETOPHYTES OF THE FERN ASPLENIUM NIDUS

The purine analogs, 8-azaadenine, 8-azaguanine, 8-azaxanthine and 8-azahypoxanthine, and the pyrimidine analogs, 2-thiocytosine, 5-fluorouracil, 2-thiouracil and 6-azauracil, inhibited the induction of 2-dimensional growth in the gametophytes of the fern Asplenium nidus L. In contrast, thymine analogs such as 5-fluorodeoxyuridine, 2-thiothymine, 6-azathymine and 5-bromouracil caused non-specific growth inhibitions without suppressing 2-dimensional growth. Subinhibitory concentrations of 8-azaxanthine, 8-azahypoxanthine, and 2-thiouracil promoted both 1-dimensional and 2-dimensional phases of growth of the gametophytes. Inhibitory effects of the analogs were observed on treatment of the spores or of gametophytes of different ages. Gametophytes growing in the analogs for different periods of time recovered from inhibition on transfer to the basal medium.     

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.     

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.     

GROWTH INHIBITION IN GAMETOPHYTES AND OAT COLEOPTILES BY THELYPTERIN A AND B RELEASED FROM ROOTS OF THE FERN THELYPTERIS NORMALIS

Gametophytes, when grown in the immediate vicinity of a Thelypteris normalis sporophyte—in soil or in sterile culture on agar—showed a reduced number of cells and an altered gross morphology. This is attributed to the action of the thelypterins, which are inhibitors released from T. normalis sporophytes. Growth inhibition of the gametophytes was greatest when thelypterins were added during early stages of gametophyte development. Removal of thelypterin A permitted resumption of growth. Thelypterin A noncompetitively inhibited auxin-enhanced elongation of A vena coleoptiles. The growth of T. normalis root segments was not affected by thelypterins. The growth of young sporophytes of T. normalis was inhibited by mature sporophytes.     

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.     

ROLE OF NUCLEIC ACID AND PROTEIN METABOLISM IN THE INITIATION OF GROWTH AT GERMINATION

When dry decotyledonized embryos of Raphanus are supplied withwater, a brief period of water absorption (phase A) is followedby a period of no fresh weight increase (phase B) which lastsfor 8 hr at 30°. In this period, embryos become ready toadvance into the period of fresh weight increase (phase C). When embryos were exposed to various concentrations of thiouracilor actinomycin D solution from 0 hr of water supply, increasesin fresh weight and in RNA content measured at 13 hr were inhibitedin parallel with each other. Chloramphenicol and puromycin inhibitedthe fresh weight increase without affecting the RNA increase.When embryos were exposed to thiouracil or puromycin for 2,4 and 6 hr, beginning at 0 hr of water supply, the start ofphase C delayed 2, 4 and 6 hr, respectively. When these drugswere given after phase B had progressed at least for 2 hr, thedelay of the start of phase C was shorter than the period ofthe drug treatment. If given at the end of phase B, thiouraciland actinomycin D inhibited the incorporation of ¹⁴C-uracilbut not the fresh weight increase, while chloramphenicol andpuromycin inhibited the latter without inhibiting the former. During phase B, protein content per dry weight of embryo didnot increase, but the rate of ¹⁴C-leucine incorporation increasedremarkably to reach the level in phase C. Incorporation of labeledleucine was inhibited if embryos were subjected to thiouracilor actinomycin D action during phase B, but not if the drugswere given when phase B had been completed. Puromycin and chloramphenicolinhibited the incorporation whenever they were given. The increase in respiratory activity during phase B was inhibitedrelatively little by the above mentioned four drugs. In conclusion syntheses of RNA and protein seem to be essentialfor the progress of phases B and C, protein synthesis havinga more direct effect. (Received September 17, 1965; )     

UNUSUAL DARK-GROWTH AND ANTHERIDIAL DIFFERENTIATION IN SOME GAMETOPHYTES OF THE FERN ONOCLEA SENSIBILIS

A fertile frond of O. sensibitis was found which yielded spores with unusual growth characteristics. About 25% of the gametophytes derived from the spores were able to undergo 2-dimensional development in darkness, in contrast to normal plants which are filamentous in darkness. When the aberrant spores were cultured in darkness under conditions of reduced ethylene concentration, the proportion of 2-dimensional plants rose to 75%, and, moreover, up to 50% of the gametophytes produced antheridia within 2 weeks. Under comparable conditions normal gametophytes produced no antheridia. The medium from antheridial cultures of the aberrant spores failed to induce the formation of antheridia in other plants.     

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.     

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.     

THE TRANSITION FROM FILAMENTOUS TO TWO-DIMENSIONAL GROWTH IN FERN GAMETOPHYTES. III. INTERACTION OF CELL ELONGATION AND CELL DIVISION

Periodic cell divisions were induced in gametophytes of Pteridium aquilinum by daily irradiation with white light. In white-dark cycles, the rate of cell division was promoted by increased time in white light; cell elongation was not affected. The time of transition to two-dimensional growth (days to 5% 2-D) was closely associated with the mitotic rate. For white-red cycles, the rate of elongation was controlled by the intensity of red light (wavelengths over 550 nm). This increased elongation delayed the initiation of 2-D development. In both cases the rate of transition to 2-D growth was correlated with the amount of elongation per division.