Poly(a)+ rna during vegetative development ofacetabularia peniculus

Summary In the juvenile stage, the diploid giant-celled green algae Acetabularia spp. are differentiated into an upright stalk and an irregularly branched rhizoid. Early amputation and grafting experiments as well as biochemical and molecular analyses have shown that mRNA (as poly(A)⁺ RNA) is continuously supplied from the primary nucleus in the rhizoid and accumulates in the stalk apex. In the present study, localization of poly(A)⁺ RNA in the juvenile stage of theAcetabularia peniculus was investigated by fluorescent in situ hybridization using oligo(dT) as a probe. The signal was localized in the apical cytoplasm and, in addition, multiple longitudinal striations throughout the stalk and rhizoid cytoplasm. A large portion of the poly(A)⁺ RNA striations exhibited structural polarity, broadened at one end and gradually thinned toward the other end. Some of the striations in the rhizoid cytoplasm were continuous with a zone of signal in the area of the perinuclear rim. The poly(A)⁺ RNA striations were associated with thick bands of longitudinal actin bundles which run through the entire length of the stalk. Cytochalasin D caused fragmentation of the actin bundles and irregular distribution of the fluorescent signal. We suggest that the poly(A)⁺ RNA striations constitute a hitherto unknown form of packaged mRNA that is transported over large distances along the actin cytoskeleton to be stored and expressed in the growing apex.     

Polyadenylated RNA from Acetabularia

Polyadenylated RNA was isolated from whole cells and from anucleate cytoplasm of Acetabularia mediterranea. It is synthesised in nucleate but not in anucleate cells. This RNA has a high molecular weight ranging from 0.5 to 3.0 × 10⁶ daltons. In contrast to the RNA of 80 S ribosomes the synthesis of polyadenylated RNA is only slightly enhanced in nucleated cell fragments during the initial phase of regeneration. Newly synthesised polyadenylated RNA migrates from the nucleus through the stalk at about 5 mm a day. The data suggest that polyadenylated RNA migrates independently of 80 S ribosomes.     

RIBOSOME SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The cellular site of synthesis of ribosomal RNA in Tetrahymena pyriformis was studied by analyzing the purified nuclear and cytoplasmic RNA from cells pulse labeled with uridine-³H. The results of studies using zonal centrifugation in sucrose density gradients show that the ribosomal RNA is synthesized in the nucleus as a large precursor molecule sedimenting at 35S. The 35S molecule undergoes rapid transformation through two main nuclear intermediates, sedimenting at about 30S and 26S. The smaller ribosomal RNA (17S) appears first in the cytoplasm and it seems to be absent from the nucleus. The apparent delay in the appearance of the larger ribosomal RNA (26S) in the cytoplasm is due to the presence of a larger pool of its precursors in the nucleus as indicated by pulse-chase experiments. The newly synthesized ribosomal RNA's appear in the cytoplasm as discrete 60S and 45S ribonucleoprotein particles, before their incorporation into the polysomes.     

Translation of tobacco mosaic virus RNA In Acetabularia cell cytoplasm.

Isolated Acetabularia nuclei were microinjected with Tobacco Mosaic Virus RNA and then implanted into an anucleate posterior fragment of an Acetabularia cell. Injected RNA was translated in the Acetabularia cytoplasm from the first to twelfth day after implantation of the nuclei. The production of specific virus proteins was detected and localized in the Acetabularia cytoplasm by an immunofluorescence precipitation technique.     

RNA metabolsim during vegetative growth and morphogenesis of the cellular slime mold Dictyostelium discoideum

During vegetative growth of the cellular slime mold Dictyostelium discoideum, RNA is rapidly labeled by radioactive precursor and both the 25 S and the 17 S ribosomal RNA species appear in the cytoplasm 6–7 min after the onset of labeling. Thirty minutes after further incorporation of radioactive RNA precursors has been blocked, less than 10% of the label in RNA is associated with the nuclear fraction. After aggregation of the slime mold amoebae, RNA appears in the cytoplasm at a reduced rate, the small ribosomal subunit appearing in the cytoplasmic fraction more slowly than the larger ribosomal subunit. Some labeled RNA remains in the nuclei of developing cells long after the incorporation of ³H-uridine is blocked.     

Development and organization of the central vacuole of Acetabularia acetabulum

* Here we analyzed the shape of the central vacuole of Acetabularia acetabulum by visualizing its development during diplophase (from juvenility through reproduction) and haplophase (from meiosis through mating). * Light microscopy and whole-organism applications of a pH-sensitive dye, neutral red, were used to visualize the anatomy of the central vacuole. We studied connectivity within the thallus by locally applying dye to morphologically distinct regions (rhizoid, stalk, apex, hairs) and observing dye movements. * In vegetative thalli most of the rhizoid, stalk and young hairs stained with dye. In reproductive structures (caps, gametangia) dye also stained the majority of the interiors. When applied to small areas, dye moved at different rates through each region of the thallus (e.g. within the stalk). Dye moved from younger hairs, but not from older hairs, into the stalk. Errors in incorporation of central vacuole into gametangia occurred at <10(-5). * These data indicate that the central vacuole of A. acetabulum is a ramified polar organelle with, potentially, a gel-like sap that actively remodels its morphology during development.     

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12.

The biosynthesis of ribosomal ribonucleic acid (rRNA) In wild-type Neurospora crassa growing at 25 degrees C was investigated by continuous-labeling and pulsechase experiments using [5-3H]uridine. The results of these experiments suggest the following precursor-product relationships: the first RNA molecule to be synthesized in significant quantities is the 2.4 X 10(6)-dalton (2.4-Mdal) ribosomal precursor RNA. This RNA is cleaved to produce two species of RNA with weights of 0.7 and 1.4-Mdal. The former is the mature 17S rRNA of the 37S ribosomal subunit. The 1.4-Mdal RNA is subsequently cleaved to produce the mature 1.27-Mdal (25S) and 61,000-dalton (5.8S) rRNA's of the 60S ribosomal subunit. In the maturation process, approximately 15 to 20% of the 2.4-Mdal ribosomal precursor rRNA molecule is lost. As in other eukaryotes that have been examined, 5S rRNA is not derived from this precursor molecule.     

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

Localization of proteins L4, L5, L20 and L25 on the ribosomal surface by immuno-electron microscopy

Summary Ribosomal proteins L4, L5, L20 and L25 have been localized on the surface of the 50S ribosomal subunit of Escherichia coli by immuno-electron microscopy. The two 5S RNA binding proteins L5 and L25 were both located at the central protuberance extending towards its base, at the interface side of the 50S particle. L5 was localized on the side of the central protuberance that faces the L1 protuberance, whereas L25 was localized on the side that faces the L7/L12 stalk. Proteins L4 and L20 were both located at the back of the 50S subunit; L4 was located in the vicinity of proteins L23 and L29, and protein L20 was localized between proteins L17 and L10 and is thus located below the origin of the L7/L12 stalk.     

Characterization of 5.8S ribosomal ribonucleic acid in Neurospora crassa.

Neurospora crassa ribosomes contain a species of ribonucleic acid (RNA) of molecular weight 54,000, similar to 5.8S ribosomal RNA previously described for other eukaryotic organisms. The 5.8S RNA from N. crassa was found to be released by heat treatment at 60 C from 25S ribosomal RNA but not from 18S ribosomal RNA. The base composition of N. crassa 5.8S RNA was similar to that of 5.8S RNA from Saccharomyces cerevisiae, but differed from animal 5.8S RNA. During the course of this study, it was discovered that N. crassa 25S ribosomal RNA had a number of internal cleavages that may exist in vivo.     

Asymmetric subcellular mRNA distribution correlates with carbonic anhydrase activity in Acetabularia acetabulum

The unicellular green macroalga Acetabularia acetabulum L. Silva is an excellent system for studying regional differentiation within a single cell. In late adults, physiologically mediated extracellular alkalinity varies along the long axis of the alga with extracellular pH more alkaline along the apical and middle regions of the stalk than at and near the rhizoid. Respiration also varies with greater respiration at and near the rhizoid than along the stalk. We hypothesized that the apical and middle regions of the stalk require greater carbonic anhydrase (CA) activity to facilitate inorganic carbon uptake for photosynthesis. Treatment of algae with the CA inhibitors acetazolamide and ethoxyzolamide decreased photosynthetic oxygen evolution along the stalk but not at the rhizoid, indicating that CA facilitates inorganic carbon uptake in the apical portions of the alga. To examine the distribution of enzymatic activity within the alga, individuals were dissected into apical, middle, and basal tissue pools and assayed for both total and external CA activity. CA activity was greatest in the apical portions. We cloned two CA genes (AaCA1 and AaCA2). Northern analysis demonstrated that both genes are expressed throughout much of the life cycle of A. acetabulum. AaCA1 mRNA first appears in early adults. AaCA2 mRNA appears in juveniles. The AaCA1 and AaCA2 mRNAs are distributed asymmetrically in late adults with highest levels of each in the apical portion of the alga. mRNA localization and enzyme activity patterns correlate for AaCA1 and AaCA2, indicating that mRNA localization is one mechanism underlying regional differentiation in A. acetabulum.     

Rna transport from the nucleus to the cytoplasm in Acetabularia mediterranea]

The biosynthesis of nuclear RNA, its transport from the nucleus to the cytoplasm and distribution in the cytoplasm were studied in Acetabularia mediterranea under different light conditions. It was shown that the nuclear RNA incorportate 3H-uracil more rapidly in the darkness and the transport of labeled RNA from the nucleus slowed down after the transfer of plants in the cold medium in the darkness. To study the distribution of nuclear RNA in the cytoplasm, the 3H-uracil labeled nuclei were transplanted in the rhizoids of unlabeled plants, the dikaryons obtained were kept for different time in the light and in the darkness and the content of 3H-RNA was determined in different stem regions. It was shown that the transport of 3H-RNA in the cytoplasm is slowed down in the darkness and it is distributed by the basal-apical gradient. RNA is rapidly accumulated in the apical stem zone in the light and redistributed afterwards in the basal stem zones. The problem of relationship between the polarity and nuclear RNA distribution in Acetabularia is discussed.     

Wounds incurred in routine cell culture prolong the duration of the life cycle ofAcetabularia acetabulum and require K+ to heal

Summary We have examined the pressure-wound healing response inAcetabularia acetabulum (L.) Silva (Chlorophyta). Commonly incurred in routine cell culture, these wounds induce disruption of the vacuole and translocation of the cytoplasm away from the wound site. Daily wounding of individual cells retarded cytoplasmic healing over time, but had no effect on the rate of membrane healing. The position of the wound along the cell stalk also affected the ability of the cell to heal: cells wounded near the rhizoid healed at least 1.9 times more slowly and were only half as likely to achieve reproduction as were cells wounded either near the apex or at mid-stalk. The 50% mortality of cells wounded at the rhizoid suggests the existence of a physical structure near the primary nucleus which is important to cell viability. The impact of wounding on reproductive potential and time to heal differed with the phase of cell development: juvenile and early adult cells healed 2–2.5 times more quickly but were less likely to achieve reproduction than late adult or reproductive cells. Growth at very high population densities (2.5 cells/ml) impaired the ability of the cells to heal. Growth of cells in seawater containing a range of potassium concentrations revealed that healing depends on potassium and is optimal at a concentration of — 1.51ogM potassium.Abbreviations SE standard error of the mean - LD light/dark - NEM N-ethylmaleimide     

Phenol Extraction Op 28 S Ribosomal RNA Prom Rat Liver Cytoplasm

A simple and reproducible phenol method for the isolation of 28 S ribosomal RNA from rat liver cytoplasm, free from poly(A)-RNA is described. The procedure is based on the observation that at lower pH of the homogenate (pH 5.5) 28 S ribosomal RNA is extracted, while 18 S ribosomal RNA remains in the interphase layer.

Isolation of pure 28 S or 18 S ribosomal RNA in preparative amounts requires density gradient cen-trifugation or preparative gel electrophoresis. In this communication a rapid and reproducible method for the isolation of 28 S ribosomal RNA is proposed.     

Improved visualization of F-actin in the green algaAcetabularia by microwave-accelerated fixation and simultaneous FITC-Phalloidin staining

Summary By employing a new procedure we have been able to visualize a highly intense actin cytoskeleton in the unicellular green algaAcetabularia acetabulum Silva. The protocol described in this study involves microwave-accelerated simultaneous permeabilization with 10% dimethyl sulphoxide, fixation with 1% glutaraldehyde and incubation with 0.5 μM fluorescein-isothiocyanateconjugated Phalloidin. Comparison of the images of the actin cytoskeleton of the stalk, as visualized by methods used previously, with those obtained in our own experiments shows that the actin filaments were preserved completely in an excellent condition. The required time for each procedure could be reduced from 12 h for the most commonly used immunofluorescence technique to 35 min. Moreover, it has been possible to observe the actin filament system of hair whorls, rhizoid and tip. Previously, the actin cytoskeleton of these parts of the cell could not be visualized by conventional techniques. It is shown that each region of the cell-stalk, tip, rhizoid and sidebranches-displays characteristic degrees of actin bundling and regularity of actin alignment.     

Nucleic acids of Entamoeba histolytica

This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 17S (0.8 kDa) and 5S rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 25S rRNA; guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 25S RNA relative to 17S RNA. The 25S RNA is "nicked" (apparently during nuclear processing) and dissociates readily into 17S (0.7 kDa) and 16S (0.6 kDa) species, and a more rigidly bound 5.8S species. A small amount of "unnicked" 25S RNA was recovered with guanidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : I. Modified Processing of 45S RNA

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20°C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15°C, and the processing of 32S RNA to 28S RNA is inhibited near 25°C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27°C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37°C.     

Cytoplasmic methylation of mature 5.8 S ribosomal RNA

Levels of 2-O-methylation were determined in ribosomal 5·8 S RNAs from whole cells and both the nuclear and cytoplasmic fractions of rat liver, rat kidney cells in culture (NRK) and HeLa cells. All 5·8 S RNA molecules contained the alkali stable Gm-Cp dinucleotide at position 77 but only whole cell rat liver RNA contained large amounts (0·7 mol) of Um at position 14. All nuclear 5·8 S RNA fractions were largely undermethylated at this site. In contrast, cytoplasmic 5.8 S RNA from rat liver and, to a lesser degree, NRK cells contained significantly more Um; up to 80% of the molecules from rat liver contained the methylated residue. These results indicate that mature 5·8 S RNA can be methylated in the cytoplasm. When labeling kinetics were examined in NRK cells, the methylation at residue 14 was found to increase as a function of the time spent in the cytoplasm, confirming that this modification is, unlike other ribosomal RNA methylations, in part or largely cytoplasmic.