Ribosomal ribonucleic acid and ribosomal precursor ribonucleic acid in Anacystis nidulans

The RNA of the blue-green alga Anacystis nidulans contains three ribosomal RNA species with molecular weights of 0.56x10(6), 0.9x10(6), and 1.1x10(6) if the RNA is extracted in the absence of Mg(2+). The 0.9x10(6)mol.wt. rRNA is extremely slowly labelled in (32)P-incorporation experiments. This rRNA may be a cleavage product of the 1.1x10(6)mol.wt. rRNA from the ribosomes of cells in certain physiological states (e.g. light-deficiency during growth). The cleavage of the 1.1x10(6)mol.wt. rRNA during the extraction procedure can be prevented by the addition of 10mm-MgCl(2). (32)P-pulse-labelling studies demonstrate the rapid synthesis of two ribosomal precursor RNA species. One precursor RNA migrating slightly slower than the 1.1x10(6)mol.wt. rRNA appears much less stable than the other precursor RNA, which shows the electrophoretic behaviour of the 0.7x10(6)mol.wt. rRNA. Our observations support the close relationship between bacteria and blue-green algae also with respect to rRNA maturation. The conversion of the ribosomal precursor RNA species into 0.56x10(6)- and 1.1x10(6)-mol.wt. rRNA species requires Mg(2+) in the incubation medium.     

Ribonucleic acid synthesis in chloroplasts

Chloroplasts isolated from young spinach leaves incorporate [(3)H]uridine into RNA. This incorporation shows an absolute requirement for light and does not occur in lysed chloroplasts. Fractionation by polyacrylamide-gel electrophoresis of the RNA synthesized in vitro reveals a major discrete product of molecular weight 2.7x10(6) and two minor products of molecular weight 1.2x10(6) and 0.47x10(6). These discrete products are super-imposed on a background of polydisperse RNA. The incorporation of (32)P(i) into chloroplast rRNA species (mol.wt. 1.05x10(6) and 0.56x10(6)) in excised spinach leaves proceeds after a distinct lag period compared with the incorporation into cytoplasmic rRNA species (mol.wt. 1.34x10(6) and 0.7x10(6)). Incorporation of (32)P(i) into chloroplast RNA species of molecular weight 2.7x10(6), 1.2x10(6), 0.65x10(6) and 0.47x10(6) proceeds without such a time-lag. The kinetics of labelling of the individual RNA components is consistent with the rapidly labelled RNA species of molecular weight 1.2x10(6) and 0.65x10(6) being precursors to the more slowly labelled rRNA species of molecular weight 1.05x10(6) and 0.56x10(6) respectively.     

The molecular weights of yeast ribosomal precursor RNAs

The molecular weights of the predominant rRNA precursors as well as those of 26-S and 17-S mature rRNA from Saccharomyces carlsbergensis were determined by polyacrylamide gel electrophoresis in the presence of formamide. Mature 26-S + 5.8-S rRNA was found to have a molecular weight of 1.24 X 10(6) while their immediate precursor, 29-S RNA, had a molecular weight of 1.52 X 10(6). Values of 0.70 X 10(6) and 0.82 X 10(6) were obtained for the molecular weights of mature 17-S rRNA and its 18-S precursor. Finally the 37-S precursor, common to both 29-S and 18-S RNA, was found to have a molecular weight of 2.80 X 10(6). Each precursor rRNA, therefore, contains extra sequences not found at the next stage of maturation.     

The synthesis and origin of chloroplast low-molecular-weight ribosomal ribonucleic acid in spinach.

Chloroplasts isolated from young spinach leaves incorporate [3H]uridine into RNA species which co-electrophorese with 5-S rRNA and tRNA, but show very little incorporation into 4.5-S rRNA. Chloroplast 4.5-S rRNA is labelled in vivo after a distinct lag period relative to 5-S rRNA and tRNA. The kinetics of labelling in vivo of chloroplast 5-S rRNA are similar to those of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, whereas the kinetics of labelling of the 4.5-S rRNAare similar to those of mature 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs. Chloramphenicol inhibits the labelling of chloroplast 4.5-S rRNA in vivo, and concomitantly inhibits the processing of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, but has little effect on the appearance of label in chloroplast 5-S rRNA. DNA/RNA hybridization using 125I-labelled RNAs suggests that chloroplast DNA contains a 2--3-fold excess of 4.5-S and 5-S rRNA genes relative to the high-molecular-weight rRNA genes. Competition hybridization experiments show that the immediate precursor to the 1.05 x 10(6)-Mr rRNA effectively competes with 125I-labelled 4.5-S rRNA for hybridization with chloroplast DNA, and is therefore a likely candidate for a common precursor to both the 1.05 x 10(6)-Mr and 4.5-S rRNAs.     

Molecular Weight Determination of Sendai and Newcastle Disease Virus RNA

The molecular weights of Sendai and Newcastle disease virus RNA were estimated by sedimentation in sucrose gradients and by length measurements in the electron microscope under both denaturing and nondenaturing conditions. Sedimentation analyses under denaturing conditions yielded molecular weight estimates of 2.3 x 10(6) to 2.6 x 10(6), whereas length measurements yielded estimates of 5.2 x 10(6) to 5.6 x 10(6) for both denatured and nondenatured viral RNA. It would appear that the conditions of denaturation used (99% dimethyl sulfoxide at 26 C, and reaction with 1.1 M formaldehyde for 10 min at 60 C) do not equally denature parainfluenza virus RNA and other RNAs, such as cellular rRNA, 45S rRNA precursor, and R17 RNA.     

Ribosomal RNA Synthesis in the Eastern North-American Newt, Notophthalmus viridescens

Ribosomal RNA (rRNA) synthesis, the initiation of which is an early major event during the transformation of iris into lens in the newt, was characterized in the TVI cell-line derived from the eastern North-American newt Notophthalmus viridescens. Employing the technique of polyacrylamide gel electrophoresis, molecular-weight measurements were made on newt rRNAs using Xenopus laevis and E. coli rRNAs as standards. The molecular weights of N. viridescens 28S and 18S rRNA were found to be 1.4 × 10⁶ and 0.7 × 10⁶ respectively. The precursor to these RNAs had a molecular weight of 3.1 × 10⁶. Three probable intermediates in the processing of precursor to mature rRNA were also identified. On the basis of the molecular weights of all species of RNA identified, a processing pathway, similar to that of Xenopus , has been suggested.
Some unusual features in the kinetics of precursor rRNA labelling and processing suggest the possibility that newt-cell rRNA synthesis may be controlled by the availability of essential amino acids in a manner similar to that observed in mammalian cells. A possible relationship between the availability of essential amino acids, the initiation of rRNA synthesis in the newt iris, and the control of lens regeneration is discussed.     

The synthesis of ribonucleic acid in immature rat uterus responding to oestradiol-17β

Stimulation of incorporation of labelled precursors into the RNA of immature rat uterus is an early result of oestradiol-17beta action. However, the extent of the increased incorporation varies with the mode of administration of the labelled precursors and with the weight of the rat. At the age and weight range normally used response is maximal at ten times control incorporation, 4h after the administration of 0.3mug or more of oestradiol-17beta. Under these conditions the stimulation of incorporation into the acid-soluble fraction is only 2-2.5-fold. When the purified RNA is separated on polyacrylamide gels the major increase in incorporation of radioactive precursor is found in rRNA and 4S RNA; the formation of the former has been followed from the 45S precursor. Preceding these events by at least 30min, however, is an increase in the incorporation of precursor into RNA species of very high molecular weight, which remained in the first few slices of the gel. The possible significance of these findings is discussed. The increased synthesis of rRNA in response to oestradiol-17beta is more strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke.

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 X 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 X 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 X 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 X 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.     

Ribonucleic acid synthesis and loss of viability in pea seed

Summary RNA synthesis and protein synthesis in embryonic axis tissue of viable pea (Pisum arvense L. var. N.Z. maple) seed commences during the first hour of germination. Protein synthesis in axis tissue of non-viable pea seed is barely detectable during the first 24 h after the start of imbibition. Nonviable axis tissue incorporates significant levels of [³H]uridine into RNA during this period but the level of incorporation does not increase significantly over the first 24 h of imbibition. In axis tissue of non-viable seed during the first hour of imbibition most of the [³H]uridine was incorporated into low molecular weight material migrating in advance of the 4S and 5S RNA species in polyacrylamide gels but some radioactivity was incorporated into a discrete species of RNA having a molecular weight of 2.7×10⁶. After 24 h, non-viable axis tissue incorporates [³H]uridine into ribosomal RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and a heterogeneous RNA species of molecular weight ranging from 2.2×10⁶ to 2.7×10⁶. No 4S or 5S RNA synthesis is detectable after 24 h of imbibition in non-viable axis tissue. Axis tissue of viable pea seed synthesises rRNA, 4S and 5S RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and the rRNA precursor species at both periods of germination studied. Loss of viability in pea seed appears to be accompanied by the appearance of lesions in the processing of rRNA precursor species and a significant loss of RNA synthesising activity.Abbreviations rRNA ribosomal RNA - TCA trichloroacetic acid - SLS sodium lauryl sulphate - PPO 2,5 Diphenyloxazole - POPOP 1,4-Bis-2-(4-methyl-5-penyloxazolyl)-benzene     

Direct evidence for the lack of methylation of two pulse labeled plant RNAs

A product of the processing of precursor rRNA ("excess" RNA)has been indirectly found to be unmethylated in mammalian systems,but direct measurement was precluded because of its instability.The "excess" RNA of duckweeds is relatively stable allowingdirect estimation of its methylation by polyacrylamide gel electrophoresis.The "excess" RNA with an apparent molecular weight 0.5?10⁶ wasunmethylated. A pulse labeled RNA with an apparent molecularweight of 1.2?10⁶ (presumably from chloroplasts) was also unmethylated.Under similar conditions the presumed cytoplasmic rRNA precursors,and the mature cytoplasmic and chloroplast rRNAs were methylated. ¹Present address: Department of Biological Sciences, S.U.N.Y.Binghamton, N.Y. 13901, U.S.A. (Received May 9, 1974; )     

Synthesis of ribonucleic acid in the venom gland of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom

1. The incorporation of [5-(3)H]uridine into RNA of the venom gland of Crotalus durissus terrificus was studied after manual extraction (;milking') of the venom. The labelled precursor was injected immediately after milking. 2. The RNA was extracted 1, 2, 4, 6 and 8h after injection of the label and analysed by sucrose-density-gradient centrifugation. 3. The sedimentation analysis showed that 18S rRNA synthesis is higher than 28S rRNA at all time-intervals. The specific radioactivities of both ribosomal components did not reach a plateau even at 8h after injection. 4. An RNA fraction was detected sedimenting between 18S rRNA and 4S tRNA and was called the 10-14S fraction. The specific radioactivity was always higher than that of both classes of rRNA and reached the maximum value at 6h of labelling. 5. The incorporation of the precursor was also studied by radioautography, which helped to elucidate the intracellular origin of the RNA analysed by sucrose-density-gradient centrifugation.     

Molecular-weight determination of animal-cell RNA by electrophoresis in formamide under fully denaturing conditions on exponential polyacrylamide gels.

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA - RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45 degrees and 55 degrees C, respectively. In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 10(4) - 10(7). Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights the size of animal cell was reexamined. Precursor tRNA from HeLa cells migrates according to a molecular weight of 4.1 x 10(6). Nascent precursor mRNA has molecular weights of up to 5 x 10(6) in the case of duck erythroblasts and of up to 10(7) in HeLa cells. This seems to represent the largest size of non-viral animal-cell RNA molecules.     

The synthesis of chloroplast high-molecular-weight ribosomal ribonucleic acid in spinach.

Illuminated suspensions of chloroplasts isolated from young spinach leaves show incorporation of [3H]uridine into several species of RNA. One such RNA species of Mr 2.7 x 10(6) shows sequence homology with both the chloroplast 23-S rRNA (Mr = 1.05 x 10(6)) and 16-S rRNA (Mr = 0.56 x 10(6)), as judged by DNA/RNA competition hybridization. Leaves labelled in vivo with [32P]orthophosphate in the presence of chloramphenicol accumulate labelled RNAs of Mr 1.28 x 10(6), 0.71/0.75 x 10(6) and 0.47 x 10(6). The 1.28 x 10(6)-Mr RNA shows 80.5% sequence homology with the 1.05 x 10(6)-Mr rRNA and the 0.71/0.75 x 10(6)-Mr RNA mixture shows 76% sequence homology with the 0.56 x 10(6)-Mr rRNA. We conclude that the pathway of rRNA maturation in spinach chloroplasts is similar to that of Escherichia coli.     

The ribosomal RNA of the trypanosomatid protozoan Crithidia fasciculata: physical characteristics and methylated sequences.

When extracted and analyzed under conditions which maintain noncovalently associated RNA-RNA complexes, the bulk cellular RNA of Crithidia fasciculata contains species of apparent molecular weights 1.3, 0.825, 0.08, 0.065, and 0.045 x 10(6) in addition to 5S rRNA and tRNA. Heat denaturation results in the disappearance of the 1.3 x 10(6) dalton RNA and the appearance of three new species having molecular weights of 0.67, 0.575, and 0.059 x 10(6). In addition, the apparent molecular weight of the 0.825 x 10(6) dalton component is reproducibly lowered to 0.81 x 10(6) after heat treatment. With the exception of tRNA, all of the RNA species are present in close to equimolar amounts in either undenatured or heat-denatured C. fasciculata bulk cellular RNA. On the basis of previous observations on the ribosomal RNA of the closely related organism, Crithidia oncopelti (Spencer, R. & Cross, G.A.M. (1976) J. Gen. Microbiol. 93, 82-88), the 1.3 and 0.825 x 10(6) dalton RNA's are considered to be components of the large and small subunits, respectively, of C. fasciculata ribosomes, but the subunit localization of the other RNA's described here has not yet been determined. O2'-Methylnucleosides account for about 1.4 mol% of the total nucleoside constituents of unfractionated C. fasciculata rRNA. Quantitative analysis suggests that the rRNA molecules in a C. fasciculata ribosome contain a total of 95-100 O2'-methyl groups, distributed in 80-85 Nm-Np sequences (including four 'hypermodified' Nm-Np, each containing a modification of a base or base-sugar linkage in addition to sugar methylation), six different Nm-Nm-Np sequences, and one Nm-Nm-Nm-Np sequence. While the specific pattern of O2'-methylation in the rRNA of C. fasciculata is distinct, both qualitatively and quantitatively, from the pattern observed in other organisms, Crithidia rRNA does contain certain 'universal' O2'-methylated sequences which appear to have been extensively conserved in evolution. The base-methylated nucleoside, N6,N6-dimethyladenosine (m26A), has been isolated from both C. fasciculata and wheat embryo rRNA in the form of the alkali-resistant dinucleotide, m26A-m26Ap. This dinucleotide and its enzymatic degradation products have been characterized by examination of their ultraviolet absorption spectra and electrophoretic and chromatographic properties.     

The action of alpha-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

alpha-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of alpha-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of alpha-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10-20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by alpha-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.