Chloroplast ribosomal ribonucleic acid synthesis in cultured spinach leaf tissue

Chloroplast rRNA synthesis was studied in spinach leaf tissue cultured under sterile conditions which eliminate bacterial rRNA synthesis. The synthesis was inhibited by darkness, but concomitant cytoplasmic rRNA synthesis was unaffected. A complex pattern of labelled rRNA precursors was found in extracts from cultured leaf tissue by using polyacrylamide-gel electrophoresis. However, differences between the precursor profiles of leaf tissue cultured in the light and in the dark could not be correlated with chloroplast rRNA synthesis since large amounts of high-molecular-weight precursors of cytoplasmic rRNA dominated the pattern in both cases. A double-isotope-labelling technique was used, which enabled light-stimulated rRNA synthesis to be studied in whole leaf tissue. Two rapidly labelled RNA species of molecular weights 1.15x10(6) and 0.65x10(6) were detected, which were thought to have possible precursor significance in the synthesis of mature chloroplast rRNA of molecular weights 1.04x10(6) and 0.56x10(6) respectively. Cycloheximide treatment resulted in the accumulation of RNA of molecular weight 1.8x10(6), whose function is unknown.     

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.     

Kern-Plasma-Transport neusynthetisierter rRNS in Zellen einer Suspensionskultur von Petroselinum sativum

Summary A rapidly labelled rRNA precursor can be detected in callus cells of Petroselinum sativum grown on a liquid synthetic medium. Its molecular weight has been calculated to be 2.3×10⁶. This value agrees with that of the rRNA precursor from other plant material. In order to follow the synthesis and processing of rRNA in time and to correlate single steps in this process with cell organelles it was necessary to obtain pure fractions of nuclei and ribosomes. The isolation method for nuclei is given in detail. The nucleic acids are separated on polyacrylamide gels of low acrylamide concentration. Pulse-chase experiments show that the rRNA precursor is split into two fragments within the nucleus: an 18S and a 25S component. The 18S RNA leaves the nucleus rapidly. It is already found quantitatively in the ribosomal fraction after 30–60 min chase. At that time the 25S RNA is still within the nucleus; it appears much later in the ribosomes. Since the increase in ribosomal label occurs simultaneously with the decrease in nuclear label, it is concluded that there is no degradation of 18S RNA within the nucleus. Apparently there are two distinct transport mechanisms with different kinetics for the two RNA components.     

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 Turnover in Contact Inhibited Cells

CONTACT inhibition of animal cell growth is accompanied by a decreased rate of incorporation of nucleosides into RNA^1–3. Contact inhibited cells, however, transport exogenously-supplied nucleosides more slowly than do rapidly growing cells^4,5, suggesting that the rate of incorporation of isotopically labelled precursors into total cellular RNA may be a poor measure of the absolute rate of RNA synthesis by these cells. Recently, Emerson⁶ determined the actual rates of synthesis of ribosomal RNA (rRNA) and of the rapidly labelled heterogeneous species (HnRNA) by labelling with ³H-adenosine and measuring both the specific activity of the ATP pool and the rate of incorporation of isotope into the various RNA species. He concluded that contact inhibited cells synthesize ribosomal precursor RNA two to four times more slowly than do rapidly growing cells, but that there is little if any reduction in the instantaneous rate of synthesis of HnRNA by the non-growing cells. We have independently reached the same conclusion from simultaneous measurements on the specific radioactivity of the UTP pool and the rate of ³H-uridine incorporation into RNAs (unpublished work of Edlin and myself). However, although synthesis of the 45S precursor to ribosomal RNA is reduced two to four times in contact inhibited cells, the rate of cell multiplication and the rate of rRNA accumulation are reduced ten times. This suggests either “wastage”⁷ of newly synthesized 45S rRNA precursor, or turnover of ribosomes in contact inhibited cells Two lines of evidence suggest that “wastage” of 45S RNA does not play a significant role in this system. (1) The rate of synthesis of 45S RNA in both growing and contact inhibited cells agrees well with that expected from the observed rates of synthesis of 28S and 18S RNAs (unpublished work of Edlin and myself). Emerson has made similar calculations⁶. (2) 45S RNA labelled with a 20 min pulse of ³H-uridine is converted in the presence of actinomycin D to 28S and 18S RNAs with the same efficiency (approximately 50%) in both growing and contact inhibited cells. These results indicate that, in order to maintain a balanced complement of ribosomal RNAs, contact inhibited cells must turn over their ribosomes. We present evidence here that rRNA is stable in rapidly growing chick cells, but begins to turn over with a half-life of approximately 35–45 h as cells approach confluence and become contact inhibited.     

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; )     

Intracellular symbiont as a major source of the ribosomal RNAs in the aphid mycetocytes.

When the aphid mycetocytes were incubated in Grace's medium, they synthesized preferentially unique RNAs with molecular weights of 1.2 × 10⁶ and 0.6 × 10⁶. The RNA synthesis was completely inhibited by a low concentration of rifampicin. Actinomycin D did not inhibit selectively the synthesis of these RNAs. Based on these findings it was assumed that in the aphid mycetocyte the rRNA genes of the prokaryotic symbionts are preferentially expressed. The exceptional properties observed with the integrity and synthesis of the aphid rRNA may be possibly interpreted by assuming the intracellular symbionts to play an important role.     

Molecular weights,poly(A)-content,and partial separation of chick globin mRNAs

Poly(A)-containing 9S RNA from chick reticulocytes was electrophoresed on formamide-polyacrylamide gels. The molecular weight was determined to be 211 000±10 000 daltons. The RNA was separated into three different fractions with respect to molecular weight. These RNAs were translated in a wheat germ cell-free system. The lower molecular weight RNA directed up to 95% -chain synthesis, compared to 60% for the higher molecular weight RNA. This was accompanied by a relative increase for -chain synthesis with increasing molecular weight. It could also be shown by hybridization with labelled poly(U) that the average poly(A) length decreased from about 83 nucleotides for fraction I to 36 nucleotides for fraction III. Our results suggest that fractionation of avian 9 S globin mRNA by electrophoresis on formamide-polyacrylamide gels is dependent upon two parameters, namely differences in the lengths of the non-poly(A)-containing portion of the and mRNAs and differences in the poly(A) lengths.     

High molecular weight RNAs from Rous sarcoma virus and Moloney murine leukemia virus contain two subunits.

The molecular weights of the large genomic RNAs from Rous sarcoma and Moloney murine leukemia viruses were determined by a combination of sedimentation coefficients and retardation coefficients from gel electrophoresis. Six RNA standards, ranging from 0.7 X 10(6) to 5.3 X 10(6) daltons, were employed. Studies in the presence of varying concentrations of Mg2+ showed that the method provided valid molecular weights for RNAs of differing amounts of ordered structure. The molecular weight (X 10(-6)) of the high molecular weight RNA complexe from Rous sarcoma virus was 7.6 (+/-0.3) and from murine leukemia virus was 6.9 (+/-0.3). The molecular weights (X 10 (-6) of their Subunits were 3.3 (+/-0.1) and 2.8 (+/-0.2), respectively. Hence, the large complexes consisted of two, not three or more, subunits plus small associated RNAs. The high molecular weight RNA from cloned Rous sarcoma virus was heterogenous in molecular weight although the apparent molecular radius was constant; stuides were performed on subfractions of the RNA as well as on RNA from virus harvested at various time intervals. The preparations with lowest molecular weight approached a mass equal to twice that of the subunit, with hydrodynamic properties approaching those expected of normal single-stranded RNA.     

In-vitro translation of messenger-RNA from developing bean leaves. Evidence for the existence of stored messenger-RNA and its light-induced mobilisation into polyribosomes

Poly(A)-containing messenger RNA was purified from polyribosomes isolated from the primary leaves of 7-day-old dark-grown seedlings of Phaseolus vulgaris var. Masterpiece. Analysis of the messenger RNA on 2.4% polyacrylamide gels showed that it consists of a heterogeneous population of molecules with an average molecular weight of 500,000. The nucleotide composition of the RNA was 16.0% cytidylic acid, 39.4% adenylic acid, 21.3% guanylic acid and 23.2% uridylic acid. Based on the degree of resistance of the RNA to digestion with ribonucleases A and T₁ the average length of the poly(A) sequence was calculated to be 120 nucleotides. No significant differences in mobility in polyacrylamide gels, nucleotide composition or polyadenylic acid content were found between the poly(A)-containing mRNA from polyribosomes of primary leaves of dark-grown plants and those given a 16 h white light treatment. Purified poly(A)-containing mRNA was shown to direct the incorporation of [³⁵S]methionine into proteins in an in vitro protein-synthesising system from wheat germ. The protein products were fractionated according to molecular size by electrophoresis in 15% polyacrylamide/urea/SDS gels and the protein bands were detected by fluorography. Messenger RNAs directing the synthesis of three polypeptides with molecular weights of 34,000, 32,000 and 25,000 were detected in polyribosomes of plants following white light treatment. These messenger RNAs were absent, or present in much lower amounts, in polyribosomal messenger RNA from leaves of dark-grown plants, although they were present in total cell poly(A)-containing RNA. This indicates that certain messenger RNAs may be stored in the dark and that light stimulates these RNAs to engage in polyribosome formation. Continuous far-red (730 nm) irradiation for 4 h also caused the appearance of these messenger RNAs in the polyribosomes although 5 min red light followed by 4 h darkness had little effect. This suggests that phytochrome acting in the high energy mode, may be the photoreceptor responsible for initiating the response.Abbreviations mRNA messenger-RNA - rRNA ribosomal RNA - oligo (dT) oligo (deoxythymidylic acid) - poly(A) polyadenylic acid - EDTA ethylenediamine-tetra-acetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - SDS sodium dodecyl sulphate     

Messenger and ribosomal RNA hydrolysis by ribonucleases I. The action of two barley leaf ribonucleases on the messenger and ribosomal RNA of isolated polysomes

When barley (Hordeum vulgare L.) leaf polysomes are incubatedwith two RNase fractions (the pH 5 insoluble and soluble RNases)under limit digestion conditions, the two enzymes exhibit characteristicpreference for messenger and ribosomal RNA (mRNA and rRNA) hydrolysis.The pH 5 insoluble RNase from a cultivar of barley, Prior, andthe corresponding enzyme from two near-isogenic lines (M1622and M1623) cleave polysomal mRNA at specific sites and generatepolysome profiles that are unique to the cultivar. By contrast,the soluble RNase from barley leaves, although a typical endoribonuclease,catalyzes no detectable hydrolysis of polysomal mRNA. Both of these barley leaf RNases hydrolyze rRNA when eitherpolysomes or monosomes are treated with these enzymes. Withpolysomes as substrate, the pH 5 insoluble RNase hydrolyzesthe high molecular weight RNA component of both large and smallsubunits of chloroplast and cytoplasmic ribosomes. The solubleRNase preferentially hydrolyzes the high molecular weight RNAcomponent of the small subunit of chloroplast and cytoplasmicribosomes. Analytical gel electrophoresis of the RNA of theRNase-treated monosomes has revealed that both enzymes hydrolyzerRNA into very small fragments. However, despite scission inrRNA at multiple sites, the RNase-treated monosomes remain activein polyuridylate-directed polyphenylalanine synthesis. (Received January 31, 1980; )     

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.     

Properties of a discrete high molecular weight poly(A) containing mitochondrial RNA

Pulse-labeled mitochondrial RNA from hamster cells contains a number of discrete high molecular weight poly[A(+)] and poly[A(?)] RNAs. Characterization of the largest and most plentiful of the poly[A(+)] RNAs, the “20S_E RNA,” showed it to be a labile, unmethylated component with a molecular weight ～- 730,000. Hybridization of the 20S_E RNA to mtDNA was 60–70% inhibited in the presence of excess 17S rRNA, suggesting a significant degree of primary sequence homology between these RNA species. In vitro treatment with RNAse III converted the 20S_E RNA to a poly[A(?)] “17S” product, while similar treatment of mitochondrial 17S rRNA or a poly[A(+)] 12S_E RNA had no effect on these RNAs. These data support the proposition that the 20S_E RNA is a precursor to the 17S rRNA.     

Ribonucleic acid from the higher plant Matthiola incana. Molecular weight measurements and DNA-RNA hybridisation studies.

The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA. This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.