Studies on ribonucleic acids from Chlorella protothecoides with special reference to the degradation of chloroplast RNA during the process of "glucose-bleaching"

By growing Chlorella protothecoides under certain nutritionaland light conditions the following three different types ofalgal cells were obtained: (i) normal "green" cells grown ina medium rich in a nitrogen source (urea) and poor in glucoseunder illumination, (ii) "etiolated" cells cultivated in thesame medium in darkness, and (iii) "glucose-bleached" cellsgrown, in the light or in darkness, in a medium rich in glucoseand poor in the nitrogen source. The "glucose-bleached" cellscontain profoundly degenerated plastids, and the "etiolated"cells have only partially organized plastids. From these algalcells RNA was extracted by the cold phenol method, and fractionatedby MAK column chromatography and sucrose density gradient centrifugation,making use of ³²P-labelled E. coli RNA as the internal marker.It was found that in comparison with the green cells that arerich in chloroplast ribosomal RNA as well as in nonchloroplastic("cytoplasmic") ribosomal RNA, the etiolated cells possess acomparable amount of "cytoplasmic" rRNA but a significantlylesser amount of chloroplast rRNA. Both types of rRNA existat extremely low levels in the glucose-bleached cells. During the process of bleaching (chloroplast degeneration) ofthe green cells induced by the addition of a high concentrationof glucose, marked changes were observed in the patterns offractionation of RNA as followed by the above procedures. Itwas disclosed that the chloroplast rRNA is rapidly degradedduring an early phase of the bleaching process, while the quantityof "cytoplasmic" rRNA remained almost unaltered. ¹Part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965, and at the Symposium on Biogenesisof Subcellular Particles, the 7th Internatl. Congress of Biochemistry,Tokyo, 1967. ²Present address: Faculty of Pharmaceutical Sciences, Universityof Hokkaido, Sapporo.     

Changes in Levels and Integrity of Ribosomal RNA During Seed Maturation and Germination in Carrot (Daucus carota L.)

Thompson, S., Bryant, J. A. and Brocklehurst, P. A. 1987. Changesin levels and integrity of ribosomal RNA during seed maturationand germination in carrot (Daucus carota L.).—J. exp.Bot. 38: 1343–1350. Amounts and integrity (percentage of complete un-nicked molecules)of ribosomal RNA (rRNA) during germination in carrot seed lotsof differing vigour and viability were examined using aqueousand formamide gel electrophoresis. In unaged seed, amounts ofrRNA per seed in dry seed are not related to maturity, vigouror viability. However, rRNA in immature seed is more vulnerableto damage by ageing than rRNA in mature seed; thus, amountsof rRNA per seed in aged seed are indicators of vigour and viability.On imbibition of aged or unaged seed, more rRNA fragmentationoccurs in immature seed than in mature seed, and the time atwhich the effects of synthesis outweigh the effects of degradationis earlier during germination in mature than in immature seed. Key words: rRNA, seed maturation, seed germination, Daucus carota L.     

RIBONUCLEIC ACIDS APPEARING DURING THE PROCESS OF CHLOROPLAST REGENERATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Investigations were made on the modes of synthesis of differentspecies of RNA which appear during the greening (chloroplastregeneration) of the "glucose-bleached" cells of Chlorella protothecoidescontaining profoundly degenerated plastids.
2. RNAs were extractedfrom the algal cells which had been labelledwith ³²P for 1hr before harvesting at different stages of thegreening inthe light and in darkness, and subjected to columnchromatographywith methylated albumin-coated kieselguhr. Itwas found that,during the greening process, the elution profilesof RNAs, interms of the optical density at 260 mµ and³²P-radioactivity,changed profoundly.
3. Based on these and other results, it wasconcluded that duringan early phase of the chloroplast regenerationin the glucosebleachedalgal cells, there occurs an active formationof both ribosomalRNAs (rRNAs) and the RNAs corresponding tosoluble RNA (sRNA),the formation coming, however, later toa standstill when thesynthesis of chlorophyll has proceededto a certain level. Thequantity ratio of sRNA to rRNA was foundto be constant (30:70)at different stages of the greening (bothin the light and indarkness), with a few exceptions. The synthesisof the chloroplastribosomal RNA is markedly accelerated bylight, and its maximumrate is observed sometime later thanthat of the non-chloroplast("cytoplasmic") ribosomal RNA. Itwas suggested that there areat least two different sites ofsynthesis of ribosomal RNAs,one in the plastid and the otheroutside of it (most probablyin the nucleus).

¹A part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965. ² Present address: Institute for Plant Virus Research, Ministryof Agriculture and Forestry, Aoba-cho, Chiba.     

Effect of inhibitors of nucleic acid and protein synthesis on the reappearance of "light interruption rhythm" in a long-day duckweed, Lemna gibba G 3

Effects of several inhibitors of DNA, RNA and protein synthesison the reappearance of a once faded-out light interruption rhythmin a long-day duckweed, Lemna gibba G 3, were studied. The reappearancewas not affected by inhibitors of RNA and protein synthesis;i.e., 2-thiouracil, 8-azaguanine, ethionine and chloramphenicol,but was suppressed by inhibitors of DNA synthesis; i. e., 5-fluorodeoxyuridine,5-fluorouracil and mitomycin C only when these were appliedduring the light period for perturbation. We concluded that synthesis of a new DNA species during thelight period was required for the recurrence of this rhythm. (Received September 25, 1968; )     

Cytochemical and Biochemical Analysis of Development of Urechis Oocytes

The echiuroid marine worm Urechis caupo is uniquely suited forthe study of oogenesis. A relatively large quantity of oocytesat various developmental stages can be obtained and subjectedto coordinated cytochemical and biochemical analysis Oocytesat the cluster, early diplotene, mid-diplotene, and diffusediplotene or lampbrush stages are active in the synthesis andaccumulation of ribosomal RNA, several proteins, carbohydrates,lipids, and also, perhaps, yolk constituents. Only corticalgranule formation, which occurs during later stages of oogenesis,appears to be stage specific. Ribosomal RNA genes are also transcribedin the nucleolus of the mature oocytes or unfertilized eggs.However, the rate of production in these eggs appears to beregulated at the level of maturation of rRNA precursor molecules.     

Synthesis and maturation of ribosomal ribonucleic acids in isolated HeLa cell nuclei. A tracer study on the topology of the 45S precursor of ribosomal ribonucleic acids

The synthesis and processing of RNA by isolated HeLa cell nuclei was studied at low ionic strength in the presence of alpha-amanitin. The RNA polymerase reaction, with endogenous template and enzyme, rapidly reaches a plateau dependent on the amount of nuclei. Evidence is presented that incorporation of [(3)H]UMP proceeds only in growing RNA chains, whereas initiation of new RNA chains is arrested. The product formed contains all the main components of the 45S pre-rRNA (precursor of rRNA) maturation pathway (45S, 32S and 20S pre-rRNA; 28S and 18S rRNA). Most of the labelled material is in the mature rRNA components and their immediate precursors, even at very short times of incubation (2min). Small, but definite, 5S and 4S RNA peaks are also observed. At shorter incubation times a substantial amount of [(3)H]UMP is incorporated into RNA molecules in the 24S and 10-16S zones. This RNA material is considered to represent the non-conserved segments of 45S pre-rRNA in the process of nucleolytic degradation. A model for the tracer study of the topology of 45S pre-rRNA, on arrest of rRNA initiation, is discussed. The experimental evidence obtained supports the following structure of 45S pre-rRNA: 5'-end-28S rRNA unit-18S rRNA unit-nonconserved segment-3'-end.     

Electron microscopy of secondary structure in partially denatured precursor and mature Escherichia coli 16 S and 23 S rRNA

The secondary structure of 16 S and 23 s rRNA sequences in 30 S preribosomal RNA of Escherichia coli was analyzed by electron microscopy after partial denaturation and compared to mature 16 S and 23 S rRNA examined under the same conditions. The sequences in the pre-rRNA notably lack the specific loops that dominate the 5'-terminal regions of mature 16 S and 23 S rRNA. In other respects, the sizes and locations of loops in the 23 S rRNA sequence are qualitatively very similar in mature and pre-rRNA. Eleven of 12 loops outside of the 5'-terminal domain correspond, with the most frequent features in the 3'-half of the molecule. In contrast, the sizes and locations of loops in the 16 S rRNA sequence differ between precursor and mature forms. In the pre-rRNA, instead of the 370-nucleotide 5'-terminal loop of mature rRNA, some 1000-nucleotide terminal loops are observed. The pre-rRNA also shows a frequent 610-nucleotide central loop and a large 1240-nucleotide loop not seen in the mature rRNA. Also, in the 3'-region of the sequence, the largest loops in pre-rRNA are 120 nucleotides shorter than in mature rRNA. We suggest that the structure of pre-rRNA may promote some alternate conformational features, and that these could be important during ribosome formation or function.     

Nucleic acid synthesis accompanying the recovery of cell division and chloroplast development in "giant" cells of the Emerson strain of Chlorella

The light-induced recovery of cell division and chloroplastdevelopment in "giant", "bleached", cells of the Emerson strainof Chlorella takes place without any increase in DNA and isrelatively insensitive to mitomycin C and 5-bromouracil. 5-Fluorouracilinhibits cell division only when it is supplied during the earlystages of recovery, perhaps by interfering with that phase ofRNA synthesis which occurs during the first 6 hr of recovery.This early burst of RNA synthesis is more sensitive to chloramphenicolthan is the second phase of RNA synthesis, suggesting that asignificant proportion of it may originate in the chloroplast.Evidence is presented which suggests that 5-fluorouracil interfereswith chloroplast development primarily through an effect onchlorophyll synthesis. The possible significance of these observationsin relation to nuclearchloroplastic interractions is discussed. (Received December 15, 1972; )     

A Kv channel with an altered activation gate sequence displays both "fast" and "slow" activation kinetics

The Kv1–4 families of K⁺ channels contain a tandem proline motif (PXP) in the S6 helix that is crucial for channel gating. In human Kv1.5, replacing the first proline by an alanine resulted in a nonfunctional channel. This mutant was rescued by introducing another proline at a nearby position, changing the sequence into AVPP. This resulted in a channel that activated quickly (ms range) upon the first depolarization. However, thereafter, the channel became trapped in another gating mode that was characterized by slow activation kinetics (s range) with a shallow voltage dependence. The switch in gating mode was observed even with very short depolarization steps, but recovery to the initial "fast" mode was extremely slow. Computational modeling suggested that switching occurred during channel deactivation. To test the effect of the altered PXP sequence on the mobility of the S6 helix, we used molecular dynamics simulations of the isolated S6 domain of wild type (WT) and mutants starting from either a closed or open conformation. The WT S6 helix displayed movements around the PXP region with simulations starting from either state. However, the S6 with a AVPP sequence displayed flexibility only when started from the closed conformation and was rigid when started from the open state. These results indicate that the region around the PXP motif may serve as a "hinge" and that changing the sequence to AVPP results in channels that deactivate to a state with an alternate configuration that renders them "reluctant" to open subsequently. voltage-gated potassium channel     

Escherichia coli 16S rRNA 3''-end formation requires a distal transfer RNA sequence at a proper distance.

The 16S rRNA species in bacterial precursor rRNAs is followed by two evolutionarily conserved features: (i) a double-stranded stem formed by complementary sequences adjacent to the 5' and 3' ends of the 16S rRNA; and (ii) a 3'-transfer RNA sequence. To assess the possible role of these features, plasmid constructs with precursor-specific features deleted were tested for their capacity to form mature rRNA. Stem-forming sequences were dispensable for both 5' and 3' terminus formation; whereas an intact spacer tRNA positioned greater than 24 nucleotides downstream of the 16S RNA sequence was required for correct 3'-end maturation. These results suggest that spacer tRNA at an appropriate location helps form a conformation obligate for pre-rRNA processing, perhaps by binding to a nascent binding site in preribosomes. Thus, spacer tRNAs may be an obligate participant in ribosome formation.     

RNA synthesis required for DNA replication in Vicia seed embryos

The synthesis of DNA and RNA during germination of Vicia seedswas examined. Incorporation of ³H-thymidine into DNA reacheda maximum at about 32 hr after the beginning of imbibition,and RNA synthesis was shown to precede DNA replication. Sedimentationanalyses of ³H-uridine-labeled RNAs indicated that the embryossynthesize all types of rRNA, heterodisperse RNA and 4–5SRNA before and also during the phase of DNA replication. Actinomycin-treatments at lower concentrations (50 or 100 µg/ml)resulted in the specific inhibition of rRNA synthesis. Suchinhibition did not lead to a large reduction in ³H-thymidineincorporation during the replication phase. However, DNA synthesiswas drastically inhibited by a higher level (200 µg/ml)of actinomycin D. The results strongly suggest the involvementof synthesis of heterodisperse RNA in DNA replication. (Received May 28, 1976; )     

Non-ribosomal nucleotide sequences in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA in yeast.

The topography and the length of the non-ribosomal sequences present in 7-S RNA, the immediate precursor of 5.8-S ribosomal RNA, from the yeast Saccharomyces carlsbergensis were determined by analyzing the nucleotide sequences of the products obtained after complete digestion of 7-S RNA with RNase T1. The results show that 7-S RNA contains approximately 150 non-ribosomal nucleotides. The majority (90%) of the 7-S RNA molecules was found to have the same 5'-terminal pentadecanucleotide sequence as mature 5.8-S rRNA. The remaining 10% exhibited 5'-terminal sequences identical to those of 5.9-S RNA, which has the same primary structure as 5.8-S rRNA except for a slight extension at the 5' end [Rubin, G.M. (1974) Eur. J. Biochem. 41, 197--202]. These data show that the non-ribosomal nucleotides present in 7-S RNA are all located 3'-distal to the mature 5.8-S rRNA sequence. Moreover, it can be concluded that 5.9-S RNA is a stable rRNA rather than a precursor of 5.8-S rRNA. The 3'-terminal sequence of 5.8-S rRNA (U-C-A-U-U-UOH) is recovered in a much longer oligonucleotide in the T1 RNase digest of 7-S RNA having the sequence U-C-A-U-U-U-(C-C-U-U-C-U-C)-A-A-A-C-A-(U-U-C-U)-Gp. The sequences enclosed in brackets are likely to be correct but could not be established with absolute certainty. The arrow indicates the bond cleaved during processing. The octanucleotide sequence -A-A-A-C-A-U-U-C- located near the cleavage site shows a remarkable similarity to the 5'-terminal octanucleotide sequence of 7-S RNA (-A-A-A-C-U-U-U-C-). We suggest that these sequences may be involved in determining the specificity of the cleavages resulting in the formation of the two termini of 5.8-S rRNA.     

The distribution of plastid ribosomes and the integrity of plastid ribosomal RNA during the greening and maturation of Spirodela fronds

Young and mature fronds of Spirodela oligorrhiza cultured inthe light or dark were analyzed for their plastid ribosome distributionby electron microscopy. This distribution was correlated tothe loss of integrity (hidden nicking) of the plastid heavyribosomal RNA. In young fronds there were relatively more plastidribosomes as well as plastid rRNA in the light than in the dark.Plastid rRNA was synthesized mainly when fronds were young.Chloroplast ribosomes disappeared from the stroma of light grownfronds upon maturation, but the relative amount of chloroplastRNA increased. In the dark the relative etioplast ribosomaldensity did not change upon maturation. The nicked productsof the plastid heavy rRNA were detected by a modified two-dimensionalgel electrophoresis. There is already some nicking of the plastidheavy rRNA at a very early stage of frond and plastid development.The rate of hidden nicking of plastid heavy rRNA was greaterin the light than in the dark. The rate of nicking in frondstransferred from the dark to the light increased to the levelof light grown plants at about the time of the appearance ofchlorophyll. The data are discussed in the context of the relationshipbetween plastid ribosomal RNA changes during frond maturationin light and dark grown fronds, as related to protein synthesisand of ribosome binding to thylakoids. (Received May 25, 1974; )     

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.     

Nucleic Acid Chemistry of the Ilyanassa Embryo

A morphological study of Ilyanassa embryogenesis was made fromsections of several stages and the nucleic acid content wasmeasured throughout the course of development. The kineticsof uptake and incorporation of radioactive precursors into RNAwas determined. The RNAs synthesized at different stages ofembryogenesis were fractionated by methylated albumin kieselguhrchromatography, and the polyadenylation of RNA during developmentwas measured. From these studies it was concluded that the Ilyanassa embryo,beginning with the 4-cell stage, synthesizes dRNA, rRNA, 4SRNA, and 5S RNA throughout all stages of development. The extensivesynthesis of dRNA and the higher proportion of RNAs polyadenylatedby the pregastrula embryo was considered to be of particularsignificance. The localization of nucleic acids and nucleic acid precursorsin the polar lobe and the effect of removing the polar lobeon nucleic synthesis was reviewed. Recent work on the ultrastructure of the Ilyanassa egg was reviewedin relation to the problem of ooplasmic segregation. The discoveryof a new cytoplasmic organelle in the Ilyanassa oocyte was reviewed.