Precursors of chloroplast ribosomal RNA in Euglena gracilis

Incorporation of ³²P into mature chloroplast rRNA species of MW 1.1 × 101 and 0.56 × 10⁶ has been followed in Euglena gracilis by pulse and pulse chase experiments. Mature rRNA species have precursors of MW 1.16 × 10⁶ ± 0.01 × 10⁶ and 0.64 × 106 ± 0.01 × 10⁶ resp. These precursors have base composition and hydridization properties similar to those of the mature, rRNA species. No evidence of a single common precursor to these molecules was found. Rifampicin did not affect the synthesis of chloroplast rRNA.     

The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10⁶ (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10⁶. On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10⁶-dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10⁶, present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10⁶, present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10⁶, independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo.     

Properties of a new strain of tobacco streak virus from Clematis vitalba (Ranunculaceae)

A distinct strain of tobacco streak virus (TS V/Cle), isolated in Yugoslavia from wild Clematis vitalba showing chlorotic spots or yellow netting of the leaves and from many symptomless shrubs, is described. TSV/Cle was seed transmitted in C. vitalba (70%), and in the experimental hosts Chenopodium quinoa (80%), Nicotiana benthamiana and N. megalosiphon. It was also detected in the pollen of infected C. quinoa. Purified virus preparations mostly contained quasi-spherical particles measuring 24–26 × 28, 26–28 × 28–30 and 28–31 × 32–36 mn, and sedimented in sucrose density gradient and analytical centrifugation as three components with sedimentation coefficients of 76S, 87S and 98S. The virus contained a single polypeptide species of mol. wt of c. 25 000. Unfractionated TSV/Cle preparations contained four RNA species with mol. wts, estimated by gel electrophoresis in agarose, of 1.1 × 10⁶, 0.9 × 10⁶, 0.7 × 10⁶ and 0.3 × 10⁶. In comparative experiments, TSV/Cle differed from four reference strains of TSV (TSV/B, TSV/HF, TSV/RN, and TSV/Ro) in host range and in symptoms induced in some common hosts. In agar gel double diffusion tests it was more closely related to TSV/B and TSV/M (SDI = 5) than to TSV/HF (SDI = 7), TSV/RN (SDI = 7) or TSV/Ro (SDI = 5–8). Immunoelectrophoresis experiments clearly distinguished TSV/Cle from the reference strains. TSV/Cle strain was detected in C. vitalba plants from distant and climatically different regions in Yugoslavia.     

Isolation of ribosomal RNA precursors from Physarum polycephalum

Ribosomal RNA synthesis in Physarum polycephalum was studied by labeling intact microplasmodia with [³H]uridine. Labeled, high-molecular-weight RNA species were found in a 30,000 S structure released by phenol extraction at room temperature. RNA was released from the structure by further phenol extraction at 65–70 °C. If the labeling period was 15 min or longer, the labeled RNA was seen by polyacrylamide gel electrophoresis to be of two major types, a heterodisperse collection of 45-35 S molecules and a 26 S species. If the labeling was carried out for 30 min in the presence of cycloheximide, the major labeled species had an electrophoretic mobility corresponding to 40 S. Studies of the labeling kinetics, methylation, and base composition of these RNA molecules indicate that they are precursors to ribosomal RNA. The molecular weights of the homogeneous 40 and 26 S precursors are 3.0 × 10⁶ and 1.45 × 10⁶ daltons, respectively, in comparison with molecular weights of 1.29 × 10⁶ and 0.68 × 10⁶ daltons for the completed ribosomal RNA's.     

Genetic structure and regulation of a replicon of plasmid lambdadv.

Strains of Saccharomyces cerevisiae carrying a small double-stranded RNA species (the killer plasmid) secrete a toxin which is lethal only to strains not carrying this plasmid.We have isolated mutants in eight chromosomal genes essential for replication or maintenance of the killer plasmid, called mak1 through mak8. Seven of these genes have been mapped. mak4 and mak5 are on chromosome II; mak1 and mak8 are on chromosome XV; mak3 and mak6 are on chromosome XVI; and mak7 is on chromosome VIII. We have not yet located mak2. Two other chromosomal genes, m and pets, have been shown to be required for replication or maintenance of the killer plasmid.One allele of mak1 results in temperature sensitivity for host growth. Two independent pets isolates also result in the petite phenotype, as well as temperature sensitivity for growth.Wild-type killer strains have been reported to carry two species of doublestranded RNA of 2.5 × 10⁶ and 1.4 × 10⁶ molecular weight (designated L and M, respectively); wild-type non-killers carried only L. We estimate the size of the L and M species at 3.0 × 10⁶ and 1.7 × 10⁶ daltons, respectively. We have also detected a third species of double-stranded RNA of molecular weight 3.8 × 10⁶ (XL) present in all killer and non-killer strains examined.Mutation of any of mak1 through mak8 results in loss of the killer-associated species of double-stranded RNA (M; 1.7 × 10⁶). These mutants retain both the L species (3.0 × 10⁶) and the XL species (3.8 × 10⁶) of double-stranded RNA, and have acquired two new minor RNA species.     

Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry

A mechanically transmissible virus obtained from symptomless plants of a red raspberry selection imported into Scotland from Quebec, Canada was indistinguishable serologically from a cherry isolate of cherry rasp leaf virus (CRLV). The raspberry isolate, CRLV-R, was graft transmitted to several virus indicator species and cultivars of Rubus without inducing noticeable symptoms. In Chenopodium quinoa sap, CRLV-R lost infectivity after dilution to 10^-5 or heating for 10 min at 60°C but was infective after 16 days (the longest period tested) at 18°, 4° or - 15°C. The virus particles are isometric, c. 28 nm in diameter, and were purified with difficulty from infected C. murale and C. quinoa plants. The particles comprise two nucleoprotein components with sedimentation coefficients of 89 and 115 S and are prone to aggregate during purification. When centrifuged to equilibrium in CS₂SO₄ solution, purified virus preparations formed two major components with p= 1·28 and 1·36 g/cm³. Virus particles contained two RNA species which, when denatured in glyoxal and electrophoresed in agarose gels, had estimated mol. wt of 2·56 × 10⁶ (RNA-1) and 1·26 × 10⁶ (RNA–2). Infectivity of CRLV-R RNA was abolished by treatment with proteinase K, suggesting that the RNA is linked to protein necessary for infectivity; RNA molecules contained polyadenylate. In reticulocyte lysates, CRLV-R RNA stimulated the incorporation of ³H-leucine, mainly into two polypeptides of estimated mol. wt 200 000 and 102 000. When electrophoresed in polyacrylamide gels, protein obtained from CRLV-R particles purified by centrifugation to equilibrium in Cs₂SO₄ separated into three bands with estimated mol. wt 26 000 , 23 000 and 21 000.     

Properties of broad bean yellow band virus, a possible new tobravirus

A virus was transmitted from broad bean plants in Apulia (Southern Italy) with leaves showing yellow rings, line patterns or yellow vein banding and malformations and necrosis of pods. Symptoms in some, but not all, test plants were similar to those induced by tobraviruses. Purified virus preparations contained two classes of rod-shaped particles containing c. 5% nucleic acid with sedimentation coefficients of 186S and 276S. After centrifugation to equilibrium in CsCl gradients, two components were resolved, with buoyant densities of 1·298 and 1·316 g/cm³. Unfractionated virus preparations contained two species of single-stranded RNA with mol. wts of c. 1·06 × 10⁶ and 2·48 × 10⁶ and one species of coat protein with mol. wt of c. 21 300. The modal lengths of the two classes of particles, both in plant sap and in purified preparations, were 77 nm (S particles) and 202 nm (L particles). L particles accumulated in infected cells in paracrystalline aggregates, whereas S particles were randomly distributed in the cytoplasm of cells. The virus was serologically unrelated to two isolates of tobacco rattle virus and two isolates of pea early-browning virus. The virus, named broad bean yellow band, is considered a distinct tobravirus.     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

A selective temperature-sensitive defect in viral RNA expression in cells infected with a ts transformation mutant of murine sarcoma virus

We investigated the nature of the defect in the temperature-sensitive mutant of Moloney murine sarcoma virus (Mo-MuSV), termed ts110. This mutant has a temperature-sensitive defect in a function required for maintenance of the transformed state. A nonproducer cell clone, 6m2, infected with ts110 expresses P85 and P58 at 33°C, the transformed temperature, but only P58 is detected at the restrictive temperature of 39°C. Shift-up (33°C → 39°C) and in vitro experiments have established that P85 is not thermolabile for immunoprecipitation. Previous temperature-shift experiments (39°C → 33°C) have shown that P85 synthesis resumes after a 2–3 hr lag period. Temperature shifts (39°C → 33°C) performed in the presence of actinomycin D prevented the synthesis of P85, whereas P58 synthesis did not decline for 5 hr, suggesting that P58 and P85 are translated from different mRNAs. The shift-up experiments also indicated that, once made, the RNA coding for P85 can function at the restrictive temperature for several hours. MuSV-ts110-infected cells superinfected with Mo-MuLV produced a ts110 MuSV-MuLV mixture. Sucrose gradient analysis of virus subunit RNAs revealed a ～28S and a ～35S peak. Electrophoresis of the ～28S poly(A)-containing RNA from ts110 virus in methyl mercuric hydroxide gels resolved two RNAs with estimated sizes of 1.9 × 10⁶ and 1.6 × 10⁶ daltons, both smaller than the wild type MuSV-349 genomic RNA (2.2 × 10⁶ daltons). RNA in the ～28S size class from virus preparations harvested at 33°C was found to translate from P85 and P58, whereas, the ～35S RNA yielded helper virus Pr63^gag. In contrast, virus harvested at 39°C was deficient in P85 coding RNA only. Peptide mapping experiments indicate that P85 contains P23 sequences, a candidate Moloney mouse sarcoma virus src gene product. Taken together, these results suggest that two virus-specific RNAs are present in ts 110-infected 6m2 cells and rescued ts110 pseudotype virions at 33°C, one coding for P85, whose expression can be interfered with by shifting the culture to 39°C; the other coding for P58, whose expression is unaffected by temperature shifts. P85 is a candidate gag-src fusion protein, while P58 contains gag sequences only.     

On the size relationship between nuclear and cytoplasmic RNA in sea urchin embryos

The approximate sizes of heterogeneous nuclear (HnRNA) and cytoplasmic RNA of sea urchin embryos were determined by DMSO density gradient centrifugation and acrylamide-formamide gel electrophoresis. The data suggest that the sizes of these molecules are smaller than those estimated under nondenaturing conditions. The size of most of the nuclear RNA ranges from 0.5 to 3 × 10⁶ daltons, while that of the cytoplasmic RNA ranges from 0.1 to 2 × 10⁶ daltons. Both nuclear and cytoplasmic RNA of sea urchin embryos may have a minor fraction (5–10%) of very large species with molecular weights up to 4 to 5 × 10⁶ daltons.The idea that the size of HnRNA may be larger in organisms higher on the evolutionary scale is discussed.     

Evidence for SV40 Specific RNA containing Virus and Host Specific Sequences

AFTER infection of monkey kidney cells with simian virus 40 (SV40), several species of SV40 specific RNA are synthesized¹. Most SV40 RNA have a molecular weight of about 6×10⁵ and 8×10⁵ as measured by polyacrylamide gel electrophoresis¹. In addition to these classes of RNA, a large heterogeneous SV40 specific RNA species of up to three times the length of the monomeric SV40 DNA molecule has been observed^1–4. Nothing is known about the structure of this large heterogeneous virus specific RNA.     

Methionine methyl group metabolism in lemna

To provide information upon the ways in which Lemna paucicostata uses the methyl group of methionine, plants were grown for various periods (from 1 minute to 6.8 days) in the presence of a tracer dose of radioactive methyl-labeled methionine. Protein methionine accounted for approximately 19% of the accumulated methyl moieties; other methylated products, about 81%. The latter group included (percent of total methyl in parentheses): methylated ethanolamine derivatives (46%); methyl esters of the pellet (chiefly, or solely, pectin methyl esters) (15%); chlorophyll methyl esters (8%); unidentified neutral lipids (6%); nucleic acid derivatives (2-5%); methylated basic amino acids (2%). No other major methylated compounds were observed in any plant fraction. Available evidence suggests that little, if any, oxidation of the methyl group of methionine, directly or indirectly, occurs in Lemna. Our results indicate that S-methyl-methionine sulfonium is formed relatively rapidly, but does not accumulate at a commensurate rate, probably being reconverted to methionine. To our knowledge, this is the first time a reasonably complete accounting of the metabolic fate of methionine methyl has been obtained for any plant. The extent to which the results with Lemna may be representative of the situation for other higher plants is discussed.     

Properties and relationships of broad bean stain virus and Echtes Ackerbohnenmosaik-Virus

The sedimentation coefficients (^s0₂₀, w) of the two sedimenting nucleoprotein components of broad bean stain virus (BBSV) were 92 S and 113 S, and of Echtes Ackerbohnenmosaik-Virus (EAMV) were 93 S and 114 S. Particles from each of these sedimenting components contained a single RNA species and two polypeptides. Estimates of the molecular weights of these constituents obtained by electrophoresis in polyacrylamide gels were: 42000 and 22200 (BBSV) and 41400 and 21800 (EAMV) for the polypeptides; and 2–64 and 1·62 × 10⁶ (BBSV) and 271 and 175 × 10⁶ (EAMV) for the RNAs. In mixtures, the protein and RNA components of BBSV and EAMV were indistinguishable from those obtained from particles of the yellow strain of cowpea mosaic virus. In freshly made virus preparations each of the sedimenting components of BBSV contained two, and those of EAMV contained three electrophoretic components. After storage for 7–10 days, BBSV preparations contained only the component migrating fastest towards the anode. Both BBSV and EAMV are distantly related serologically to cowpea mosaic but, whereas BBSV reacted only with antiserum to the severe strain, EAMV reacted only with antiserum to the yellow strain.     

The species composition and quantitative distribution of the diatom genus Pseudo-nitzschia H. Peragallo, 1990 in Russian waters of the Sea of Japan and the Sea of Okhotsk

This paper studies the species composition and quantitative distribution of diatoms that belong to the genus Pseudo-nitzschia in the Russian waters of the Sea of Japan and the Sea of Okhotsk. In total, 11 species of this genus were found in the area, including 7 that are known as being potentially toxic. The highest concentrations of Pseudo-nitzschia microalgae (1.4 × 10⁶–2.7 × 10⁶ cells/L) were found in the summer and autumn in the Peter the Great Bay of the Sea of Japan and the lowest concentrations (2.5 × 10²–1 × 10⁴ cells/L) were found in the Sakhalinsky and Akademiya bays of the Sea of Okhotsk. The species diversity of potentially toxic diatoms was greatest (seven species) and the cell concentrations highest (over 6 × 10⁵ cells/L) in the Peter the Great Bay, Sea of Japan, and in the Aniva Bay, Sea of Okhotsk. The density of potentially toxic species was highest near the northeastern coast of Sakhalin Island, in the Amur River estuary, and in adjacent waters. This paper also presents geographical distribution maps of Pseudo-nitzschia species and maps of the density distribution of potentially toxic microalgae over the studied area and identifies potential amnesic shellfish poisoning areas.     

Occurrence in vivo of "hidden breaks" at specific sites of 26 S ribosomal RNA of Musca carnaria.

A fragmentation process occurs in 26 S ribosomal RNA of mature cytoplasmic ribosomes of Musca carnaria. It consists of the sequential appearance of three “hidden breaks” that fragment 26 S rRNA (M_r = 1.42 × 10⁶) into four pieces with approximate molecular weights of 0.68 × 10⁶, 0.35 × 10⁶, 0.29 × 10⁶ and 0.096 × 10⁶, respectively. This fragmentation was not observed in 17 S rRNA (M_r = 0.74 × 10⁶).Extremely mild treatment of newly assembled ribosomes with pancreatic RNAase reproduces the 26 S rRNA fragmentation phenomenon in vitro in the same way as it occurs in vivo.This evidence is discussed in relation to the secondary structure of 26 S rRNA and its binding with specific ribosomal proteins.     

Synthesis of RNA in isolated polytene nuclei from salivary gland cells of Chironomus thummi

The incorporation of [³H]UTP into RNA by isolated polytene salivary gland nuclei of Chironomus thummi was investigated under different incubation conditions; the labeled RNA fractions were characterized by electrophoresis. The results suggested that at two characteristic ionic conditions most of the RNA synthesized was the product of RNA polymerase I or RNA polymerase II as distinguished by their differential sensitivities to α-amanitin. Electrophoretical analysis of the RNA synthesized under conditions favouring polymerase I showed that this RNA population consisted mainly of four distinct molecular weight fractions within a range between 2.8 × 10⁴ and 2.5 × 10⁶. Under conditions favouring polymerase II two fractions were detected: one with a broad molecular weight distribution around 0.4 × 10⁶ containing considerable amounts of poly(A)-bearing RNA molecules, and a second with a peak at a molecular weight of 2.8 × 10⁴.     

The early synthesis and possible function of a 0.5 X 10(6) Mr RNA after transfer of dark-grown Spirodela plants to light.

A 0.5 × 10⁶$\text{M}$_r RNA found in plastids of the aquatic angiosperm $\text{Spirodela}$, is synthesized at a much higher rate than any other rapidly labeling RNA species about $\text{3–3}\text{1}\text{2}$ h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 10⁶$\text{M}$_r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in $\text{Spirodela}$, poly(A) sequences are not detected in the 0.5 × 10⁶$\text{M}$_r RNA; yet a sucrose gradient fraction which includes RNA of this $\text{M}$_r stimulates amino acid incorporation by an $\text{E. coli}$ cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 10⁶$\text{M}$_r RNA as a chloroplast messenger is discussed.     

Characterization of sub-genomic double-stranded RNAs from virus-infected plants

Double-stranded RNAs (sub-RFs) smaller than the double-stranded RNAs (RFs) corresponding to genomic RNAs of tobacco mosaic (TMV) and cowpea chlorotic mottle (CCMV) viruses were isolated from infected plants and characterized. Seven of the 12 sub-RFs of TMV that were found ranging in size from 3.00 – 0.42 × 10⁶ daltons corresponded to twice the size of the 7 sub-genomic mRNAs reported by Goelet and Karn (8). Six sub-RFs of CCMV were found ranging from 0.98 – 0.39 × 10⁶ daltons with the most abundant species corresponding to twice the size of RNA 4. The kinetics of incorporation of ³H-uridine into sub-RFs were different from that into RFs. Incorporation into sub-RFs was slow and linear whereas that into RF turned over rapidly.