The nature of polydisperse ribonucleic acid in plants

The aggregation of ribosomal RNA species during chromatography on methylated albumin on kieselguhr was decreased from 50 to 15% by the lower salt concentrations made possible by the use of higher pH values. The polydisperse RNA was resolved into two fractions. About 50% was eluted with the rRNA whereas the remainder was bound to the column, and was recovered only by solubilization of the methylated albumin. Both fractions of polydisperse RNA were similar in size range, but the bound fraction was considerably richer in AMP. No D-RNA (DNA-like RNA) peak was resolved under these conditions of column fractionation. However, the properties of the bound RNA were consistent with it containing both D-RNA and TB-RNA (tenaciously bound RNA). The relationship between these two fractions of AMP-rich RNA was considered. The bound RNA and ribosomal RNA responded differently to various treatments. The salt concentration required to elute ribosomal RNA was halved by increasing the pH of the fractionation, but the amount of bound RNA was in fact increased. Denaturation by hot urea decreased the binding of ribosomal RNA to the methylated albumin, but did not facilitate elution of bound RNA. The high affinity between the AMP-rich polydisperse RNA and the methylated albumin does not therefore appear to arise for the secondary structure conferred by the high AMP content.     

Fractionation of nuclear and cytoplasmic ribonucleic acids from rat tissues on the methylated albumin–kieselguhr column

1. The conformation of RNA was found to affect its behaviour on methylated albumin-kieselguhr chromatography. The less regular the secondary structure of RNA, the more tightly it binds to the methylated albumin-kieselguhr column. 2. The presence of various denaturing agents (such as urea or perchlorate) in the medium while RNA was adsorbed on the column increased the resolving power of the technique as exemplified by the separation of rat liver rRNA into two distinct peaks. A special procedure for selective adsorption of the cytoplasmic DNA-like RNA on the preparative scale has been developed. Polyribosomal mRNA (rapidly labelled RNA formed in the presence of small doses of actinomycin D) can also be adsorbed selectively by the column. 3. A type of tissue specificity was detected in nuclear RNA from rat liver, kidney, thymus and spleen by using a modified salt and temperature gradient for the chromatographic fractionation (Lichtenstein, Piker & Shapot, 1967; Shapot, Lichtenstein & Piker, 1967). It was also found that cytoplasmic RNA from the different rat tissues contained no tenaciously bound fraction at all, whereas it constituted about 50% of the nuclear RNA. The problem of the possible biological function of the tenaciously bound fraction is discussed.     

STUDIES ON RAPIDLY LABELLED NUCLEAR RNA OF RAT BRAIN

—Methyl albumin kieselguhr chromatography (MAK) has been employed to separate rat brain nuclear RNA, labelled in vivo with [³H]uridine, into three major fractions. The first fraction (QI RNA) is ribosomal in nature for it has a high G + C/U ratio and is methylated by [methyl-³H] methionine. The other two fractions (Q2 RNA and TD RNA) are DNA-like for they exhibit a low G + C/U ratio and are labelled minimally by methionine. Pure ribosomal RNA chromatographs almost entirely in the Q1 RNA fraction. Labelling studies indicate that ribosomal RNA and DNA-like RNA behave differently. Initially, the label in the DNA-like RNA fractions increases rapidly and in a linear fashion for the first 30 min, but thereafter decreases rapidly and reaches a steady state level by 1 h and remains so up to at least the 2 h period. In contrast, the labelling of ribosomal RNA is much slower than that of DNA-like RNA during the first 30 min; however, unlike DNA-RNA, the labelling of ribosomal RNA still continues to increase linearly thereafter. Thus, during longer labelling periods, ribosomal RNA is labelled more rapidly than DNA-like RNA. It appears that the labelling of ribosomal RNA relative to DNA-like RNA is more rapid in liver than in brain.     

Nucleic Acid and protein changes in relation to cold acclimation and freezing injury of korean boxwood leaves

Quantitative and qualitative differences in nucleic acids of Korean boxwood (Buxus microphylla var. Koreana) leaves were determined by methylated albumin kieselguhr chromatography at different levels of cold hardiness. During cold acclimation there was an increase in RNA, mainly ribosomal RNA, with little or no change in DNA. The increase in ribosomal RNA was closely paralleled by an increase in water soluble and membrane bound proteins. As cold hardiness increased, ribonuclease activity declined.     

Study of messenger-like RNA extracted from HeLa cells polysomes. II. Identification of the types of RNA eluted from methylated albumin kieselguhr columns.

Together with the elution pattern of pure messenger RNA molecules of various origin, the labelling kinetics of rapidly labelled heterogeneously sedimenting RNA (HSRNA) extracted from polysomes of HeLa cells have been studied by chromatogrphy on columns made of methylated bovine serum albumin adsorbed on kieselguhr. HSRNA is eluted within three peaks-IP, Q2P and TDP-following in that order the increase of NaC1 concentration in the eluting buffer. Besides peak TDP which results from an experimental artefact, our data suggest that the appearance of peaks IP and Q2P reflects the absence and presence respectively of polyadenylic acid stretches in these molecules. Within peak Q2P, the critical factor affecting the order of elution is the size of the polyadenylic acid stretch.     

Denatured helical sites and ribonuclease resistant RNA associated with DNA from the dormant seeds of a higher plant

Denatured helical regions in the DNA of dormant cotton seeds were detected by means of ribonuclease interaction, methylated albumin kieselguhr chromatography and sedimentation analysis. Ribonuclease resistant RNA was also found associated with the dormant seed DNA. The implications of these two findings were discussed with regard to possible binding sites for RNA that stabilizes folded DNA.     

The Selective Effect of Abscisic Acid on Ribonucleic Acid Components

As determined by methylated albumin kieselguhr (MAK) fractionation, GA₃ (gibberellic acid) significantly increased and ABA (abscisic acid) decreased RNA levels. In the case of ABA this effect was selective, the ribosomal RNA manifesting the typical decrease; while the sRNA peak was markedly increased. The pattern of labelled uridine incorporation into RNA resembles the MAK absorbancy profile and here as well, ABA although causing an overall decrease, increases labelling in the sRNA peak. The results are interpreted as a possible selective effect of ABA or alternatively as an accumulation in the sRNA peak of rRNA breakdown products. From in vitro experiments it was furthermore evident that ABA mediated RNA hydrolysis probably does not involve a direct activation of RNase by ABA. The in vivo effect would probably be devious.     

Changes in the patterns of synthesis of ribonucleic acid species in immature rat uterus in response to oestradiol-17β

1. After treatment of immature rats with diethylstilboestrol, the wet weight and RNA content of uterine tissue increased rapidly, reaching a peak at 40hr. After an initial lag of a few hours, the acid-soluble ribose and protein contents also rose to maxima at 40hr. No increase in DNA content occurred until at least 24hr. after treatment. 2. The RNA from immature rat uterus isolated at various times up to 6hr. after administration of oestradiol-17beta was labelled by injecting [(3)H]uridine and [(3)H]guanosine intraperitoneally 30min. before the animals were killed. It was fractionated on columns of kieselguhr coated with methylated serum albumin and the radioactivity in fractions corresponding to transfer RNA, 7s RNA, ribosomal RNA, Q(1)-RNA, Q(2)-RNA and DNA-like RNA was determined. 3. The radioactivity of the whole RNA increased steadily for 6hr. after hormone treatment. The earliest changes occurred in the Q(1)-RNA (ribosomal RNA precursor), whereas at longer time-intervals the radioactivity of the ribosomal RNA, 7s RNA and transfer RNA increased by four- to five-fold. The radioactivity of the DNA-like RNA increased by about 50%, but only at the longer time-intervals. 4. It is concluded that one of the earliest changes in response to oestradiol treatment is a major increase in synthesis of ribosomal RNA followed later by a similar increase in synthesis of transfer RNA and by a much smaller increase in synthesis of DNA-like RNA. The change in synthesis of ribosomal RNA in immature rat uterus may represent one of the most important responses to oestradiol treatment.     

Characterization of messenger-like ribonucleic acid from Saccharomyces cerevisiae by the use of chromatography on methylated albumin–kieselguhr

Chromatography on methylated albumin–kieselguhr of RNA from Saccharomyces cerevisiae was used to separate stable RNA from a tenaciously bound DNA-like RNA fraction. The tenaciously bound RNA, which was eluted with a dilute solution of sodium dodecyl sulphate, was characterized as messenger-like RNA by its sedimentation behaviour, nucleotide composition, lack of methylated bases and labelling kinetics. Chromatography of purified ribosomal RNA indicated a minor contamination of the tenaciously bound fraction with ribosomal RNA. On the other hand, a large portion of pulse-labelled polyribosomal RNA from protoplasts of Saccharomyces cerevisiae was tenaciously bound to the columns. The `chase' of isotopic label from the messenger-like RNA was found to be retarded during inhibition of protein synthesis both by cycloheximide and by starvation for a carbon source.     

Metabolism of Adenine in Healthy and Blighted Potato Leaves

Analysis of the distribution of ¹⁴C in extracts prepared fromleaf tissue which had been exposed to labelled adenine by petiolaruptake revealed that this purine is extensively metabolizedin both healthy and Phytophthora-infected potato leaves. Incorporationof labelled adenine into the major ribonucleic acid speciesof the leaf was also extensive as determined by radioactiveassays performed on individual fractions which were separatedon columns of methylated albumin kieselguhr. Examination ofindividual nucleotides released by alkaline hydrolysis showedthat both the adenylic and guanylic acid moieties were labelled.Although the labelling patterns were similar for RNA from healthyand infected leaf tissue, the specific activity of the latterwas consistently higher than the former. When partially purified leaf extracts were assayed for phosphoribosyltransferase,they exhibited relatively high levels of activity with adenineas substrate, but were virtually devoid of activity with hypoxanthineand guanine. However, direct petiolar uptake of labelled hypoxanthineresulted in highly labelled RNA. A comparison of adenine phosphoribosyltransferase activity inextracts from healthy and blighted leaves failed to reveal measurabledifferences. Therefore, it was concluded that the differentialincorporation of labelled adenine into the RNA of healthy andinfected leaves was due neither to increased activity of thisenzyme in response to infection nor to its differential activation. Apart from its role in the recovery of preformed purines fornucleic acid synthesis, adenine phosphoribosyltransferase mayfunction as part of a mechanism for regulating levels of adeninein the potato leaf.     

Abscission: the role of RNA synthesis

Ethylene stimulated the incorporation of ³²P into RNA in the abscission zone of bean explants (Phaseolus vulgaris L. var. Red Kidney). The enhancement was observed in all fractions separated by methylated albumin kieselguhr column chromatography, although the magnitude of the increase was not the same for each fraction. Differential extraction of the nucleic acids indicated that the ethylene stimulation was confined to the fraction extracted with sodium lauryl sulfate, with the increase mainly in Fraction III (Ribosomal RNA) and Fraction IV (Messenger RNA). Actinomycin D, which blocks ethylene-stimulated abscission, inhibited ³²P incorporation into all column fractions. 5-Fluorouracil, which blocked 50% of the ethylene-enhanced ³²P incorporation, did not inhibit ethylene-enhanced abscission. The results indicate that ethylene may regulate abscission through control of specific RNA's.     

The origin of the polydispersity in sedimentation patterns of rapidly labelled nuclear ribonucleic acid

1. A study was made of the sedimentation properties of purified preparations of the rapidly labelled RNA in the nucleus and the cytoplasm of the HeLa cell. The sedimentation of the rapidly labelled nuclear RNA was very sensitive to changes in ionic strength and bivalent cation concentration. Under the conditions usually used in sucrose-density-gradient centrifugation the rapidly labelled nuclear RNA showed extreme polydispersity, and much of it sedimented more rapidly than the 28s RNA. At low ionic strength and after removal of Mg(2+), however, the rapidly labelled nuclear RNA sedimented as a single peak at about 16s. The conversion of the polydisperse material into the 16s form did not involve degradation of the RNA, since the effect could be reversed by increasing the ionic strength of the solution. 2. The cytoplasm did not contain any RNA that showed polydisperse sedimentation under the usual conditions of sucrose-density-gradient centrifugation, or that had the same sensitivity as the rapidly labelled nuclear RNA to changes in ionic strength. All the radioactivity in the cytoplasmic RNA sedimented with the 28s, 16s and 4s components over a wide range of physical conditions, but these components did contain a labelled fraction with some of the features of the rapidly labelled nuclear RNA on columns of methylated albumin on kieselguhr. 3. In both nucleus and cytoplasm the RNA detected by ultraviolet absorption could also be converted into a 16s form by removal of bivalent cations at low ionic strength; this effect was again, within certain limits, reversible. The nuclear RNA as a whole was more susceptible to changes in ionic strength than the cytoplasmic RNA. 4. It thus appears that all the RNA in the cell, except the 4s RNA, can be prepared, without degradation, as a single peak sedimenting at about 16s. The relationship of these various 16s components to each other is discussed.     

COMPARISON OF NUCLEIC ACIDS IN MAIZE SHOOTS AND PEA EPICOTYLS

A nucleic acid component (x-RNA) has been found in high concentration in maize shoots. It is eluted from a MAK (methylated albumin kieselguhr) column at about the same position as messenger RNA. The amount of x-RNA in pea epicotyls is absent or very low. It is suggested that x-RNA is long-lived messenger RNA and is found in high concentration in monocotyledonous plants, especially in the case of plants of the Gramineae family. Dicotyledonous plants, typically, contain little or no detectable x-RNA as observed by ultraviolet absorbancy. In the case of corn shoots, x-RNA is in highest concentration in the ribosomal fraction (78,500 × g, 70 min). In both maize shoots and pea epicotyls the newly synthesized nucleic acids were confined to the nuclear fraction (10,000 × g, 10 min).     

Properties of Single- and Double-stranded Ribonucleic Acid from Barley Plants Infected with Bromegrass Mosaic Virus

Incorporation of ³²P into nucleic acids in barley plants infected with bromegrass mosaic virus (BMV) was analyzed by chromatography on methylated albumin kieselguhr (MAK) columns. Treatment with actinomycin D reduced the synthesis of ribosomal ribonucleic acid (RNA) to low levels and allowed the detection of the three components of BMV-RNA in vivo. The kinetic study on ³²P incorporation into these BMV-RNA components suggested that a single cleavage occurred in some of the intact RNA shortly after completion of its synthesis, giving rise to the small and medium components. Chromatographic analyses also revealed a double-stranded, ribonuclease-resistant RNA which has been purified by differential extraction, sucrose-density gradient centrifugation, and MAK column chromatography. This RNA sediments at approximately 14S, is alkali-labile, and has a sharp thermal transition with a T_m of 96 C in 0.1 × standard saline citrate buffer, as determined by susceptibility to ribonuclease. The RNA is absent in uninfected barley plants.     

Immunofractionation of chromatin regions associated with histone H1o

Two monoclonal antibodies, which were elicited against histone H5, bind to purified rat liver chromatin and to rat liver H1o but not to rat liver H1. The monoclonal antibodies were immobilized on CNBr-Sepharose and the resulting immunoaffinity column was used to fractionate rat liver oligonucleosomes. Enzyme-linked immunoabsorbant assay (ELISA) and immunoblotting experiments indicate that the nucleosomes bound to the column were tenfold enriched in their content of H1o. Oligonucleosomes, prepared from the livers of either untreated or 3-methylcholanthrene-treated adult rats, were fractionated on the anti-H1o affinity column. The DNA purified from the unfractionated nucleosomes, from the unbound nucleosomes and from the nucleosomes which were bound to the column was examined with various 32P-labeled probes. A slight enrichment in H1o was detected in the coding region of the rat albumin gene. In contrast DNA which was bound to the column was significantly depleted in sequences hybridizing with total cellular RNA (which contains mostly ribosomal RNA) and with sequences hybridizing to the 3'-terminal region of a cytochrome P-450 gene, which is inducible by the chemical carcinogen 3-methylcholanthrene, regardless of whether isolated from control or from carcinogen-treated rat livers. Our experiments clearly demonstrate that chromatin can be efficiently immunofractionated. The results suggest that the H1o content of chromatin regions containing genes which are constitutively transcribed is not necessarily different from that of regions containing non-transcribed genes and that highly inducible genes may be segregated into chromatin regions which are depleted of H1o.     

Tight interaction between densely methylated DNA fragments and the methyl-CpG binding domain of the rat MeCP2 protein attached to a solid support.

In a previous report we have found that a number of short DNA fragments methylated at CpG sequences bound more tightly to a methyl-CpG binding column than DNA fragments having a larger number of methyl-CpG sequences. The column consists of a polypeptide comprising the DNA binding domain of the rat MeCP2 protein attached to a solid support. In the present study, we have investigated the features of short DNA fragments which bind tightly to a methyl-CpG binding column. Tight binding was observed when the DNA fragment had a high density of methyl-CpG sequences. Many of these fragments, derived from human genomic DNA, contained Alu repeated sequences supporting the previous observation that the highly-abundant Alu sequences are highly methylated. Our results suggest that methyl-CpG density is an important factor in the interaction between DNA fragments and the DNA binding domain of MeCP2 attached to a solid support.     

Studies on the biosynthesis of TMV

Summary RNA synthesis was investigated at different stages of systemic infections in which TMV formation approaches synchrony. The phenol-extracted nucleic acids were chromatographed on methylated albumin coated kieselguhr columns. At the onset of the rapid phase of virus particle formation RNA with the same chromatographic properties as TMV-RNA was predominantly synthesized when the leaves were labeled for 1.5 hours with ¹⁴C-uridine. Only a small amount of label was found in the double-stranded replicative form. At late stages of infection proportionally more replicative form was synthesized.When leaves at the start of the rapid phase of virus particle formation were labeled for 3.5 min only, a considerable amount of label was found in the replicative form. The proportion of radioactivity in this structure decreased with increasing labeling time, suggesting an intermediary function of this RNA.     

Inhibition of Host Cell Ribosomal Ribonucleic Acid Methylation by Foot-and-Mouth Disease Virus

A study of protein and ribonucleic acid (RNA) synthesis in cells infected by foot-and-mouth disease virus has indicated possible mechanisms of viral control over host cell metabolism. Foot-and-mouth disease virus infection of baby hamster kidney cells resulted in 50% inhibition of host cell protein synthesis at 180 min postinfection. A viral-induced interference with host cell RNA methylation was observed to be more rapidly inhibited than protein synthesis. To determine the nature of methylation inhibition, the kinetics of several host cell methylated RNA species were examined subsequent to virus infection. Data from sucrose zonal centrifugation and methylated albumin kieselguhr chromatography showed that methylation of nuclear RNA was inhibited 50% at 60 min postinfection. Inhibition of nuclear ribosomal RNA precursors and formation of nascent ribosomes correlated with inhibition kinetics of nuclear RNA methylation. It is suggested that the viral interference with the host nuclear RNA methylation is directly responsible for the observed loss of nascent ribosome formation. Moreover, early in the infectious cycle, methylation inhibition of host cell RNA could, in part, account for the cessation of host protein synthesis.     

Control of Deoxyribonucleic Acid and Ribonucleic Acid Synthesis in Pyrimidine-Limited Escherichia coli

The effects of pyrimidine limitation on chromosome replication and the control of ribosomal and transfer ribonucleic acid syntheses were investigated. Chromosome replication was studied by autoradiography of (3)H-thymine pulse-labeled cells. Pyrimidine limitation did not affect the fraction of cells incorporating radioactive thymine during a short pulse, indicating that when growth is limited by the supply of pyrimidine, the time required for chromosome duplication increases in proportion to the time required for cell duplication. Control of ribosomal RNA and transfer RNA syntheses was examined by chromatographing cell extracts on methylated albumin kieselguhr columns. When growth was controlled by carbon-nitrogen limitation, the ratio of tRNA to total RNA remained roughly constant at growth rates above 0.5 doublings per hour. During pyrimidine limitation, however, the control of rRNA synthesis was apparently dissociated from the control of tRNA synthesis: the ratio of tRNA to total RNA increased as the growth rate decreased.     

The binding of radioactive label from labelled phenacetin and related compounds to rat tissues in vivo and to nucleic acids and bovine plasma albumin in vitro

1. acetyl-(3)H- and ethyl-(14)C-labelled derivatives of phenacetin and related compounds are described. 2. Radioactive label from the ethyl-(14)C-labelled derivatives of 4-nitrophenetole, 4-phenetidine and phenacetin binds in vitro to various extents to bovine plasma albumin, salmon sperm DNA and yeast RNA; the extent of binding is increased in the presence of a rat liver microsomal hydroxylating system and further increased when the microsomal enymes are induced by prior treatment of rats with 3-methylcholanthrene. 3. The ratios of the bound radioactive labels in vitro from [ethyl-(14)C]phenacetin, N-acetoxy[ethyl-(14)C]phenacetin, [acetyl-(3)H]phenacetin and [diacetyl-(3)H]N-acetoxyphenacetin per g-atom of DNA P, RNA P and per mol of protein in the absence of the microsomal system are approximately 1:60:11:863, 1:68:41:1835 and 1:88:713:2399 respectively. 4. Radioactive label from labelled phenacetin binds in vitro to all tissues examined, including the spleen, intestines, kidney and bladder; about 80% of the radioactivity bound to the liver is concentrated in the RNA and proteins. 5. Comparison of the relative extents of binding of radioactive label derived from equimolar amounts of labelled phenacetin, ethanol or acetate shows that the incorporation of labelled C(2) units into tissues and biological macromolecules in vivo and in vitro may account for only a part of the total bound radioactive label derived from phenacetin and not at all from the incorporation of radioactive acetate into nucleic acids. 6. Some implications of these findings are discussed.