SELECTIVE RETENTION AND FILTRATION OF BRAIN NUCLEIC ACIDS IN AGAROSE GELS

Abstract— Total nucleic acids of rat brain have been separated by agarose gel chromatography at 2 m -NaCl into DNA. transfer RNA plus low molecular weight RNA. and high molecular weight RNA fractions. The DNA fraction contained less than 1 per cent RNA by weight judged by either short-term or long-term labelling with ortho[³²P]phosphate. The high molecular weight RNA fraction contained 28 s and 18 s ribosomal RNAs and a heterogeneous population of 20-60 s RNAs, apparent after short-term labelling and characterized by a high content of nearest-neighbour-labelled uridylic acid. The rapidly sedimenting (>30 s ) portion of these RNAs could be largely separated from ribosomal RNAs by gel filtration using 4% agarose. The ribosomal RNAs could be fully resolved into 28 s and 18 s components by agarose gel chromatography at 0.5 m -0.6 m -NaCl, as shown by analysis of their sedimentation and nucleotide composition.     

Transport of different RNA species from the nucleus to the cytoplasm in Xenopus laevis neurula cells

Isolated cells from Xenopus laevis neurulae were labeled, and the RNAs extracted from their nuclear and soluble cytoplasmic fractions were analyzed on polyacrylamide gels. In the soluble cytoplasm, 4S RNA emerged very rapidly, and this was immediately followed by the emergence of poly(A)-containing RNA and 18S ribosomal RNA. In contrast, the emergence of 28S ribosomal RNA was delayed by about 2 hr. The size distribution of cytoplasmic poly(A)-containing RNA was much smaller as compared to that of nuclear poly(A)-containing RNA. These results indicate that the newly synthesized RNAs in Xenopus neurula cells are transported from the nucleus to the cytoplasm in a characteristic sequence.     

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.     

Low molecular weight RNAs hydrogen-bonded to nuclear and cytoplasmic poly(a)-terminated RNA from cultured chinese hamster ovary cells

A group of RNAs 90–100 nucleotides long were isolated by melting them from poly(A)-terminated nuclear or cytoplasmic RNA from cultured Chinese hamster ovary cells. Conditions that favor hydrogen bond formation allowed the reassociation of these low molecular weight RNAs with poly(A)-terminated RNA. The nuclear poly(A)-terminated molecules contained 1.3 moles of the low molecular weight RNAs per mole of poly(A), while the cytoplasmic poly(A)-terminated RNA contained only one seventh as much. These low molecular weight RNAs were also isolated from the total 4S RNA of either the nucleus or cytoplasm by polyacrylamide gel electrophoresis. They formed a prominantly labeled band of RNA in the gels after cells had been labeled with H₃³²PO₄ for 4 hr. The low molecular weight RNAs melted from the nuclear poly(A)-terminated RNA were slightly different (although not necessarily in primary nucleotide sequence) from those melted from the cytoplasmic poly(A)-terminated RNA.     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

Small RNAs of Tetrahymena thermophila

Highly purified nuclear and cytoplasmic RNAs were obtained from Tetrahymena thermophila BVII containing only a minimal amount of cross-contamination. In the nuclear RNA fraction we have detected at least 6 distinct snRNAs. Some of the RNA species showed microheterogeneity. SnRNAs of Tetrahymena thermophila are very similar to rat snRNAs, as far as length is concerned. Our cytoplasmic small RNA fraction contained two RNAs, 7S and T7, reported recently (18) as nuclear, particularly nucleolar RNAs. Moreover, we could detect only one cytoplasmic small RNA species Tc1, Tc2 was not observed.Neither the nuclear nor the cytoplasmic small RNA species are degradation products of ribosomal RNA as was shown by Northern blotting and following hybridization with pGY17 containing the entire transcribed region of the ribosomal DNA of Tetrahymena thermophila.     

KINETICS OF GROWTH OF HAEMOPOIETIC COLONY CELLS IN AGAR

Cell kinetic parameters of mouse granulocytic and mononuclear cells growing in colonies in agar cultures have been measured. Analysis of flash and continuous labelling studies with ³H-thymidine together with determinations of colony size, growth fraction and mitotic indices, gave the following values for the phases of the cell cycle: G₁= 6·3 1·6 hr, S = 5·8 ± 1·4 hr, G₂= 1·7 ± 0·1 hr and M = 0·7 ± 0·1 hr (42 ± 8 min). No difference in the cell cycle parameters of granulocytic and mononuclear cells were found in this study.
Colonies of different size from cultures of the same age group had similar labelling indices, indicating that the size of a colony is not a function of the rate of proliferation of cells in the colony. Rather, variation in colony size is probably representative of an initial delay in the onset of colony development.     

Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

DNA fiber replication in chromosomes of a higher plant (Pisum sativum).

Ribosomal precursor particles were extracted from the yeast Saccharomyces carlsbergensis and analysed. After a brief labelling of yeast protoplasts with ³H-uridine, three basic ribonucleoprotein components were detected, sedimenting at approx. 90S, 66S and 43S in sucrose gradients containing magnesium. The 90S particles contained the 37S ribosomal precursor RNA as a major component and a small though variable amount of 29S ribosomal precursor RNA. The 66S and 43S particles contained 29S and 18S ribosomal precursor RNA, respectively. Kinetic data indicate a precursor-product relationship between the 90S particles and the two other ribonucleoprotein components, consistent with the conversion: 90S → 66S + 43S. The 90S and 66S preribosomes appeared to be present exclusively in the nucleus, whereas the 43S particles were mainly present in the cytoplasmic fraction. Apparently, the final maturation step in the formation of the 40S ribosomal subunits takes place in the cytoplasm. The 90S and 66S precursor particles have a relatively higher ratio of protein to RNA than the mature large ribosomal subunits, as judged from their buoyant densities in CsCl gradients. This finding suggests that also in a primitive eukaryotic organism, like yeast, ribosome maturation involves, in addition to a decrease in the size of the RNA components, an even stronger decrease in the amount of associated protein. In contrast, the 43S particles appeared to have the same buoyant density as the 40S ribosomal subunits.     

RNA SYNTHESIS IN THE DIFFERENT PHASES OF CELL CYCLE OF CHICK EMBRYO CELLS

RNA synthesis was studied at different phases of the cell cycle of chick embryo fibroblasts, which were synchronized by medium replacement in the confluent phase. The synthesis of DNA started at 4 hr and continued for 8 hr. RNA synthesis increased with time after medium change. The ratio of total amount of radioactivity in nuclear RNA prepared at 0, 2 and 8 hr was 1.0:1.03:5.05. The distribution of radioactive RNA in the sedimentation pattern was similar, showing remarkable incorporation in 45S region of ribosomal precursor RNA. The base composition of newly synthesized RNA, however, varied at different time intervals after medium replacement. Even within the G₁ phase, the molar percentage of G and C was quite different. Treatment with actinomycin D at a concentration of 0.02 μg/ml for 1 hr specifically inhibited ribosomal RNA synthesis. At 2 hr after medium change, ribosomal and AU-rich RNA including larger than 28S were synthesized in about equal amounts.     

Variability in Complementarity for Chloroplastic and Cytoplasmic Ribosomal Ribonucleic Acid among Plant Nuclear Deoxyribonucleic Acids

The nuclear DNAs from a number of angiosperm species were tested for hybridization to the RNAs contained in 70 S (chloroplastic) and 80 S (cytoplasmic) ribosomes. All of the DNAs contained regions complementary to RNAs from chloroplastic as well as cytoplasmic ribosomes. DNAs from closely related plants varied widely in their proportion of coding for these RNAs. About 0.15% of the DNAs from a number of different species of Nicotiana were found to be complementary to the RNAs of each kind of ribosome; however, DNAs from some other members of this genus had more than three times this proportion of coding for ribosomal RNAs. These and other data suggest that hybridization percentage for ribosomal RNA is not a familial or generic characteristic.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

Intranuclear distribution of rat liver ribosomal RNA]

A method is described for the isolation of pure liver nuclei with minimal cytoplasmic contaminants, loss of nuclear RNA and degradation of nuclear RNA. The RNA components are extracted in three distinct fractions by subsequent treatment with phenol at 4 degrees, 50 degrees and 85 degrees C. The total and 14C-orotate labelled RNA components in the three nuclear RNA fractions are characterized by nucleotide composition, poly(A)-RNA content and agar-gel electrophoresis. The results show that the RNA in three fractions correspond to the nucleosol, nucleolus and chromatin compartments of the nucleus. The nuclear HnRNA components are exclusively in the 85 degrees C RNA. Nuclear ribosomal RNA is extracted in the 4 degrees C and 50 degrees C RNA fractions. These two nuclear RNA fractions are distinct in constituent pre-rRNA species and the rate of labelling of their rRNA components. The amount of the pre-rRNA and rRNA species is determined. The results show that the nucleolus-nucleosol and nucleosol-cytoplasm transitions of ribosomal subparticles are markedly slower processes than the preceeding steps of ribosome biogenesis.     

THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

Biochemical studies of mammalian oogenesis: synthesis and stability of various classes of RNA during growth of the mouse oocyte in vitro

The synthesis of various classes of RNA in mouse oocytes at different stages of growth has been examined after incubating follicles in medium containing radiolabeled uridine. After fractionation on poly(U)-Sepharose of radiolabeled oocyte RNA, of which about 83% is associated with the nucleus after a 5-hr labeling period, revealed that about 40–50% of the radiolabeled RNA behaved as poly(A)-containing RNA. This value remained fairly constant during the period of oocyte growth in which oocyte diameter increased from about 35 to about 55 μm. After a 5-hr labeling, the percentage of radiolabeled poly(A)-containing RNA in either the fully grown dictyate oocyte, metaphase II oocyte, or one-cell embryo was about 20%. After a 5-hr labeling, agarose gel electrophoretic analysis of the radiolabeled species of oocyte RNA obtained after fractionation on poly(U)-Sepharose revealed the presence of a putative ribosomal RNA precursor, ribosomal (28 and 18 S) RNA, transfer plus 5 S RNA and heterodisperse poly(A)-containing RNA. A significant fraction of the radiolabeled RNA species was quite large (>40 S). The ratios of the relative proportions of the radiolabeled ribosomal RNAs and transfer plus 5 S RNA remained essentially constant during oocyte growth. The stability of various classes of RNA was examined by incubating follicles with radiolabeled uridine, washing the follicles free of radioactivity and culturing the follicles under conditions which support oocyte growth in vitro (Eppig, 1977). Under these conditions, total oocyte radiolabeled RNA was quite stable as determined by retention of acid-insoluble radioactive material ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 28}\text{days}$). However, under conditions in which oocytes are viable but do not grow, the half-life of total RNA was about 4.5 days. Poly(A)-containing RNA was also very stable; after 8 days in culture, about 50% of the radiolabeled poly(A)-containing RNA present after 5 hr of labeling was still present. Agarose gel electrophoretic analysis of radiolabeled RNA in oocytes after 4 days of culture and after fractionation on poly(U)-Sepharose revealed the presence of ribosomal (28 and 18 S) RNA, transfer plus 5 S RNA, and heterodisperse poly(A)-containing RNA. At this time, these RNAs are located in the oocyte cytoplasm. In addition, the molecular weight distribution of poly(A)-containing RNA was significantly lower than that after 5 hr of labeling. The ratios of the relative proportions of radiolabeled ribosomal RNAs and transfer plus 5 S RNA were quite similar to those after 5 hr of labeling.     

Effects of 5-azacytidine on nucleolar RNA and the preribosomal particles in Novikoff hepatoma cells.

Examination of nucleolar RNA from cultured Novikoff hepatoma cells treated for 3 hr with 5 x 10-4 M 5-azacytidine shows that significant amounts of analog-substituted 45S RNA are processed to the 32S RNA species, but 28S RNA formation is completely inhibited. Under these conditions of analog treatment 37% of the cytidine residues in the 45S RNA is replaced by 5-azacytidine. During coelectrophoresis of nucleolar RNA from 5-azacytidine-treated and control cells, the analog-substituted 45S RNA and 32S RNA display reduced mobilities compared to the control 45S RNA and 32S RNA. Coelectrophoresis of analog-substituted and control RNA after formaldehyde denaturation shows no differences in electrophoretic mobility between the two RNA samples, suggesting that 5-azacytidine incorporation may alter the secondary structure of the 45S RNA and the 32S RNA. 5-Azacytidine at 5 x 10-4 M severely inhibits protein synthesis in Novikoff cells by 3 hr. After this length of treatment, however, CsCl buoyant density analysis reveals no difference in density of either the 80S or 55S preribosomal ribonucleoprotein particles when compared to normal particles. Also 5-azacytidine treatment does not appear to cause major changes in the polyacrylamide gel electrophoresis patterns of the proteins in the 80S and 55S preribosomal particles. These results together with previous findings suggest that 5-azacytidine's inhibition of rRNA processing is possibly related to its alteration of the structure of the ribosomal precursor RNAs and is not a consequence of a general inhibition of ribosomal protein formation.     

Small molecular weight RNA components in sea urchin embryos

Polyacrylamide gel electrophoresis of RNA from Paracentrotus lividus embryos has shown this material to contain five RNA components of small molecular weight. The components are synthesized early in sea urchin development, simultaneously with tRNA and heterodisperse RNA. After the blastula stage, when synthesis of ribosomal RNA is activated, the labeling incorporated into small molecular weight RNA components constitutes a relatively decreasing proportion of the total labeling in RNA. When labeling is performed prior to the blastula stage, three of the small molecular weight RNA components are labeled to a similar or greater extent than “5” S RNA and the 26-ass RNA. The gel electrophoretic mobilities of the small molecular weight RNA components have been compared with those in Ehrlich ascites cells and found to be different.     

Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus: I. Patterns of Ribonucleic Acid Synthesis in Productively Infected Cells

HEp-2 cells were pulse-labeled at different times after infection with herpes simplex virus, and nuclear ribonucleic acid (RNA) and cytoplasmic RNA were examined. The data showed the following: (i) Analysis by acrylamide gel electrophoresis of cytoplasmic RNA of cells infected at high multiplicities [80 to 200 plaque-forming units (PFU)/cell] revealed that ribosomal RNA (rRNA) synthesis falls to less than 10% of control (uninfected cell) values by 5 hr after infection. The synthesis of 4S RNA also declined but not as rapidly, and at its lowest level it was still 20% of control values. At lower multiplicities (20 PFU), the rate of inhibition was slower than at high multiplicities. However, at all multiplicities the rates of inhibition of 18S and 28S rRNA remained identical and higher than that of 4S RNA. (ii) Analysis of nuclear RNA of cells infected at high multiplicities by sucrose density gradient centrifugation showed that the synthesis and methylation of 45S rRNA precursor continued at a reduced but significant rate (ca. 30% of control values) at times after infection when no radioactive uridine was incorporated or could be chased into 28S and 18S rRNA. This indicates that the inhibition of rRNA synthesis after herpesvirus infection is a result of two processes: a decrease in the rate of synthesis of 45S RNA and a decrease in the rate of processing of that 45S RNA that is synthesized. (iii) Hybridization of nuclear and cytoplasmic RNA of infected cells with herpesvirus DNA revealed that a significant proportion of the total viral RNA in the nucleus has a sedimentation coefficient of 50S or greater. The sedimentation coefficient of virus-specific RNA associated with cytoplasmic polyribosomes is smaller with a maximum at 16S to 20S, but there is some rapidly sedimenting RNA (> 28S) here too. (iv) Finally, there was leakage of low-molecular weight (4S) RNA from infected cells, the leakage being approximately three-fold that of uninfected cells by approximately 5 hr after infection.     

Synthesis and transport of various RNA species in developing embryos of Xenopus laevis.

Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.