Intracellular distribution and sedimentation properties of virus-specific RNA in two clones of BHK cells transformed by polyoma virus.

The virus-specific RNA in two independently derived clones of polyoma virus-transformed hamster cells was studied by hybridizing labeled RNA, with excess purified polyoma DNA, immoblized on filters. In one clone (PyBHK1), less than 25% of the total labeled virus-specific RNA was found in the cytoplasm, irrespective of the labeling time. In the other clone (PyBHK2), it was estimated that 39% of the total virus-specific RNA was present inthe cytoplasm after labeling for 3 h. Both the proportion of radioactive label incorporated into virus-specific RNA and the sedimentation pattern of total virus-specific RNA differed markedly between PyBHK and PyBHK2. Most of the virus-specific RNA of PyBHK1 sedimented in the range 25S-35S, whereas a prominent 18S component was present in PyBHK2. Most of the cytoplasmic virus-specific RNA in both clones sedimented at 18S-19S. The sedimentation patterns of virus-specific RNA from whole cells and from washed nuclei of PyBHK1 were closely similar: it was estimated from sedimentation analysis in dimethyl sulfoxide that the molecular weight of 50% of this RNA was within the range 1.1 X10(6) to 2.9 X 10(6). These results, demonstrating the accumulation of virus-specific RNA within the nucleus in at least one virus-transformed cell line, indicate that the large virus-specific RNA previously described in the nuclei of transformed cells may not have represented precursors of virus-specific mRNA.     

Structure and Function of Bacteriophage R17 Replicative Intermediate Ribonucleic Acid II. Properties of the Parental Labeled Molecule

Replicative intermediate ribonucleic acid (RNA), designated RI, which contained parental RNA labeled with (32)P was separated by filtration through agarose from the nucleic acids prepared from (32)P-labeled RNA phage-infected Escherichia coli. A larger amount of ribonuclease-sensitive parental label was found in the rapidly sedimenting forms of RI than in the slower sedimenting forms, indicating that parental RNA is displaced to form a single-stranded tail. This result indicates that some phage RNA is generated by asymmetric semiconservative replication of RI, but it does not mean that a portion of the RI duplexes cannot be conserved during generation of phage RNA. Parental RNA was also found in double-stranded RNA with no apparent tails which sedimented with an S value of 13. This RNA was soluble in 2 m NaCl, and its sedimentation rate was unaffected by ribonuclease; nevertheless, single-strand scissions were produced by ribonuclease and were detected after the duplex was converted to its component single strands.     

A precursor of globin messenger RNA

The size of pulse-labeled globin messenger RNA nucleotide sequences was investigated, to determine whether newly transcribed globin mRNA molecules are larger than steady-state globin mRNA. Molecular hybridization techniques were used to compare directly the sedimentation of steady-state (unlabeled) and pulse-labeled (radioactive) globin mRNA sequences in the same analytical sucrose gradient. In gradients containing 98% formamide, radioactive globin mRNA sequences from mouse fetal liver cells labeled for 15 to 20 minutes with [³H]uridine sediment in a broad band with a peak at approximately 14 S, while steady-state globin mRNA sediments at 10 S. The large radioactive RNA can be recovered from one gradient and recentrifuged in a second gradient, in which it again sediments in a broad band with a peak at 14 S. The large radioactive RNA is cleaved to 10 S during a 75-minute “chase” with either actinomycin D or unlabeled uridine plus cytidine. The estimated half-life of the precursor is 45 minutes or less under these conditions. A covalent RNA precursor larger than 18 S with a similar turnover rate is not observed.     

RNA metabolsim during vegetative growth and morphogenesis of the cellular slime mold Dictyostelium discoideum

During vegetative growth of the cellular slime mold Dictyostelium discoideum, RNA is rapidly labeled by radioactive precursor and both the 25 S and the 17 S ribosomal RNA species appear in the cytoplasm 6–7 min after the onset of labeling. Thirty minutes after further incorporation of radioactive RNA precursors has been blocked, less than 10% of the label in RNA is associated with the nuclear fraction. After aggregation of the slime mold amoebae, RNA appears in the cytoplasm at a reduced rate, the small ribosomal subunit appearing in the cytoplasmic fraction more slowly than the larger ribosomal subunit. Some labeled RNA remains in the nuclei of developing cells long after the incorporation of ³H-uridine is blocked.     

Synthesis of Saint Louis Encephalitis Virus Ribonucleic Acid in BHK-21/13 Cells

Infection of baby hamster kidney cells (BHK-21/13) with Saint Louis encephalitis (SLE) virus depressed the rate of protein and ribonucleic acid (RNA) synthesis until viral RNA synthesis began 6 hr postinfection (PI). Virus-directed RNA synthesis was subsequently inhibited until 12 hr PI when virion maturation began. The rate of protein synthesis reached a peak 6 hr PI and was subsequently depressed until just before the onset of virion maturation. Density gradient analysis of phenol-extracted RNA from actinomycin-treated infected cells indicated that, at 6 to 8 hr and again at 12 to 20 hr PI, three species of viral-specific RNA were synthesized. The most rapid sedimenting form (43S) was ribonuclease-sensitive and had a base composition similar to the RNA isolated from mature virions. The 20S RNA species was ribonuclease-resistant and had a sedimentation coefficient and base composition similar to the replicative form associated with other arbovirus infections. The 26S RNA was ribonuclease-resistant (0.2 mug/ml, 0.1 m NaCl, 25 C, 30 min) and had a nucleotide base composition closer to the 20S form than to the values for 43S RNA. Five-minute pulse labeling of infected cultures during the period viral RNA synthesis was maximal resulted in labeling of only the 20S to 22S RNA fractions. With pulse-labeling periods of 10 min, both the 20S and 26S RNA species were radioactive. Periods of radioactive labeling of as long as 15 min were required before the 43S form was radioactively labeled. These results suggest that the 20S and 26S RNA may be intermediate forms in the synthesis of 43S viral RNA.     

Studies on 5-hydroxyuridine (isobarbituridine): X. Isolation and tentative identification of 5-hydroxymethyluridine from ribosomal RNA.

Yeast ribosomal RNA was hydrolyzed to its constituent nucleosides with the aid of snake venom and bacterial alkaline phosphatase. Lyophilized hydrolysate was labeled with radioactive 5-hydroxyuridine and applied to partition chromatography. It was found that some components of rRNA are held on the column and can be eluted with water. Eighty-nine percent of the label, a large portion of cytidine, and several unidentified compounds were found in the water wash. The direct application of the wash concentrate to ascending paper chromatography in saturated butanol-H2O resulted in the separation of three distinct UV-absorbing bands. Further resolution and characterization of one band of unidentified material revealed the presence of an additional nucleoside. On the basis of chromatographic and electrophoretic behavior and UV-absorption spectra, it was tentatively identified as 5-hydroxymethyluridine.     

RIBOSOME PRECURSOR PARTICLES IN NUCLEOLI

Ribonucleoprotein (RNP) particles containing the precursors of ribosomal RNA were extracted from L cell nucleoli and analyzed under conditions comparable to those used in the characterization of cytoplasmic ribosomes. Using nucleoli from cells suitably labeled with ³H-uridine, we detected three basic RNP components, sedimenting at approximately 62S, 78S, and 110S in sucrose gradients containing magnesium. A fourth particle, sedimenting at about 95S, appears to be a dimer of the 62S and 78S components. When centrifuged in gradients containing EDTA, the 62S, 78S, and 110S particles sediment at about 55S, 65S, and 80S, respectively. RNA was extracted from RNP particles which were prepared by two cycles of zonal centrifugation. The 62S particles yielded 32S RNA and a detectable amount of 28S RNA, the 78S structures, 32S RNA and possibly some 36S RNA, and the 110S particles, a mixture of 45S, 36S, and 32S RNA's. When cells were pulsed briefly and further incubated in the presence of actinomycin D, there was a gradual shift of radioactivity from heavier to lighter particles. This observation is consistent with the scheme of maturation: 110S → 78S → 62S. The principal buoyant densities in cesium chloride of the 110S, 78S, and 62S particles are 1.465, 1.490, and 1.545, respectively. These densities are all significantly lower than 1.570, which is characteristic of the mature large subunit of cytoplasmic ribosomes, suggesting that the precursor particles have a relatively higher ratio of protein to RNA, and that ribosome maturation involves, in addition to decrease in the size of the RNA molecules, a progressive decrease in the proportion of associated protein.     

Ribosomal-type ribonucleic acid from rodent mitochondria

1. Highly purified mitochondria containing 3.0mug of RNA/mg of mitochondrial protein were prepared from rat liver by differential centrifugation. 2. RNA, labelled with [(32)P]P(i) or [(3)H]orotate, was isolated from these mitochondria by a phenol extraction method. The RNA sedimented at 15S and 13S on sucrose density gradients. Its nucleotide composition was 23% uridylate, 30% adenylate, 22% guanylate and 25% cytidylate. 3. RNA from mouse L cells was labelled with [(3)H]-uridine in the presence of 0.1mug of actinomycin D/ml to suppress the synthesis of cytoplasmic rRNA. The RNA isolated from crude L-cell mitochondria by a cold-phenol-sodium dodecyl sulphate method had components sedimenting at 15S and 12.5S. These components had an electrophoretic mobility on agarose-acrylamide gels of 21 and 12S(E) compared with 28 and 18S(E) for cytoplasmic rRNA. The nucleotide composition was 26% uridylate, 34% adenylate, 18% guanylate and 22% cytidylate. 4. RNA extracted from crude L-cell mitochondria by a hotphenol-sodium dodecyl sulphate method had an additional component sedimenting at 21S and having an electrophoretic mobility of 18S(E). It was probably DNA because of its sensitivity to deoxyribonuclease and its insensitivity to ribonuclease and alkali. It was present in nuclear fragments contaminating the crude mitochondrial fraction and could be removed by deoxyribonuclease or isopycnic-gradient centrifugation.     

In Vitro Product of a Ribonucleic Acid Polymerase Induced by Influenza Virus

The ribonucleic acid (RNA)-dependent RNA polymerase induced in the microsomal fraction of cells infected with influenza virus synthesized a mixture of single-and double-stranded RNA in vitro. The single-stranded RNA sedimented mainly in the 8S region on sucrose density gradients, with a smaller proportion of the RNA sedimenting at 18S. This sedimentation pattern corresponds closely to that of incomplete influenza virus RNA. The double-stranded RNA formed in vitro sedimented at 11S, but molecules which may be replicative intermediate, sedimenting at 14 to 20S, were also detected in the in vitro reaction product. Similar species of RNA were detected in vivo by pulse-labeling infected cells at the time of polymerase harvest, but the proportion of each RNA species was different, most of the RNA being single-stranded and sedimenting in the 18S region. An 11S double-stranded RNA was also synthesized in vivo. Pulse chase analysis of the double-stranded RNA synthesized in vitro showed that most is stable, and only a small proportion turns over during the reaction. A proportion of the RNA formed in vitro could be annealed to RNA formed in infected cells and to RNA extracted from purified virus.     

Autoradiography ofCoelopa frigida (Diptera) embryos,after labeling with3H-uridine during various phases of oogenesis

Summary The distribution of tritiated (³H) uridine or its derivatives in embryos of the kelp flyCoelopa frigida is analyzed by autoradiography. The radioactive label is introduced into the growing oocyte, after injectio into females at different time periods post eclosion. The majority of the label remains in the cytoplasm for the entire embryonic development and is presumably incorporated into stable RNA. Only when the precursor is injected toward the final stages of oogenesis, can presence of label be detected in the nuclei, for a short time period during the blastodern stage. Since no preferential location in nucleoli is found, it appears possible that labeled cytidine could have been derived from the original precursor which would be available for incorporation into DNA.     

Sedimentation Properties of Simian Virus 40-Specific Ribonucleic Acid Present in Green Monkey Cells During Productive Infection and in Mouse Cells Undergoing Abortive Infection

The size distribution of polyribosome-associated simian virus 40 (SV40) ribonucleic acid (RNA) was examined at various times after productive infection. Eight hours after infection, virus-specific RNA was detected in the 14 to 17S region of a sucrose gradient by deoxyribonucleic acid (DNA)-RNA hybridization; RNA present in fractions sedimenting more rapidly did not react with SV40 DNA. At successively later times, SV40 RNA was detected in more rapidly sedimenting regions. By 24 hr, a portion of the SV40 RNA was detected in the 28S region, sedimenting slightly more rapidly than a MS2 RNA marker. Nuclear SV40 RNA, prepared from cells 48 hr after infection, was distributed in more rapidly sedimenting regions of the gradient, peaking at about 32 to 34S. Some nuclear virus-specific RNA could be detected in the 45 to 50S region. During the abortive infection of mouse cells, the sedimentation profile of SV40 RNA was very similar to that observed during the early phases of the lytic cycle.     

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.     

Structural Rearrangement and Subunit Composition of RNA from Released Soehner-Dmochowski Murine Sarcoma Virions

Two types of genomic, high-molecular-weight RNA species were found in Soehner-Dmochowski murine sarcoma virions released from virus-induced rat tumor cells grown in tissue culture. The type of RNA species observed depended on the length of exposure of the tumor cells to radioactive precursor. Early RNA of virions labeled up to 4 h with radioactive uridine had a sedimentation coefficient of 50S, and late RNA of virions labeled for 24 h had a sedimentation coefficient of 58S. Thermal transitions of early and late RNA indicated a difference in the configuration or structure of these two types of RNA. The late RNA may represent either a different configurational state of the early RNA or an aggregate molecule of two early RNA components joined together. Heat dissociation revealed that the major subunit of both RNA types was a 28S species, which was not susceptible to degradation by the addition of micrococcal nuclease to virions. A transitional, intermediate RNA species with a sedimentation coefficient of 37 to 40S was detected when early RNA was dissociated by dimethyl sulfoxide or heat at temperatures suboptimal for complete conversion. No free RNA subunit components were detected in virions harvested at intervals as short as 30 s or 5 min. A model for the assembly of genomic RNA from 28S RNA subunits is proposed.     

RNA synthesis in the ovary and oocytes of the starfish

Qualitative studies on the in vitro uptake and incorporation of tritiated uridine into RNA of the somatic and germinal elements of the starfish ovary were carried out prior to and during hormone-induced oocyte maturation and spawning.Autoradiography of nonhormone-treated ovaries indicated that the outer ovarian wall contained the highest concentration of label, with lesser amounts in the follicle cells and least in the oocytes. Oocytes and follicle cells localized at the periphery of the ovary were labeled first, and both cells became progressively labeled throughout the ovary with time; the label first appeared localized in the nucleolus of the oocyte.Sucrose gradient analysis of the separated cellular components of prelabeled hormone-treated ovaries indicated that RNA synthesis occurred in all segments of the ovary and that the spawned oocyte fraction was the least active. Synthesis of ribosomal RNA was detectable after a lag period of approximately 4 hr. Oocytes incubated in ³H-uridine during and subsequent to 1-methyladenine-induced spawning and maturation synthesized 15–19 S and low molecular weight RNA but not ribosomal RNA. Synthesis of the 15–19 S RNA was inhibited with ethidium bromide and to a limited extent by actinomycin D. Isolated mitochondrial fractions contained most of the labeled 15–19 S RNA. These data suggest the mitochondrial origin of most, if not all, of this intermediate-weight RNA. On the basis of these studies, it appears that starfish oocytes and follicle cells are metabolically active at the transitional period from growth to maturational stages in oocytes. Synthesis of RNA furthermore apparently continues in the cytoplasm subsequent to germinal vesicle breakdown and spawning.     

RNA synthesized in calicivirus-infected cells is atypical of picornaviruses.

RNA labeled with [3H]uridine from Vero cells infected with San Miguel sea lion virus in the presence of actinomycin D was analyzed by glycerol density gradient sedimentation and polyacrylamide gel electrophoresis. The predominant single-stranded RNA (36S, 2.6 x 10(6) molecular weight) was genome size. There was also a prominent 22S, 1.1 x 10(6)-molecular weight, single-stranded component and one or more double-stranded or partially double-stranded classes. Replicative forms, sedimenting at 18S, contained single-stranded RNA corresponding to the larger-molecular-weight class. All classes of intracellular RNA and virion RNA were polyadenylated. These findings and results with pig kidney cells infected with vesicular exanthema of swine virus and feline cells infected with feline calicivirus indicate that caliciviruses exhibit a strategy of replication different from typical picornaviruses and supports removal of the caliciviruses from the family Picornaviridae.     

Content of N-6 methyl adenylic acid in heterogeneous nuclear and messenger RNA of HeLa cells.

With the aid of a suitable thin layer chromatographic procedure, the N-6 methyl adenylic acid (m6A), content of a variety of 32P labeled RNA species from HeLa cells has been measured. Poly(A)-containing (poly(A)+) cytoplasmic RNA has on the average one m6Ap per 800 to 900 nucleotides. This value is independent of the length of the molecules. The proportion of m6Ap in poly(A)+ cytoplasmic RNA does not change between 4 and 18 hours of labeling with 32P, suggesting that the majority of the messenger RNA molecules may have a similar level of internal methylation regardless of their half-life. The non-polyadenylated, non-ribosomal cytoplasmic RNA fraction sedimenting from 10S TO 28S is less methylated with approximately one m6A per 2,700 nucleotides. Heterogeneous nuclear RNA molecules (DMSO treated) which sediment from 28S to 45S have approximately one m6Ap per 3,000 nucleotides. The hnRNA molecules sedimenting from 10S to 28S have one m6Ap per 1,800 nucleotides. Poly(A)+ nuclear RNA is enriched in m6A, containing 1 residue of m6A per 700 to 800 nucleotides, a value close to that obtained for the polyadenylated cytoplasmic RNA.     

Formation, size, and solubility in chloroform/methanol of products of protein synthesis in isolated mitochondria of rat liver and Zajdela hepatoma.

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incroporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HC1) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [3H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [3H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions.     

The metabolism of isocytidine in Escherichia coli

Intact cells and cell-free extracts of E. coli convert isocytidine to isocytosine and uracil. The radioactive label of 5-[³H]isocytidine is incorporated into RNA and, DNA of growing bacteria at a rate equal to about 1.4% of that of cytidine under similar conditions; the radioactivity is found in uridylic, cytidylic and 2′-deoxythymidylic acids, while less than 0.4% of incorporated radioactive material might be due to possible incorporation of intact isocytidine. Uridine phosphorylase and cytidine deaminase apparently do not participate in the metabolic conversion of isocytidine.     

Evidence for en bloc incorporation of exogenous oligonucleotides into HeLa cell RNA.

3H uridine and 3H guanosine labeled oligonucleotides were separately produced by degradation of high molecular weight RNA by endogenous intracellular ribonucleases of HeLa cells. After incubation of this low molecular weight RNA with HeLa cells under conditions limiting labeled mononucleotide incorporation, significant label was found in high molecular weight 18s RNA, but not in 28s RNA. The label in the 18s RNA was alkali labile and remained with the 18s RNA peak under denaturation conditions. Actinomycin in the incubation mixture prevented the appearance of label in the 18s RNA. These results raise the possibility that exogenous oligonucleotide segments can be incorporated en bloc into RNA.