Molecular weights of some HeLa ribosomal RNA's

The size of virus-specific RNA synthesized in cultured mouse kidney cells infected with polyoma virus was estimated by electrophoresis and sedimentation analysis of RNA extracts from whole cells. Newly synthesized "late" polyoma-specific RNA appears as "giant" molecules of heterogeneous size, up to several times larger than a strand of polyoma DNA (1.5 X 106 daltons). Treatment with dinethylsulfoxide or urea showed that the large size of these molecules is not due to aggregation. Giant polyoma-specific RNA is strikingly similar in size distribution to "nuclear messenger-like" RNA ("heterogeneous nuclear" RNA) of the host cell. Subsequent to its synthesis, some of the giant polyoma-specific RNA appears to be cleaved to at least three smaller species.During lytic infection of mouse kidney cell cultures with polyoma virus, virus-specific RNA can be detected by hybridization with polyoma DNA (1, 2). Viral RNA transcribed after the onset of polyoma DNA replication has been designated as "late" RNA (2). The rate of synthesis of late RNA increases rapidly beyond 12 hr after infection and approaches a maximum at about 30 hr.Earlier experimental results suggested that late polyomaspecific RNA is the transcript of most or all of the genetic information contained in a strand of polyoma DNA (2). We undertook the present experiments to define more precisely the size of late virus-specific RNA found in polyoma-infected cells. Total RNA was extracted about 30 hr after infection from ['HJuridine-labeled cultures under conditions that minimize breakdown, and was analyzed by gel electrophoresis and sucrose or Me2SO-sucrose gradient centrifugation. RNA from each gel or gradient fraction was hybridized with an excess of polyoma DNA to determine the amount of virus-specific RNA present. The results show that the bulk of late polyomaspecific RNA is synthesized as "giant" molecules larger than the viral genome, and that some of this giant RNA is subsequently cleaved to smaller RNAs of specific sizes. Within 10-30 sec of lysis, total cellular RNA was extracted with redistilled phenol; this treatment was repeated twice, for a total of 8 min at 60-650C (5). Nucleic acids were precipitated with ethanol, redissolved, and incubated at 00C for 30 min with 10 jhg/ml of DNase I (ribonuclease-free, electrophoretically purified; Worthington Biochemical Corp.) inl 0.01 M sodium acetate (pH 5.1)-2 mM MnCl2. Macaloid, 100 Mg/ml, was added to adsorb DNase and was removed by centrifugation; the purified RNA was reprecipitated. One Petri dish yielded 100-150 ug RNA (taking 1 Am0 Unit to equal 42 ug RNA.) DNA-RNA hybridization Highly purified polyoma DNA I, prepared as the 53 S form by sedimentation at pH 12.5 (6), was converted into single strands by boiling for 30 min in 0.1 X SSC [SSC: 0.15 M NaCl-0.015 M Na citrate (pH 7.4)], cooled rapidly by dilution in ice-cold 6 X SSC, and fixed onto 25-mm membrane filters (Schleicher and Schuell, B6) by filtration (7). Filters 3.5 mm in diameter were cut from dried and baked (800C, 2 hr) filters. A ...

Section: Methodsmentioning

confidence: 89%

Transcription of the Polyoma Virus Genome: Synthesis and Cleavage of Giant Late Polyoma-Specific RNA

Acheson¹,

Buetti²,

Scherrer³

et al. 1971

“…Cytoplasmic polyribosomes were prepared with Nonidet P-40 followed by separation in linear 7.5-45% sucrose gradients as described (9). An alternative method, centrifugation through 50% sucrose in RSB [0.01 M NaCl, 1 20-to 25-,ug Samples of RNA in 0.01 ml of 0.01 M NaCl were diluted with 0.09 ml of Me2SO and 0.01 ml of dimethylformamide and layered on the above gradients, which were centrifuged in an SW65 rotor for 15 hr at 50,000 rpm at 270. Sevendrop fractions were collected from the bottom of the gradient with a probe and a Buchler pump.…”

Section: Methodsmentioning

confidence: 99%

Adenovirus Messenger RNA in Mammalian Cells: Failure of Polyribosome Association in the Absence of Nuclear Cleavage

McGuire

Swart

Hodge

1972

The nuclear synthesis of adenovirus-specific RNA late in the infectious cyclein the presence of toyocamycin (an adenosine analogue) has been investigated. There is reduced synthesis of viral RNA with an accumulation of virus-specific RNA in the molecular weight range of at least 4 to 8 X 106. No new viral RNA associates with cytoplasmic polyribosomes. In addition, hybridization competition experiments indicate a 70% competition between these large nuclear transcripts and polyribosomeassociated viral RNA that was synthesized in the absence of inhibitor. These data are consistent with the following interpretations: complete nuclear processing of viral RNA is necessary for polyribosome association, and precursor viral message(s) contain sequences that are lost normally during post-transcriptional processing.Evidence has been presented that messneger RNA(mRNA) in mammalian cells may undergo a process of cleavage after transcription. A precursor-product relationship has been suggested between nuclear heterogeneous RNA of high molecular weight and the mRNA of lower molecular weight that is associated with polysomes in HeLa cells (1). In addition, cells either lytically infected or transformed by DNA nuclear viruses contain larger virus-specific RNA transcripts in the nucleus than in the cytoplasm (2, 3). To examine in more detail the nature of this cleavage of mRNA and its possible implications in mammalian cells, it would be helpful if the processes of synthesis and cleavage could be separated. This separation has been achieved by the use of toyocamycin in studies on processing of ribosomal RNA (4). When used in relatively low concentrations, this inhibitor is incorporated into the newly synthesized 45S RNA resulting in the subsequent failure of processing. In the present study, the effect of toyocamycin on processing of viral RNA has been investigated. HeLa cells, at 22 hr after infection with adenovirus 2, were incubated with this inhibitor and the synthesis, accumulation, and fate of nuclear viral transcripts were ascertained. The data demonstrate that in the presence of inhibitor large virus-specific transcripts are synthesized and accumulate in the nucleus, but fail to be processed. Under these conditions, no virus-specific RNA becomes associated with cytoplasmic polyribosomes. MATERIALS AND METHODSMaintenance of Cells. HeLa S3 cells were maintained in suspension culture in Eagle's medium (5) supplemented with 7% calf serum.Preparation of Virus and Infection of Cells. Stocks of adenovirus type 2 were prepared and stored as described by Maizel et al. (6). Virus preparations were used within 1 month and were free of identifiable, small, adeno-associated virus. Cells were infected at a concentration of 2000 viral particles per cell as described (7).Preparation of Nuclear RNA. Nucleoplasm was prepared from HeLa cells by the procedure of Penman (8). Nucleic acid and protein were precipitated at -20°for 16 hr with 0.15 M sodium chloride, 0.1% of 2-mercaptoethanol, and 2.5 volumes of ethanol. This precipitate ...

“…As seen in Fig. 5c, 10-15% of the singlestranded, 3H-labeled RNA sedimented at about 18 S (molecular weight = 0.71 X 106) (23). Since hidden breaks in the RNA were probably introduced during the RNase treatment to which the molecules had been subjected during preparation, the size and amount of the high molecular weight RNA should be considered as minimum figures.…”

Section: Methodsmentioning

confidence: 99%

Extensive Symmetrical Transcription of Simian Virus 40 DNA in Virus-Yielding Cells

Aloni

1972

Rapidly labeled RNA was extracted from monkey cells after infection with Simian Virus 40 (SV40) and exposure to short pulses of [5-3Hjuridine late in infection. When this RNA was self-annealed, it became resistant to digestion with ribonuclease. The fraction of RNA that resisted the ribonuclease treatment decreased with increased labeling time, or when a short pulse of radioactivity was followed by incubation with unlabeled uridine and actinomycin D. The RNase-resistant RNA was isolated by chromatography on Sephadex G-100 and shown to be double-stranded by its susceptibility to ribonuclease as a function of salt concentration and temperature. This behavior was not due to RNA-DNA hybrid formation, since deoxyribonuclease had no effect upon the double-stranded molecules, even after their denaturation. The relation of the double-stranded RNA to SV40 was demonstrated by the hybridization of about 50%7 (corrected value, >90%7,) of the separated RNA strands with component I of SV40 DNA from plaque-purified virus. After self-annealing in formamide at low temperature, about 10%O of the rapidly labeled, viral RNA sedimented at 13 S. This value corresponds in size to about 60%/c of the SV40 DNA.These observations indicate that late in infection of monkey cells, SV40 DNA is transcribed symmetrically over a considerable portion of its length, and that subsequently some sequences from one or both of the RNA strands are degraded.It has been generally accepted that transcription is asymmetric, in the sense that only one of the two DNA strands of a given gene serves as a template for RNA (1). However, the possibility that both DNA strands are transcribed, and that some sequences from one or both of the RNA strands are subsequently degraded, has not been excluded. Indeed, it has recently been found that transcription of mitochondrial DNA in HeLa cells operates according to the latter possibility (2. 3). SV40 DNA has circular configurations similar to those of mitochondrial DNA from HeLa cells (4). Early after infection, and in SV40-transformed cells, the viral genome is only partially transcribed, while at later stages in the infection process most or all of the genetic information contained in the SV40 genome is transcribed (5, 6). I show that late after infection of monkey cells with SV40, the viral DNA is transcribed symmetrically over a considerable portion of its length and, subsequently, some sequences from one or both of the RNA strands are degraded. MATERIALS AND METHODSGrowth of SV40 (strain 777) not purified by plaque formation on monkey cells of the BS-C-1 line, as well as concentration and purification of the virus from the tissue culture lysates, were described (5). In all experiments, cultures (4 X 106 cells) were infected with 1.0 ml of the same stock of SV40 [4 X 108 plaque-forming units (PFU)/ml] except in the experiment described in Table 2, where both plaque-purified and nonpurified virus was used for infection of the cells. DNA was extracted from the purified virus with phenol (7) In each case, RNA...