Poly A associated with SV40 Messenger RNA

The template properties of chromatin were studied in a system in which transcription of unique genes could be examined. Chromatin and DNA were isolated from SV3T3 cells, a line of simian virus 40 (SV40)-transformed mouse cells, and used as templates for Escherichia coli RNA polymerase. The SV4O sequences in the RNA transcribed in vitro and in RNA isolated from the transformed cells were analyzed by hybridization to radioactive RNA transcribed from SV40 DNA. The results indicate that the same sequences of the SV40 "-" strand (DNA strand which serves as a template for early RNA) are transcribed in vitro from chromatin as are found in transformed cells. In contrast, when the DNA isolated from SV3T3 chromatin by removal of proteins is used as a template, the entire SV40 "-" strand is transcribed. It is concluded that the specific template restriction observed with chromatin is due to the presence of proteins associated with the DNA.Much of the DNA of eukaryotic cells is bound to proteins (1). One mechanism for regulation of gene expression in vivo may be that these proteins prevent transcription of the region of DNA to which they are bound. Indeed, the proteins bound to the DNA of purified chromatin do seem to inhibit the ability of specific sequences in the DNA to act as template for transcription by Escherichia coli RNA polymerase in vitro (5,7,12,13), presumably by rendering these sequences inaccessible to the polymerase. Moreover, the DNA sequences of chromatin that are available for transcription in vitro are the same sequences that are transcribed in vivo, at least with respect to families of related or reiterated sequences (8,12,15). Whether such a mechanism operates to repress transcription of unique sequences is not known. If so, it may represent a general mechanism by which gene expression is regulated in eukaryotic cells. Chromatin would thus be an appropriate system for studying eukaryotic gene regulation in cell-free systems.We have attempted to clarify the template properties of chromatin by examining the transcription in vitro of the simian virus (SV)40 genome in SV3T3 cells, a line of mouse fibroblasts transformed by SV40 virus. The use of this system to study chromatin has the following unique advantages. (1) The SV40 genome is integrated into cellular DNA (9) at a frequency of about one copy per cell (4) and is not part of a family of related sequences. Consequently, RNA that hybridizes to SV40 DNA must be a transcript of the integrated SV40 genome, and not of a related sequence. (2) Although the entire viral genome is present (17), less than 70% of the genome is transcribed in transformed cells (10). Consequently, one may determine whether the same regions of the genome are available for transcription in chromatin as are transcribed in vivo. (3) When purified SV40 DNA (covalently closed double-stranded circles) is used as a template for E. coli RNA polymerase, the entire "-" strand (DNA strand that serves as a template for early RNA) is transcribed (19). Therefore, an inability of the poly...

Section: Methodsmentioning

confidence: 99%

In Vitro Transcription of Simian Virus 40 Sequences in SV3T3 Chromatin

Astrin

1973

“…Unlike many viruses, SV40 does not depress cellular RNA and protein synthesis, and the viral capsid structural proteins do not represent more than 10% of the total cellular spectrum of polypeptides even late in lytic infection (3,4). However, in this report we will show that by combining a sensitive cell-free translation system from wheatgerm extracts (5) with relatively simple methods of purification of viral messenger RNA (mRNA) based both on the presence of polyadenylate [poly(A) ] sequences (6,7) and selective hybridization to SV40 DNA (8), it is possible to synthesize in vitro the major SV40 capsid protein, VP-1. The cell-free translation of oncogenic virus mRNA, selected by hybridization, can provide the basis for identifying additional virus-specified products in permissive and transformed cells.…”

mentioning

confidence: 99%

Cell-Free Translation of Messenger RNA of Simian Virus 40: Synthesis of the Major Capsid Protein

Prives¹,

Aviv²,

Paterson³

et al. 1974

Extracts of wheat germ are capable of synthesizing the major capsid protein of simian virus 40. Poly(A)-containing RNA from BS-C-1 cells infected with simian virus 40 directed the synthesis of a novel polypeptide that migrates in polyacrylamide gels together with the major capsid polypeptide of simian virus 40, VP-I. The patterns of the major tryptic peptides of purified VP-1 and the novel polypeptide synthesized in vitro were identical after two-dimensional paper electrophoresis. The novel polypeptide was not synthesized in response to poly(A)-rich RNA from uninfected cells or from virus-infected cells treated with cytosine arabinoside. Messenger RNA from infected cells purified by selective hybridization to DNA of simian virus 40 directs the synthesis of a major polypeptide of electrophoretic mobility similar to that of VP-1 of simian virus 40. This approach should prove useful in identifying additional products specified by DNA tumor viruses.Depending on the host cell, the oncogenic simian virus 40 (SV40) can produce either a lytic response accompanied by the production of viral progeny, or cell transformation. In either case, the number of changes wrought in the cell by viral infection appears to be too numerous to be directly accounted for by the relatively small (3 X 106 daltons) viral genome (1, 2). It follows that these changes may represent a pleotropic response initiated by one or very few viral gene products. One decisive way to identify a virus-specific product is by means of the cell-free synthesis of proteins directed by purified viral genetic material. Unlike many viruses, SV40 does not depress cellular RNA and protein synthesis, and the viral capsid structural proteins do not represent more than 10% of the total cellular spectrum of polypeptides even late in lytic infection (3, 4). However, in this report we will show that by combining a sensitive cell-free translation system from wheatgerm extracts (5) with relatively simple methods of purification of viral messenger RNA (mRNA) based both on the presence of polyadenylate [poly(A) ] sequences (6, 7) and selective hybridization to SV40 DNA (8), it is possible to synthesize in vitro the major SV40 capsid protein, VP-1. The cell-free translation of oncogenic virus mRNA, selected by hybridization, can provide the basis for identifying additional virus-specified products in permissive and transformed cells. MATERIALS AND METHODS Cells and virusesA plaque-purified stock of SV40 (strain 777) was grown in the BS-C-1 line of African green-monkey kidney cells as described (9). For production of radioactive virus, [IS]methionine (3 ,Ci/ml of culture medium; 170 Ci/mmol, The Radiochemical Center, Amersham, England) was added 24 hr after infection to cells in medium containing 7.5 ,ug/ml of methionine and 2% calf serum. The labeled virus was purified by two consecutive bandings in CsCl density gradients (9) and dialyzed against 0.01 M Tris* HCl buffer, pH 7.9 containing 0.15 M NaCl. The purified virus was dissociated by heating at 1000 for 1 min in a ...

“…In HeLa cells, the majority of mitochondrial poly(A) is covalently linked to mitochondrial DNA (mit-DNA)-coded RNA (2). Inasmuch as poly(A) in eukaryotic cells appears to be associated only with cytoplasmic messenger RNA (mRNA) and its presumptive nuclear precursors (5-10), and with viral or viral-directed RNA with messenger function (5,(11)(12)(13)(14)(15), the above evidence strongly suggests that at least a portion of the mRNA translated by mitochondrial polysomes is coded for by mit-DNA.Previously (2), no clear-cut evidence of discrete components was observed in the sedimentation patterns of poly(A)-containing RNA from HeLa-cell mitochondria. The main source of difficulty was probably the tendency of poly(A)-containing molecules to aggregate and sediment with heavier molecules.…”

mentioning

confidence: 99%

Identification of Discrete Polyadenylate-containing RNA Components Transcribed from HeLa Cell Mitochondrial DNA

Ojala¹,

Attardi²

1974

Polyacrylamide gel electrophoresis and sedimentation analysis under denaturing conditions of poly(A)-containing RNA from the polysome region of the sedimentation pattern of a HeLa-cell mitochondrial lysate has revealed the occurrence of a discrete RNA component, which sediments in the native state with a sedimentation constant of about 7 S. From the sedimentation behavior under native and denaturing conditions and the poly(A) content, a molecular weight of about 9 X 104 has been estimated for this component. RNA-DNA hybridization experiments have indicated that this component is coded for by the light strand of mitochondrial DNA. Evidence for the occurrence of a poly(A)-containing RNA component sedimenting at about 9 S and coded for by the heavy strand has also been obtained.Poly(A) sequences of a size corresponding to 60-80 nucleotides occur in mitochondrial RNA from HeLa cells (1, 2); a poly(A)-synthesizing enzyme has been identified in rat-liver mitochondria (3,4). In HeLa cells, the majority of mitochondrial poly(A) is covalently linked to mitochondrial DNA (mit-DNA)-coded RNA (2). Inasmuch as poly(A) in eukaryotic cells appears to be associated only with cytoplasmic messenger RNA (mRNA) and its presumptive nuclear precursors (5-10), and with viral or viral-directed RNA with messenger function (5,(11)(12)(13)(14)(15), the above evidence strongly suggests that at least a portion of the mRNA translated by mitochondrial polysomes is coded for by mit-DNA.Previously (2), no clear-cut evidence of discrete components was observed in the sedimentation patterns of poly(A)-containing RNA from HeLa-cell mitochondria. The main source of difficulty was probably the tendency of poly(A)-containing molecules to aggregate and sediment with heavier molecules. Thus, RNA from the polysome region of the sedimentation pattern of a mitochondrial lysate, selected for poly(A) by two passages through poly(dT)-cellulose and analyzed in sucrose gradient in the native state, showed a fairly uniform distribution of material in the region from 6 S to about 16 S, with only a hint of a 7S component emerging over a background of heterogeneous RNA. After formaldehyde denaturation and sedimentation in the presence of formaldehyde, most of the poly-(A)-containing RNA sedimented more slowly than 12 S RNA, and a pronounced sharp peak sedimenting about 20% faster than denatured 5S RNA and 40% slower than denatured 12S RNA was observed. ,ug/ml of ethidium bromide (eih. brom.), and passed through poly(dT)-cellulose. The mitochondrial 12S RNA and cytoplasmic 5S RNA were treated as the experimental samples and run in parallel gels. RESULTSA Triton X-100 mitochondrial lysate from HeLa cells labeled for 2 hr with [8-8H]adenosine in the presence of 0.1 ug/ml of actinomycin D was run through a sucrose gradient, and RNA was extracted from the polysome region (74-180 S) of the gradient (21) and passed through a poly(dT)-cellulose column. It had been shown (1, 2) that poly(dT)-cellulose or poly(U) filters bind mitochondrial poly(A)-containing RNA, wh...