Electron microscopic evidence for splicing of SV40 late mRNAs

Bratosin, Susan; Horowitz, Mia; Laub, Orgad; Aloni, Yosef

doi:10.1016/0092-8674(78)90228-3

Cited by 37 publications

(21 citation statements)

References 15 publications

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“…(21). We suggest that the three RNA species correspond to 19S, 18S and 16S viral mRNAs described recently by Siddell and Smith (18) (5). Another conclusion of the present st'udies is that the three late polyoma RNAs are spliced, namely, the leader sequences at the 5' ends of the RNAs are not coded at a site immediately adjacent to the main portion of the RNAs.…”

Section: Sharp (19) (Personal Communication)supporting

confidence: 56%

“…Using this technique, it has recently been shown that in adeno and SV40 mRNAs, sequences close to the 5' end of the mRNA (called "leader sequences") are coded for by DNA which is not contiguous with the main coding sequences (6,22,7,8,5).…”

Section: Introductionmentioning

confidence: 99%

“…The cells were labeled at 30 hrs post-infection for times as indicated with 5,6[3H]-uridine (200 pci/6 ml of medium per culture, 40 ci/mmol: The Radiochemical Center, Amersham, England). Preparation of cytoplasmic RNA: At the end of the labeling period the cultures were washed and cytoplasmic fraction was prepared by lysing the cells with phosphate-buffered saline containing 0.5% Nonidet-P40 (10).…”

Section: Introductionmentioning

confidence: 99%

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Polyoma infected cells contain at least three spliced late RNA s

1978

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Poly(A)-containing polyoma cytoplasmic RNA was hybridized with linear double-stranded polyoma DNA and RNA displacement loops (R-loops) were formed. The structures visualized in the electron microscope are consistent with the conclusion that there are at least three late polyoma specific RNAs and that the leader sequences at the 5' ends of these viral RNAs are not coded immediately adjacent to the bodies of the RNAs. Measurements carried out on the R-loop structures have provided the locations on the physical map of pblyoma DNA, for the bodies and leaders of the RNAs and the length of the bodies, leaders and the corresponding intervening DNA sequences.

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Section: Sharp (19) (Personal Communication)supporting

confidence: 56%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Polyoma infected cells contain at least three spliced late RNA s

1978

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“…In contrast, we estimate that not more than 30% of the nucleoprotein complexes are capable of yielding FOII DNA upon in vitro incubation. One might conceive, therefore, that other important biological properties of this region of the genome may-account for the structural specificities of the chromatin such as transcriptional activities that are likely to be initiated here close to the leader sequences (29,31).…”

Section: Methodsmentioning

confidence: 99%

Origin of DNA replication in papovavirus chromatin is recognized by endogenous endonuclease.

Waldeck¹,

Föhring²,

Chowdhury³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

116

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Isolated simian virus 40 (SV40) and polyoma nucleoprotein complexes contain endonuclease that, under in vitro conditions, converts part (up to 30%) of the covalently closed superhelical DNA to full-length linear rods. The positions of the cleavage sites within the genomes of SV40 and polyoma were determined by digestion with various single-cut restriction endonucleases and subsequent agarose gel electrophoresis of the cleavage products. Both SV40 and polyoma covalently closed superhelical DNA were cleaved open at their respective origins of DNA replication (±75 base pairs). The full-length linear DNA rods whose ends map adjacent to the origin of DNA replication could also be isolated by sodium dodecyl sulfate/phenol extraction both from SV40-infected permissive cells and from purified SV40 virions. These data reveal the resence of a unique structure of the papovavirus chromatin cose to the initiation site of DNA replication.Isolated papovavirus chromatin has been used as a simple model for the study of the fine structure of chromosomes (1-5). A close similarity between the structural organizations of viral and eukaryotic chromatins was revealed with the main feature being that beaded globular structures termed "nucleosomes" (6), which consist of cellular histones (5, 7) are aligned along the DNA filaments on a small number of distinct alternative positions (4).Viral chromatin also has been successfully used to determine the in vitro factors and conditions that are required for DNA replication. The availability of soluble nucleoprotein complexes derived from the nuclei of simian virus 40 (SV40)-infected cells (8) has fostered such studies (8,9). A number of enzymes such as a and y DNA polymerases (10,11) and RNA polymerase (12) are associated with papovavirus nucleoprotein complexes. As will be shown in this report, there is, in addition, an endonuclease activity present that introduces one double-strand break into the viral DNA. In the case of both SV40 and polyoma DNA, the position that is susceptible to the endonuclease activity within the chromatin was shown to be located at the origin of replication. This finding has revealed a specific structure of the papovavirus chromatin in a biologically important region of the genome. MATERIALS AND METHODSVirus and Cells. SV40 strain Rh 911 was grown on CV-1 cells as described (13). Propagation of polyoma virus and infection of 3T6 cells were carried out as described (14). For isolation of nucleoprotein complexes the cells were infected at a multiplicity of 10 plaque-forming units per cell.Preparation of Nucleoprotein Complexes. Nuclei were isolated from SV40-infected CV-1 cells (107 cells in a 15-cm petri dish, 38 hr after infection) and from polyoma-infected 3T6 cells (2 X 107 cells in a 15-cm petri dish, 26 hr after infection) as described (14). For preparation of nuclear extracts as initially described by Su and De Pamphilis (8) the nuclei were suspended in 2 ml of modified hypotonic extraction buffer (10 mM Hepes, pH 8.0/1 mM MgCl2/0.5 mM CaCl2/1 mM dithio...

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“…(13) or the urea/formamide technique (14). Grids were rotary shadowed with platinum/palladium and examined by using a Philips 300 electron microscope.…”

Section: Methodsmentioning

confidence: 99%

Mechanism for circularization of linear DNAs: circular parvovirus MVM DNA is formed by a "noose" sliding in a "lasso"-like DNA structure.

Bratosin¹,

Laub²,

Tal³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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During an electron-microscopic survey with the aim of identifying the parvovirus MVM transcription template, we observed previously unidentified structures of MVM DNA in lysates of virus-infected cells. These included doublestranded "lasso"-like structures and relaxed circles. Both structures were of unit length MVM DNA, indicating that they were not intermediates formed during replication; they each represented about 5% of the total nuclear MVM DNA. The proportion of these structures was unchanged after digestion with sodium dodecyl sulfate/Pronase and RNase and after mild denaturation treatment. Cleavage of the "lasso" structures with EcoRI restriction endonuclease indicated that the "noose" part of the "lasso" structure is located on the 5' side of the genomic single-stranded MVM DNA. A model is presented for the molecular nature of the circularization process of MVM DNA in which the "lasso" structures are identified as intermediates during circle formation. This model proposes a mechanism for circularization of linear DNAs.Animal viruses having linear DNAs provide useful model systems for the study of eukaryotic DNA replication and somatic rearrangement in animal cells. For example, the parvoviruses have linear single-stranded DNA genomes with an Mr of about 1.5 X 106. The genomic DNA contains a stable hairpin duplex structure at its 5' terminus and a 3'-terminal hairpin structure suitable for priming complementary strand synthesis in vitro. Both hairpins are believed to be involved in the initiation of subsequent cycles of replication, generating complex doublestranded intermediates with a y-shaped appearance, as seen in the electron microscope (1).Based on the Cavalier-Smith model for replication of linear DNA (2), Tattersall and Ward (3) have suggested the "rolling hairpin" model to explain how the structures at the ends of the linear MVM DNA are replicated. The central process in this model is the synthesis and rearrangement of the palindromic terminus to produce a "rabbit-eared" structure. Circular structures are not considered in this model because no circular forms of MVM DNA have been detected in lysates from infected cells.The parvoviruses, because of their relative simplicity, also provide one of the best model systems to date for studying transcription and posttranscriptional modifications (4), specifically, the mechanism of splicing (5-7) and the physiological significance of this process in animal cells.In a study with the aim of determining the MVM template of transcription, we have lysed MVM-infected cell nuclei with Triton X-100 and Sarkosyl, a procedure that we have found suitable for the analysis of simian virus 40 transcriptional complexes (8), and analyzed the lysates by electron microscopy.We report the occurrence in this lysate of "lasso"-like structures and circles of MVM DNA. A model is presented for the molecular nature of the circularization process of MVM DNA in which the "lasso" structures are considered as intermediates during circle formation. This model incorporates a ...

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Electron microscopic evidence for splicing of SV40 late mRNAs

Cited by 37 publications

References 15 publications

Polyoma infected cells contain at least three spliced late RNA s

Polyoma infected cells contain at least three spliced late RNA s

Origin of DNA replication in papovavirus chromatin is recognized by endogenous endonuclease.

Mechanism for circularization of linear DNAs: circular parvovirus MVM DNA is formed by a "noose" sliding in a "lasso"-like DNA structure.

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