Localization of Adenovirus 2 Messenger RNAs to Segments of the Viral Genome Defined by Endonuclease R·R1

Tal, Jacov; Craig, Elizabeth A.; Zimmer, Stephen G.; Raskas, Heschel J.

doi:10.1073/pnas.71.10.4057

Cited by 28 publications

(37 citation statements)

References 25 publications

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“…Of the nine Ad2-transformed cell lines examined, all contain DNA from the left end of the R * EcoRI A fragment but none has the complete genome (15,26). This is also a region of the Ad2 DNA transcribed early in permissive infection (11)(12)(13)(14)(15). Determination of which of the Ad2 early polypeptides is located in this region of the genome would implicate that protein in the maintenance of the transformed cell phenotype by Ad2 genetic information.…”

Section: Discussionmentioning

confidence: 99%

“…Discrete size classes of viral mRNA have been demonstrated by fractionation on a sucrose gradient and correlated with their polypeptide products (5). In addition, the mRNA homologous to Ad2 DNA has been characterized by electrophoretic mobility (11,12) and by its hybridization with specific DNA fragments (11)(12)(13)(14)(15), but in neither of these latter cases have the RNA classes been correlated with their polypeptide products. Thus, the pattern of transcription of Ad2 is well characterized but the relationship of this map to the location of specific genes is unknown.…”

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confidence: 99%

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Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

Lewis

Atkins

Anderson

et al. 1975

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

112

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Cytoplasmic RNA, isolated from cells late after infection by adenovirus type 2 and fractionated by hybridization to specific fragments of adenovirus DNA produced by cleavage with the endonuclease R EcoRI, was used as template for protein synthesis in cell-free mammalian extracts. Each of the R-EcoRI fragments of DNA selects RNA that encodes specific subsets of the viral polypeptides, From the known order of the R.EcoRI fragments, the following pgrtial map is deduced: (III, Hila, IVa2, V, P-VII, IX), (II, P-VI), lOOK, IV-where the relative order of the components enclosed in parentheses has not yet been determined.A general technique for isolation of conditional lethal mutants in conjunction with identification of the altered protein products has not been developed for eukaryotes to the degree that led to such success in the genetic analysis of prokaryotes. However, the availability of enzymes that specifically fragment DNA, and the demonstration that RNA selected by hybridization to DNA can be translated in cell-free extracts (1, 2), have opened up alternative ways to do genetic mapping by biochemical means.One system that is amenable to such an analysis is the infection of mammalian cells by adenovirus. Adenovirus 2 (Ad2) is a human virus capable of transforming certain nonpermissive cells in vitro. In the productive infection of permissive cells, two phases have been defined, an early phase which includes events occurring before the onset of DNA synthesis (8 hr) and a subsequent late phase when most of the genome is expressed (for a review see ref.3). Approximately 18 virusspecific proteins have been found in extracts of infected cells (4), and at least 10 of these have been identified among the products of cell-free translation programmed by cytoplasmic RNA from adenovirus-infected cells (5-10). Discrete size classes of viral mRNA have been demonstrated by fractionation on a sucrose gradient and correlated with their polypeptide products (5). In addition, the mRNA homologous to Ad2 DNA has been characterized by electrophoretic mobility (11, 12) and by its hybridization with specific DNA fragments (11-15), but in neither of these latter cases have the RNA classes been correlated with their polypeptide products. Thus, the pattern of transcription of Ad2 is well characterized but the relationship of this map to the location of specific genes is unknown.We report here a partial map of the localization on the viral DNA of the genes encoding adenovirus polypeptides. This map was obtained by programming cell-free protein synthesis with cytoplasmic RNA, prepared from human cells late after Ad2 infection, that had first been fractionated by hybridization to the separated specific fragments of Ad2 DNA obtained after digestion with the restriction endonuclease RLEcoRI.The order of the six R-EcoRI fragments on Ad2 DNA is known (16) and so we can deduce the location of Ad2 genes. This technique provides a general method to locate genes, provided mRNA and suitable specific fragments of DNA can be obtained. MATERIAL...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

Lewis

Atkins

Anderson

et al. 1975

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

112

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“…(ii) A second observation pertains to sequences contained in the EcoRI-D fragment. This fragment specifies 20S and 13S early mRNA's (43). However, late in infection, the early sequences appear to be part of RNA species that are different in size from the species synthesized before the onset of DNA synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…Expression of the adenovirus type 2 genome during productive infection is under temporal control (18,47). At early times, before viral DNA replication begins, seven to eight viral mRNA species are transcribed (8,12,43). These mRNA's include transcripts from limited regions of both strands of the genome (33,38).…”

Section: I'mentioning

confidence: 99%

Species Identification and Genome Mapping of Cytoplasmic Adenovirus Type 2 RNAs Synthesized Late in Infection

McGrogan

Raskas

1977

J Virol

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Adenovirus type 2 cytoplasmic RNAs synthesized late in productive infection were resolved by electrophoresis on formamide gels. Regions of the adenovirus 2 genome specifying RNAs of distinct size were determined by hybridization to specific DNA fragments generated by cleavage with endo R EcoRI and endo R * SmaI. From these studies 13 distinct viral RNA species were identified. A 26S to 28S size class and a 21S to 23S size class were each found to consist of four distinct RNA species. Three RNA species were identified in a 16S to 18S size class, and a fourth size class, 11S to 13S, was resolved into two components. The SmaI-D region (0.38 to 0.51 on the unit genome) and the EcoRI-F, D region (0.70 to 0.83) of the genome were found to code for multiple transcripts. Three RNAs (28S, 22S, and 18S) are specified by SmaI-D, and four components, 28S, 22S, 18S, and 16S, are encoded by EcoRI-F,D. The RNA represented by each set of multiple transcripts exceeds the coding capacity ofthe respective region, and the species within each set of RNAs appear to contain common sequences. The relationship between the cytoplasmic RNA species synthesized at late times and early cytoplasmic RNAs was determined by hybridization-inhibition experi-240

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“…Restriction endonuclease fragments of adenovirus 2 DNA were prepared according to published procedures (15,16,(18)(19)(20)(21). The fragments used in this study were generated by cleavage of adenovirus 2 For gel electrophoresis the eluted RNA was precipitated with 0.1 M NaCl and 2 volumes of 95% ethanol.…”

Section: Methodsmentioning

confidence: 99%

Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution

et al. 1979

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A method is described for the preparative isolation of highly purified adenovirus RNA species. Cytoplasnic RNAs frcn cells infected with adenovirus 2 were selected by hybridization to viral DNA fragments bound to nitrocellulose membranes. A series of washes at elevated temperatures (50-700) determined conditions at which the true hybrids were stable but non-specific RNA was removed. This tenWerature has been found to correlate with the base composition of the DNA fragment. After wash at this predetemined tesxerature, the specific RNA was eluted at 85 . The purity of the eluted RNA was greater than 95% as determined by size, sequence specificity, and template activity in an in vitro protein synthesizing system. The method described should be generally useful for purification of specific RNAs. JlN CONCharacterization of individual mRNA species has become increasingly important in the study of eukaryotic gene expression and has increased the understanding of the extensive processing mechanisms involved in mRNA synthesis. In light of recent findings that demonstrate the presence of multiple inserts within the coding sequences of a gene (1-4) and show that a specific gene sequence may express different products by synthesizing overlapping mRNAs(5-8), it is often necessary to identify functional genes by purifying the mRNA coding for the gene product. Several methods have been described for the preparative isolation of specific RNA molecules by hybridization to DNA immobilized on a matrix (9-12), or by hybridization to DNA in liquid and purification of the DNA-RNA hybrid (13). The nitrocellulose membrane technique has several advantages: DNA can be bound to membranes by rapid and efficient methods (14) C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

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Localization of Adenovirus 2 Messenger RNAs to Segments of the Viral Genome Defined by Endonuclease R·R1

Cited by 28 publications

References 25 publications

Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

Species Identification and Genome Mapping of Cytoplasmic Adenovirus Type 2 RNAs Synthesized Late in Infection

Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution

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