Preparative isolation and mapping of adenovirus 2 early messenger RNA species

Büttner, Werner; Veres-Molnár, Zsuzsanna; Green, Maurice

doi:10.1016/s0022-2836(76)80020-4

Cited by 48 publications

(20 citation statements)

References 21 publications

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“…m R N A s which could be detected by the primer pairs are shown at the top. We detected three, one, and one c D N A bands in lanes (7,8), and 1 and 5 (9, 10) (see Fig. 1).…”

Section: Relevance Of the Pcrmentioning

confidence: 83%

“…W According to Allard and Wadell [1], M Steinthordottir and Mautner [34]. The primer pairs, [3,6] and [3,7], are identical with those used in these papers determined by comparing their sequences with that of Ad40 genomic DNA. In some cases, PCR products eluted from agarose gels were purified and sequenced directly without cloning into vectors as described by Fujinaga et al [12].…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

“…-transformed cells [5,7,9,16,23,31,33,37], although they are expressed in low abundance. However, splice sites in the E1A-E1B cotranscripts based on the nucleotide sequences remain to be established.…”

Section: Introductionmentioning

confidence: 99%

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Unusual splice sites in the E1A?E1B cotranscripts synthesized in adenovirus type 40-infected A549 cells

Ishida

Fujinaga

et al. 1994

Archives of Virology

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The adenovirus E1 DNA region consists of two transcription units, E1A and E1B. In this paper we report that the E1A-E1B cotranscripts containing sequences of both the E1A and E1B regions are synthesized during adenovirus type 40 (Ad40) infection of A549 cells. Cytoplasmic RNA was isolated from Ad40-infected A549 cells at 24, 72, and 100 h post infection (p.i.). The complementary (c) DNA was synthesized by reverse transcription using an oligo-dT primer and then amplified by the polymerase chain reaction (PCR) using primers derived from the E1A and E1B regions. The cDNAs thus amplified were sequenced either directly or after cloning into bacteriophage M13 vectors. Analysis of cDNA indicated that the E1A-E1B cotranscripts are synthesized at 72 h p.i., but not at 24 or 100 h p.i. Nucleotide sequences of three cDNAs of the E1A-E1B cotranscripts indicated that the cotranscripts originate from the E1A promoter and lack sequences for both the E1A poly(A) site and E1B cap site. The splices create open reading frames for E1A-E1B fused polypeptides around the E1A-E1B junctions in these mRNAs. Most interestingly, the sequence analysis showed that the 5' and 3' splice junctions in the two E1A-E1B cotranscripts do not conform to the splice consensus GT-AG rule. Our results thus suggest that factor(s) which lead to unusual splicing in the E1 mRNAs are present in Ad40-infected A549 cells.

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“…m R N A s which could be detected by the primer pairs are shown at the top. We detected three, one, and one c D N A bands in lanes (7,8), and 1 and 5 (9, 10) (see Fig. 1).…”

Section: Relevance Of the Pcrmentioning

confidence: 83%

Section: Polymerase Chain Reactionmentioning

confidence: 99%

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Unusual splice sites in the E1A?E1B cotranscripts synthesized in adenovirus type 40-infected A549 cells

Ishida

Fujinaga

et al. 1994

Archives of Virology

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“…A different example of the multiple use of a set of DNA sequences is found early in adenovirus infection. The transforming regioif of the genome specifies several small mRNAs that do not overlap each other but do share sequences with a single large mRNA (5,18). In bacteriophage systems, multiple molecular forms have been found for T7 lysozyme mRNA (19).…”

Section: Discussionmentioning

confidence: 99%

“…Transcriptional maps have been derived from two types of experiments: The number of nucleotides transcribed into mRNA has been determined by hybridization of nonradioactive RNAs with separated strands of radioactive DNA fragments (1,2). mRNA species transcribed from various regions of the genome have been identified by hybridization analysis of radioactive RNAs separated by size (4)(5)(6).…”

mentioning

confidence: 99%

Two regions of the adenovirus 2 genome specify families of late polysomal RNAs containing common sequences.

McGrogan¹,

Raskas²

1978

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

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Late cytoplasmic RNAs specified by two regions of the adenovirus 2 genome (39.3-51.8 and 70.7-83.4 The availability of specific DNA fragments has enabled the formation of transcriptional maps for adenovirus 2 RNAs synthesized both early and late in productive infection. By late times, after the onset of viral DNA replication, greater than 90% of the coding capacity of the virus is represented in cytoplasmic transcripts (1, 2). The viral mRNAs synthesized at typical late times, 18 hr after infection, are derived more than 99% from the r strand of the genome (3), the strand transcribed in the rightward direction on the conventional map (1). Transcriptional maps have been derived from two types of experiments: The number of nucleotides transcribed into mRNA has been determined by hybridization of nonradioactive RNAs with separated strands of radioactive DNA fragments (1, 2). mRNA species transcribed from various regions of the genome have been identified by hybridization analysis of radioactive RNAs separated by size (4-6).Mapping experiments with cytoplasmic viral RNAs synthesized late in infection revealed an unexpected size distribution for the RNAs transcribed from two separate regions of the viral genome (6). Each region specified a series of cytoplasmic RNAs whose collective sequence content exceeded the coding capacity of the region at least 2-fold. These two regions included the sequences contained in map positions 39.3-51.8 (Sma I-D DNA fragment) and 70.7-83.4 (EcoRI-F and -D DNA fragmepts). The region 39.3-51.8 appears to code forThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 625 several virion proteins, penton (85,000 daltons) pVII (20,000 daltons) and V (48,400 daltons) (7). The late proteins identified as products of the second region, 70.7-83.4, are pVIII (26,000 daltons) and a 100,000-dalton polypeptide.To better define the relationship between the series of RNAs transcribed from each of these regions, it was necessary to establish the presence of each of these RNAs in polyribosomes and to map in greater detail the sequences specifying each species. The mapping data presented here establish that, within each set of RNAs, sequences towards the 3' ends are shared, and the longer the molecule, the further it extends in the 5' direction. MATERIALS AND METHODSVirus Infection, Labeling of Cultures, and RNA Purification. Maintenance of KB cell suspension cultures and infections with adenovirus type 2 were performed as described previously (4,6). Cytoplasmic RNA was purified from cultures labeled with [3H]uridine (40 Ci/mmol; New England Nuclear at 50 ,uCi/ml. [3H]RNA labeled 12-14 hr after infection was utilized in mapping the cytoplasmic RNAs from the EcoRI-F,D region, and [3H]RNA labeled from 17-19 hr was used in analyzing the Sma I-D transcripts.Polyribosomes were isolated by sedimentation through a 7-47% sucrose gradient...

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Regulation of Adenovirus Gene Expression

Persson

Philipson

1982

Current Topics in Microbiology and Immunology

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Preparative isolation and mapping of adenovirus 2 early messenger RNA species

Cited by 48 publications

References 21 publications

Unusual splice sites in the E1A?E1B cotranscripts synthesized in adenovirus type 40-infected A549 cells

Unusual splice sites in the E1A?E1B cotranscripts synthesized in adenovirus type 40-infected A549 cells

Two regions of the adenovirus 2 genome specify families of late polysomal RNAs containing common sequences.

Regulation of Adenovirus Gene Expression

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