Unique species of mRNA from adenovirus type 7 early region 1 in cells transformed by adenovirus type 7 DNA fragment

A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters.

Section: Resultsmentioning

confidence: 98%

“…and on the 1 strand at 96.3 m.u. The former may account for minor mRNA's detected from Ad2-, Ad5-, Ad7-, and Adl2-infected and -transformed cells (5,12,30,41). No Ad2 mRNA has been mapped with a 5' terminus at 96.3 m.u.…”

Section: Discussionmentioning

confidence: 99%

In vitro transcription of adenovirus

Fire¹,

Baker²,

Manley³

et al. 1981

“…(pH 6.4), and 0.001 M EDTA (5), essentially by the method of Berk and Sharp (3) as previously described (33). The melting temperature (Tm) irreversible in this solvent was 46 to 47°C as determined by susceptibility of the Adl2 EcoRI-C fragment to nuclease S1 (Y. Sawada, unpublished data).…”

Section: Pipes [Piperazine-nn'-bis(2-ethanesulfonic Acid)]mentioning

confidence: 99%

“…In the former case, mRNA transcription starts by the 1B promoter, continues beyond the viral-cellular DNA junction, and terminates at an appropriate cellular signal. mRNA transcription starts by the 1B promoter and terminates at the 1A signal in the adjacent viral DNA, which is integrated in a tandem array (33). The band at 1,480 nucleotides detected with the HindIII-G fragment in the neutral gel by the nuclease Si gel procedure ( Fig.…”

Section: Figmentioning

confidence: 99%

Mapping of adenovirus 12 mRNA's transcribed from the transforming region

Sawada¹,

Fujinaga²

1980

Self Cite

Adenovirus 12 mRNA's transcribed from the transforming region were analyzed and mapped on viral DNA by the nuclease Si gel and diazobenzyloxymethyl paper blot techniques in cells transformed by the EcoRI-C (leftmost 16.5%) and HindmI-G (leftmost 6.8%) fragments of adenovirus 12 DNA and in cells early and late after lytic infection with adenovirus 12. Two initiation sites of mRNA transcription and two kinds of splicing were found in each of early regions 1A and 1B. For early region 1A mRNA's, four species were found in lytically infected cells. Three of them were commonly found in cells transformed by either HindIII-G or EcoRI-C. Cells transformed by HindllI-G contained two additional 1A transcripts, which could be the 3' portions of chimeric mRNA's of cellular-viral or viral-viral sequences. Transcription in the 1B region diverged among the above cell lines. Early after lytic infection, no appreciable amount of 1B mRNA was detected, whereas two species of mRNA's, one corresponding to protein IX mRNA and another having splicing, were found at the late stage. In cells transformed by EcoRI-C, a distinct mRNA species with splicing was observed. Cells transforned by HindIII-G contained a transcript from the leftmost part of the 1B sequence at the 5' portion of chimeric mRNA species, suggesting the presence of tandem integration of viral DNA in the cells. Other mRNA species in the 1A and 1B regions were also detected in both transformed cell lines. The results are discussed in relation to the nucleotide sequence of the HindIII-G fragment of adenovirus 12 DNA.

“…In addition to early genes, some of the late genes appear also to be transcribed into mRNA in Adl2-induced rat brain tumor cells (13). Virusspecific mRNA has been mapped in a number of Ad2-transformed rat cells (20) and in rat cells transformed by the left end of Ad7 DNA (31).…”

mentioning

confidence: 99%

Expression of Viral DNA in Adenovirus Type 12-Transformed Cells, in Tumor Cells, and in Revertants

Schirm

Doerfler

1981

The expression as cytoplasmic RNA of integrated human adenovirus type 12 (Ad12) DNA in transformed and tumor cell lines and in revertants was investigated. The transformed and tumor cells contained multiple copies of the viral genome, 3 to 22 copies per cell in different cell lines. The integrated Ad12 DNA molecules persisted intact or nearly intact and in most cases colinear with the virion DNA. In the revertant cell lines, which were derived from cell line T637 (22 copies of Ad12 DNA per cell), all of the Ad12 DNA molecules were lost (line F10) or only one copy and a fraction of a second copy persisted (line TR12). The size classes and map locations of Ad12-specific cytoplasmic RNAs in three Ad12-transformed hamster cell lines (T637, HA12/7, and A2497-3), in two revertant lines (F10 and TR12), in one Ad12-induced hamster (CLAC3), and in one rat brain tumor line (RBT12/3) were determined. Cytoplasmic RNA from uninfected B3 hamster cells and from human KB cells productively infected with Ad12 served as controls. In the latter control experiments, the RNA was isolated early or late postinfection. With respect to the amounts of Ad12-specific RNAs detected in cytoplasmic RNA from various Ad12-transformed or Ad12-induced tumor cell lines, we could not establish any correlations to the number of Ad12 genome copies integrated into the cellular DNAs. Thus, the expression of the integrated viral genomes in these lines was regulated by mechanisms more complicated than simple gene dosage effects. Using cloned fragments of Ad12 DNA as hybridization probes, we analyzed the cytoplasmic RNAs from the cell lines mentioned by electrophoresis on agarose gels, blotting, and DNA-RNA hybridization. For each transformed and tumor cell line, except for the revertants, several size classes of Ad12-specific cytoplasmic RNA were detected for the early E1, E2, and E4 regions of Ad12 DNA. Some of these size classes were similar but not identical to those observed in cytoplasmic RNA isolated early from human KB cells productively infected with Ad12. Only cell lines A2497-3, T637, and RBT12/3 contained several size classes of cytoplasmic RNA homologous to the E3 region of Ad12 DNA. Weak homologies to the E1 region of Ad12 DNA were also detected in the revertant lines F10 and TR12. Late regions of Ad12 DNA were expressed as cytoplasmic RNA in cell lines CLAC3 and RBT12/3. Weak homologies were detected between certain segments of the Ad12 genome (the Eco RI-B, -C, and -D fragments) and the cytoplasmic RNA from uninfected hamster cells. These homologies had no apparent counterpart at the level of DNA, perhaps because these homologies could be detected only due to an overrepresentation of RNA sequences. In preliminary experiments, we failed to detect the expression as cytoplasmic RNA of the so-called virus-associated RNA in transformed and tumor cell lines. Virus-associated RNA represents a population of low-molecular-weight RNAs that map at around 30 fractional length units on the viral genome.