Cis and trans activation of adenovirus IVa<sub>2</sub>gene transcription

Natarajan, V.; Salzman, Norman P.

doi:10.1093/nar/13.11.4067

Cited by 19 publications

(24 citation statements)

References 46 publications

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“…Data from four separate run-on experirpents with nuclei from cells infected with mids has proved to be an inefficient experimental method because of the low basal activity of the wild-type MLP in the absence of an enhancer or trans-activator (26,37,51). However, in experiments performed with a chloramphenicol acetyltransferase (CAT) plasmid assay, mutant MLP 14 consistently had lower CAT activity than the wild type and some other viable MLPs (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…After DNA replication, transcription from the MLP is apparently activated to higher levels in a cis-acting manner (45). Although ElA transactivation of the MLP has been demonstrated in plasmid transfection experiments (26,37), the temporal DNA replication-dependent regulation of this promoter has not been reconstituted in vitro or in transient transfection experiments with replicating plasmids (28).…”

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confidence: 99%

See 1 more Smart Citation

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

Brunet¹,

Babiss²,

Young³

et al. 1987

Mol. Cell. Biol.

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We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up-and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit 0-globin sequences placed in the dispensable El region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.The adenovirus major late promoter (MLP) has been studied extensively as a model for eucaryotic promoters. It contains a classic TATA box, necessary for correct and quantitative transcription in vitro (11,22,49), to which binds a transcription factor termed IID (41). As with many other eucaryotic promoters, a second sequence upstream from the cap site also affects the efficiency of transcription. This sequence element, centered at -58, is necessary for efficient transcription in vitro and in vivo (20,23,31) and is capable of binding a second transcription-promoting HeLa cell protein (7,32,41) termed USF.In addition to these well-characterized upstream sequence elements, the MLP contains striking direct repeats of nonalternating GC pairs flanking the TATA box (see Table 1). It has been suggested, on theoretical grounds, that these repeats and similar sequences in cellular promoters may form a structure which acts as a "trap" for...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

Brunet¹,

Babiss²,

Young³

et al. 1987

Mol. Cell. Biol.

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“…6 and 12). Previous in vitro studies have established that the organization of the IVa 2 promoter is unusual: it lacks a TATA sequence (25)(26)(27), or indeed, any upstream sequence that strongly influences the efficiency of transcription in vitro (27)(28)(29), or in infected cells (C.S.H. Young, A. Timko, and S.J.F., unpublished observations).…”

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confidence: 99%

Viral DNA synthesis-dependent titration of a cellular repressor activates transcription of the human adenovirus type 2 IVa ₂ gene

Iftode

Flint²

2004

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

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Synthesis of progeny DNA genomes in cells infected by human subgroup C adenoviruses leads to several changes in viral gene expression. These changes include transcription from previously silent, late promoters, such as the IV a2 promoter, and a large increase in the efficiency of major-late (ML) transcription. Some of these changes appear to take place sequentially, because the product of the IV a2 gene has been implicated in stimulation of ML transcription. Our previous biochemical studies suggested that IV a2 transcription is regulated by viral DNA synthesis-dependent relief of transcriptional repression by a cellular protein that we termed IV a2-RF. To test the relevance of such a repressor-titration mechanism during the viral infectious cycle, we introduced into the endogenous IV a2 promoter two mutations that impair in vitrobinding of IV a2-RF, but introduce no change (Rep7) or one conservative amino acid substitution (Rep6) into the overlapping coding sequence for the viral DNA polymerase. The results of run-on transcription assays indicated that both mutations induced earlierthan-normal and more efficient IV a2 transcription. Both mutations were also observed to result in modest increases in the efficiency of viral DNA synthesis. However, measurement of the concentration of IV a2 transcripts as a function of IVa2 template concentration demonstrated that the Rep mutations increased by up to 60-fold the efficiency with which IV a2 templates were used during the initial period of the late phase of infection, as predicted by the repressor titration hypothesis. These mutations also increased the efficiency of ML transcription in infected cells.late transcription ͉ repressor titration T he infectious cycles of animal viruses with DNA genomes are characterized by transcription of viral genes in a stereotyped temporal sequence, regardless of whether transcription is carried out by cellular or viral DNA-dependent RNA polymerases. The complexity of such temporal regulation of transcription increases with viral genome size. Nevertheless, transcription of viral late genes encoding structural proteins invariably depends on synthesis of progeny viral DNA genomes in the infected cell (reviewed in ref. 1). In some cases, the activities of a single viral protein control both viral DNA synthesis and late gene transcription. For example, the simian virus 40 early protein large T antigen is not only the origin-recognition protein required for initiation of viral DNA synthesis but also an activator of late transcription (see refs. 2-5). The transition from the early to the late transcriptional program is considerably more intricate in cells infected by DNA viruses with larger genomes, such as human adenoviruses.Synthesis of viral DNA in cells infected by human subgroup C adenoviruses, such as adenovirus type 2 or 5 (Ad2 or Ad5), results in transcription by cellular RNA polymerase II from three previously inactive viral promoters, those of the IX and IVa 2 genes and the E2 late promoter (reviewed in ref. 6) Such off-to-on swit...

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“…The plasmid DNAs examined in the present study are pARKR (22), pARIVa2, pARMLP, pSVOCAT242 (29), and pSVOCATMLP (29 Fig. 2.…”

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“…Cell monolayers at 70% confluency were transfected with DNA by the calcium phosphate method followed by treatment with 15% glycerol 4 h after transfection as described earlier (29 (22) and analyzed by Southern blotting and hybridization to nick-translated pARKR as described by Maniatis et al (28).…”

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Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

Vasudevachari¹,

Natarajan²,

Salzman³

1987

Mol. Cell. Biol.

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Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus Ela gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the Ela region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides Ela functions in trans to regulate transcription.By considering the timing of appearance of gene products, the process of lytic infection by human adenoviruses has been divided into four temporal stages of gene expression: pre-early, early, intermediate, and late (3, 25, 36). Pre-early and early genes are expressed before the onset of DNA replication, which occurs at 6 to 7 h postinfection. Inhibition of protein synthesis at early times blocks DNA synthesis and leads to a general increase in the level of many.early RNAs (5,6,34,35). Although the promoter for the major late transcription unit is active at early times, formation of several late mRNAs is prevented by premature transcription termination (1,33,37), implying that full expression of late genes requires DNA replication. The expression of intermediate genes like IVa2 promoter (IVa2) and IX, begins about the same time as viral DNA synthesis, but the peak level of accumulation of these mRNAs is found after DNA replication (5,7,27,38,42 with a deletion between 1.5 and 4.5 map units (31). The viruses were purified, and their DNAs were isolated by digestion with proteinase K (1 mg/ml) in the presence of 0.6% sodium dodecyl sulfate followed by phenol and chloroform extractions and ethanol precipitation (13,30).Plasmids. The plasmid DNAs examined in the present study are pARKR (22), pARIVa2, pARMLP, pSVOCAT242 (29), and pSVOCATMLP (29). Plasmids pSVOCAT242 and 10...

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Cis and trans activation of adenovirus IVa₂gene transcription

Abstract: The transcriptional control region of the adenovirus IVa2 promoter was analyzed by cloning this promoter

Cited by 19 publications

References 46 publications

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

Viral DNA synthesis-dependent titration of a cellular repressor activates transcription of the human adenovirus type 2 IVa ₂ gene

Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

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Cis and trans activation of adenovirus IVa2gene transcription

Abstract: The transcriptional control region of the adenovirus IVa2 promoter was analyzed by cloning this promoter

Cited by 19 publications

References 46 publications

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

Viral DNA synthesis-dependent titration of a cellular repressor activates transcription of the human adenovirus type 2 IVa 2 gene

Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

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Cis and trans activation of adenovirus IVa₂gene transcription

Viral DNA synthesis-dependent titration of a cellular repressor activates transcription of the human adenovirus type 2 IVa ₂ gene