A cis-acting element 7 bp upstream of the ESF-1-binding motif is involved in E1A 13S autoregulation of the adenovirus 12 TS2 promoter.

Pützer, Brigitte M.; Gnauck, J; Kirch, Hans-Christoph; Brockmann, Dieter; Esche, Helmut

doi:10.1099/0022-1317-78-4-879

Cited by 4 publications

(11 citation statements)

References 43 publications

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“…Nuclear proteins were incubated with a radiolabelled oligonucleotide containing either the E2F motif upstream of TS1 of the Ad12 E1A promoter (designated E2FI) or the E2F site within the second Ad12 E1A promoter region (TS2) (designated E2FII). Previous studies identified the first E2F motif as a DNA binding site for the E2F-1 protein (31) and the second one as an E2F-5 binding site (46). As shown in Fig.…”

Section: Resultsmentioning

confidence: 74%

“…Preparation of nuclear extracts and GSA. An A549 nuclear extract was prepared as described previously (46). GSA were carried out as described by Barrett et al (3) with minor modifications: the end-labelled oligonucleotide (0.1 to 0.5 ng) was incubated with nuclear proteins (1 to 2 g) and unspecific DNA [1,000fold molar excess of poly(dA-dT)] for 30 min in a 15-to 20-l binding reaction mixture consisting of 20 mM HEPES and 150 mM NaCl (pH 7.4).…”

Section: Cell Lines and Virusesmentioning

confidence: 99%

“…Primers used for amplification of the Ad12 E1A promoter construct TS1-prom-CAT (nt positions 199 to 340 upstream of TS1) were described previously (32). The Ad12 E1A promoter construct TS2-prom-CAT (nt positions 349 to 456 upstream of TS2) as well as construct TS1/ϩTS2-prom-CAT, containing both the TS1 and the TS2 promoter regions (nt positions 199 to 456 of Ad12 E1A), were generated as described earlier (46). The PCR products were amplified with a PCR thermocycler (Perkin-Elmer Cetus).…”

Section: Cell Lines and Virusesmentioning

confidence: 99%

“…For cotransfections, 10 g of the expression vectors (Invitrogen) pRc/ RSV-13S-WT and pRc/RSV-13S-CS-1, described earlier by Brockmann et al (5), and pRc/RSV-⌬CR2, containing the 13S cDNAs of Ad12-WT and of the mutants Ad12-CS-1 and Ad12-⌬CR2, respectively, was used. Electroporation of A549 cells was performed as described previously (46). CAT assays were carried out by the diffusion technique described by Neumann et al (43).…”

Section: Cell Lines and Virusesmentioning

confidence: 99%

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E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter

Pützer

Rumpf

Rega

et al. 1997

J Virol

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The transformation-defective Vero cell host range mutant CS-1 of the highly oncogenic adenovirus type 12 (Ad12) (Ad12-CS-1) has a 69-bp deletion in the early region 1A (E1A) gene that removes the carboxy-terminal half of conserved region 2 and the amino-terminal half of the Ad12-specific so-called spacer that seems to play a pivotal role in the oncogenicity of the virus. Despite its deficiency in immortalizing and transforming primary rodent cells, we found that the E1A 13S protein of Ad12-CS-1 retains the ability to bind p105-RB, p107, and p130 in nuclear extract binding assays with glutathione S-transferase-E1A fusion proteins and Western blot analysis. Like wild-type E1A, the mutant protein was able to dissociate E2F from retinoblastoma-related protein-containing complexes, as judged from gel shift experiments with purified 12S and 13S proteins from transfection experiments with an E1A expression vector or from infection with the respective virus. Moreover, in transient expression assays, the 12S and 13S products of wild-type Ad12 and Ad12-CS-1 were shown to transactivate the Ad12 E1A promoter containing E2F-1 and E2F-5-motifs, respectively, in a comparable manner. The same results were obtained from transfection assays with the E2F motif-dependent E2 promoter of adenovirus type 5 or the human dihydrofolate reductase promoter. These data suggest that efficient infection by Ad12 and the correlated virus-induced reprogramming of the infected cells, including the induction of cell cycle-relevant mechanisms (e.g. E2F activation), can be uncoupled from the transformation properties of the virus.

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Section: Resultsmentioning

confidence: 74%

Section: Cell Lines and Virusesmentioning

confidence: 99%

Section: Cell Lines and Virusesmentioning

confidence: 99%

Section: Cell Lines and Virusesmentioning

confidence: 99%

See 2 more Smart Citations

E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter

Pützer

Rumpf

Rega

et al. 1997

J Virol

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“…All viruses were propagated, purified and titrated by standard methods (Graham and Prevec 1995;Pützer et al 1997). The peptide VPTQSSG was covalently linked to Ad vectors by a bifunctional crosslinker NHS-PEG 4 -maleimide (Thermo Scientific, Germany).…”

Section: Methodsmentioning

confidence: 99%

Targeting of neural stem cells in the hippocampus of adult rats by custom-made Ad vectors

Hildebrandt

Schmidt²,

Stoll³

et al. 2010

Brain Struct Funct

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Adult hippocampal neural stem cells (NSC) are an intriguing source for cell replacement or could serve as delivery vehicles for therapeutic genes. We recently reported selective transduction of adult mouse NSC in the DG by in vivo injection of GFP encoding adenoviral (Ad) vectors engineered to bind NSC-specific peptides. Here, we investigated the specificity of these peptide-tagged vectors in the adult rat DG, and whether they can be used to follow differentiation of infected cells over time. The virus-containing solution was injected into the DG by stereotaxic surgery. Specific transduction of NSC was demonstrated by the radial glia-like morphology of GFP-expressing type-1 cells and co-labeling with nestin or glial fibrillary acidic protein. Three days post-injection more than 82% of GFP-containing cells were nestin-immunoreactive, as revealed by unbiased stereology and no GFP-expressing neurons were observed. However, 30 days after injection, the amount of GFP and nestin-containing cells declined (56%), whereas now neurons that contained NeuN or possessed the typical granular nerve cell morphology expressed GFP, indicating that they were derived from initially transduced NSC. Importantly, still more than 20% of nestin-immunoreactive NSC was found to be GFP-positive 90 days after infection, but unfortunately at this time point no GFP-containing neurons were detectable. Our results demonstrate that Ad vectors tagged with NSC-specific ligands can be used to target type-1 NSC, the low-proliferating cell population, in the rat hippocampus. They are a valuable tool to monitor the differentiation of their descendants, at least over short time periods.

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Two Domains within the Adenovirus Type 12 E1A UniqueSpacerHave Disparate Effects on the Interaction of E1A with P105-Rb and the Transformation of Primary Mouse Cells

1999

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Transformation of primary rodent cells by functions of the adenovirus type 12 (Ad12) early region 1 (E1) is reduced severalfold compared with transformation by E1 of Ad2. We analyzed whether the unique spacer region of Ad12 E1A that borders the conserved region (CR) 2 and represents an oncogenic determinant of Ad12 E1A is involved in this impaired transformation property, putatively by modulating transformation-relevant biological E1A functions. We show that a mutant (E1ASpm1) that lacks 12 amino-terminal residues of the spacer binds p105-Rb and p130 as Ad12 E1A wild type (E1Awt), whereas a second spacer mutant (E1ASpm2) that lacks an adjacent stretch of six alanines exhibits highly reduced binding to p105-Rb. The binding of this mutant to the p130 pocket protein is, however, little impaired. E1ASpm1 diminishes the formation of the p105-Rb-E2F complex more efficiently than E1Awt or, least efficient, E1ASpm2. These properties of the spacer mutants to target and to disintegrate the p105-Rb-E2F complex correspond with their ability to transform primary mouse cells in combination with E1B: E1ASpm1 (plus Ad12 E1B)-transfected cells could be easily established as cell lines, comparable to Ad12 E1Awt- or Ad2 E1Awt-transfected cells. In contrast, cells transfected with E1ASpm2 or Ad12 E1AdelCR2 (lacking the entire CR2) died within 6-10 weeks after replating, although foci were formed in all cases. Of note, the E1ASpm1-transformed cells grow as fast as the Ad2 E1Awt-transformed cells, with a doubling rate of 15 h, whereas the doubling of the Ad12 E1Awt-transformed cells takes approximately 120 h. Moreover, in the established cell lines, the affinity of E1ASpm1 to p105-Rb was higher than with that of E1Awt. Our data suggest the presence of a transformation-suppressing domain within the carboxyl-terminal 12 residues of the Ad12 E1A-unique spacer, whereas the hydrophobic stretch of six alanines in the spacer is required for stable transformation.

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A cis-acting element 7 bp upstream of the ESF-1-binding motif is involved in E1A 13S autoregulation of the adenovirus 12 TS2 promoter.

Cited by 4 publications

References 43 publications

E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter

E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter

Targeting of neural stem cells in the hippocampus of adult rats by custom-made Ad vectors

Two Domains within the Adenovirus Type 12 E1A UniqueSpacerHave Disparate Effects on the Interaction of E1A with P105-Rb and the Transformation of Primary Mouse Cells

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