Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells

Belt, Peter B. G. M.; Groeneveld, Herman; Teubel, Wilma; Putte, Pieter van de; Backendorf, Claude

doi:10.1016/0378-1119(89)90515-5

Cited by 60 publications

(29 citation statements)

References 25 publications

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“…This mutated peptide has no affinity for DNA in vitro as determined by Southwestern blot experiments (10). The HpECV control cell line was obtained by pooling the clones selected after transfection of the pECV23Xho vector (11) blotting experiments (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Schreiber

Hunting

Trucco

et al. 1995

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

189

122

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leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version ofthe DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNAbinding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

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Section: Resultsmentioning

confidence: 99%

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Schreiber

Hunting

Trucco

et al. 1995

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

189

122

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“…The GD25 cell line, derived from ␤1-deficient embryonic stem cells (Wennerberg et al, 1996), was transfected with ␤1B, ␤1A, ␤1TR, and ␤1COM expression constructs, together with the plasmid pECV6-hyg (Belt et al, 1989), by electroporation. Resistant populations were selected in medium containing 300 g/ml hygromycin ␤ (Boehringer Mannheim, Mannheim, Germany).…”

Section: Cells and Transfectionsmentioning

confidence: 99%

β1-Integrin Cytoplasmic Subdomains Involved in Dominant Negative Function

Retta

Balzac

Ferraris

et al. 1998

MBoC

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The ␤1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used ␤1A and ␤1B isoforms as well as four mutants lacking the entire cytoplasmic domain (␤1TR), the variable region (␤1COM), or the common region (␤1⌬COM-B and ␤1⌬COM-A). By expressing these constructs in Chinese hamster ovary and ␤1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that ␤1B, ␤1COM, ␤1⌬COM-B, and ␤1⌬COM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn ϩϩ ions, however, ␤1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that ␤1B interferes in a dominant negative manner with ␤1A and ␤3/␤5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the ␤1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the ␤1B isoform. INTRODUCTIONIntegrins are ␣/␤ heterodimeric transmembrane cell surface receptors that mediate cell adhesion and migration and also the bidirectional transfer of information across the plasma membrane. These properties are essential in regulating several biological processes, including morphogenesis, immune response, cell growth, differentiation, and survival (Hynes, 1992;Ruoslahti and Reed, 1994).The cytoplasmic domains of integrin subunits are required for these functions (Hibbs et al., 1991;Yamada and Miyamoto, 1995;Dedhar and Hannigan, 1996). In vitro, the isolated ␤1 cytoplasmic domain was shown to bind talin, ␣-actinin, and paxillin, three cytoskeletal proteins that mediate the anchorage of actin filaments to the plasma membrane (Chen et al., 1995;Otey et al., 1993;Schaller et al., 1995). Binding of the ␤1 cytoplasmic sequence to focal adhesion kinase (FAK), a tyrosine kinase specifically localized to focal adhesions, and to the serine/threonine kinase integrin-linked kinase has also been reported Hannigan et al., 1996). In vivo, the ␤1 cytoplasmic domain is sufficient for its localization to preformed focal adhesions (LaFlamme et al., 1992, Akiyama et al., 1994 and for the initiation of signaling to FAK (Lukashev et al., 1994). A model has been proposed in which the ␤1 subunit cytoplasmic domain ‡ Corresponding author.© 1998 by The American Society for Cell Biology 715contains a default signal for interaction with cytoskeletal molecules that is masked by the ␣ subunit cytoplasmic domain (Briesewitz et al., 1993;Ylanne et al., 1993). In response to matrix ligands or to multivalent antibody binding, the inhibitory effect of the ␣ subunit can be release...

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“…The ShPrP allelic variants PrP VQ , PrP AQ , and PrP AR were cloned and analyzed as described previously (10). PrP ORFs were subcloned between the ␤-globin intron and ␤-globin polyadenylylation sequences downstream the human cytomegalovirus (hCMV) promoter of expression vector pECV7, a derivative of expression vector pECV6 (27) in which the Rous sarcoma virus promoter has been substituted for the hCMV promoter. Mouse neuroblastoma cells (N2a cells; Hubrechts Laboratory, Utrecht, The Netherlands) were stably transfected with these constructs by electroporation (28), hygromycine B (500 g͞ml)-resistant single-cell clones were isolated, and high PrP-expressing clones were selected by immunoperoxidase monolayer assay using the antipeptide antibody R521-7 (peptide corresponds to amino acids 94-105 of the ShPrP sequence) (29).…”

Section: Methodsmentioning

confidence: 99%

Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms

Bossers¹,

Belt²,

Raymond³

et al. 1997

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

161

118

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Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells

Cited by 60 publications

References 25 publications

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

β1-Integrin Cytoplasmic Subdomains Involved in Dominant Negative Function

Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms

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