The replication-competent bovine leukemia virus (BLV) has been modified for use as a vector for foreign genes. The gag, pol, env, and pX regions of the virus were replaced by an exogenous nuclear location signal LacZ (nlsLacZ) or SVnlsLacZ gene. Transfection of the ovine cell line FLK-BLV, which expresses all BLV proteins from a wild-type provirus, with this viral DNA resulted in a viral titer of 10(4) CFU/ml. The inclusion of a large portion of the gag region did not significantly increase the titer. Both activator-dependent and activator-independent retroviruses were constructed. In activator-dependent vectors, the expression of the insert was dependent on the presence of the Tax protein, which activated the BLV long terminal repeat. In activator-independent vectors, the expression of the insert was constitutive because of the presence of an internal promoter. Infections with the recombinant retrovirus were inhibited by specific neutralizing antibodies. The structure of the transduced genetic material was not rearranged. BLV vectors encoding a reporter nlsLacZ gene, the product of which can be detected in single cells, greatly simplified studies of their biological properties. Determination of the host range of BLV vectors established that BLV-based recombinant retroviruses are effective in the transduction of genes in a variety of species and cell types.
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| http://dx.doi.org/10.1128/JVI.65.4.1938-1945.1991 | DOI Listing |
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Mediator, known as a coactivator, can act in transcription initiation in an activator-independent manner in vivo.
Biochim Biophys Acta Gene Regul Mech
August 2018
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France. Electronic address:
Mediator is an evolutionarily conserved complex best known for its role as a coactivator responsible for transducing regulatory signals from DNA-bound activators to the basal RNA polymerase II (Pol II) machinery that initiates transcription from promoters of protein-encoding genes. By exploiting our in vivo activator-independent transcription assay in Saccharomyces cerevisiae, in combination with new temperature sensitive (ts) mutants of Med14 N-terminal half exhibiting widespread transcriptional defects, and existing ts mutants of Kin28 and Med17, we show that, in the absence of activator: (i) Mediator can associate with a promoter as a form devoid of the Cyclin-dependent kinase 8 (CDK8) module, and this association remains regulated by Kin28; (ii) Mediator can stimulate the assembly of the entire Pol II initiation machinery. Although the literature emphasizes the role of the interaction between activators and Mediator, together our results support the view that Mediator is able to act through a dual mechanism in vivo, activator-dependent but also activator-independent, therefore not always as a coactivator.
View Article and Find Full Text PDFGene looping is conferred by activator-dependent interaction of transcription initiation and termination machineries.
J Biol Chem
September 2009
Department of Biological Science, Wayne State University, Detroit, Michigan 48202, USA.
Gene looping juxtaposes the promoter and terminator regions of RNA polymerase II-transcribed genes in yeast and mammalian cells. Here we report an activator-dependent interaction of transcription initiation and termination factors during gene looping in budding yeast. Chromatin analysis revealed that MET16, INO1, and GAL1p-BUD3 are in a stable looped configuration during activated transcription.
View Article and Find Full Text PDFStructural and functional characterization of PC2 and RNA polymerase II-associated subpopulations of metazoan Mediator.
Mol Cell Biol
March 2005
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Ave., #166, New York, NY 10021, USA.
The coactivator complexes TRAP/SMCC and PC2 represent two forms of Mediator. To further understand the implications of the heterogeneity of the cellular Mediator populations for regulation of RNA polymerase II (Pol II) transcription, we used a combination of affinity and conventional chromatographic methods. Our analysis revealed a spectrum of complexes, including some containing significant proportions of Pol II.
View Article and Find Full Text PDFRequirement of TRAP/mediator for both activator-independent and activator-dependent transcription in conjunction with TFIID-associated TAF(II)s.
Mol Cell Biol
April 2002
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA.
The multiprotein human TRAP/Mediator complex, which is phylogenetically related to the yeast SRB/Mediator coactivator, facilitates activation through a wide variety of transcriptional activators. However, it remains unclear how TRAP/Mediator functions in the context of other coactivators. Here we have identified a previously uncharacterized integral subunit (TRAP25) of the complex that is apparently metazoan specific.
View Article and Find Full Text PDFp300 forms a stable, template-committed complex with chromatin: role for the bromodomain.
Mol Cell Biol
June 2001
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
The nature of the interaction of coactivator proteins with transcriptionally active promoters in chromatin is a fundamental question in transcriptional regulation by RNA polymerase II. In this study, we used a biochemical approach to examine the functional association of the coactivator p300 with chromatin templates. Using in vitro transcription template competition assays, we observed that p300 forms a stable, template-committed complex with chromatin during the transcription process.
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