Functional analysis of the papilloma virus E2 trans-activator in Saccharomyces cerevisiae.

Lambert, Paul F.; Dostatni, Nathalie; McBride, A A; Yaniv, Moshe; Howley, Peter M.; Arcangioli, Benoı̂t

doi:10.1101/gad.3.1.38

Cited by 60 publications

(61 citation statements)

References 44 publications

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“…It is worth noting that several transcription initiation factors and regulators from yeast and animal cells can function in cells from nonhomologous species (Buratowski et al 1988;Chodosh et al 1988;Kakidani and Ptashne 1988;Struhl 1988;Lambert et al 1989); this implies that our strategy may provide a general approach for fine structure analysis of other gene products from organisms with complex or inaccessible genetics. The procedure appears particularly well suited to the facile isolation and preliminary characterization of a large number of mutations; indeed, the mutants described here were all obtained after treatment of only one strand of the DNA double helix with a single mutagen.…”

Section: Discussionmentioning

confidence: 99%

Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities.

Schena¹,

Freedman²,

Yamamoto³

1989

Genes Dev.

229

125

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Mammalian glucocorticoid receptors bind specifically to glucocorticoid response element (GRE) DNA sequences and enhance transcription from GRE-linked promoters in mammalian cells and in yeast. We randomly mutagenized a segment of the receptor encompassing sequences responsible for DNA-binding and transcriptional regulation and screened in yeast for receptor defects. The mutations all mapped to a 66-aminoacid subregion that includes two zinc fingers; in general parallel phenotypes were observed in yeast and animal cells. Mutants defective for DNA binding also failed either to enhance or to repress transcription. However, several mutations in the second finger selectively impaired enhancement; we suggest that such 'positive control' mutants may alter protein-protein contacts required for transcriptional activation.

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Section: Discussionmentioning

confidence: 99%

Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities.

Schena¹,

Freedman²,

Yamamoto³

1989

Genes Dev.

229

125

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“…The amino terminus of E2 (amino acids 1-220) is the trans-SLCtiyation domain and is believed to belong to the amphipathic helix class of transcription factors (Giri and Yaniv 1988;Haugen et al 1988;McBride et al 1989). Like these acidic activators, E2 functions are operative in Saccharomyces cerevisiae (Lambert et al 1989;Morrissey et al 1989;Stanway et al 1989). The DNA-binding/dimerization and trans-activation domains are separated by a poorly conserved sequence that is -100 amino acids in BPV.…”

mentioning

confidence: 99%

Amino acids necessary for DNA contact and dimerization imply novel motifs in the papillomavirus E2 trans-activator.

et al. 1992

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The bovine papillomavirus E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT. The dimerization and DNA-binding properties are localized within its carboxy-terminal 85 amino acids (325-410). Utilizing random mutagenesis coupled with phenotypic selection in yeast, functionally important amino acids in the DNA-binding domain were identified. Four trans-activation defective point mutants within a short segment (amino acids 337-344) were DNA binding defective but dimeric. The mutation of a conserved tryptophan to serine also eliminated DNA binding, but loss of dimerization was implicated because addition of dimeric monoclonal antibody complemented this defect. A simple assay for £2 dimerization was developed using UV irradiation to produce an interchain cross-link within a dimer. No heterodimeric complexes were formed when pools of E2 of varying lengths were mixed, and only proteins with tryptophan at position 360 could be UV cross-linked. Peptide mapping of irradiated E2 protein localized the cross-link to an 18-amino-acid region bracketing this tryptophan. Substitutions for this tryptophan demonstrated the requirement for a hydrophobic residue at this position, but surprisingly, even alanine was functional. Replacement of this tryptophan with three polar amino acids or glycine eliminated DNA-binding activity, but addition of dimeric monoclonal antibody restored this function. The amino acids that were identified as being involved in DNA contact and dimerization imply that these functions are mediated by novel binding motifs.

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“…The efficiency and rapidity of yeast culture methods allow recovery of approx 2.5 × 10 10 yeast cells from a 250-mL culture grown in 24 h, which argues that production of infectious HPV in yeast could be made extremely efficient by use of this system. The virion 9 Use of a galactose-inducible promoter to express L1 and L2 allows better control of protein stoichiometry during the virus assembly experiments. Our experience in the lab indicates that over-expression of certain trans-factors such as E1, and to a far lesser extent E2, can lead to recombination of plasmids, and therefore should be carefully controlled.…”

Section: Hpv Pseudovirion Isolation From Yeastmentioning

confidence: 99%

“…The pPD2-16E2 plasmid also contains a yeast 2-μ origin, the Leu2 biosynthetic gene, a coli 1 origin, and an ampicillin marker. A complete description of the construction of this plasmid is given in (9). For expression of HPV-16 L1 and L2, a bidirectional galactose-inducible promoter was used; the vector is referred to here as pL1L2 (Fig.…”

Section: Hpv-orf Expression Vectorsmentioning

confidence: 99%

Replication and Encapsidation of Papillomaviruses in <I>Saccharomyces cerevisiae</I>

Angeletti

Human Papillomaviruses

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SummaryImprovements in methodologies to recapitulate and study particular biological functions of the papillomavirus life cycle have led to great advances in our knowledge of these viruses. Described in this chapter are techniques that allow low-copy and high-copy replication of full-length human papillomavirus (HPV) genomes, as well as assembly of virus-like particles, in Saccharomyces cerevisiae (yeast). This system has several distinct advantages that make it an attractive complement to the well-established raft-culturing system. First, yeast are inexpensive, rapid, and simple to culture in the lab. Second, they provide an ever-widening array of genetic tools to analyze HPV functions -most recently notable, the yeast ORF-deletion library. Third, yeast provide a potentially highefficiency means to produce large quantities of infectious virus in a short time frame. Fourth, assembly of HPV virus in yeast allows encapsidation of mutant genomes, since previous studies have shown that no viral ORF is required for replication of full-length HPV in yeast.

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Functional analysis of the papilloma virus E2 trans-activator in Saccharomyces cerevisiae.

Cited by 60 publications

References 44 publications

Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities.

Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities.

Amino acids necessary for DNA contact and dimerization imply novel motifs in the papillomavirus E2 trans-activator.

Replication and Encapsidation of Papillomaviruses in <I>Saccharomyces cerevisiae</I>

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