Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expressionvectors with bi-directional promoters for use in Saccharomyces cerevisiae.
The pBEVY (bi-directional expression vectors for yeast) plasmids were designed with constitutive and galactose-induced bi-directional promoters to direct the expression of multiple proteins in Saccharomyces cerevisiae . Using human estrogen receptor as a test gene, relatively balanced expression levels from each side of a bi-directional promoter were observed. Expression of a functional heterodimeric transcription factor composed of human aryl hydrocarbon receptor (Ahr) and aryl hydrocarbon receptor nuclear translocator (Arnt) proteins was accomplished using a single pBEVY plasmid. Previous studies suggest that inhibitory cross-talk between the estrogen receptor and the Ahr/Arnt complex may occur and that Hsp90-Ahr complex formation is important for Ahr-mediated signal transduction. Evidence for functional interaction among these proteins was investigated using pBEVY plasmids in a yeast system. No inhibitory cross-talk was observed in signaling assays performed with yeast that co-expressed Ahr, Arnt and estrogen receptor. In contrast, Ahr/Arnt-mediated signal transduction was reduced by 80% in a temperature-sensitive Hsp90 strain grown under non-permissive conditions. We conclude that pBEVY plasmids facilitate the examination of multiple protein interactions in yeast model systems.
Miller
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CA
Environmental Health Sciences Department and Tulane-Xavier Center for Bioenvironmental Research,Tulane University School of Public Health and Tropical Medicine, 1430 Tulane Avenue, New Orleans,LA 70112, USA.
Martinat
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MA
Hyman
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LE
D019281:Dimerization; D015870:Gene Expression; D005822:Genetic Vectors; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D011401:Promoter Regions, Genetic; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D018841:HSP90 Heat-Shock Proteins; D018336:Receptors, Aryl Hydrocarbon; D011960:Receptors, Estrogen