Expression of functional chicken oviduct progesterone receptors in yeast (Saccharomyces cerevisiae).
The cDNAs encoding full-length chicken oviduct progesterone receptor B (PRB) and a truncated receptor (C1C2) lacking the amino-terminal domain were expressed in yeast (Saccharomyces cerevisiae) using a ubiquitin fusion system. The expression of the fusion protein is under the control of a copper-responsive yeast metallothionein promoter, and the fusion protein is subsequently cleaved by the yeast host enzyme to produce receptor protein. Western immunoblot analyses of yeast extracts containing full-length PRB revealed a polypeptide co-migrating with authentic chicken oviduct PRB. Using a polyclonal antibody (907) directed against the "hinge" region of the authentic chicken progesterone receptor, a 42-kDa polypeptide was detected by Western analysis in yeast extracts containing C1C2 receptors. Standard hormone binding assays indicated that these receptors produced in yeast cells exhibited steroid binding affinity and specificity characteristic of the authentic chicken progesterone receptor. To test for progesterone receptor-mediated activation of transcription in yeast, reporter plasmids were constructed to transform yeast cells expressing PRB or C1C2 receptors. The reporter gene contained two copies of a progesterone response element upstream of the yeast proximal CYC1 promoter fused to the beta-galactosidase gene of Escherichia coli. The induction of beta-galactosidase activity by PRB and C1C2 was strictly dependent on specific ligand and the presence of a progesterone response element. However, overproduced C1C2 receptors had an adverse effect on the transcription of the lacZ gene. It was found that when overproduced C1C2 was activated by progesterone, an inhibitory effect on normal yeast cell growth was evident. These observations suggest that C1C2 is a potent trans-acting factor in yeast and that the amino-terminal domain of the chicken progesterone receptor may play a role in selective modulation of target gene activation.
Mak, P; McDonnell, DP; Weigel, NL; Schrader, WT; O'Malley, BW
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