Biology Reference
In-Depth Information
recruitment of the VP16 activation domain to the promoter of the luciferase reporter
gene, thereby inducing transcription and luciferase protein production that is quan-
titated in a luminometer. The following protocols are examples given to determine by
a mammalian two-hybrid system the effect of the single mutation introduced into the
dimerization interface of RXR a (tyrosine 402 to alanine in helix H9) on the dimer-
ization function of this receptor, namely, the formation of heterodimers with RAR a
and TR a (NR1A1). The study led to the conclusion that the RXR a Y402A mutant
exhibits loss of heterodimerization abilities ( Vivat-Hannah, Bourguet, Gottardis, &
Gronemeyer, 2003 ; Fig. 2.6 ).
FIGURE 2.6
Example of mammalian two-hybrid analysis that reveals impaired heterodimerization of
mRXRaY402A with RARa and TRa in live cells. While the heterodimerization of RXRa-RARa
(A) and RXRa-TRa (B) is obvious from the strong two-hybrid signals (wt bars), this signal is
95% decreased for RARa when Gal-RXRaY402A is used (Y402A bars). TRa shows a
significant, albeit 65% reduced, interaction with the Y402 mutant. The control corresponds to
the reporter gene activity transfected alone. The transcriptional activity exerted by each
heterodimer is expressed as fold induction of reporter gene activity. The results shown are the
averages
standard errors of the means of three independent experiments.
Search WWH ::




Custom Search