Identification of an activator which specifically binds to thyroid hormone receptor b

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We tried to identified novel proteins which can specifically bind to thyroid hormone receptor (TR) b using yeast two-hybrid system.{Method}Yeast two-hybrid Matchmaker System 3 (Clontech, Palo Alto, CA, USA) was used to screen for novel TR-interacting proteins. A human TR b full length cDNA was cloned into pGBKT7 plasmid vector which contained GAL4 DNA binding domain. We screened a human liver cDNA library (Clontech) using the human TR cDNA as bait in the yeast two hybrid system.{Results}The mating efficiency and number of clones screened corresponded to the values indicated in the manufacture's instructions.1) In the 1st screening we got 131 clones on the plates supplemented with T3 (+T3). The additional screening was yeast clones were streaked both on the +T3 and -T3 plates. Ninety one clones grew in the T3 dependent manner. The plasmids of these yeast clones were purified and the inserts were sequenced. Two unknown sequences were identified ; No. 60 and 93.2) The full length of 2 clones were cloned from human liver total RNA by RT-PCR. Three different mRNAs of the clone No. 60 and a mRNA of No.93 were found in the liver. To determine whether TR physically interact with either the products of clone 60 or 93, the plasmid for GST fusion proteins were constructed.