Construction Of Yeast Two-hybrid System Of The BHLH-PAS Domain Of HPER1

The physiology and behavior of all living organisms from bacteria to humans are controlled by the circadian rhythms driven by internal oscillators, the circadian clock. The circadian clock is composed of the positive and negative loops that consist of coock genes and proteins. A common theme underlying circadian rhythmicity is that oscillations of clock gene transcription are the consequence of intracellular transcriptional-translational feedback loops consisting of series of clock genes and proteins. During the past several years, a large number of clock genes were discovered in several species, including humans. But it is still difficult to describe the mechanism of the generation and regulation of circadian rhythms precisely.Among the clock proteins, PER1 is expressed widely in brain regions, non-neuronal tissues and cell lines. It plays an important role in the circadian clock. As many other clock proteins, PER1 consists of bHLH-PAS domain. The bHLH-PAS domain is the key region of many transcriptional regulators. To find proteins intecting with hPER1 and study the human circadian system, we construct the yeast two-hybrid system of the bHLH-PAS domain of hPERl.The sequence encoding bHLH-PAS domain of hPERl was amplified by RT-PCR. The amplified fragment was ligased with the vector pGBKT7 to construct recombinant bait plasmid pGBKT7/hPer1bHLH-PAS. The recombinant bait plasmid is tested for Transcriptional Activation and Toxicity. RNA was isolated from the human brain tissue belong to the ventral hypothalamus. Messenger RNA transcripts were efficiently copied into ds cDNA using BD SMART â…¢ technology. The cDNA library of human brain was constructed. The recombinant plasmid, ds cDNA and pGADT7-Rec were transfected intoyeast strain AH109 by LiAc Method. The yeast two-hybrid AH109 were screened with Triple Dropout Medium and Quadruple Dropout Medium. The positive clones were also checked through X-gal and PCR Colony Screening.Digested with endonuclease and sequenced, the recombinant plasmid was constructed correctly. The DNA-BD Fusion is inactive and nontoxic. Twenty-six positive colonies were selected by the yeast two-hybrid system. The yeast two-hybrid system of the bHLH-PAS domain of hPER1 was constructed.The positive colonies paved the way for further screening cDNA library, finding unknown proteins interacting with hPERl, and studying the molecular mechanism of human circadian machinery.