Of Per1 Interacting Protein Rack1 Circadian Rhythm Related Genes

ObjectsTo identify novel PERIOD1 interacting proteins by Yeast two hybrid screening, and find the Circadian related role of RACK1. There are two parts in the research: 1 Selecting new interplaying molecule of PERI-PAS domain; 2 investigate RACK1 function in the Circadian rhythm.MethodExperiment 1: Selecting novel PER1 interacting proteins by Yeast two-hybrid.cDNA fragment which encode PER1-PAS domain was directionally cloned into pGBKT7 bait vector to construct recombinant expressional plasmid hPer1-PAS/pGBKT7. Construct human brain cDNA library by homologous recombination and establish Yeast two-hybrid system by sequence transfection. Then select positive clones by report genes tests. Amplify the inserts of pGADT7-Rec plasmid in the positive clones for sequencing and bioinformatics analysis to identify novel PER1 interacting protein. Verify the interplaying between proteins by co-immunoprecipitation with the products from in vitro transcription-translation.Experiment 2 Investigation of RACK1 function in the Circadian system:Half-quantitate RACK1 expression by RT-PCR detecting Gnb2l1 mRNA level with the NIH3T3 cell samples induced by PMA and with the BALB/C-mouse brain samples under condition of 12D/12L or DD. Record the free running wheeling of BALB/C mice simultaneously. Stimulate the BALB/C mice with light pulse which can induce phase shift of behavioral Circadian to observe photosensitized expression of Immediate Early Gene Gnb2l1. Observe distribution of RACK1 in peak time and valley time of PER1 protein.ResultExperiment 1: Constructed hPer1-PAS/pGBKT7 recombinant bait plasmid and human brain cDNA library and then established Yeast two-hybrid system...