Detection Of The Transcriptional Regulator RPS601 And Its Downstream Target Gene Ace2 By Speculation Of Fission Yeast Ribosomal Protein Gene Rpl32-2

Early, our lab found that Schizosaccharomyces pombe’s ribosomal protein gene rpl32-2 expression level is constantly changing in the different stages of the cell cycle, which explained that the expression of rpl32-2 is tightly regulated. Further,our lab has screened the ribosomal protein RPS601 in Yeast one-hybrid screening system which used the promoter of rpl32-2 as bait to screen the target from the building cDNA library of the wild strains of Schizosaccharomyces pombe, which illustrates RPS601 may be a potential a transcription activating factor of rpl32-2.This article first uses EMSA (Electrophoretic mobility shift assay) experiment to verificat it in vitro. First constructe heterologous expression strains which express RPS601(His tag), get high purity RPS601 protein after Ni-NTA agarose, verify the correctness of the His-RPS601 protein by Western Blot, at the same time get the fluorescent tagged five rpl32-2 upstream promoter fragment which length are 200 bp. Then process the EMSA experiment in which incubate the promoter fragment and purified RPS601 protein.The result is that there were not hysteresis bands in EMSA that made by RPS601 successfully combined with a purpose of promoter fragment. Then process further verification about this topic with CHIP-PCR (chromatin immunoprecipitation assay) experiment in vivo. Constructe mutant strains of yeast SP-Q01S which contain His tagged RPS601 and G418 resistance by using the method of homologous recombination.Then fixed SP-Q01S using formaldehyde, decrosslinked it, and used ultrasonic to shear chromatin etc. Immunoprecipitate the compounds of RPS601 and anti-His and combination of chromatin DNA fragments by Protein G Agarose. Western Blot confirms the.RPS601-His.Then enriched and purified DNA combining with RPS601 and use the purified DNA as template with the designed specific primers to PCR. The result is that there were not amplifying the aim DNA sequence which is upstream promoter sequence of rpl32-2 in CHIP-PGR experiments. The two experiments hint us that RPS601 had no effect to rpl32-2. It tells us that you can not be sure the interaction between proteins and genes only through single hybrid screening experiment.Our lab early found that RPL32-2 can activate the vital to cell division gene ace2 to promote cell division, by analyzing the microarray results of the wild type and Schizosaccharomyces pombe of overexpression RPL32. This article used the Yeast one-hybrid screening system, constructing the strain containing the ace2 gene promoter and reporting gene and cDNA library of the wild type yeast.We screen the transcription factors of ace2 from cDNA library by the Yeast one-hybrid screening system. But among the positive clone we haven’t screened the strain of RPL32-2 cDNA and maybe RPL32-2 has no effect to the promoter of ace2. Otherwise,this system has screened 10 other strains of binding proteins cDNA. Whether these proteins could be the potential transcription factor of ace2, we will be verificate the speculation in the subsequent experiment by EMSA, CHIP-PCR.