Exploring The Function Of Saccharomyces Cerevisiae Gene PHO85 And Homo Sapiens Novel Gene BRI3

PHO85 is a versatile gene in Saccharomyces cerevisiae, which involves inmetabolism of inorganic phosphate and usage of carbon source, accumulation ofglycogen, regulation of protein stability and cell cycle control. The viability of wildtype budding yeast strain YPH499 and its derivatives, pho85? mutant, pho80?mutant and pap1 (pcl7)? mutant in different cations were investigated and theirtolerance of the cations (LC50) was measured. The results showed that both thedeletion of PHO85 or PHO80 gene increase growth sensibility of Sacchromycescerevisiae to ion K+, Mg2+, Zn2+, Ca2+ and Mn2+ while the deletion of pap1 (pcl7)gene does not. The difference among the patterns of relative growth curve of themutants and YPH499 in the ions mentioned above also implied that Pho80 may bethe unique Pcl in complex with Pho85 CDK contributed to K+ and Mg2+ iontolerance and some other Pcls besides Pho80 were involved in Zn2+, Ca2+ and Mn2+tolerance regulation as cyclin of Pho85 CDK. Furthermore, the measurement of thetotal cellular calcium of pho85? mutant, pho80? mutant and YPH499 indicated thatthe ability of calcium accumulation of pho85? mutant, pho80? mutant was impaired.Calcineurin is a well-known phosphotase involved in metal ions homeostasisregulation of Saccharomycese cerevisiae, whose loss of function leads to somephenotype similar to pho85? mutant such as hypersensitive to ion Na+, Mn2+,pheromone and hygromycin B. We investigate the sensitivity of YPH499, pho85?mutant, pho80? mutant, and pap1 (pcl7)? mutant to Na+, Mn2+, Ca2+ with or withoutaddition of cyclosporin A, a specific inhibitor of calcineurin. It was found thatpho85? and YPH499 in cyclosporin A YPD medium show the same Na+ sensitivityas that of pho85? in YPD medium without cyclosporin A. That means Pho85 kinaseand calcineurin were involved in salt tolerance with an identical target protein or in 中科院上海生化与细胞所 博士学位论文 摘要 the same pathway. Inhibition of calcineurin decrease the YPH499, pho80? mutant,and pap1 (pcl7)? mutant Mn2+ tolerance but not that of pho85? mutant and thepho85? mutant was more sensitive to Mn2+ than YPH499, pho80? mutant, and pap1(pcl7)? mutant even with the addition of cyclosporin A. Therefore, the conclusioncould be drawn that PHO85 gene played a dominant role in Mn2+ homeostasisregulation in compare with calcineurin. As for Ca2+ tolerance, cyclosporin A canincrease the tolerance to Ca2+ of all the mutant mention above, that means Pho85kinase and calcineurin function antagonistically in regulation of Ca2+ homeostasis.In Bioinformatics, BRI3 gene is a novel gene without any function clue but givehigh conservation in mammalian. To exploring its function, we screen human fetalbrain cDNA library for interaction protein by Two Hybrid System 3. The CAML genewas 5 time screened out as positive clone in 0.75×106 transformants. It has beenreported that in Jurcat cell line, overexpression of CAML gene could activatecalcineurin in the presence of PMA by elevate intracellular calcium to turn on manyimportant cytokine transcription downstream TCR activation such as IL-2, 3, 4, INFγand TNFα. We measured HeLa cell intracellular Ca2+ transfected with constructsbearing overexpressing BRI3 and CAML respectively. The results showed that BRI3overexpression could increase the cytoplasmic Ca2+ as well as CAML gene did. Thatimplied the involvement of the BRI3 gene in calcineurin regulation by intracellularcalcium modulation.