To Explore The Effect Of Yeast Gene Containing Multiple Introns On The Splicing And Expression Of Introns

Regulation of gene expression is a highly conservative cellular process in eukaryotic cells,and it has been accuratly coupled and cordinate.Multiple factors regulate this complex process.Recent reprots shown that proteins which concerning chromatin structure can not only regulate transcript initiation,but also control the mRNA maturation process?the stability of mRNA,protein folding and modification as well.In early analysis in yeast database,we found some rules of its genome:First,only nearly 4%of genes of the total genes contain introns,and most of these intron-contain genes are very improtant in cellular growing development regulation;Second,for these intron-contain genes,most of their introns occupanced in the upstream of the genes,nearby 5' site and these genes almost have one intron only;Third,the length of yeast introns are most between 50 to 200 base-pair(highly expressed genes);Finally,in the localization of 5'UTR of most genes containing a Nuclear Free Region,and the occupancy of the nucleosome has been weeker from gene's 5' site to 3' site.Current studies about splicing mechanism have not only show many splicing factors and modification enzyme concerning splicing process,but also find that gene structure such as nucleosome or promoter plays important role in intron splicing.Our work try to find some protein that regulate chromatin structure can slso affect intron deffinition and splicing process.We try to answer these three question in evolutional oppiniori:Why the Saccharomyces cerevisiae have low efficiency in splice downstream introns;Why most of the intron-contain genes have one intron only;Why the intron-contain genes usually have signifficant roles in cellular development.Based on preious analysis and experimental data,We decide to use the Arab RPL36B gene and Yeast RPL36B gene as object gene,and we cloned the Arab RPL36B gene with a flag tag in yeast plasmid pRS413 so that it can be expressed in Saccharomyces cerevisiae cell,the Arab RPL36B can act as yeast RPL36B when the yeast endogenous RPL36B has been knocked out.In order to investigate the efficiency of different intron splicing,we insert three different yeast intron in the orign intron site of Arab RPL36B in each position.In our results,the efficiency of intion spling show a lower level when the intron locallized in downstream of gene or gene have two or three introns.We also show different promoter have vary efficiency of intron deffinition and splicing.Next step we plan to use yeast genome cDNA library to find some protein factors have effect on intron deffinition and splicing process.Our subject will show us the mechanism of regulation of gene expression in a novel way.