Plasmid And Gst-exo84 And Exo84-2 Of Saccharomyces Cerevisiae Mutant Strains,

Objective1.We managed to construct the Exo84p expressed plasmid, in order to perform theexperiment to conform that if acetylated histone H3 interact with exo84p in vitro.2.We also made the temperature sensitive exo84-2 mutant for strain of Saccharomycescerevisiae , and tested the ratio of inclusion of second exon in DYN2 gene(YDR424C) andSCR1. The minigene for DYN2 gene were constructed by us previously.Methods1. We chose the pGEX-2T as a vector and designed a pair of primers for the EXO84gene which could be amplified from budding yeast genomic DNA. The amplified EXO84 geneDNA were subcloned into pGEX-2T via xmaI site.2.We designed a pair of primers for amplifying the Leu gene , and transformed the PCRproduct into budding yeast strain which containing the DNY2 minigene. The transformants werescreened by Sc-Trp-Leu. After we got the mutant, The yeast strain were cultured into permisivetemperature(30°C )and then put into non permisive temperature(37°C) for 1.5h and 3h. Afterharvesting the yeast cells we extracted the mRNA and perform the RT-PCR to detecte the ratio ofinclusion of second exon of DYN2 gene znd Scr1 mRNA .ResultsWe got the plasmids containing the exo84 gene. We also make the exo84-2 mutant strain andobserved the inclusion/exclusion of second exon of DYN2 gene in the exo84-2 mutationbackground.ConclusionsThe efficiency of mRNA splicing for DYN2 gene becomes low in exo84-2 background.