Molecular Mechanisms Of Histone Modification Influencing The Faithful Inner Exon Inclusion In S.cerevisiae.

Objective: The first aim of the project is to Establish (DYN– 2) gene artificial alternative splicing model in S.cerevisiae and detect the second exon inclusion/exclusion status of DYN - 2 gene by SYBR fluorescent real-time PCR method. The second one is to explore how histone post translational modification influence the pre-mRNA alternative-splicing events in DYN2 gene.Methods: In our previous work, we got the plasmid containing DYN-2 minigene. To identify the right construction of the plasmid we transformed the shuttle plasmid in to E.coli , we extracted the plasmid DNA and sequenced it. After the right construction was identified, the plasmid DNA was transformed into the blank yeast, and screened out the positive clones on CAA-Trp plat. The positive clones were marked as X yeast. We have tested the histone wild type and H3K14A, H3K18A/H4K5A and H4K8A mutant to see if there are any different between wild type and mutants. The approaches are as follows:Extract their plasmid DNAs, and enzyme cut to detect whether these plasmid DNAs are correct or not. Then transferred these plasmid DNAs into X yeast, and screened out the positive clones on 5-FOA plats. The positive clones were marked as O yeast, AA yeast, L yeast and Q yeast. Then we extracted the total mRNA from O, AA, L, Q yeasts and X, Y yeasts. Using DNase I deal with the RNAs to eliminate DNA pollution, then RT. The cDNAs were Preserved at - 20℃or -80℃. Design and synthesize primers specific for DYN - 2 gene and SCR - 1 gene, use the SYBR fluorescent real-time PCR method to detect the second exon expression of DYN - 2 gene.Results: This paper successfully established the effects of histone modification on the DYN - 2 gene pre - mRNA alternative splicing in vitro. Used yeast double hybrid system to transduce the DYN-2 gene and histone mutated genes into the same yeast cells. We successfully established a SYBR fluorescent Real - time PCR detection model, to detect relative expression level of the second exon on DYN - 2 gene.Conclusion: The reconstruction of histone modifications influence the alternative splicing model in vitro, can reflect the influences of histone modifications on pre-mRNA splicing more directly and efficiently; Real-time PCR can be used as a practical method for studying pre-mRNA splicing.