Studies On The Mitochondrial DNA Stability And Histone Acetylation In Saccharomyce Cerevisiae

Mitochondria are the energy factories of cells, control cell growth, apoptosis and other life activities.The mitochondrial DNA (mtDNA) often associated with a variety of proteins to form DNA-protein complex, ie, mitochondrial nucleoids. Mitochondria nucleoprotein has an important role in the maintenance of mtDNA copy number and structure stability. The mitochondrial genome instability will lead to the disorder induced heart disease, cancer, metabolic diseases and degenerative diseases of oxidative phosphorylation. SLSlp (Sigma-like sequence protein1)Saccharomyces cerevisiae nuclear gene encoding a mitochondrial inner membrane integrated protein involved in mitochondrial DNA transcription and translation. Clarify SLS1p regulation of mitochondrial function and downstream nuclear gene expression mechanism is of great significance.By mean of constructed sls1knockout yeast strain (W1021-7C the△SLS1), SLS1recovery (W1021-7C of△SLS1with pDB20-SLS1) yeast strain and SLS1overexpression (W1021-7C with pDB20-SLS1) found that the strain cannot grow in glycerol as the carbon source GlyYP medium and the doubling time was significantly longer than wild-type yeast strains. Southern Blot hybridization showed that the sls1gene knockout lead to mitochondrial genes COX1, COX2, COX3, ATP6, ATP9, CYTB,15S rRNA gene and21S rRNA content has decreased in varying degrees. Northern Blot hybridization showed that the mitochondria to COX1, COX2, COX3, ATP6, ATP9, CYTB,15S rRNA and21S rRNA transcription level decreased significantly, to COX1, ATP6and CYTB decreased very significantly. Subsequently, we tested the knockout yeast strains, the ATP level, knockout yeast strains of the entire ATP levels significantly lower than the wild-type yeast cells. And added the2-D-G inhibition of glycolytic ATP production, sls1null-mutant yeast strains ATP levels have a very significant decrease. SLS1recovery strain growth conditions, the doubling time, content and transcription level of the mitochondrial genome, as well as the ATP level has been restored, no significant difference between the wild type strain. SLS1 overexpression strains with wild-type strain was no significant difference. Subsequently, we use the Q-PCR detection of NAD+-dependent go the deacetylase SIR gene family in slsl knock the level of transcription in addition to yeast cells, SIR2, SIR3and SIR4transcription level was significantly lower than the wild type strain. Finally, we use the Western Blot method to detect the SIR gene family action site of H4K16acetylation levels and found that the acetylation levels of the knockout yeast strains was significantly higher than the wild type strain. That the instability of the mitochondrial genome may also be closely related and the level of histone acetylation, thereby affecting the downstream nuclear gene expression.RIM1and ABF2mitochondrial nucleoid-associated protein known to play an important role in mitochondrial DNA replication and reorganization process. Then we also build the abf2and riml gene knockout yeast cells. Experiments show that these two strains were unable to growth in glycerol as the carbon source and the doubling time was significantly longer than wild-type cells. Southern Blot and Northern Blot results show that the entire mitochondrial genome knockout yeast strains were destroyed. ATP test results prove that the knockout yeast whole-cell ATP levels were significantly lower than the wild type strain and in the case of pathways by inhibition of glycolysis to produce ATP, ATP levels very substantially decreased. This indicates that the ABF2and RIM1is essential to maintain the yeast mitochondrial genome. Finally, we use the Real-time PCR detection of NAD+-dependent deacetylase SIR gene family knocking at the transcriptional level in addition to the yeast cells. Also found that the transcription level of SIR2, SIR3and SIR4were significantly lower than the wild type strain. That may close relationship between the mitochondrial genome stability and histone acetylation.These results suggest that ABF2, RIM1and SLS1gene were associated with the stability of mitochondrial genome closely. And knockout of the yeast cells at the same time lead to mitochondrial dysfunction, may also on the level of histone acetylation of the yeast cells affected. So we combined these three factors:environmental (energy), mitochondria and epigenetic. May have mitochondrial dysfunction or apparent dysfunction caused by disease treatment and diagnosis of opening up new ideas.