A technology for detecting unselected mutational spectra in human genomic DNA

Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 1999.Includes bibliographical references (leaves 186-205).by Xiaocheng Li-Suckoleiki.Ph.D

Oxidative Stress Impairs the Heat Stress Response and Delays Unfolded Protein Recovery

Background: Environmental changes, air pollution and ozone depletion are increasing oxidative stress, and global warming threatens health by heat stress. We now face a high risk of simultaneous exposure to heat and oxidative stress. However, there have been few studies investigating their combined adverse effects on cell viability. Principal Findings: Pretreatment of hydrogen peroxide (H2O2) specifically and highly sensitized cells to heat stress, and enhanced loss of mitochondrial membrane potential. H 2O 2 exposure impaired the HSP40/HSP70 induction as heat shock response (HSR) and the unfolded protein recovery, and enhanced eIF2a phosphorylation and/or XBP1 splicing, land marks of ER stress. These H2O2-mediated effects mimicked enhanced heat sensitivity in HSF1 knockdown or knockout cells. Importantly, thermal preconditioning blocked H 2O 2–mediated inhibitory effects on refolding activity and rescued HSF1 +/+ MEFs, but neither blocked the effects nor rescued HSF1-/- MEFs. These data strongly suggest that inhibition of HSR and refolding activity is crucial for H2O2–mediated enhanced heat sensitivity. Conclusions: H2O2 blocks HSR and refolding activity under heat stress, thereby leading to insufficient quality control and enhancing ER stress. These uncontrolled stress responses may enhance cell death. Our data thus highlight oxidative stres

Mutant Versions of the S. cerevisiae Transcription Elongation Factor Spt16 Define Regions of Spt16 That Functionally Interact with Histone H3

In eukaryotic cells, the highly conserved FACT (FAcilitates Chromatin Transcription) complex plays important roles in several chromatin-based processes including transcription initiation and elongation. During transcription elongation, the FACT complex interacts directly with nucleosomes to facilitate histone removal upon RNA polymerase II (Pol II) passage and assists in the reconstitution of nucleosomes following Pol II passage. Although the contribution of the FACT complex to the process of transcription elongation has been well established, the mechanisms that govern interactions between FACT and chromatin still remain to be fully elucidated. Using the budding yeast Saccharomyces cerevisiae as a model system, we provide evidence that the middle domain of the FACT subunit Spt16 – the Spt16-M domain – is involved in functional interactions with histone H3. Our results show that the Spt16-M domain plays a role in the prevention of cryptic intragenic transcription during transcription elongation and also suggest that the Spt16-M domain has a function in regulating dissociation of Spt16 from chromatin at the end of the transcription process. We also provide evidence for a role for the extreme carboxy terminus of Spt16 in functional interactions with histone H3. Taken together, our studies point to previously undescribed roles for the Spt16 M-domain and extreme carboxy terminus in regulating interactions between Spt16 and chromatin during the process of transcription elongation