cWe identified genes encoding components of the Hap complex, CbHAP2, CbHAP3, and CbHAP5, as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. We found that the Cbhap2⌬, Cbhap3⌬, and Cbhap5⌬ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae, the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii. Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.
The Hap complex of the methylotrophic yeast Candida boidinii was found to be required for methanol-regulated gene expression. In this study, we performed functional characterization of CbHap3p, one of the Hap complex components in C. boidinii. Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from Saccharomyces cerevisiae (ScHap3p), but is found in Hap3p proteins of methylotrophic yeasts. Deletion of the C-terminal region of CbHap3p (D256-292 or D107-237) diminished activation of methanol-regulated genes and abolished the ability to grow on methanol, but did not affect nuclear localization or DNA-binding ability. However, deletion of the N-terminal region of CbHap3p (D1-20) led to not only a growth defect on methanol and a decreased level of methanol-regulated gene expression, but also impaired nuclear localization and binding to methanol-regulated gene promoters. We also revealed that CbHap3p could complement the growth defect of the Schap3D strain on glycerol, although ScHap3p could not complement the growth defect of a Cbhap3D strain on methanol. We conclude that the unique C-terminal region of CbHap3p contributes to maximum activation of methanol-regulated genes, whilst the N-terminal region is required for nuclear localization and binding to DNA.
We present a novel study on electromigration (EM) phenomena in Cu interconnects using a newly developed multi-scale simulator that consists on a combination of a device scale simulator based on a kinetic Monte Carlo (KMC) method and an atomic scale simulator based on ultra accelerated quantum chemical molecular dynamics (UA-QCMD). We have firstly demonstrated the simulation of the lifetime of Cu interconnects using the newly developed device scale simulator setting some suitable KMC probabilities for the void movement according to the regions in which it can be divided, i.e., the crystal grain and the grain boundary. The simulated values are shown to be in good agreement with experimental values. In an attempt to connect the device scale studies to quantum chemical instances of the system -since the correlation of probability of the void movement with, for example, activation energies or diffusion coefficients is important -we have developed an atomic scale simulator based on our original UA-QCMD method. In this atomic scale simulation, the electron wind force was evaluated using our original electrical conductivity prediction simulator based on KMC method which uses the electronic states from tight-binding quantum chemical (TBQC) calculation. Using this atomic scale simulator under the conditions of 475 K of temperature and 2:5 Â 10 10 A/m 2 of current density, we were able to successfully simulate the migration of a Cu atom from a lattice site to a vacant site by evaluating the electron wind force.
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