Thèses sur le sujet « RSC complex, Rsc2 »

Faithful chromosome segregation during mitosis is fundamental for cell viability and genome stability. For a correct division, all kinetochores must be attached to the mitotic spindle and cohesion must be timely removed. Anaphase is triggered by the Anaphase Promoting Complex bound to its regulatory subunit Cdc20 (APC-Cdc20) that polyubiquitylates securin (Pds1 in budding yeast), whose role is to maintain inactive the protease separase (Esp1 in budding yeast) until anaphase onset. Once active, separase cleaves cohesin, thus triggering sister chromatid separation. Separase also promotes cyclinB proteolysis and mitotic exit due to its involvement in the Cdc14-early anaphase release (FEAR) pathway that promotes a partial activation of the Cdc14 phophatase, which is in turn key for CDK inactivation and mitotic exit. Cdc14 is maintained inactive throughout most of the cell cycle bound to its inhibitor Net1/Cfi1 and trapped in the nucleolus. At the beginning of anaphase Cdc14 is released from the nucleolus into the nucleus by the FEAR pathway; subsequently, Cdc14 is released also in the cytoplasm by the MEN (Mitotic Exit Network) pathway. In this way Cdc14 is fully active and can trigger mitotic exit by cyclinB-CDK inactivation. The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism conserved in all eukaryotic organisms that ensures the correct segregation of the genetic material. In fact, it inhibits the metaphase to anaphase transition until all kinetochores are properly attached to the mitotic spindle by inactivating the APC-Cdc20 complex, thus providing the time for error correction. Cells do not arrest indefinitely upon SAC activation. After a variable period of time cells escape from the metaphase arrest also in the presence of a damaged mitotic spindle or faulty kinetochore attachments to spindle microtubules. This process is referred to as adaptation or mitotic slippage and is often involved in the resistance to chemotherapeutic compounds that target the mitotic spindle. In spite of its importance, the adaptation process is still little known. Within this context, the goals of my Ph.D. were: (1) to characterize the molecular mechanisms underlying SAC adaptation and (2) to search for factors involved in this process. For these purposes we used the yeast Saccharomyces cerevisiae as a model organism. (1) We characterized the adaptation process in either the presence or the absence of mitotic spindle perturbations. We depolymerized spindles by using two different drugs that alter microtubule dynamics, i.e. nocodazole and benomyl, whereas we induced SAC hyperactivation without spindle damage by overproducing Mad2 (GAL1-MAD2 cells), one of the key proteins for SAC signal generation and maintenance. We observed that in all the conditions cells are able to adapt, but with different kinetics. In particular, cells adapt faster in benomyl, while in nocodazole and with high levels of Mad2 cells need more time to slip out of mitosis. The few data available about SAC adaptation in higher eukaryotes indicate that SAC adaptation is accompanied by chromatid separation, a decrease in mitotic CDK activity and mitotic exit. Indeed, like in mammalian cells, yeast securin and cyclinB are degraded and sister chromatids are separated during adaptation. In addition, cyclinB stabilization, as well as Cdc20 and Cdc5 (polo kinase) inactivation, markedly delay adaptation, while the only yeast CKI (Sic1) is not involved in this process. Finally, when yeast cells adapt the SAC is likely to be turned off, as shown by the disassembly of the Mad1/Bub3 checkpoint complex. (2) To search for factors involved in SAC adaptation, we performed a genetic screen using GAL1-MAD2 cells. In particular, we screened for mutants that would remain arrested for prolonged times in mitosis upon MAD2 overexpression. We identified Rsc2, a non-essential component of the RSC chromatin remodelling complex, as a regulator of SAC adaptation in yeast. We demonstrated that RSCRsc2 is involved in fine tuning mitotic exit during the unperturbed cell cycle. Its activity becomes particularly important in conditions that would activate the SAC, as it contributes to cyclinB degradation. In the absence of Rsc2 Net1 phosphorylation and the early anaphase release of Cdc14 from the nucleolus are impaired, whereas expression of a dominant allele of CDC14 that loosens Net1 inhibition (CDC14TAB6-1) is sufficient to restore mitotic exit in conditions where Rsc2 becomes essential for this process. We further demonstrated that the ATPase activity of RSC is required for mitotic exit regulation, suggesting that its chromatin-remodelling activity is involved in this process. By studying possible genetic interactions between the RSC2 deletion and FEAR or MEN mutations, we found that RSC2 deletion confers synthetic lethality or sickness to MEN but not to FEAR mutants. Altogether, our data suggest that RSCRsc2 is a novel component of the FEAR pathway. Finally, we demonstrated that Rsc2 interacts in vivo and in vitro with the polo kinase Cdc5, which controls mitotic exit at different levels. Since RSC binds to acetylated histone tails, it is possible that histone transacetylases are also involved in SAC adaptation. We tested if the SAGA (Spt-Ada-Gcn5 Acetyltransferase) complex is involved in SAC adaptation by deleting ADA2 or GCN5 in yeast. Indeed, SAGA seems involved in adaptation, although the contribution of Ada2 and Gcn5 in the process differs depending on the conditions used to activate the SAC. Finally, since we found that upon treatment with benomyl (a microtubule destabilizer) cells adapt dividing nuclei, we wondered if SAC adaptation could be linked to the presence of cytoplasmic microtubules that are still partially detectable in these conditions. We therefore asked whether motor proteins and microtubule regulators are involved in mitotic slippage. Indeed, we found that in the absence of Kip2 and Bik1, which specifically bind to cytoplasmic microtubules, cells divide nuclei and exit mitosis slower than wild type cells, demonstrating that cytoplasmic microtubules and associated proteins could accelerate SAC adaptation. In conclusion, SAC adaptation is a very complex process whose timing probably depends on the interplay between different mechanisms. An important aim for a complete comprehension of this process, as well as for the development of new and more efficient cancer therapies, will be to identify novel factors implicated in adaptation and clarify how their function might be linked to one another.

Le complexe RSC est un des facteurs de remodelage de la chromatine capables de lever la barrière nucléosomale notamment lors de la transcription. Ce processus est effectué chez les eucaryotes par trois ARN polymérases (pol). Nous avons montré que le complexe RSC interagit avec les pol I et III. La protéine Rsc4 interagit par son domaine C-terminal avec la protéine ABC27, commune aux trois ARN polymérases. Nous avons isolé une mutation de la sous-unité Rsc4 qui abolit cette interaction. Les profils d'expression génomiques, établis par puces à ADN, ont permis de caractériser ses effets sur la transcription par la pol II. Curieusement, la majorité des gènes induits sont répartis sur le chromosome XII de manière non polaire. La présence de l'ADN ribosomique sur ce chromosome suggère un lien avec ce comportement particulier. Par ailleurs, la maturation de l'ARN 35S, transcrit par la pol I, est altérée, mais nous n'avons pas pu caractériser des défauts de transcription par les pol I et III<br>The RSC complex is one of the chromatin remodeling complexes that helps the transcripiton machinery to overcome the nucleosomal barrier. Eukaryotic transcription is carried out by three RNA polymerases. We have demonstrated that RSC complex interacts with pol I and III. The Rsc4 protein interacts by its C-teminal domain with the ABC27 protein, a subunit shared by the three eukaryotic RNA polymerases, We have isolated a mutation in the Rsc4 subunit that ablolish thi interaction. We performed genome profiling experiments using DNA microarrays to characterise pol II transcription defects. Surprisingly, the vast majority of the upregulated genes localised to the chromosome XII, spreading all along in a non-polar manner. We propose that the presence of the rDNA cluster on chromosome XII could be responsible for this peculiar transcriptional pattern. We have seen defects in the 35S RNA maturation but have been unable to clearly establish defects on pol I and pol III transcription

Regional Security Complex Theory (RSCT) innefattar analysenheter som är nära kopplade till försvarsmateriella handelavtal och har därför ett teoretiskt ramverk som kan hjälpa förstå och förklara avtalen i detalj. Det är dock oklart exakt hur detta ska göras eftersom det inte tas upp i Regions and Powers: The Structure of International Security. Syftet med denna uppsats är att med hjälp av RSCT förklara Mistralaären för att sedan undersöka om det finns en underliggande process gemensamt för liknande fall.

New corrole–BODIPY conjugates have been synthesized in high yield under mild conditions. Upon excitation at the absorption maximum of the BODIPY antenna chromophore, the fluorescence intensity of the free base corrole–BODIPY conjugate increases by ca. 300%, and significant phosphorescence intensity is observed for the iridium(III) complex of the conjugate, while almost no phosphorescence is observed for the parent iridium(III) corrole, due to through-bond energy transfer from the BODIPY antenna-chromophore to the corrole core.<br>Original publication is available at http://dx.doi.org/10.1039/c5ra07250f<br>Arbortext Advanced Print Publisher 9.1.510/W Unicode<br>Acrobat Distiller 8.1.0 (Windows); modified using iText� 5.3.3 �2000-2012 1T3XT BVBA (AGPL-version)

Cette thèse présente la reconstruction de l’histoire tectono-thermique du massif varisque des Jebilet (Maroc) à fort potentiel minier, depuis son évolution pré-orogénique au Dévonien supérieur-Carbonifère inférieur jusqu’à sa structuration pendant l’orogénèse varisque-alléghanienne au Carbonifère supérieur-Permien inférieur. Pour répondre à cette problématique, ce travail s’organise autour de deux approches : (1) l’une métrologique appliquée à la géothermométrie Raman sur la matière carbonée (RSCM) et (2) l’autre intégrant une étude structurale, géochronologique et une analyse de la thermicité.L’approche métrologique a permis de valider l’applicabilité du géothermomètre RSCM (1) dans un contexte de métamorphisme polyphasé, (2) pour des roches carbonatées et des skarns des Jebilet et (3) de proposer un nouveau paramètre Raman RSA permettant de mieux préciser les températures supérieures à 500°C et d’étendre l’applicabilité de la méthode jusqu’à des températures maximales qui atteignent les 700°C.A partir de l’approche intégrée, trois épisodes tectono-thermiques ont été mis en évidence. Le premier épisode D₀ correspond à une tectonique extensive permettant l’ouverture du bassin des Jebilet au Dévonien supérieur-Carbonifère inférieur. Cette tectonique extensive est accompagnée par une anomalie thermique supérieure à 500°C déduites des mesures de géothermométrie RSCM (TRSCM) et par une importante activité magmatique bimodale et granodioritique datée dans ce travail entre 358 ± 7 et 336 ± 4 Ma. Au Carbonifère supérieur débute la phase compressive structurant le massif des Jebilet avec la mise en place de nappes superficielles au Namuro-Westphalien (D₁), suivie par la phase varisque majeure (D₂). L’analyse structurale a permis de montrer une évolution progressive du régime de déformation de D2 depuis une compression coaxiale à une transpression dextre compatible avec un raccourcissement horizontal WNW-ESE à NW-SE. D₂₁ est associée à deux événements thermiques, le premier syn-tectonique de moyenne température (300°<br>This thesis presents the reconstruction of the tectono-thermal history of the Paleozoic Jebilet massif (Morocco), from its pre-orogenic evolution at the Upper Devonian-Lower Carboniferous to its structuration during the variscan-alleghanian orogeny at the Upper Carboniferous-Lower Permian. To address this issue, this work is organized around two approaches: (1) one metrological applied to the Raman Spectroscopy of Carbonaceous Matterial (RSCM) and (2) the other integrating a structural and geochronological study and a thermicity analysis.The metrological approach allowed to validate the applicability of the RSCM geothermometer (1) in a context of polyphase metamorphism, (2) for carbonate rocks and skarns of Jebilet and (3) to propose a new parameter Raman RSA allowing to better specify temperatures above 500°C and extend the applicability of the method to maximum temperatures of up to 700°C.From the integrated approach, three tectono-thermal episodes were highlighted. The first episode D₀, corresponds to an extensive tectonic allowing the opening of the Jebilet basin at the Upper Devonian-Lower Carboniferous. This opening is accompanied by a HT thermal anomaly as shown by the important bimodal and granodioritic magmatic activity dated between 358 ± 7 Ma and 336 ± 4 Ma and the TRSCM higher than 500°C recorded by the rocks. During Upper Carboniferous, the compressive phase structuring the Jebilet massif begins with the emplacement of superficial nappes (D₁), followed by the variscan major phase (D2). Structural analysis showed a gradual evolution of D₂ deformation regime from coaxial compression to dextral transpression consistent with WNW-ESE to NW-SE horizontal shortening. D₂ is associated with two thermal events, the first is syn-tectonic with TRSCM between 300 and 400°C, and the second is syn- to post-tectonic with TRSCM between 600 and 660°C.This tectono-thermal context would be the expression of geodynamic processes involving from the Upper Devonian a delamination of the Rheic lithosphere by "slab break-off" or "slab roll-back" which would induce (1) the rise of hot asthenospheric current, and (2) the clockwise rotation of Gondwana and its gradual amalgamation with Laurussia structuring the variscan-alleghanian belt during the Upper Carboniferous-Lower Permian

Les cordillères Bético-Rifaines sont situées en Méditerranée occidentale, où elles forment un arc étroit. Comparée à d’autres segments orogéniques, leur évolution géodynamique reste extrêmement discutée en raison d’une histoire tectono-métamorphique polyphasée depuis le rifting Mésozoïque, jusqu’à l’histoire alpine incluant subduction,collision et effondrement tardi-orogénique. Cette thèse s’est focalisée sur deux problématiques restant activement débattues et qui concernent le complexe Alpujárride, appartenant aux Zones Internes (ZI) des Cordillères Bétiques.Deux zones d’étude ont été ciblées, avec (i) les massifs péridotitiques affleurant à l’ouest et dont les modalités de mise en place restent énigmatiques et (ii) les unités tectoniques affleurant à l’est et dans lesquelles les paragenèses de haute pression-basse température (HP/BT) en lien avec l’épisode de subduction alpine, sont les mieux préservées mais dont les contraintes temporelles sont largement incertaines.Ce travail de recherche s’est attaché à utiliser une approche multi-échelles, depuis celle de l’affleurement jusqu’à celle de la région, et pluridisciplinaire, avec un travail de cartographie détaillée, des observations structurales et pétrographiques, de la thermométrie Raman et des datations 40Ar/39Ar. Nos résultats mettent en évidence(i) l’exhumation du massif péridotitique de Ronda dans un contexte d’hyper-amincissement de la croûte continentale résultant d’un épisode de rifting et (ii) la fin de l’épisode métamorphique de HP/BT vers 38 Ma avec l’initiation du retrait du panneau plongeant et l’ouverture d’un bassin d’arrière-arc, associée à un métamorphisme de haute température-basse pression (HT/BP). C’est dans ce contexte extensif que le complexe Alpujárride s’exhume après une phase majeure d’amincissement crustal. Cet épisode de métamorphisme de HT/BP prend fin avec la mise en place des ZI des Cordillères Bético-Rifaines sur les marges Ibérie et Afrique, vers 20 Ma<br>The Betic-Rif Cordillera, located in the western Mediterranean region, forms a narrow, arcuate orogenic belt. By comparison with other orogenic belts in the Mediterranean realm, its geodynamic evolution is higly controversial because of a long and complex tectono-metamorphic history, including the Mesozoic rifting and the Alpineorogenesis where subduction, collisional and post-orogenic extensional events are successively recorded. This Ph.D.thesis aims to address two major issues about the geodynamic evolution of the Alpujárride Complex, a group of metamorphic units that belong to the Internal Zones of the Betic Cordillera. Two study areas have been investigated with (i) the peridotitic massifs located in the western part of this complex whose mechanisms and timing of exhumation remain unclear and (ii) the tectonic units located further east that display well-preserved high pressure low temperature (HP/LT) mineral parageneses related to the Alpine subduction episode but with high uncertainties regarding the age of this metamorphic event.This work has been carried out by following a multi-scale and multi-disciplinary approach, from the outcrop- to the regional-scale, including detailed field mapping, structural measurements, petrographic observations, Ramanthermometry and 40Ar/39Ar dating. Our results evidence (i) the exhumation of the Ronda peridotite controlled by anhyper-extension event associated with a rifting stage and (ii) the end of the HP/LT peak conditions around 38 Ma,juste before the inception of the slab roll-back and back-arc opening associated with a high temperature-lowpressure (HT-LP) metamorphic overprint. The Alpujárride Complex is exhumed during this stage, while the crust ishigly stretched. This HT-LP metamorphic event ended with the thrusting of the Internal Zones of the Betic-RifCordillera onto the Iberian and African margins, around 20 Ma ago

碩士<br>國立陽明大學<br>遺傳學研究所<br>92<br>RSC complex, an essential chromatin-remodeling complex in Saccharomyces cerevisiae, performs central roles in transcriptional regulation and cell cycle progression. RSC complex has been found to interact with Htl1p. To study the relationship between Htl1p and RSC complex, a series of analysis, including ion exchange column chromatography, sucrose gradient assay, multiple copy suppressor and partial protease, were performed in this thesis. By ion exchange column chromatography and sucrose gradient assay, the eluted protein and the distribution of subunits of RSC complex with or without DNase treatment were different between htl1 mutant and wild type cells. According to the analysis of sucrose gradient distribution in wild type and htl1 deletion strains at 37℃, we found Htl1p may play a role in maintaining stability of RSC complex, especially in the interaction between Rsc8p and Sth1p. Therefore, the instability of RSC complexes might be the main reason that causes temperature sensitivity. To further characterize if the interaction of Sth1p and Rsc8p is crucial for the temperature sensitive phenotype. A fusion protein experiment was designed. We constructed STH1-LINKER-RSC8 (SLR) fusion gene and inserted it into the strain without HTL1. So far that Ura+ spore can grow at 30℃ and 37℃. Partial protease digestion demonstrated that Rsc8p exhibits endopeptidase Glu-C sensitivity in htl1 deletion mutant. In order to further characterize HTL1 gene, we screened suppressors of Benomyl sensitivity of htl1 deletion strain by multicopy suppressor scheme. However, no suppressor of htl1 deletion strain was obtained.

碩士<br>國立陽明大學<br>生命科學系暨基因體科學研究所<br>105<br>Eukaryotes evolved a specific strategy for DNA packaging. A repeating unit, nucleosome, consists of 147 base pairs of DNA segment wrapped histone core proteins. Nucleosome plays an important role in the regulation of DNA-dependent biosynthesis and gene expression by its structure. Chromatin structure remodeling complex (RSC, Remodel the Structure of Chromatin) is essential for the regulation of nucleosome structure in vivo. Previous researches indicated that RSC complex can hydrolyze ATP to perturb nucleosome structures resulting in nucleosome sliding and histone octamer ejection. However, the mechanisms and the transition state of RSC-mediated nucleosome remodeling are still unclear. Here we utilize tether particle motion (TPM) experiments to investigate the RSC-mediated nucleosome remodeling process in detail. RSC has been known to play an important role in DNA-dependent biosynthesis pathways. Therefore, we study the interplay between RSC and other factors, acceptor DNA and nucleosome assembly protein (Nap1). By using TPM experiments, the histone octamer ejection was observed during RSC-mediated nucleosome remodeling. Moreover, the high percentage of the RSC-mediated histone octamer ejection was observed under saturated ATP and in the presence of acceptor DNA. In contrast, the ratio of RSC-mediated histone octamer ejection decreased in the presence of Nap1. In this project, the whole reaction scheme of RSC-mediated nucleosome remodeling process, including nucleosome sliding and histone octamer ejection, can be depicted based on TPM results.

碩士<br>國立陽明大學<br>遺傳學研究所<br>90<br>HTL1 is essential for high temperature growth in yeast. By yeast two hybrid screening, Rsc8p was identified to have physical interaction with Htl1p. By the synthetic lethal scheme, STH1、RSC30 and RSC58 were found. By multi-copy suppression, RSC3 was able to rescue the temperature-sensitive lethalits of htl1 deletion mutant. Rsc8p、Sth1p、Rsc58p、Rsc30p and Rsc3p are members of RSC complex, mediating chromatin remodeling. Reports have demonstrated that specific sth1-ts and rsc8 strains exhibit G2/M cell cycle arrest and diversed cellular morphologies at non-permissive temperature. Similar phenotype was also observed in the htl1 deletion strain. To study the relationship between Htl1p and RSC complex , a series of analysis, including in vitro binding assay, coimmunoprecipitation analysis, sucrose gradient fractionation analysis and the statistical analysis of cell morphology were performed. Statistical analysis of cell morphology demonstrated that the size of htl1 deletion strain is significantly larger than that of the wildtype strain. By in vitro binding assay to further dissect the interaction region of Rsc8p, the region between a.a. 206 to a.a.356 of Rsc8p was defined to interact with Htl1p. This region contains a ZZ-zinc finger domain and a Myb-DNA binding domain. Furthermore, coimmunoprecipitation analysis showed that Htl1p was co-precipitated with Rsc8p. To examine the intactness of RSC complex in htl1 deletion mutant, sucrose gradient analysis was performed using Sth1p and Rsc8p as indicators. In wild-type yeast cells, Rsc8p and Sth1p were cofractionated in the high molecular weight fractions. However, Rsc8p also appeared in the low molecular weight fractions in which Sth1p was absent. The distribution of Htl1p in sucrose gradient was also examined. Three peaks were observed, one in the low molecular weight fractions and the second in the high molecular weight fractions similar to that of Rsc8p. However, the third peak appeared in fractions with ever higher molecular weight, indicating that Htl1p might possesses different cellular functions. In htl1 deletion strains, unlike Sth1p, the distribution of rsc8p was altered. The high molecular weight peak diminished. Our results suggest that by interacting with Rsc8p, Htl1p may play a role in the formation of stable RSC complex.

碩士<br>國立陽明大學<br>遺傳學研究所<br>91<br>Yeast Htl1p was found to be functionally related to RSC complex by genetic analysis. In addition to the association of Htl1p and Rsc8p, other components may also interact with Htl1p. Using HA tagged strain; we show that Sth1p and Rsc8p can interact with Htl1p by co-immunoprecipitation. By immunofluorescent staining, Htl1p was found in the nucleus. In recent report, STH1 and RSC8 have been shown to be required for proper transcriptional regulation and nucleosome positioning in CHA1 promoter. Here we show that the htl1 disruption strain prolonged the repression state in the absence of serine. In the presence of serine, the de-repression of the CHA1 expression is similar to that of wild-type yeast. The observation that htl1 disruption strain was demonstrated to be sensitive to calcofluor white dye which binds to chitin of the cell wall and the temperature sensitive phenotype of htl1 mutant can be partially rescued by sorbitol suggests that HTL1 involved in the mutation of the cell wall integrity.

碩士<br>國立陽明大學<br>生命科學暨基因體科學研究所<br>96<br>The interaction of proteins with DNA packaged into chromatin may be blocked by the nucleosome arrays and the higher order structure of chromatin. To solve this problem, in yeast Sacchromyces cerevisiae, ATP-dependent chromatin remodeling complex, such as SWI/SNF and its homolog RSC, have been proven to be involved in the perturbation of nucleosome structure. RSC8 encodes a polypeptide of 558 amino acids residues and is 30% identical and 52% similar to that of Swi3p in the SWI/SNF complex. Unlike Swi3p, Rsc8p is essential for cell viability. The SWIRM domain of Swi3p has been shown to be a DNA- and possibly a protein-binding domain. To investigate if SWIRM domain of Rsc8 could bind DNA and affects complex integrity, the influence of SWIRM domain on DNA-binding property of Rsc8 and the RSC complex integrity are evaluated , using the site-directed mutagenesis to generate substitution mutations on Rsc8p A158 and K162 relative to Swi3p K383 and K387 on the SWIRM domain. To examine if the substitution mutations on A158 and K162 of Rsc8p affect its function, the phenotypes of the Rsc8p mutants are examined and were compared with that of the wild type cells. The results demonstrated that, (1) A158D, A158K, K162A, K162D and the double mutants on A158/K162 showed no significant growth defect, (2) the chromatin association have not changed and (3) the RSC complex integrity are not affected.

碩士<br>國立陽明大學<br>遺傳學研究所<br>94<br>The RSC complex, which consists of at least fifteen proteins, alters the histone-DNA interactions and nucleosome mobility by ATP hydrolysis, allowing transcription factors accessing to chromatin for a broad range of gene regulation in yeast genome. Htl1p is a component of RSC complex. In the absence of Htl1p, the integrity of RSC complex is disturbed. To better understand the role of Htl1p in the RSC complex, here we examine the components of RSC complex by tandem affinity purification and compare the differences of RSC components purified from RSC2-TAP, which chromosome RSC2 gene was fused with a TAP-tag at the c-terminus and its htl1 deletion derivative RH2-TAP. We observed that the Rsc58p seems to be unstable in RH2-TAP. Moreover, in the presence of Htl1p mutant protein (Htl1-7), the protein profile is different from that of RSC2-TAP and RH2-TAP. By immunoblot analysis, we observed that the relative quantity of Rsc58p and Rsc8p in the presence of Htl1-7 protein is decreased in the purified protein profile. Htl1p and Rsc58p associated proteins were also purified by tandem affinity purification. They are essential RSC components. However, in comparison of the protein profiles purified by the rsc8 mutants derived from RSC2-TAP and RH2-TAP, there’s no difference among them. Our preliminary data showed that deletion of htl1 affects the interaction between Rsc8p and RSC complex. To further understand the interaction between Rsc8p and RSC complex, we design a fusion protein SLR, using a linker containing the GGMP repeats of Hsc70 to connect Sth1p and Rsc8p. Yeast cells contain SLR with deletion of htl1 failed to survive. But SLR could rescue the lethal phenotype caused by down-regulation of RSC8 expression in tet-off system, indicating that SLR may replace the function of both RSC8 and STH1. Overall speaking, above results further confirm that Htl1p may be crucial for the stability of RSC complex.

博士<br>國立陽明大學<br>生命科學暨基因體科學研究所<br>100<br>The Saccharomyces cerevisiae RSC (remodel the structure of chromatin) complex is a chromatin remodeler that hydrolyzes ATP as energy to mediate the chromatin structure. RSC plays a role in the cellular function of transcriptional regulation, sister chromatids cohesion, DNA damage repair and cell wall integrity. The HTL1 (high temperature lethal) gene encodes a nonessential subunit of the RSC complex. Deletion of HTL1 causes increased ploidy at the permissive temperature, and the ∆htl1 mutant accumulates in the cell cycle at the G2/M phase at restrictive temperatures. In this thesis, we investigated the cellular function of the HTL1 gene. The results are described in three chapters. In chapter 1, we explored the defect in cell wall integrity in the ∆htl1 mutant. The HTL1 gene has been shown to genetically interact with the PKC1 pathway, the signaling pathway important for the biogenesis and stress response of yeast cell wall; however, a characteristic defect in cell wall integrity has not been explored in the ∆htl1 single mutants. We show that the ∆htl1 mutant is sensitive to agents that cause cell wall stress; in addition, various phenotypes of htl1 cells, including a defect in cell wall integrity and the mitotic arrest are rescued by the presence of osmotic stabilizer such as sorbitol. We also show that the aberrant structure of the RSC complex in the ∆htl1 mutant is not consistent with the increasing severity of the phenotypes of ∆htl1 cells as the temperature increases. In chapter 2, we explored the mechanism of mitotic arrest in the ∆htl1 mutant. The results show that the mitotic arrest in ∆htl1 cells is dependent on the spindle assembly checkpoint; in addition, the mitotic arrest in the ∆htl1 mutant is partially caused by the defect in the cell wall integrity. In chapter 3, we explored the regulation of CHA1 gene, which encodes the catabolic L-serine deaminase. A previous study showed that the transcriptional expression of CHA1 gene was de-repressed when Rsc8p and Sth1p, two major RSC components, had been depleted. However, our results show that CHA1 is persistently repressed in the ∆htl1 mutant during prolonged incubation under the repressive condition. Furthermore, the RSC complex associates with the CHA1 locus whether serine is present or not, and the association between the RSC complex and the CHA1 locus is enhanced in ∆htl1 cells. Taken together, our present findings suggest that HTL1 may play a role different from that of other RSC components in terms of cell wall integrity and the G2/M transition. The results also suggest that the defects in cell wall integrity and the G2/M transition of the ∆htl1 mutant are interconnected. Moreover, the functional and structural effects of the ∆htl1 mutant on the RSC complex do not seem to be coordinated.