Browsing by Subject "cell cycle"
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Item Regulation of DNA Replication During Conventional and Unconventional Cell Cycles in Tetrahymena(2014-02-06) Lee, Po-HsuenAs the nucleating protein for pre-replicative complex (pre-RC) assembly, the conserved Origin Recognition Complex (ORC) specifies where replication initiates in eukaryotic chromosomes. During the vegetative cell cycle of Tetrahymena thermophila, previously published work has shown that DNA replication initiates from defined chromosomal sites in an ORC-dependent manner. Tetrahymena exhibits nuclear dimorphism, a polyploid somatic macronucleus (MAC), which is transcriptionally active and maintains vegetative growth, and a diploid germline micronucleus (MIC) responsible for the transmission of genetic information during conjugation. In order to provide more information about the fundamental mechanisms of micro- and macro- nuclear replication programs, I study the impacts of changing in ORC protein contents on the fate of micro- and macro- nuclear chromosomes during the vegetative cell cycle and development in Tetrahymena. I examined the effect of down-regulation of ORC1 on genome stability and intra- S phase checkpoint activation by disrupting ORC1 gene in the macronucleus. Partial depletion of Orc1p leads to genome instability in the diploid mitotic micronucleus, abnormal division of the polyploid amitotic macronucleus, and failure to mount a robust intra-S phase checkpoint response. In addition, the ORC1 knockdown strain fails to execute two developmentally- regulated DNA replication programs, endoreplication and ribosomal DNA (rDNA) gene amplification. I also examined the regulation of ORC and MCM during development. Remarkably, the result suggests that the demand on the ORC-dependent replication machinery differs during development and the vegetative S phase. To further gain new insights into fundamental mechanisms that protect chromosomes from replication stress, I examined the impact of replication stress on the regulation of ORC and MCM. This study led to the discovery of a novel DNA replication program that is activated under HU treatment. While Orc1p and Mcm6p were selectively degraded in response to HU, cells were competent to complete S phase in the absence of Orc1p and Mcm6p after HU was removed. In addition, the rDNA origin used exclusively during the S phase of vegetative cell cycle and developmentally programmed gene amplification is suppressed when these replication proteins are selectively degraded under HU treatment. Instead, an alternative program was used to resume the cell cycle progression. These data provide compelling evidence for an ORC-independent DNA replication program in cells recovering from replication stress.Item Regulation of initiation of division in Saccharomyces cerevisiae: characterization of the role of DCR2, GID8, and KEM1 in completion of START(Texas A&M University, 2007-04-25) Pathak, RituThe decision to initiate division is very important, as once cells have initiated division they are committed to complete it. In Saccharomyces cerevisiae, commitment to a new round of cell division occurs at a regulatory point in late G1 called START. Progression through START requires the activation of the cyclin dependent kinase Cdc28p by the G1 cyclins. G1 cyclins in complex with Cdc28p activate the transcription of approximately 100 genes involved in the G1 to S transition and degradation of Sic1p, an inhibitor of B type cyclins, and thus are important for initiation of DNA replication. Despite the widely studied role of regulatory cyclins and cyclin dependent kinase in the G1 to S transition, how cells determine when to initiate DNA replication is poorly understood. We have identified several gene products, which when overexpressed, cause cells to initiate DNA replication faster than wild type. Here we discuss the role of DCR2 (Dosage dependent Cell cycle Regulator), GID8 (Glucose Induced Degradation) and KEM1 (Kar-Enhancing Mutation) in the regulation of START. Over expression of DCR2 and GID8 accelerates initiation of DNA replication. Cells lacking both these genes delay initiation of DNA replication. Genetic analysis suggests that Gid8p functions upstream of Dcr2p to promote START. Further, we show that DCR2, which codes for a metallo-phosphoesterase, might regulate completion of START by affecting degradation of Sic1p. Over expression of DCR2 lowers the half-life of Sic1p without altering the expression of Cln2p. The evidence suggests that Dcr2p affects START completion through dephosphorylation of Sic1p. KEM1 is a Saccharomyces cerevisiae gene, conserved in all eukaryotes, which codes for a 5??????-3?????? cytoplasmic exonuclease. This exonuclease is involved in exiting mitosis, by degrading the mRNA of the mitotic cyclin CLB2. Besides its role in mitotic exit, an enzymatically inactive version of Kem1p can accelerate the G1 to S transition and initiation of DNA replication when over expressed. This result suggests that Kem1p might have a previously unrecognized role in the G1 to S transition independent of its exonuclease activity, and supports the notion that Kem1p is a multifunctional protein with distinct and separable roles.Item The role of 4-hydroxynonenal in cellular signaling mechanisms(2007-04-03) Brad Allen Patrick; Yogesh C. Awasthi, Ph.D.; Xiaodong Cheng, Ph.D.; Piotr Zimniak, Ph.D.; Naseem Ansari, Ph.D.; Cornelis ElferinkOne of the critical steps involved in cellular oxidative stress is the peroxidation of membrane lipids. The downstream effects of the autocatalytic lipid peroxidation (LPO) cycle are widespread and involve several processes. Using lens epithelial cells (HLE B-3) and retinal pigment epithelial cells (RPE28 SV4) as models, in proposed studies we addressed the hypothesis that a physiological level of LPO product 4-hydroxynonenal (HNE) is necessary for maintaining key proliferation, adhesion, and survival signaling by regulating expression of genes involved in these pathways, and that alteration of that level either up or down will result in induction of genes pro-apoptotic or pro-carcinogenic signaling pathways, respectively.\r\n After stable transfection of hGSTA4 in HLE B-3, cells underwent a morphological transformation becoming smaller and rounded, and lost anchorage dependence. Additionally they were observed to divide much more rapidly and acquired resistance to apoptosis via oxidative stress. In an attempt to provide a mechanistic explanation for this phenotypic set of changes, cells were assayed for changes in expression for a wide set of genes via gene microarray studies. Results showed that over 6900 genes were strongly modulated in hGSTA4-transformed cells compared to controls. This prompted further verification in a subset of well-known and important genes involved in cell cycle control, survival, and cell adhesion via quantitative RT-PCR and Western blot studies. In these cells, considerably lower levels of transcription and translation of key genes regulating those affected processes were observed. \r\n Further exploration of HLE B-3 showed that expression of Fas correlated with HNE concentration, an observation found both before and after HNE treatment. This is strengthened by observations that transiently-transfected HLE B-3 overexpress hGSTA4 along with a depletion of intracellular HNE and depletion of Fas expression and further strengthened in a mouse mGSTA4¬ knockout model with high steady-state HNE tissue levels, where Fas expression is found to be elevated in several tissues above WT. Together these studies implicate HNE as an important mediator of expression of several key genes responsible for processes underlying cell cycle regulation, survival, and adhesion, and lay a foundation upon which further investigation can be performed.Item Zinc ion homeostasis in cellular physiology and experimental models of traumatic brain injury(2009-03-06) Yuan Li; Jonathan B. Ward; Wolfgang Maret; Robert A. Colvin; Ping Wu; Karl E. Anderson; Douglas S. DeWittA major yet unsolved quest in treating traumatic brain injury (TBI) is the understanding of the secondary cellular injury that contributes to cell death. Whether zinc ions are toxic or protective in TBI is controversial. As an essential human micronutrient, zinc is needed for the structure and function of at least 3,000 proteins, and thus affects almost any aspect of cellular function. Although extremely low, intracellular zinc ion concentrations, [Zn2+]i, are tightly controlled to ensure optimal physiology and to avoid toxicity. Furthermore, zinc ions are now believed to be signaling ions, especially in neuronal systems. This dissertation addresses the dynamics of [Zn2+]i and quantitatively defines its safe range in particular cell types. [Zn2+]i was measured to be pico- to nanomolar in undifferentiated and differentiated rat pheochromocytoma (PC12) cells and in rat glioma (C6) cells. When PC12 cells proliferate, [Zn2+]i undergoes precisely controlled fluctuations with two peaks within one cell cycle. These results demonstrate that the already established requirement for zinc in the cell cycle and in differentiation relates to the availability of zinc ions. In a mechanical model of cellular injury, namely rapid stretch injury (RSI), nitric oxide induces an increase in [Zn2+]i that subsequently may protect cells by repressing the generation of ROS. A peak at one hour was followed by decreased [Zn2+]i. In PC12 cells, [Zn2+]i dropped below its normal level, indicating that these cells were in a state of ¡°zinc ion deficiency¡± hours after RSI. In an in vivo model of neural injury, namely fluid percussion TBI of rats, changes of [Zn2+]i were indirectly demonstrated by measuring the levels and states of the zinc-binding protein, metallothionein/thionein, in the hippocampus and the cortex. These results demonstrate that [Zn2+]i as well as zinc buffering dynamically fluctuate to adapt to the requirements of cellular functions, even when [Zn2+]i is extremely low inside the cell. They suggest that toxicity occurs when [Zn2+]i falls outside the safety thresholds. Therefore, when, where, how much and in which form zinc is present determine whether chelation or supplementation is an option for treatment. These new concepts provide new leads for developing strategies to treat TBI.