No description available.

<p>Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCF<super>Cdc4</super> ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that in the budding yeast,<italic> Saccharomyces cerevisiae</italic>, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APC<super>Cdh1</super>). I have determined, however, that the Kip1 and Cin8 proteins are actually present at wild-type levels in yeast in the absence of Clb/Cdc28 kinase activity. Here, I show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28, and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's <italic>in vivo</italic> localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, I have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCF<super>Cdc4</super> target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, I propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors. </p><p>In addition to the positive regulation of kinesin-5 function in spindle assembly, I have also found evidence that suggests CDK phosphorylation of kinesin-5 motors at different sites negatively regulates kinesin-5 activity to prevent premature spindle pole separation. I have also begun to characterize a novel putative role for the kinesins-5 in mitochondrial genome inheritance in <italic>S. cerevisiae</italic> that may also be regulated by CDK phosphorylation. </p><p>In the course of my dissertation research, I encountered problems with several established molecular biology tools used by yeast researchers that I have tried to address. I have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in <italic>S. cerevisiae</italic> that can be propagated in the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes <italic>HIS2</italic> and <italic>ADE1</italic>, respectively. My new pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed in order to increase the availability of unique restriction sites within their polylinker regions. Hence, my pRSII plasmids are a complete set of integrating, centromere and 2&#61549; episomal plasmids with the biosynthetic marker genes <italic>ADE2</italic>, <italic>HIS3</italic>, <italic>TRP1</italic>, <italic>LEU2</italic>, <italic>URA3</italic>, <italic>HIS2</italic> and <italic>ADE1</italic> and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, I have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin and bialaphos. Finally, in the process of generating the new plasmids, I have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using updated plasmid sequences, I constructed pRS plasmid backbones with a unique restriction site for inserting new markers in order to facilitate future expansion of the pRS/pRSII series.</p> / Dissertation

No description available.

CDC73, also known as HRPT2, is a tumour suppressor gene whose expression is lost or downregulated in parathyroid, renal, breast, uterine and gastric cancers. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. As part of the Paf1 complex, it remains associated with ribonucleic acid (RNA) polymerase II and is involved in transcript site selection, transcriptional elongation, histone H2B ubiquitination, histone H3 methylation, poly(A) length control and, coupling of transcriptional and posttranscriptional events. It has been reported to negatively regulate cellularproliferation by targeting oncogenes CCND1 (cyclin D1) and MYC (c-Myc). Moreover, it has also been indicated to inhibitβ-catenin-mediated transcription. Taken together, these findings strongly suggest that it contributes to the expression of genes whose products have an important role in the suppression of tumor development and cell death. In this study, we have attempted to study the transcriptional and posttranscriptional regulation of CDC73 and its role in OSCC. The main findings of the present study are listed below. 1. To begin with, the expression analysis of CDC73 was performed both at the RNA and the protein levels by qRT-PCR and IHC, respectively. As expected, a majority of the OSCC samples showed downregulation of CDC73 both at the RNA and the protein levels compared to their normal oral tissues. 2. Loss-of-heterozygosity (LOH), mutation and promoter methylation are the hallmarks of a tumor suppressor gene (TSG). Therefore, to characterize CDC73 as a TSG in OSCC and to look into the mechanisms that could be the cause of CDC73 downregulation in OSCC, LOH, mutation and promoter methylation of CDC73 were studied. The results showed that LOH, mutation and promoter methylation are not the major causes of CDC73 downregulation in OSCC. 3. To identify the alternate mechanisms as the cause of CDC73 downregulation in OSCC, a combination of bioinformatics and molecular approaches were used. The results showed that the upregulation of an inhibitory transcription factor WT1 (Wilms tumor protein 1) and an oncogenic microRNA-155 are the major causes of its downregulation in OSCC. 4. The luciferase reporter assay of SCC131 cells co-transfected with a WT1 construct and a CDC73 promoter construct showed that WT1 over expression represses CDC73 expression in a dose-dependent manner. 5. Due to the presence of zinc fingers in its C-terminal half, WT1 has been found to be a potent transcriptional regulator of genes. Therefore, to determine if WT1 down regulates CDC73 via binding its promoter, the chromatin immunoprecipitation (ChIP) assay was performed. The results showed the binding of WT1 to the CDC73 promoter in vivo. Binding of WT1 to the CDC73 promoter was further confirmed in vitro by the electrophoretic mobility shift assay (EMSA). 6. The 5-aza-2’-deoxycytidine (AZA) treatment of SCC131 cells led to upregulation of WT1 with a concomitant downregulation of CDC73. The COBRA technique demonstrated that the upregulation of WT1 upon the 5-AZA treatment was due to its promoter methylation. 7. To determine if the WT1-mediated reduction of CDC73 expression has a functional relevance in cell growth and proliferation, we knocked down CDC73 expression by transient over expression of WT1 in SCC131 cells and quantitated cell proliferation by the MTT assay. As expected, the results demonstrated that the reduced CDC73 level was associated with an increased cell proliferation. Cotransfection of CDC73 with WT1 in SCC131 cells attenuated the pro-oncogenic effect of WT1 by apoptosis induction. 8. After validating CDC73 as the target of WT1 by bioinformatics and in vitro assays, we quantitated the expression levels of WT1 and CDC73 by qRT-PCR in OSCC samples and their matched normal oral tissue samples. The results showed an inverse correlation between the expression levels of WT1 and CDC73 in a majority of the samples. To exclude the possibility of alternate mechanisms as the cause of CDC73 downregulation in OSCC, we selected a subset of OSCC samples with downregulated level of CDC73 and analysed them for LOH at the CDC73 locus and promoter methylation. Further, some of these OSCC samples were also analyzed for mutations in CDC73. The results showed that these OSCC samples did not have LOH, promoter methylation or any mutation, again validating the fact that CDC73 is a biological target of oncogenic WT1, and the transcriptional repression of CDC73 by WT1 could be a major mechanism for CDC73 downregulation in OSCC. 9. Recent studies have shown that a growing class of noncoding RNAs called microRNAs (miRNAs) is involved in posttranscriptional regulation of genes. There is a growing body of literature supporting the potential role of miRNAs in tumorigenesis. The importance of CDC73 in orchestration of several cellular functions and its role in tumorigenesis make it an attractive candidate for miRNA-mediated regulation of cell growth and proliferation. Using bioinformatics approaches, we identified an oncogenic microRNA-155 (miR-155) that could posttranscriptionally regulate CDC73 expression. 10. Consistent with its oncogenic role, miR-155 was found dramatically upregulated in OSCC samples and was found to be another mechanism for downregulation of CDC73 in a panel of human cell lines and a subset of OSCC samples in the absence of LOH, mutations and promoter methylation. 11. miRNAs regulate posttranscriptional gene expression generally via binding to their cognate sites in the 3’UTR. Therefore, a luciferase reporter construct was made by cloning the 3’UTR of CDC73 downstream to the luciferase reporter gene and the reporter assay was performed. Our experiments clearly indicated that the mature miR-155 regulates CDC73 expression by interacting with its 3’UTR in a site specific manner. 12 Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells over expression miR-155 resulted in increased CDC73 level, decreased cell viability, increased apoptosis and marked regression of engrafts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3’UTR in CDC73. In nutshell, the knockdown of CDC73 expression due to over expression of WT1 and miR-155 not only adds a novelty to the list of mechanisms responsible for its downregulation in different tumors, but the restoration of CDC73 levels by the use of inhibitors to WT1 and antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.