Siu Yau Lung, Philip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 99-119). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.iv / Declaration --- p.v / Scientific publication --- p.vi / Abbreviations --- p.vii / Genetic symbols --- p.ix / Table of contents --- p.x / List of tables --- p.xiv / List of figures --- p.xv / Chapter Chapter One --- Introduction / Chapter 1.1 --- Thesis outline --- p.1 / Chapter 1.2 --- Candida albicans --- p.2 / Chapter 1.3 --- Physical characterization of C. albicans --- p.3 / Chapter 1.3.1 --- Strain identification --- p.3 / Chapter 1.3.2 --- Dimorphism --- p.5 / Chapter 1.3.3 --- Genome of C. albicans --- p.10 / Chapter 1.3.4 --- Karyotype --- p.11 / Chapter 1.4 --- Candidiasis --- p.12 / Chapter 1.4.1 --- Superficial candidiasis --- p.15 / Chapter 1.4.2 --- Systemic candidiasis --- p.16 / Chapter 1.4.3 --- Virulence --- p.16 / Chapter 1.4.4 --- Multi-drug resistance --- p.17 / Chapter 1.5 --- Parasexual genetics --- p.20 / Chapter 1.5.1 --- Mutant isolation --- p.20 / Chapter 1.5.2 --- Spheroplasts complementation --- p.21 / Chapter 1.5.3 --- Mitotic complementation --- p.22 / Chapter 1.6 --- Natural heterozygosity in C. albicans --- p.22 / Chapter 1.7 --- Adenine biosynthesis --- p.26 / Chapter 1.7.1 --- de novo pathway --- p.26 / Chapter 1.7.2 --- Salvage pathway --- p.29 / Chapter 1.7.3 --- Importance of C. albicans ADE1 and ADE2 genes --- p.29 / Chapter 1.8 --- Aim of study --- p.30 / Chapter Chapter Two --- Construction of disrupted C. albicans ADE1 gene / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and Methods --- p.34 / Chapter 2.2.1 --- Strains --- p.34 / Chapter 2.2.2 --- Construction of plasmid pGEMTE-ADEl --- p.34 / Chapter 2.2.2.1 --- Isolation of Candida genomic DNA --- p.34 / Chapter 2.2.2.2 --- Isolation of C. albicans ADE1 gene from CAM --- p.36 / Chapter 2.2.2.2.1 --- Amplification of C. albicans ADE1 gene --- p.36 / Chapter 2.2.2.2.2 --- Purification of PCR product --- p.37 / Chapter 2.2.2.3 --- Cloning of ADEl gene into pGEMT-Easy vector --- p.38 / Chapter 2.2.2.3.1 --- Cloning vector pGEMT-Easy --- p.38 / Chapter 2.2.2.3.2 --- Ligation --- p.38 / Chapter 2.2.2.4 --- Transformation of E. coli DH5a cells --- p.39 / Chapter 2.2.2.4.1 --- Preparation of competent E. coli DH5a cells --- p.39 / Chapter 2.2.2.4.2 --- Plasmid DNA transformation --- p.40 / Chapter 2.2.2.4.3 --- Isolation ofplasmid DNA from E. coli --- p.40 / Chapter 2.2.3 --- Construction of pGEMTE-ADElA-URA3 --- p.41 / Chapter 2.2.3.1 --- Isolation of C. albicans URA3 gene from plasmid pCUB-6 --- p.41 / Chapter 2.2.3.2 --- Preparation of cloning vector pGEMTE-ADE 1Δ --- p.42 / Chapter 2.2.3.2.1 --- PCR amplification of vector pGEMTE-ADElΔ --- p.42 / Chapter 2.2.3.2.2 --- Modification of PCR vector pGEMTE-ADElΔ --- p.44 / Chapter 2.2.3.2.3 --- Dephosphorylation --- p.45 / Chapter 2.2.3.3 --- Cloning and isolation of plasmid pGEMTE-ADE1Δ-URA3 --- p.46 / Chapter 2.3 --- Results and Discussion --- p.47 / Chapter Chapter Three --- Gene disruption of C. albicans CAI4 by electroporation / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.54 / Chapter 3.2.1 --- Strains --- p.54 / Chapter 3.2.2 --- Transforming DNA --- p.54 / Chapter 3.2.3 --- Purification of PCR product --- p.55 / Chapter 3.2.4 --- DNA transformation --- p.55 / Chapter 3.2.5 --- Transformation efficiency --- p.56 / Chapter 3.2.5.1 --- Pulse length --- p.56 / Chapter 3.2.5.2 --- Amount of DNA --- p.57 / Chapter 3.2.6 --- Southern analysis of transformants --- p.57 / Chapter 3.2.6.1 --- Isolation of Candida genomic DNA --- p.57 / Chapter 3.2.6.2 --- Preparation of Candida genomic DNA for Southern analysis --- p.57 / Chapter 3.2.6.3 --- Southern hybridization --- p.58 / Chapter 3.2.6.4 --- Preparation of radioactive probe --- p.60 / Chapter 3.2.6.5 --- Radioactive labelling of the probe --- p.61 / Chapter 3.2.6.6 --- Hybridization of nylon membrane --- p.62 / Chapter 3.2.6.7 --- Stringency washes --- p.62 / Chapter 3.2.6.8 --- Auto-radiography --- p.62 / Chapter 3.3 --- Results and Discussion --- p.64 / Chapter Chapter Four --- UV mutagenesis of disrupted C. albicans / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Materials and Methods --- p.76 / Chapter 4.2.1 --- Strains --- p.76 / Chapter 4.2.2 --- Generation of recombinants by UV irradiation --- p.76 / Chapter 4.2.3 --- Analyses of twin-sectored colonies --- p.77 / Chapter 4.2.3.1 --- Replica analyses of twin-sectored colonies --- p.77 / Chapter 4.2.3.2 --- Southern analysis of segregants --- p.77 / Chapter 4.3 --- Results and Discussion --- p.78 / Chapter Chapter Five --- Concluding remarks and perspectives --- p.96 / Bibliography --- p.99

Asbesto é um nome geral dado a seis tipos de fibras minerais encontradas naturalmente na crosta terrestre. Estas fibras vêm sendo exploradas industrialmente desde 1970, porém diversos trabalhadores expostos às fibras apresentaram patologias no trato respiratório, como fibroses e carcinomas. Alguns tipos de fibra foram banidos do mercado, porém o tipo de asbesto crisotila ainda pode ser comercializado na maioria dos países. Estudos in vivo e in vitro tentam elucidar as alterações causadas pela exposição à asbesto nos tecidos e nas células que possam estar relacionadas ao aparecimento de doenças, e foi verificado que a exposição às fibras leva a quebras na dupla fita de DNA, estresse oxidativo, formação de células micronucleadas e células aneuploides. O presente estudo teve como objetivo verificar a presença de alterações causadas em células em cultura expostas à crisotila por 48 h e recuperadas em meio livre de fibras por 48 h, 4 dias e 8 dias, além de observar por microscopia em tempo real divisões aberrantes após a exposição as fibras por 24 e 48h. Foram verificadas alterações que permaneceram na cultura mesmo após 8 dias de recuperação, quando não foram mais observadas fibras na cultura, como formação de células aneuploides, diminuição de frequência de células em G0/G1, aumento de células em G2/M e aumento relativo de células em metáfase quanto à porcentagem de células em fases mais tardias da fase M do ciclo. Já aumento da frequência de células micronucleadas ocorreu apenas nos períodos quando foram observadas fibras na cultura. Para análise da formação de mitoses multipolares e destinos destas células foram construídos vetores para expressão de tubulinas fusionadas a proteínas fluorescentes RFP e GFP, padronizadas as condições de transfecção e de aquisição de imagens para que as células tratadas com crisotila fossem observadas por time-lapse. Alguns destinos de mitoses multipolares causadas pelo tratamento com crisotila foram observados, como morte em metáfase, divisão gerando duas ou três células filhas, fusão de células durante a telófase e retenção em metáfase. Os dados sugerem também a indução da amplificação centrossômica, que parece ocorrer inicialmente em células interfásicas, e também devido à fusão de células. / Asbestos is a general name given to six different fibrous silicate minerals found naturally in the earth\'s crust. These fibers are being exploited industrially since 1970, but several workers exposed to the fibers developed diseases in the respiratory tract, such as fibrosis and carcinomas. Some types of fiber were banished from the market, but the type of asbestos chrysotile can still be marketed in most countries. Studies in vivo and in vitro are trying to elucidate the asbestos effects in tissues and cells that could be related to the development of diseases, and these studies verified that asbestos exposure lead to DNA double strand breaks, oxidative stress, multinucleated and aneuploid cell formation. The present work aimed to verify the alterations in culture cells exposed to chrysotile for 48 h and recovered in fiber-free medium for 48 h, 4 days and 8 days, and also observe aberrant mitosis using time-lapse microscopy after 24 h and 48 h of chrysotile exposure. Some alterations were observed and remained in cell culture even after 8 days of recovery when chrysotile fibers were no longer observed - such as aneuploid cell formation, increased frequencies of G2/M cell, decreased frequencies of G1 cells, and increased frequencies of cells in early M phases as metaphase. The induction of micronuclei occurred only during the periods that fibers were observed in cell culture. For the analysis of multipolar mitosis formation and destinies of these cells after chrysotile treatment, DNA vectors for the expression of tubulins fused to fluorescent proteins (GFP and RFP) were constructed, and the conditions for cells transfection and image acquisition for time-lapse microscopy were established. The fate of some multipolar metaphases was observed: cell retention on metaphase, cell cycle progression generating two or three daughter cells, cell fusion during cytokinesis or during telophase after a multipolar anaphase, and cell death. The centrosome amplification was not observed during the M phase of cell cycle, and may occur in interphase, and also despite cell fusion.

The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase in response to unattached kinetochores. The mechanism by which the SAC works is by inhibiting the activity of the Anaphase-promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase that targets several anaphase inhibitors for proteasome-mediated degradation, including securin and cyclin B. When the SAC is satisfied, the APC/C becomes active and this allows progression through the cell cycle. Work from the last decade identified the mitotic checkpoint complex (MCC) as the main transducer of the SAC. The MCC is composed of BubR1, Bub3, Mad2 and Cdc20 and it is a very potent inhibitor of the APC/C. When the SAC is active, the MCC binds the APC/C and it inhibits its activity. Once the SAC is satisfied, the MCC becomes disassembled, which allows APC/C activation and mitotic progression. However, the mechanisms that dictate MCC assembly and how it inhibits the APC/C remain to be understood. Here, I used a combination of cell biology and in vitro biochemistry to investigate the mechanism by which the MCC component BubR1 participates in the SAC. My data shows that through its interaction with Bub3, BubR1 localises to kinetochores and this event greatly facilitates its assembly onto the MCC and its SAC function. On the other hand, MCC formation and APC/C binding were only dependent on BubR1's N-terminus, therefore questioning the existence of a second Cdc20 binding site. Within this region, TPR domains and an N-terminal motif known as the KEN box (KEN1) mediates these interactions. By contrast, BubR1's second KEN box (KEN2) does not participate in MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for APC/C inhibition. Indeed, I show that this second KEN box promotes SAC function by blocking the interaction of the APC/C with its substrates. Thus, both KEN boxes in BubR1 participate differentially in the SAC, the first to promote MCC assembly and the second one to block substrate recruitment to the APC/C.In addition, I investigated the mechanisms that mediate MCC inactivation, following SAC silencing. I observed that p31comet and APC/C activity cooperate to promote MCC turnover. The implication of these observations in our understanding of the SAC is discussed.

Accurate chromosome segregation is critical as unequal distribution of the genomic DNA results in impaired cell function or cell death. Kinetochores, the multi-protein structures assembled on centromeric DNA, drive chromosome segregation. Chromosome segregation is under supervision of mitotic spindle checkpoint. The mitotic spindle checkpoint is a surveillance mechanism ensuring that cells enter anaphase with all kinetochores properly attached to spindle microtubules and thereby preventing missegregation. Some checkpoint proteins are localised at kinetochore where they generate and enhance the checkpoint signal. Mps1 (Mph1 in S. pombe) and Aurora B (Ark1 in S. pombe) kinases are required for precise chromosome segregation and mitotic spindle checkpoint in fission yeast. In this study we investigate the roles of Mph1 and Ark1 in regulating the S. pombe kinetochore protein Spc7, which is the homologue of human Blinkin/KNL1. We demonstrated that both kinases target the N-terminus of Spc7. Loss of phosphorylation on the candidate phosphosites results in sensitivity to microtubule depolymerizing drugs indicating mitotic defects. As Blinkin has been proposed to be a docking platform for checkpoint proteins, we tested the possibility that Mph1 kinase is involved in kinetochore targeting of checkpoint proteins, Bub1 and Bub3. Our results demonstrate that Mph1-dependent phosphorylation of Spc7 at conserved MELT motifs is required for Bub1 and Bub3 kinetochore localisation. We were able to reconstitute the interaction between Spc7 and the Bub proteins in vitro demonstrating that the Spc7 phosphorylation is sufficient for Bub1 and Bub3 association with Spc7, most likely with Bub3 making the Spc7 contact. Mimicking phosphorylation at the MELT motifs leads to constitutive Bub1 localisation at kinetochores. We also showed that the N-terminus of Spc7 has microtubule binding activity regulated by Ark1 kinase. Mimicking phosphorylation at Ark1 sites results in reduced amount of recombinant Spc7 co-precipitating with microtubules in microtubule binding assays. Moreover, two stretches of basic residues, that contribute to Spc7 microtubule binding activity, have been mapped in the extreme Nterminus of Spc7. Spc7 also interacts with PP1 phosphatase, Dis2 in S. pombe, which is required for checkpoint silencing, but the mechanism of this interactions remains to be determined. These findings allow us to speculate on Spc7 role(s) in coupling microtubule binding with spindle checkpoint activation and silencing.

Bei der Teilung einer Zelle werden das Genom und die Zellbestandteile zwischen zwei Tochterzellen aufgeteilt. Dies erfordert verschiedene fein aufeinander abgestimmte Vorgänge. Unter anderem ist eine proteinreiche Struktur beteiligt, die 1891 entdeckt wurde: der Mittelkörper. In vorliegender Arbeit wurden gezielt gekennzeichnete Mittelkörperproteine analysiert und verschiedene Phasen des Transports unterschieden. Es erfolgten erstmals Messungen unter Nutzung der ZF1-Methode. Zudem wird anhand der ZF1-Technik nachgewiesen, dass im Rahmen der Zellteilung die Trennung der interzellulären Brücke zu beiden Seiten des Mittelkörpers stattfindet, woraufhin dieser nach extrazellulär abgegeben wird und über einen der Phagozytose ähnlichen und von Aktin abhängigen Mechanismus von einer Tochterzelle oder unverwandten Nachbarzelle aufgenommen wird. / In animals, the midbody coordinates the end of cytokinesis. Using the ZF1-mediated degradation technique it is shown that midbodies are released outside the cell in C. elegans embryos. Furthermore it is shown that midbodies are released after abscission cuts on both sides of the midbody and that released midbodies are internalized via actin-driven phagocytosis.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

In the budding yeast Saccharomyces cerevisiae, the mitotic cell cycle is regulated by the cyclin-dependent kinase (CDK) Cdc28. Cdc28 is activated by binding to one of nine cyclins, which then directs Cdc28’s function and localization. Clb2 is the main mitotic cyclin, promoting entry into mitosis and progression from the metaphase to anaphase transition. In order for cells to exit mitosis, CDK activity must decrease; CDK activity is regulated through Clb2 degradation. Degradation of Clb2 is mediated by the Anaphase-Promoting Complex (APC), which is an E3 ubiquitin ligase that is regulated by the spindle assembly checkpoint. The APC has two activators: Cdc20 and Cdh1; APC-Cdc20 recognizes an N-terminal destruction box (D box) motif in Clb2 during anaphase, and APC-Cdh1 recognizes a KEN box motif at the onset of telophase. Activation of the spindle assembly checkpoint or inactivation of the APC causes cells to arrest in metaphase and, thus, results in accumulation of Clb2. While performing analysis of the function of Clb2 in regulation of mitosis, a lower molecular weight population of Clb2 was identified that seems to correspond to a cleaved form of Clb2 (p45Clb2). Both full-length Clb2 (p56Clb2) and truncated Clb2 (p45Clb2) have different accumulation patterns during either activation of the spindle assembly checkpoint or inactivation of the APC. Our current hypothesis is that p45Clb2 is cleaved between the D box and the KEN box and thus cannot be recognized by APC-Cdc20 but can still be ubiquitinated by APC-Cdh1. p45Clb2 also lacks a nuclear-export signal (NES), which causes its accumulation in the nucleus. We hypothesize that Clb2 cleavage constitutes a mechanism to ensure presence of high CDK activity to delay exit from mitosis.

In the budding yeast Saccharomyces cerevisiae, the mitotic cell cycle is regulated by the cyclin-dependent kinase (CDK) Cdc28. Cdc28 is activated by binding to one of nine cyclins, which then directs Cdc28’s function and localization. Clb2 is the main mitotic cyclin, promoting entry into mitosis and progression from the metaphase to anaphase transition. In order for cells to exit mitosis, CDK activity must decrease; CDK activity is regulated through Clb2 degradation. Degradation of Clb2 is mediated by the Anaphase-Promoting Complex (APC), which is an E3 ubiquitin ligase that is regulated by the spindle assembly checkpoint. The APC has two activators: Cdc20 and Cdh1; APC-Cdc20 recognizes an N-terminal destruction box (D box) motif in Clb2 during anaphase, and APC-Cdh1 recognizes a KEN box motif at the onset of telophase.Activation of the spindle assembly checkpoint or inactivation of the APC causes cells to arrest in metaphase and, thus, results in accumulation of Clb2. While performing analysis of the function of Clb2 in regulation of mitosis, a lower molecular weight population of Clb2 was identified that seems to correspond to a cleaved form of Clb2 (p45Clb2). Both full-length Clb2 (p56Clb2) and truncated Clb2 (p45Clb2) have different accumulation patterns during either activation of the spindle assembly checkpoint or inactivation of the APC. Our current hypothesis is that p45Clb2 is cleaved between the D box and the KEN box and thus cannot be recognized by APC-Cdc20 but can still be ubiquitinated by APC-Cdh1. p45Clb2 also lacks a nuclear-export signal (NES), which causes its accumulation in the nucleus. We hypothesize that Clb2 cleavage constitutes a mechanism to ensure presence of high CDK activity to delay exit from mitosis.

published_or_final_version / Anatomy / Master / Master of Philosophy

The Aurora kinases are highly conserved serine/threonine kinases that play essential roles throughout mitosis. In metazoans, these functions are mediated by Aurora A and B at the spindle poles and the equatorial region respectively. I show here that Dictyostelium contains a single Aurora kinase, DdAurora that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box and localizes to the spindle poles during early mitosis. Like Aurora B, DdAurora localizes to centromeres in metaphase, the central spindle during anaphase and the cleavage furrow at the end of cytokinesis. In addition to these known features of Aurora A and B, I found that DdAurora remains associated with centromeres during anaphase and telophase which has not been shown in any other organisms. INCENP is known to be an important binding partner of Aurora B. In Dictyostelium the conserved C-terminal IN-box domain of DdINCENP is essential for its interaction with DdAurora and for the localization of DdAurora to the central spindle. In contrast, the centromeric and spindle pole localization of DdAurora does not require an interaction with DdINCENP. Surprisingly, a truncated DdINCENP protein lacking the IN-box domain can still localize on centromeres and the central spindle even though it does not bind to DdAurora. I also found that the localization of DdAurora to the central spindle requires Kif12, a protein similar to mitotic kinesin like protein 2 (MKLP2). However, this requirement is suppressed by the overexpression of GFP-DdINCENP. GFP-DdINCENP can localize to the central spindle in the absence of Kif12 and it probably recruits DdAurora to the same location through their strong interaction. Finally, I demonstrated that Myosin II heavy chain is important for the proper localization of the DdAurora/DdINCENP complex to the cleavage furrow during late cytokinesis. With the exception of DdINCENP, no other binding partner or substrate of DdAurora has been identified in Dictyostelium. By performing large-scale immunoprecipitation in wild-type cells, I identified several potential binding partners/substrates of DdAurora, including topoisomerase B and HspA. Future esearch on these proteins may help to elucidate DdAurora function in different stages of M phase.