The degree of DNA supercoiling in the cell is carefully controlled by DNA topoisomerases. These enzymes catalyze the passage of individual DNA strands (Type I DNA topoisomerases), or double helices (Type II DNA topoisomerases) through one another. The purpose of the present study is to conduct a detailed analysis of the topo llα and β mRNAs expressed in several vertebrate cell lines. The final aim of this project is to analyze the relative roles of topo llα in chromatin condensation and chromosome segregation during mitosis, by performing topo llα gene targeting experiments in the DT 40 chicken lymphoblastoid cell line. The knock-out strategy was based on the observation of a high rate of homologous recombination versus random integration in the DT40 cell line. The topo llα gene was shown to be located on the chicken chromosome 2 (APM unpublished), for which the DT40 cell line is trisomic. The targeting vector completely replaced the 32 kb topo IIα genomic locus, generating a topo llα (-/+/+)cell line, which showed an increased resistance to topo II inhibitors. Paradoxically, 150 uM etoposide or 100 uM mitoxanthrone induced apoptosis within 5 hours in the topo llα (-1+1+) cell line, more rapidly as compared to the normal DT 40 cells. A topo IIα (-I-I+) cell line has also been generated. This study revealed the presence of evolutionarily conserved alternatively spliced forms of both topo llα and β isoforms between birds and humans. Hybridization screening of two chicken cDNA libraries, MSB-1 and DU249, revealed the presence of two distinct forms of both topo llα and β cDNAs. One form of topo llα, designated topo llα-1, encodes the chicken topo llα amino acid sequence previously reported (dbjiAB007445) in the database (unpublished). The second form, designated topo llα-2, encodes a protein containing an additional 35 amino acids inserted after Lysine-322 of chicken topo IIα-1 protein sequence. In the case of topo 11(3 mANA, one form, designated topo IIβ-1, encodes the protein already described (dbjiAB007446). The second form, tapa IIβ-2, would encode a protein missing the next 86 amino acids after Valine-25 in tapa II β-1 protein sequence. The tapa 11β variant is positioned similarly to one previously described in human (Hela) cells. The 5 amino acid insertion in the human tapa 11β-2 variant follows v23. In chicken cells, a longer insertion of 86 amino acids sequence follows v25, the homologous position in the tapa 11β protein. In human cells, the situation with tapa llα is more complex, as revealed by RT-PCR experiments (APM, unpublished) which generated several bands. One of these amplified species was found to contain a 36 amino acids insertion, positioned after residue K321 in the human tapa IIα cDNA, similarly to chicken tapa IIα-2 variant. The second human tapa llα spliced form cDNA was shown to contain a 26 amino acids insertion after residue A401 in the canonical human tapa llα protein sequence. The third cDNA variant isolated from human cells was described to encode a 81 amino acids insertion after residue Q355 positioned within the known human tapa IIα protein. It appears possible that the posttranslational modifications of the a-2 and β-2 isoforms may differ substantially from those of the canonical a-1 and β-1 isoforms. Such variant proteins could fulfil specialized functions, which might be tissue or cell-type specific. In summary, two novel forms of tapa llα and β cDNAs have been identified in three chicken cell lines. These spliced versions of both tapa llα and 13 isoforms seem to be evolutionary conserved, with similar forms occurring in their human counterparts. Future functional analysis of vertebrate tapa IIα and β will have to account for the existence of these novel isoforms, which might encode proteins that may exhibit different regulation of their subcellular localization, interaction with other proteins, or catalytic activity.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

The Best PhD thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing prize,2006-2007 / published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

Analyzing regions in the genome with low levels of recombination helps understand the prevalence of sexual reproduction. Here, I show that variability in regions of reduced recombination in Drosophila can be explained by interference among strongly deleterious mutations; selection becomes progressively less effective in influencing the behaviour of neighbouring sites as the number of closely linked sites on a chromosome increases. I also show that the accumulation of loss-of-function mutations on the neo-Y chromosome of Drosophila miranda is compatible with a model of selection against such mutations alone, without the need to invoke the action of selective sweeps. I describe the discovery of two new sex-linked genes in the plant Silene latifolia, SlCyt and SlX9/SlY9. SlCyt has been recently translocated from an autosome to the X and shows signs of a selective sweep. Its possible role in having caused recombination arrest between the evolving X and Y chromosome is discussed. SlX9 still has an intact Y-linked copy that is presumably functional. Nucleotide diversity at SlY9 is very low, whereas SlX9 has an unusually high diversity and shows signs of introgression from S. dioica into S. latifolia, but the effect of this seems very localized.

Skin cancer is one of the most common forms of adult solid tumour. The incidence is increasing rapidly making skin cancer a major health problem in several countries. Cutaneous Malignant Melanoma (CMM) is the least common but the most life threatening type of skin cancers and is responsible for 90% of all skin malignancy associated deaths. The precise cellular and molecular etiology of malignant melanoma is quite complex and the molecular events directly related to melanoma progression are yet to be elucidated. However, recent advances in molecular biology have resulted in a clearer understanding of the cellular and molecular events of skin cancer development. The best-characterized locus associated with CMM development is the CDKN2A that maps to chromosome 9p21 and encodes for the cell cycle regulator p16 tumour suppressor gene (TSG), and is frequently inactivated in melanoma tumours. In addition to p16, other loci located in 9p21 appear to be important in CMM development and functional evidence for the presence of TSG(s) has been provided (Parris et al., 1999). The aim of our study is to contribute to the understanding of CMM development by isolating and characterising novel TSG(s) at this location. In order to pursue identifying potential TSG(s), we have developed several monochromosome hybrids using microcell mediated chromosome transfer, and evaluated the tumourigenicity of the constructed hybrids by anchorage independent growth in soft agar. For the molecular biology aspects, expression analysis of the genes in the 9p21 region was carried out by reverse transcription PCR. Potential candidate tumour suppressor genes were then carefully evaluated by generating expression profiles via conducting real time PCR. Experimental evidence is provided which supports the candidacy of interferon alpha 1 (IFNA1) as a tumour suppressor gene for melanoma development.

Chromosome Instability (CIN) is defined as an increase in the rate at which whole chromosomes or large parts are gained or lost. CIN is not only associated with virtually all tumor types, but it is associated with aggressive tumors, tumor recurrence, acquisition of multidrug resistance and poor patient prognosis. However, the genes and molecular defects that contribute to CIN are poorly understood. I hypothesize that KIF11 is an essential gene for chromosomes integrity during mitosis and therefore any defect in KIF11 expression or function will induce CIN and contribute to tumorigenesis. Accordingly, KIF11 was either silenced using siRNA or inhibited using monastrol within two distinct human cell lines and was investigated for CIN associated phenotypes. Here, I have identified and validated KIF11 as a novel CIN gene. This study represents the first steps necessary to identify and develop novel treatments design to target origins of CIN in CIN associated cancers. / February 2017

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Sister chromatid cohesion is essential for accurate chromosome segregation. Cohesion is generated by cohesin, a conserved multi-subunit protein complex composed of four core subunits: Smc1, Smc3, Scc1, and Scc3. Cohesin holds sister chromatids together in mitotic cells starting from S-phase, when DNA replicates, until their separation at the onset of anaphase where its Scc1 subunit is cleaved. In budding yeast, most Scc1 is destroyed by cleavage at anaphase and is only re-synthesised in late G1, whereupon it associates with the unreplicated chromatin. Although sister chromatid cohesion is known to be mediated by a topological interaction of cohesin complexes around sister DNAs, the nature of cohesin`s interaction with chromatin before DNA replication remains to be elucidated. My project aims to develop a new system in order to find out whether ‘non-cohesive’ cohesin interacts with chromatin topologically. This is important for two main reasons. Firstly, understanding the physical nature of cohesin’s interaction with chromatin before DNA replication is essential for determining how cohesion is established during DNA replication. Another reason is that most cohesin in multicellular organisms is associated with the unreplicated chromatin of post mitotic cells where it regulates transcription. How cohesin mediates gene expression is unknown. Understanding how cohesin binds unreplicated chromatin may therefore bring insights into the mechanisms by which cohesin complex performs its non-canonical functions. In order to address this, we needed a situation where cohesin is already loaded onto chromosomes, but either DNA replication or cohesion establishment is prevented. Therefore, we used a temperature sensitive allele of Eco1 (required for establishment of cohesion). Quantitative measurement of cohesin levels on chromosomes in either wild type allele or temperature sensitive allele of Eco1 showed that the amount of cohesin associated with centromeric and inner pericentromeric regions in both strains are almost indistinguishable from each other throughout the whole cell cycle. Despite normal levels of cohesin, we confirmed by minichromosomal assay that no sister chromatid cohesion is established in the absence of functional Eco1 protein. If “non-cohesive” cohesin interacts with the chromatin in a topological manner when there is no sister chromatid cohesion, then its association with chromatin should be resistant to denaturing conditions in the presence of a modified version of the cohesin complex that can be covalently circularized. To test this prediction, a cross-linkable cohesin molecule was needed, which should be resistant to SDS denaturation and should not have major cohesion defects due to the modifications making it to be cross-linkable. The previously created cross-linkable cohesin molecule had cohesion defects due to the presence of Smc3-Scc1 fusion protein. In addition, this fusion alone could bypass the requirement for Eco1, and therefore we could not test how “non-cohesive” cohesin interacts with chromatin, using this version of cross-linkable cohesin complex. We tried two different methods to conditionally close Smc3/Scc1 interface in a way resistant to protein-denaturants and create a new cross-linkable cohesin complex. In our first attempt, the C-terminus of Smc3 and the N-terminus of Scc1 were fused to FRB and FKBP12 respectively, proteins that can form a complex upon addition of rapamycin. Crystal structure of the ternary complex of FKP12/rapamycin/FRB enabled us to design cysteine pairs for the crosslinking of FRB and FKBP12 only in the presence of rapamycin. A more efficient in vivo crosslinking was achieved between the Smc3 and Scc1 in our second attempt. Amino acids within the coiled coil region of Smc3 were replaced by the unnatural photo-cross-linkable amino acid ρ-benzoyl-phenylalanine that can be induced to form covalent bonds with neighbouring proteins (T.Gligoris, unpublished data). Photo and chemically cross-linkable interfaces of cohesin were then integrated with each other to generate a new version of cross-linkable cohesin molecule.