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

The long-range structure of the Y chromosome alphoid satellite DNA has been determined in the cell lines 3E7 and OXEN. Variation in alphoid DNA block size and restriction enzyme sites were observed. The alphoid block size and restriction enzyme site variations were determined for a collection of 42 normal Y chromosomes. The alphoid DNA polymorphisms observed denned 24 Y chromosome alleles. Unexpectedly, the Y alphoid DNA alleles analysed revealed two distinct groups of Y chromosomes indicating that most of the Caucasian and Asian men analysed were descended from one of two males. The structure of the alphoid DNA was determined for 25 cell lines expected to contain abnormal Y chromosomes. Six of the cell lines lacked Y chromosomes. Four lacked both alphoid DNA and Y a centromere. 13 out of the remaining 15 Y chromosomes had centromeres and Y alphoid DNA block sizes and restriction enzyme site variation similar to that of normal Y chromosome alphoid DNA. Two of the abnormal cell lines had alphoid DNA blocks significantly different from the normal Y alphoid DNA structure. These results confirm that alphoid DNA is located very close to, or at the centromere and make it a prime candidate for a functional mammalian centromere sequence.

The accurate inheritance of genetic material depends upon the establishment and maintenance of sister chromatid cohesion. Replicated chromosomes are topologically encircled by the large, tripartite protein complex cohesin, allowing bi-orientation in mitosis. To entrap and reversibly dissociate from DNA, the annular complex structure must be disrupted at either the hinge domain between Smc1 and Smc3, or the interfaces created by the kleisin subunit Scc1 bridging the two ABC-like ATPase domains. The aim of this work was to characterise the cohesin complex loading and releasing mechanisms by examining the biochemical requirements for these processes. Although the identity of a chromosomal cofactor could not be assigned, the loading reaction was found to necessitate engagement of ATPase domains in an ATP-dependent manner. Notions of allosteric modulation of ATP binding and NBD engagement by acetylation were discredited. Likewise, a direct and stable physical association of hinge domains with NBDs was shown to be an unlikely conformational intermediate in a reaction thought to promote hinge opening for loading of cohesin onto DNA. The Smc3-Scc1 kleisin interface might be exploited during the opposing process of release of cohesin from DNA. Therefore, a novel protein- protein cross-linking system was adapted for use in S. cerevisiae, with a view to (1) confirming the well-founded role of hinge dissociation in topological entrapment, and (2) validating the Smc3- Scc1 interface as the recently conjectured exit gate. Despite promising preliminary kinetics in vitro, the SpyTag-SpyCatcher system was considerably less efficient in vivo. It was thus deemed unsuitable in its current format for investigating the process of interface dissociation in live cells. Finally, the large, cohesin complex-associated HEAT repeat protein Pds5 has been either speculated or shown to participate in all of the aforementioned processes, potentially modulating the hinge and Smc-kleisin interfaces. Acting as a regulatory node in cohesin function, it was pursued as an informative target for structural studies. Although no diffractive material could be obtained, Pds5 was confirmed to bind a short, N-terminal sequence of Scc1 and was in turn bound at its N- terminus by Wapl. Together, these findings contribute to defining the structures and states of the cohesin complex.

Although the many cells within a mammal share the same DNA sequence, their gene expression programmes are highly heterogeneous, and their functions correspondingly diverse. This heterogeneity within an isogenic population of cells arises in part from the ability of each cell to respond to its immediate surroundings via a network of signalling pathways. However, this is not sufficient to explain many of the transcriptional and functional differences between cells, particularly those that are more stable, or, indeed, differences in expression between parental alleles within the same cell. This conundrum lead to the emergence of the field of epigenetics - the study of heritable changes in gene expression independent of DNA sequence. Such changes are dependent on “epigenetic modifications”, of which DNA methylation is one of the best characterised, and is associated with gene silencing. The establishment of correct DNA methylation patterns is particularly important during early development, leading to cell type specific and parental allele specific gene regulation. Besides DNA methyltransferases, various other proteins have recently been implicated in DNA methylation. The absence of these proteins leads to defects in DNA methylation and development that can be even more severe than those in DNA methyltransferase knockouts themselves. In this study I focus on three such accessory proteins: LSH (a putative chromatin remodelling ATPase), G9a (a histone lysine methyltransferase) and SmcHD1 (a structural maintenance of chromosomes protein). To compare DNA methylation between WT cells and cells knocked out for each of these proteins, I used whole genome methylated DNA affinity purification and subsequent hybridization to promoter microarrays. This enabled me to compare the requirement for each protein in DNA methylation at specific genomic regions. The absence of LSH in mouse embryonic fibroblasts (MEFs) resulted in the loss of DNA methylation at 20% of usually methylated promoters, and the misregulation of associated protein coding genes. This revealed a requirement for LSH in the establishment of DNA methylation at promoters normally methylated during pre-implantation as well as post-implantation development. Secondly, I identified hypomethylation at 26% of normally methylated promoters in G9a-/- compared to WT ES cells. Strikingly, this revealed that G9a is required for maintenance of DNA methylation at maternal as well as paternal imprinting control regions (ICRs). This is accompanied by expression defects of imprinted genes regulated by these ICRs. Finally, in collaboration with the Brockdorff lab at the University of Oxford I identified a role for SmcHD1 in establishing DNA methylation at promoters on the X chromosome normally methylated slowly during X chromosome inactivation. Interestingly, SmcHD1 was also required for DNA methylation at autosomal gene promoters, contrary to previous reports that it is mainly involved in X chromosome methylation. I conclude that different accessory proteins are required to facilitate correct DNA methylation and gene repression at distinct regions of the genome, as well as at different times during development. This function of accessory proteins may be in part dependent on the prior establishment of specific chromatin signatures and developmental signals, together comprising a precisely regulated system to establish and maintain appropriate DNA methylation throughout development.

In most societies, surnames are passed down from fathers to sons, just like the Y chromosome. It follows that, theoretically, men sharing the same surnames would also be expected to share related Y chromosomes. Previous investigations have explored such relationships but most data has been collected so far only from the British Isles. In order to provide additional in sights into the con-elation between surnames and Y chromosomes, this study focuses on the Spanish population and investigates Y chromosome SNP/STR variation by analysing a total of 1,766 DNA samples from unrelated Spanish male volunteers belonging to 37 surnames and 355 controls. The results suggest that the degree of coancestry within surnames is highly dependent on surname frequency. Within-surname genetic variation, as measured by different statistics, con-elates well with surname frequency, though a few exceptions are found. In addition, geographic distance between the individuals' place of origin influences Significantly the con-elation between Y chromosome and surnames: men with the same surname tend to have more similar Y chromosomes if their paternal grandfathers were born geographically close to each other. Therefore, it seems that Y chromosome coancestry within surnames is as much about surname frequency as it is about geographical proximity.

No description available.

A thesis submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg fulfilment of the requirements for the degree of Philosophy / Seven Y chromosome probes and thirteen restriction enzyme digests were used to examine a conservative estimate of 20000bp, and no new Y-specific polymorphisms were revealed by these systems. The Y chromosome probe 49a, which reveals a Y-specific haplotype with TaqI, was shown to reveal five new complex polymorphisms with Bglll, Hindlll, Pst I, PvuII and Sstl. The new polymorphisms exhibit great genetic diversity, and each enzyme reveals numerous haplotypes, which mostly occur infrequently and are population-specific. The haplotypes for a given enzyme do not correlate strictly with those revealed by the other enzymes, including TaqI, suggesting that each polymorphism results from a combination of restriction site mutations and rearrangement events. Association between the different 49a polymorphisms occurs only in individuals of recent common genetic origin. Y-specific 49a/TagI haplotypes were determined for 933 individuals drawn from 23 different African populations. A total of 31 new haplotypes were observed, some of which contained new alleles or allelic variants. Duplication, in addition to CpG mutation, is implicated in the generation of certain allelic variants. Cluster analysis of genetic distances between populations was calculated using the 49a/TagI haplotype frequencies. Y-specific 49a/TagI haplotype analysis of individual populations was not sufficiently sensitive to accurately distinguish between the different Bantu-speaking Negroid tribal groups. Cluster analysis of larger groupings was more stable, and with the exception of the Khoisan, resulted in a basic split between African and non-African populations. The linkage disequilibrium of the XY275 MspI Y-linked polymorphism was determined. The high allele was generally found in association with the Y chromosome, but the Y-associated low allele was found to occur in Bantu-speaking Negroids, Khoisan-speaking Negroids, the Khoisan, two groups of mixed ancestry, and the Caucasoid South African Asiatic Indian population. The discovery of Y-associated low alleles in non-African as well as African populations suggests that more than one Y chromosome gave rise to the present-day non-African population. The pDP31/EcoRI, p21Al/TagI and Y Alu polymorphisms were also studied in several southern African populations. The pDP31 duplication occurred at high frequencies in Caucasoids, and could be used to indicate Caucasoid male gene flow into hybrid populations. The p21Al/TagI point mutation showed no distinct trends in frequency in the different populations, and several Taql mutations are proposed to have occurred in the repeat unit recognized by this sequence. The Y Alu polymorphism occurred infrequently in Caucasoids, at intermediate frequency in the Khoisan, and at high frequency in Negroids. The presence of the Y Alu insertion in all three major population groups studied is interpreted to suggest that the insert predates the diversification of Homo sapiens. The relationship between the different Y-linked polymorphisms was determined in the populations studied. The Y Alu polymorphism is believed to have originated once from sequencing data, but such information is not available for the other Y polymorphisms studied. No absolute relationship was observed between the Y Alu polymorphism and the 49a/TagI, XY275 Mspl, pDP31/£coRI and p21Al/TagI polymorphisms. It is suggested that the latter polymorphisms have arisen more than once. / WHSLYP2017

A dissertation submitted to the faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine in the Division of Human Genetics. / Genetic polymorphisms within the non-recombining portion of Y-chromosome (NRY) preserve a record of human paternal genetic heritage that has persisted to the present, allowing human evolutionary inference, population affinity and demographic history, to be elucidated. To elucidate the geographic origins of the paternal ancestry of the present- day South African (SA) ‘Coloured’ population, a total sample of 167 individuals consisting of Cape Malay (N=54) and ‘Coloured’ groups from Cape Town (N=48) and Johannesburg (N=65) were analysed at 21 binary and eight short tandem repeat (STR) polymorphic loci within NRY. A SA White sample (present study, N=97) as well as other presumed parental populations were included for comparative analysis. Haplotypes constructed using both biallelic haplogroup and STR haplotype data assisted in resolving the geographic regions of origin of Y-chromosome in these groups. Altogether the proportions of African, European and Asian contributions were estimated to be 0%, 18.5% and 46.3% in the Cape Malay, 31.3%, 25% and 20.1% in the Cape ‘Coloureds’, and 24.6%, 40% and 16.9%, in the ‘Coloured’ group from Johannesburg. Those haplotypes that could not be unambiguously resolved to European or Asian origins were referred to as Eurasian lineages, and constituted 35.2%, 22.9% and 18.5% of Y- chromosomes in the Cape Malays, Cape ‘Coloureds’ and Johannesburg ‘Coloureds’, respectively. While the ‘Coloured’ groups currently residing in Cape Town and Johannesburg were not significantly different from each other, both groups were significantly different from the Cape Malay population. This was further supported from the association of these groups in population trees. For the most part, these data corroborate historical data concerning the history of ‘Coloured’ populations, but is the first study to show how males have contributed in shaping the gene pool of the ‘Coloured’ population from South Africa. / WHSLYP2016

Recent technological advances underpinned by high throughput sequencing have given new insights into the three-dimensional structure of mammalian genomes. Chromatin conformation assays have been the critical development in this area, particularly the Hi-C method which ascertains genome-wide patterns of intra and inter-chromosomal contacts. However many open questions remain concerning the functional relevance of such higher order structure, the extent to which it varies, and how it relates to other features of the genomic and epigenomic landscape. Current knowledge of nuclear architecture describes a hierarchical organisation ranging from small loops between individual loci, to megabase-sized self-interacting topological domains (TADs), encompassed within large multimegabase chromosome compartments. In parallel with the discovery of these strata, the ENCODE project has generated vast amounts of data through ChIP-seq, RNA-seq and other assays applied to a wide variety of cell types, forming a comprehensive bioinformatics resource. In this work we combine Hi-C datasets describing physical genomic contacts with a large and diverse array of chromatin features derived at a much finer scale in the same mammalian cell types. These features include levels of bound transcription factors, histone modifications and expression data. These data are then integrated in a statistically rigorous way, through a predictive modelling framework from the machine learning field. These studies were extended, within a collaborative project, to encompass a dataset of matched Hi-C and expression data collected over a murine neural differentiation timecourse. We compare higher order chromatin organisation across a variety of human cell types and find pervasive conservation of chromatin organisation at multiple scales. We also identify structurally variable regions between cell types, that are rich in active enhancers and contain loci of known cell-type specific function. We show that broad aspects of higher order chromatin organisation, such as nuclear compartment domains, can be accurately predicted in a variety of human cell types, using models based upon underlying chromatin features. We dissect these quantitative models and find them to be generalisable to novel cell types, presumably reflecting fundamental biological rules linking compartments with key activating and repressive signals. These models describe the strong interconnectedness between locus-level patterns of local histone modifications and bound factors, on the order of hundreds or thousands of basepairs, with much broader compartmentalisation of large, multi-megabase chromosomal regions. Finally, boundary regions are investigated in terms of chromatin features and co-localisation with other known nuclear structures, such as association with the nuclear lamina. We find boundary complexity to vary between cell types and link TAD aggregations to previously described lamina-associated domains, as well as exploring the concept of meta-boundaries that span multiple levels of organisation. Together these analyses lend quantitative evidence to a model of higher order genome organisation that is largely stable between cell types, but can selectively vary locally, based on the activation or repression of key loci.

by Wang Wei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (leaves 154-168). / ACKNOWLEDGMENTS --- p.v / SUMMARY --- p.vi / PUBLICATIONS --- p.viii / STATEMENT OF ORIGINALITY --- p.ix / LIST OF ABBREVIATIONS --- p.x / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.5 / Chapter 2.1 --- Chromosome --- p.6 / Chapter 2.2 --- Chromosome and Human Disease --- p.9 / Chapter 2.3 --- Chromosome and Tumour --- p.12 / Chapter 2.4 --- Chromosome in Gynaecologic Tumours --- p.17 / Chapter 2.4.1 --- Cervical tumour --- p.17 / Chapter 2.4.2 --- Uterine corpus tumour --- p.20 / Chapter 2.4.3 --- Ovarian tumour --- p.23 / Chapter 2.5 --- Methodology in cytogenetics --- p.26 / Chapter 2.5.1 --- Materials --- p.27 / Chapter 2.5.2 --- Methods of chromosome preparation --- p.28 / Chapter 2.5.3 --- Karyotype analysis --- p.34 / Chapter 2.6 --- Problems of cytogenetic analysis in solid tumour --- p.42 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.47 / Chapter 3.1 --- Chemicals and Solutions --- p.48 / Chapter 3.2 --- Chromosome preparation from solid gynaecologic tumours --- p.50 / Chapter 3.2.1 --- Solid tumour specimens --- p.50 / Chapter 3.2.2 --- Chromosome preparation --- p.54 / Chapter 3.3 --- Chromosome preparation from an established ovarian carcinoma cell line --- p.61 / Chapter 3.3.1 --- Origin of OCC1 cell line --- p.61 / Chapter 3.3.2 --- Characteristics of OCC1 cell line --- p.61 / Chapter 3.3.3 --- Maintaining of OCC1 cell line --- p.62 / Chapter 3.3.4 --- Chromosome preparation --- p.62 / Chapter 3.4 --- Karyotype analysis --- p.65 / Chapter CHAPTER 4 --- RESULTS --- p.66 / Chapter 4.1 --- Cytogenetic features of gynaecologic solid tumour --- p.67 / Chapter 4.1.1 --- Cervical cancer --- p.67 / Chapter 4.1.2 --- Uterine corpus cancer --- p.94 / Chapter 4.1.3 --- Ovarian cancer --- p.104 / Chapter 4.2 --- Cytogenetic features of OCC1 ovarian carcinoma cell line --- p.114 / Chapter CHAPTER 5 --- DISCUSSION --- p.123 / Chapter 5.1 --- Methodology of chromosome preparation in solid tumour --- p.124 / Chapter 5.2 --- Chromosome changes in gynaecologic solid tumour --- p.126 / Chapter 5.2.1 --- Cervical cancer --- p.126 / Chapter 5.2.2 --- Uterine corpus cancer --- p.132 / Chapter 5.2.3 --- Ovarian cancer --- p.138 / Chapter 5.2.4 --- In summary --- p.141 / Chapter 5.3 --- Chromosome changes in an OCC1 ovarian carcinoma cell line --- p.143 / Chapter CHAPTER 6 --- CONCLUSION --- p.148 / REFERENCES --- p.154