published_or_final_version / abstract / toc / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

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This thesis is an investigation of the concepts and tools required for the successful application of genome ecology on real genomic transposable element (TE) data. Beginning with the formation of an interdisciplinary working group to examine the distinction between ecology and evolution within genome ecology. By establishing these definitions it was possible to account for the relative effect of proxies for these processes in explaining the variation with the TE communities in a group of genomes. This resulted in the finding that ecological processes were could only explain variation in closely related groups of genomes. Thus ecological methods, developed for examining species distributions along a linear transect, were used to analyze the 30 B.taurus chromosomes. This resulted in the identification of 8 TE species responsible for explaining the spatial distribution in the B.taurus chromosomes. This successful application of ecological methods on TE data promises to inspire many other promising interdisciplinary studies. / OGS

No description available.

Every living organism is programmed to reproduce and to pass genetic information to descendants. The information has to be carefully copied and accurately transferred to the next generation.  Therefore organisms have developed the network of conserved mechanisms to survey the protection and precise transfer of the genetic information. Such mechanisms are called checkpoints and they monitor the correct execution of different cell programs. The DNA damage and the replication blocks are surveyed by the conserved Mec1-Rad53 (human ATM/ATR and Chk2, respectively) protein kinase cascade. Mec1 and Rad53 are essential for survival and when activated orchestrate the multiple cellular responses, including the activation of the ribonucleotide reductase (RNR), to the genotoxic stress. RNR is an enzyme producing all four dNTPs - the building blocks of the DNA - and is instrumental for the maintenance both proper concentration and balance of each of dNTPs. The appropriate concentration of the dNTPs should be strictly regulated since inadequate dNTP production can impede many cellular processes and lead to higher mutation rates and genome instability. Hence RNR activity is regulated at many levels, including allosteric and transcriptional regulation and the inhibition at protein level. In our research, we addressed the question of the transcriptional regulation of RNR and the consequences of dNTP malproduction in the terms of the genomic stability. In yeast S. cerevisiae, four genes encode RNR: 2 genes encode a large subunit (RNR1 and RNR3) and 2 genes encode a small subunit (RNR2 and RNR4). All 4 genes are DNA-damage inducible: transcription of RNR2, RNR3 and RNR4 is regulated via Mec1-Rad53-Dun1 pathway by targeting the transcriptional repressor Crt1 (Rfx1) for degradation; on the contrary, RNR1 gene promoter does not contain Crt1-binding sites and is not regulated through the Mec1-Rad53-Dun1 pathway. Instead, we show that intrastrand cross (X)-link recognition protein (Ixr1) is required for the proper transcription of the RNR1 gene and maintenance of the dNTP pools both during unperturbed cell cycle and after the DNA damage. Thus, we identify the novel regulator of the RNR1 transcription. Next, we show that the depletion of dNTP pools negatively affects genome stability in the hypomorphic mec1 mutants: the hyper-recombination phenotype in those mutants correlates with low dNTP levels. By introducing even lower dNTP levels the hyper-recombination increased even further and conversely all the hyper-recombination phenotypes were suppressed by artificial elevation of dNTP levels. In conclusion, we present Ixr1 as a novel regulator of the RNR activity and provide the evidence of role of dNTP concentration in the genome stability.

The fungal pathogen Ophiostoma novo-ulmi is the causal agent of Dutch elm disease (DED) and has been responsible for the catastrophic decline of elms in North America and Europe. Double-stranded RNA (dsRNA) viruses are common to all fungal classes and although these viruses do not always cause disease symptoms, the presence of certain dsRNA viruses have been associated with reduced virulence (hypovirulence) in O. novo-ulmi. A new mitovirus was found in a Canadian isolate of O. novo-ulmi (93-1224) and has been named Ophiostoma mitovirus 1c (OMV1c). The positive strand of the dsRNA of OMV1c was 3,003 nucleotides in length and when the mitochondrial codon usage pattern was employed (mitochondria use UGA to encode tryptophan rather than as a chain terminator), a single large open reading frame (ORF) was found. This ORF had the potential to encode a protein of 784 amino acids, and revealed a high degree of nucleotide identity to genes encoding RNA-dependent RNA polymerase (RdRp) in other mitoviruses. The putative RdRp region of the newly characterized virus had the highest sequence similarity to Ophiostoma mitovirus 1b. The 5’- terminal sequence of the positive strand could potentially be folded into a double-stranded stem-loop structure with a free energy of 16.6 kcal/mol. Attempts to cure the O. novo-ulmi isolate 93-1224 of virus were unsuccessful. Screening of the re-cultured isolates for the presence of OMV1c revealed that it was still present in the fungus despite repeated hyphal tip transfer, a method known to cure cytoplasmic but not mitochondrial viruses. Based on the genome size, phylogenetic analysis, and the observation that infected isolates could not be cured, it was surmised that the virus was a member of the genus Mitovirus (family Narnaviridae). To assess the distribution of the virus in O. novo-ulmi at the disease front in Winnipeg, a small sample of thirteen isolates were screened for the presence of the new mitovirus. All proved to be negative for OMV1c, which indicated this dsRNA virus was rare and that isolate 93-1224 was the only isolate identified to date infected with OMV1c. It was also discovered that the isolate O. novo-ulmi 93-1224 potentially harboured more than one virus. Electron microscopy of fractionated cells revealed the presence of two flexuous rod-shaped particles that may represent additional novel viruses. / Graduate

Thesis (Ph. D.)--University of Hawaii at Manoa, 2004. / Includes bibliographical references (leaves 147-159). / Also available by subscription via World Wide Web / xv, 159 leaves, bound ill. (some col.) 29 cm

The traditional view of the genome is based on the dogma that genetic information flows from DNA to RNA to protein. Genes have essentially been synonymous with proteins, with RNA viewed primarily as an intermediate template for protein translation. Intriguingly, only ~2% of the human genome encodes proteins, and the number of protein-coding genes (~20,000) is similar between humans and the simple nematode worm.I have been involved with the analysis of a large-scale transcriptome study, intiated by the RIKEN Genomic Sciences Centre in Japan. As part of this work, it was discovered that the genome carries instructions for tens of thousands of “non protein-coding RNAs” (ncRNAs)(please refer to Appendix A to view the original articles that appeared in Science). The significance of these ncRNAs remains a matter of intense interest and debate. Some have argued that these ncRNAs are simply transcriptional noise, while others have suggested that they comprise a critical regulatory system, which directs the complex patterns of gene expression that underlie differentiation and development.To investigate this further, I have conducted a series of studies that explore the expression and evolution of ncRNAs in mammals. Firstly, I established a comprehensive, on-line database of ncRNAs. This collection provides information on more than twenty thousand ncRNAs, and has proven a valuable resource for ncRNA studies. Secondly, I have systematically analyzed the conservation of known functional ncRNA subsets. I found that small ncRNAs (microRNAs and snoRNAs) were well-conserved similar to protein-coding sequences, whereas longer functional ncRNAs were not. These results indicate that long ncRNAs are evolving more rapidly than other functional genomic elements, and suggest that many of the recently-discovered ncRNAs – most of which are long and of unknown significance – might still be functional, despite having poor sequence conservation. Thirdly, I have shown that many ncRNAs are derived from genuine transcripts, whose expression appears regulated in a biologically-relevant manner. Fourthly, I helped develop a computational strategy to identify extremely large ncRNAs and discovered >60 novel candidates, several of which were characterized experimentally. Prior to this work, only a handful of extremely large ncRNAs had been previously described, and these play critical roles in processes such as genomic imprinting and X chromosome inactivation. This study represented the first systematic discovery of extremely large ncRNAs. Finally, I designed custom microarrays and profiled ncRNA expression across the development of CD8+ T cells. CD8+ T cells serve an important role in immunity by killing virus-infected and tumour cells, and transit through a series of functionally-distinct developmental stages. I found that ~200 novel ncRNAs are dynamically expressed during CD8+ T cell development.Taken together, my findings indicate that ncRNAs are a major, regulated output of the mammalian genome, and are consistent with the notion that ncRNAs represent an important, previously-unrecognised biological control system.

After the initiation of the human genome sequencing project and the introduction of the field of ‘bioinformatics’, interest in human genetic diversity studies has been increased. Sequence diversity has helped define differences between genes and genomic regions that were previously unknown or difficult to determine. In this thesis I have undertaken to study sequence diversity in the human genome in three areas; 1) investigated diversity in the MHC as represented by the MICA alleles with respect to the known HLA alleles, 2) investigated the structure and diversity in the intergenic region from an MHC related (paralogous) genomic region and related the structural and diversity findings to the knowledge available on the MHC and the wider genome, and 3) described the identification of three and characterization of five new MHC class I polymorphic markers (Alu) and their polymorphic characteristics in worldwide populations and their associations with skin cancer. 1. Phylogenetic analysis of MICA alpha-domain (extracellular) sequences demonstrated relationships with HLA-B cross-reactive serogroups. The HLA-B and MICA loci are in linkage disequilibrium. The data indicated that MICA and HLA-B have evolved in concert from their common ancestors and that the transmembrane polymorphisms have arisen independently and more recently. 2. Sequence analysis of the CD1 genomic region confirmed the presence of five CD1 genes and revealed that there are four unrelated intergenic regions (IGRs). The IGRs are composed mostly of retroelements including five full-length L1 PA sequences and various pseudogenes. Genomic and phylogenetic analyses support the view that the human CD1 gene copies were duplicated prior to the evolution of primates and the bulk of the HLA class I genes found in humans. 3. Five polymorphic Alu insertions (POALINs) were identified (two from previous studies) and located within the 1.8 megabase of the MHC class I genomic region. All five POALINs are polymorphic, and are positively associated with the HLA-A and HLA-B alleles. The AluyHJ insertion was found most frequently associated with HLA-A1 or A24, AluyHG with HLA-A2, AluyHF with HLA-A2, A-10 or -A26 and AluyTF showed a marginal association with HLA-A29. The AluyMICB insertion was strongly associated with HLA-B17 (HLA-B57, HLA-B58) and HLA-B13. The presence of three Alu insertions (AluyHJ, AluyHG and AluyHF) was found in only one HLA class I haplotype (HLA-A1, -B57, -Cw6) in the 10th IHW cell lines. A novel positive association between the presence of AluyMICB and the ‘MICAdel/MICBnull/HLA-B48’ haplotype was determined. The AluyMICB insertion was also associated with at least three different MICB alleles (*0102, *0107N and *0105) and three different HLA-B alleles (B13, B48 and B57). Based on the analysis of associations between different polymorphic markers within the beta block, the MICB*0102 allele was inferred to be the ancestral form of the MICB*0105 and MICB*0107N alleles. The AluyMICB polymorphism can be used to further investigate haplotype relationship and consequently their lineage origins. Some of the MHC POALINs are haplospecific and associate strongly with certain groups of HLA class I alleles and MHC ancestral haplotypes. The AluyTF frequency was significantly associated with skin cancer (p<0.005). MICA gene diversity is derived from two different evolving paths, therefore one or the other alone cannot reliably mark an ancestral haplotype. The CD1 duplicons originated well before the HLA class I duplicons. The MHC POALINs provide new lineage and linkage markers for the fine mapping study of different haplotypes and variations in linkage groups across 1.8 Mb of the MHC class I region. The POALINs may also prove useful in investigating the origins and history of human populations and in determining the role of human genetic diversity in disease risk.

Thesis (Ph.D.)--Murdoch University, 2005. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 220-245.