In Saccharomyces cerevisiae, DNA replication initiates at distinct origins termed Autonomously Replicating Sequences (ARSs). A key element in ARS is the ACS/B1 sequence, which binds the Origin Recognition Complex (ORC). During early G1 phase, the pre-replication complex is assembled by ORC. These ARSs are termed replicators. In yeast, ORC is also involved in gene silencing. These loci also contain an ACS/B1 element; however at these positions ORC recruits the silencing SIR complex. ARSs found here are termed silencers. Therefore, ARSs have a dual function. Research has also shown that origin ARSs can be substituted for silencing ARSs and vice versa. Since a mutation in ACS abolishes replication activity, studies have turned to the B1 element to account for the functional duality, but results have been mixed. I hypothesize that the B1 element plays a key role in the dual nature of ARS. To test this hypothesis, silencer and replicator ARSs were subjected to site directed mutagenesis around the WTW motif of the B1 element. Their efficiency was then tested using routine silencing and replication assays. Results reveal that the silencing ability of silencer ARSs is unaffected by these mutations; however mutations within and around the WTW motif reduce silencing efficiency of replicator ARSs. Sequence alignments have also shown that silencer ARSs have a broader WTW consensus than replicator ARSs. Preliminary replication assays are consistent with the above results and other research, contributing to a conclusion that the B1 element is not the sole determining factor in the dual nature of ARS. / NSERC

<p>Sexual development enables microbial pathogens to purge deleterious mutations from the genome and drives genetic diversity in the population. <italic>Cryptococcus neoformans</italic> is a human fungal pathogen with a defined sexual cycle. Nutrient-limiting conditions and pheromones induce a dimorphic transition from unicellular yeast to multicellular hyphae and the production of infectious spores. <italic>C. neoformans</italic> has a defined <bold>a</bold>&ndash;&alpha; opposite sexual cycle (bisexual reproduction); however, >99% of clinical and environmental isolates are of the &alpha; mating type. Interestingly, &alpha; cells can undergo &alpha;&ndash;&alpha; unisexual reproduction, even involving genotypically identical cells. A central question is why would cells mate with themselves given that sex is costly and typically serves to admix pre-existing genetic diversity from genetically divergent parents? Sexual reproduction generates abundant spores that following inhalation, they penetrate deep into the alveoli of the lung, germinate, and establish a pulmonary infection growing as budding yeast. Therefore sex has been linked with virulence; however, hyphal development has been previously associated with reduced virulence and thus the roles of morphogenesis in virulence have not been extensively analyzed. To further understand the role of unisexual reproduction in <italic>C. neoformans</italic> we will investigate the evolutionary implications of &alpha;&ndash;&alpha; mating, explore its role in pathogenesis, and we will dissect the signaling pathway that regulates sexual development.</p><p>We isolated &alpha;&ndash;&alpha; unisexual reproduction progeny from the hyperfilamentous strain XL280 and subjected to a variety of phenotypic and genotypic assays (including whole genome sequencing and CGH). We found that unisexual and bisexual reproduction frequently generates phenotypic and genotypic diversity de novo, including aneuploidy. Aneuploidy was responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments, such as the mammalian host.</p><p>Interestingly aneuploidy strains that were fluconazole resistant were as virulent as the WT parental strain XL280. Although XL280 belongs to the serotype D lineage that exhibits limited pathogenicity, in further studies we found that is hypervirulent in the murine model. It can grow inside the lung of the host, establishing a pulmonary infection, and then disseminates to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is characterized by less protective immunity and is usually observed in the highly virulent sibling species <italic>C. gattii</italic>, responsible for the Pacific Northwest outbreak. These studies: 1) provide a technological advance that will facilitate analysis of virulence genes and attributes in <italic>C. neoformans</italic> var. <italic>neoformans</italic> (serotype D), and 2) reveal the virulence potential of serotype D that is broader and more dynamic than previously appreciated.</p><p>Bisexual and unisexual reproduction are governed by shared components of the conserved pheromone-sensing Cpk1 MAPK signal transduction cascade and by Mat2, the major transcriptional regulator of the pathway. However, the downstream targets of the pathway are largely unknown, and homology-based approaches have failed to yield downstream transcriptional regulators or other targets. To address this question we applied an insertional mutagenesis via <italic>Agrobacterium tumefaciens</italic> transkingdom DNA delivery to identify mutants with unisexual reproduction defects. In addition to elements known to be involved in sexual development (Crg1, Ste7, Mat2, and Znf2), three key regulators of sexual development were identified by our screen: Znf3, Spo11, and Ubc5. Spo11 and Ubc5 promote sporulation during both bisexual and unisexual reproduction. Genetic and phenotypic analyses provide further evidence implicating both genes in the regulation of meiosis. Phenotypic analysis of sexual development showed that Znf3 is required for hyphal development during unisexual reproduction and also plays a central role during bisexual reproduction. Znf3 governs cell fusion and pheromone production through a pathway parallel to and independent of the pheromone signaling cascade. Surprisingly, Znf3 participates in transposon silencing during unisexual reproduction and may serve as a link between RNAi silencing and sexual development. In further studies we found that Znf3 is required for sex- and mitotic-induced (SIS and MIS). SIS is less efficient in <italic>znf3</italic> unilateral matings and is abolished in <italic>znf3</italic> x <italic>znf3</italic> bilateral matings, similar to the phenotypes of <italic>rdp1</italic> mutants (the RNA-dependent RNA-polymerase of RNAi pathway). Znf3 is also required for transgene-induced mitotic silencing; <italic>znf3</italic> mutations abrogate silencing of repetitive transgenes during vegetative growth. Znf3 tagged with mCherry is localized in the cytoplasm in bright, distinct foci. Co-localization of Znf3 with the P-body marker Dcp1-GFP further supports the hypothesis that Znf3 is a novel element of the RNAi pathway and operates to defend the genome during sexual development and vegetative growth. In concussion our studies provide further understanding of unisexual reproduction as an evolutionary successful strategy.</p> / Dissertation

In the eukaryotic cell nucleus, spatial organization and dynamics of the genome is important in the regulation of gene expression. This thesis describes the use of the fission yeast, Schizosaccharomyces pombe, to study chromatin regulation and dynamics. We used nitrogen starvation to induce transcription of genes in fission yeast cells. In induced genes, nucleosomes get evicted in both the promoter and in the open reading frame (ORF). In the genes with the highest expression more nucleosomes get evicted from the ORF than from the promoter. This indicates that large rearrangements of the chromatin are occurring during a drastic gene induction. Many of the genes that become expressed early after nitrogen starvation are located together in clusters. In a cell where nitrogen is present in the surrounding media the gene clusters locate close to the nuclear periphery. When the nitrogen source is removed from the media, the clusters move to a more internal position. Thus rearrangement of chromatin due to gene induction, described in the first study, is accompanied by subnuclear changes of localization. Another type of regulation is the silencing of genes. We have studied a factor necessary for correct repression of genes located in silent chromatin, in S. pombe. The protein, Clr2, is part of the SHREC complex containing a remodeler (Mit1) and a histone deacetylase (Clr3). By bioinformatic analysis of Clr2 and newly sequenced fungi genomes, three motifs were identified. To gather more information about important parts of the Clr2 protein, deletions were made. When removing from about 20 to 100 amino acids in the middle of the protein, silencing of a reporter gene inserted at the mating-type region, inner repeats of centromere 1 and at the central core of centromere 2, failed. This indicates that Clr2 has an important role in establishing silent chromatin.

Long terminal repeat (LTR) retrotransposons account for approximately 10% of mouse and 8% of human genomes and may play a role in modifying gene expression. Many species harbor retrotransposon families encompassing both autonomous and non-autonomous members. Specifically, the mouse Early Transposon (ETn) family members lack all retroviral genes but are transcriptionally and retrotranspositionally active, causing over 20 known insertional germline mutations. ETns owe their retrotransposition potential to proteins encoded by structurally intact MusD retrotransposons with whom they share LTRs. ETn elements are transcribed at a much higher level than MusD retrotransposons in embryos and undifferentiated cells, suggesting their evasion of host restriction mechanisms. However, mechanisms responsible for the replicative success of non-autonomous retrotransposon subfamilies over their coding-competent relatives are poorly understood. In the first stage of my research, I analyzed regulatory sequences in an ETn LTR responsible for its high promoter activity in the undifferentiated cell line P19. I found that three GC-boxes that may function as Sp1/Sp3 binding sites act synergistically and are indispensable for undifferentiated cell-specific promoter activity of the LTR. Sp1 binding partners may be responsible for the restricted ETn expression. Moreover, I have shown that unlike many retroviruses, ETn elements possess multiple transcription initiation sites and that they have amplified via intracellular retrotransposition in the P19 teratocarcinoma cell line. In the next step of my research, I performed analysis of epigenetic mechanisms as a means of ERV suppression. Specifically, I showed that in embryonic stem cells, autonomous MusD retrotransposons are epigenetically suppressed to a greater degree than non-autonomous ETn retrotransposons, illustrated by a higher level of DNA methylation and a lower level of active histone modifications. I hypothesize that MusD elements may be silenced by DNA methylation and repressive chromatin spreading into the LTR from the CpG-rich internal retroviral sequence absent in ETn elements. I propose that internal structure largely devoid of high CG content enables ETn elements to evade host-imposed transcriptional repression, contributing to their high mutagenic activity in the mouse germline.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor traditionally regarded as a regulator of fibrinolysis and extracellular matrix degradation. More recently, PAI-2 has been implicated in diverse processes such as keratinocyte differentiation, cell death and viral pathogenesis. Although PAI-2s limited pattern of expression in vivo generates significant interest in this molecule, little is known about the underlying mechanisms controlling its cell specific regulation. In this thesis, the function that the previously identified PAI-2 gene silencer (Antalis et al., 1996) plays in the regulation of PAI-2 gene expression was investigated. The PAI-2 upstream silencer element 1 (PAUSE-1) is located approximately 1800bp upstream of the PAI-2 transcription initiation site. By employing electrophoretic mobility shift assays and transient transfection assays with mutant PAUSE-1 sequences, the sequence that defines PAUSE-1 was identified as TCT N3 AGA N3 T4. This element was shown to bind a number of protein complexes of similar electrophoretic mobility from various cultured cell lines. Transient transfection assays with the cervical adenocarcinoma, HeLa S3 and the macrophage-like, U937 cell lines, showed that PAUSE-1 repressed transcription by approximately 2.5 fold when cloned into the SV40 promoter or the minimal PAI-2 promoter. Ultraviolet (UV)-crosslinking analyses determined that the PAUSE-1 binding protein (BP) was approximately 67kDa. Examination of several similar DNA promoter sequences, such as the human IFNb and insulin promoters, suggested that PAUSE-1 might be an example of a universal silencer with the consensus sequence TCT Nx AGA, where x=4. The PAUSE-1 sequence shows significant homology to the binding sequence of the transcriptional regulators Ski, Smad3 and Smad4. EMSAs incorporating anti-Ski, -Smad3 and -Smad4 antibodies suggested that each are members of the PAUSE-1 BP complex in HeLa S3 cells. The PAUSE-1 BP complex has been purified by employing DNA affinity chromatography using streptavidin labelled magnetic beads. Approximately nine PAUSE-1 associated proteins from HeLa S3 extracts were visualised. Amino-terminal protein sequencing identified the first eight amino acids of the PAUSE-1 BP as EIQQRAAQ. The PAUSE-1 BP fails to show significant sequence similarity to any known protein and therefore potentially represents a novel DNA binding protein.

Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that infects subterranean clover (Trifolium subterraneum) and a number of related legume species. The ultimate aim of this research was to investigate aspects of SCMoV that would support its use as a gene silencing vector for legume species, since RNA (gene) silencing is now a potential tool for studylng gene function. The ability of viruses to induce an antiviral defense system is being explored by virus-induced gene silencing (VIGS), in which engmeered viral genomes are used as vectors to introduce genes or gene ii-agments to understand the function of endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects of SCMoV host range and molecular biology needed to be studied. A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this work involved study of the host range and symptom development of SCMoV in a range of leguminous and non-leguminous plants. The aim of this work was to identify new and most suitable hosts among economically important crop and model legumes for functional genomic studies, and also to study symptom development in these hosts for comparison with host responses to any SCMoV-based viral vectors that might be used in later infection studies. A total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7 families were examined for their response to SCMoV infection, including established and new crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae, Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for symptoms and tested for primary and secondary infection by RT-PCR andlor ELISA after 2-3 weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This work considerably expanded knowledge of the host range of SCMoV. To provide the information needed to modify the SCMoV genome to develop gene vectors, the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence of SCMoV RNA is not polyadenylated. The 5' non-coding region (NCR) is 68 nt in length and the 3' NCR is 174 nt. The coding regon contains four overlapping open reading fi-ames (ORFs). The first, OW1 (nt 68-608), encodes a putative protein containing 179 amino acids with a calculated molecular mass (Ma,) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Ma, of 63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b is probably translated as part of a polyprotein by -1 ribosomal fiameshifting in ORF2a. The transfiame product (Ma, = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of RNA virus polymerases is present in ORF2b. The 3' terminal ORF3 (nt 3323-4084) encodes the 27.3 kDa coat protein (CP). Nucleotide variation between the complete sequences of the isolates was two to three orders of magnitude larger than base misincoporation rates of the polymerases used in RT-PCR. Molecular relationship analysis between all five isolates, undertaken with the complete nucleotide sequences, clearly separated them into three groups. These groups reflect similar significantly diverse groupings based on the symptoms and their severity in subterranean clover. Intra-isolate sequence variability is therefore a possible cause of the differences in symptom severity. The analysis also showed that there were more nucleotide substitutions at the 5' terminal half of SCMoV than at the 3' end. This observation was confirmed by the higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/ds ratio) estimated for the ORF1, compared to the near conservation of sequences of the other ORFs. These results, together with functional and comparative sequence analysis with other sobemoviruses, implicate the ORFl gene product in pathogenicity of SCMoV, possibly as a severity determinant or as a viral suppressor of RNA silencing in plants. Because more information on SCMoV genome function was required, the possible involvement of the ORFl gene product (PI) and the CP in movement of SCMoV was studied in cells of grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish (Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for in planta synthesis of reporter gene constructs was developed. The vector was based on pGEM-T with 35s RNA transcriptional promoter of Caulzjlower Mosaic virus (CaMV) and nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple subcloning site. A custom-made particle inflow gun was used to introduce the constructs into plant cells. The bombardment conditions were fxst optimised for efficient delivery of DNAcoated particles. Transient gene expression of GFP was monitored 24-96 hours after particle bombardment. Fluorescence from GFP alone, GFP:CP and GFP:Pl constructs was observed in the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was limited spread of these fusion proteins from one cell to another 36-48 hours after transformation. These results indicate that the P 1 and CP cannot move independently from cell to cell. Other viral/cellular components might be needed to form a complex with these proteins to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of SCMoV were identified by sequence comparison. These could be tested by mutagenesis using full-length infectious clones. To determine the possibility of gene expression of vectors based on SCMoV, three forms of a full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous transcriptional factors (pFL), a second under the control of only 35s (p35SFL) and a third with 35s and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and subterranean clover seedlings were inoculated by particle bombardment using gold particles coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR after two weeks in grasspea but not in subterranean clover and chickpea. Experiments were undertaken towards developing the SCMoV genome into a VIGS vector. Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be derived. The rationale used in developing these constructs was gene insertion andlor replacement with d p , and duplication of the putative subgenomic RNA promoter (sgPro) of SCMoV. The major constructs were as follows: pFLCPgfp, pFL with the d p gene fused to the 3' end of the CP gene, pFLP 1 gfp, pFL with gj27 gene fused to the 3 ' end of the ORF 1, pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable & gene cloned in tandem between the CP gene and the 3' NCR of SCMoV, pFLCPVsgprogf$, pFL with a putative sgPro sequence and a translatable gfp gene cloned in tandem between a truncated CP gene and the 3' NCR and pFLREPsgprog@, pFL with the ORF2b, a putative sgPro sequence and a translatable &fP gene cloned in tandem between a truncated CP gene and the 3' NCR These constructs were all made, but a detailed assessment of their vector potential could not be done because there was a delay of about one year whilst the Office of the Gene Technology Regulator processed the application for permission for glasshouse testing. Although additional work needs to be undertaken to complete development of a final RNA silencing vector, this study has contributed to new knowledge in terms of extending understanding of SCMoV host range, symptoms, sequence variation and control of gene expression. The constructs made have also laid the groundwork for development of a legume gene silencing vector based on SCMoV.

Thesis (Ph. D.)--University of New South Wales, 2000. / Also available online.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Thesis (Ph.D.)--Murdoch University, 2005. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 184-207.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 114-132) Also available in print.