Browsing by Subject "Comparative genomics"
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Item Open Access EVOLUTION OF THE MATING-TYPE LOCUS AND INSIGHTS INTO SEXUAL REPRODUCTION IN THE CRYPTOCOCCUS SPECIES COMPLEX(2010) Findley, Keisha MoniqueSexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The ascomycetes, the largest phylum of fungi, primarily possess a bipolar mating system while the basidiomycetes, the second largest group, are mostly tetrapolar. The human fungal pathogen and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system that encodes homeodomain (HD) and pheromone/receptor (P/R) genes. The MAT locus of C. neoformans is unusually large, spans greater than 100 kb, and encodes more than 20 genes. To understand how the pathogenic Cryptococcus species complex evolved this unique bipolar mating system, we investigated the evolution of MAT in closely and distantly related species and discovered an extant sexual cycle in Cryptococcus amylolentus.
Phylogenetic analysis using a six-gene multi-locus sequencing (MLS) approach identified the most closely related species to the pathogenic Cryptococcus species complex that are currently known. The two non-pathogenic sibling species, Tsuchiyaea wingfieldii and Cryptococcus amylolentus, and the more distantly related species Filobasidiella depauperata define the Filobasidiella clade. We also resolved the phylogeny of the species located in the sister clade, Kwoniella. A comprehensive tree dendrogram revealed that the 15 Tremellales species examined suggests a common saprobic ancestor. Moreover, the pathogenic Cryptococcus species have a saprobic origin but later emerged as pathogens. We further characterized the mating-type locus for T. wingfieldii and C. amylolentus by cloning and sequencing two unlinked genomic loci encoding the HD and P/R genes. Interestingly, linked and likely divergently transcribed homologs for SXI1 and SXI2 are present in T. wingfieldii and C. amylolentus, while the P/R alleles contain many genes also found in the MAT locus of the pathogenic Cryptococcus species. Also, hypothetical genes present in C. neoformans MAT are also MAT-linked in both species and indicate a possible translocation event between chromosomes 4 and 5 of C. neoformans. Our analysis of MAT in the sibling species indicates that T. wingfieldii is likely tetrapolar, and the C. amylolentus sequence comparison of the dimorphic SXI1 and SXI2 region and the pheromone receptor, STE3, suggests that C. amylolentus is also tetrapolar. The examination of MAT in these sibling species confirms the model for MAT evolution previously proposed in which this structure in C. neoformans and C. gattii evolved from an ancestral tetrapolar mating system. Moreover, the organization of MAT in these sibling species mirrors key aspects of the proposed intermediates in the evolution of MAT in the pathogenic Cryptococcus species, and for sex chromosomes in plants, animals, and alga in general.
We discovered an extant sexual cycle for C. amylolentus, a species previously thought to be asexual. Matings between two strains of opposite mating-types produce dikaryotic hyphae with fused clamp connections and uni- and bi-nucleate basidiospores. Genotyping of basidiospores using markers linked and unlinked to MAT revealed that genetic exchange (recombination) occurs during the sexual cycle of C. amylolentus, and it is likely that either aneuploids are generated during sex or more than one meiosis event occurs within each basidium. This is in contrast to C. neoformans, where only one meiotic event per basidium has been observed. Uniparental mitochondrial inheritance has also been observed in C. amylolentus progeny; similar to the pathogenic Cryptococcus species, mtDNA is inherited from the C. amylolentus MATa parent. Analysis of sex in C. amylolentus has provided insight into the mechanisms that phylogenetically related fungi employ in orchestrating sexual reproduction.
We also extended our analysis to include the distantly related tetrapolar basidiomycete Tremella mesenterica. We completed comparisons of MAT-specific genes between five strains of T. mesenterica and identified the regions that define its mating-type system. The HD locus is limited to the SXI1- and SXI2-like genes while the P/R locus is defined by STE3, STE12, STE20, and the pheromone gene, tremerogen a-13. Interestingly, many of the genes associated with the MAT locus of the pathogenic Cryptococcus species flank the HD and P/R locus and are not incorporated in MAT in T. mesenterica. The MAT region includes transposons and C. neoformans hypothetical genes also present in T. wingfieldii and C. amylolentus. The mating-type system in T. mesenterica reflects an ancestral intermediate in the evolution of the MAT locus in the pathogenic Cryptococcus species. In conclusion, this study provides an in-depth analysis on the structure, function, and evolution of an unusual mating-type locus with broader implications for the transitions in modes of sexual reproduction in fungi that impact gene flow in populations.
Item Open Access The Animal-fungi Hybrid Cell Cycle of the Zoosporic Fungus Spizellomyces punctatus - a New Model to Understand Evolution of Eukaryotic Cell Cycle Control(2019) Medina Tovar, Edgar MauricioThe cell cycle is arguably one of the most conserved regulatory networks within Eukaryotes. Despite the animals and fungi are sibling “kingdoms” within the Opisthokont supergroup, the core transcription factors that control commitment to cell division (E2F and SBF, respectively) and their repressors (Rb and Whi5, respectively) do not appear to have a shared molecular origin. My thesis work has focused on understanding how the networks that regulate cell cycle decisions have changed and rewired through evolutionary time.
By using comparative genomics, I found that the main fungal regulator (SBF) was acquired very early in the evolution of fungi by horizontal gene transfer from a viral origin. I also showed that this viral-derived transcription factor still coexists with the ancestral E2F in the zooporic fungus Spizellomyces punctatus, forming a hybrid cell cycle control network. I hypothesize a viral-derived regulator (SBF) hijacked cell cycle control in the dawn of Fungi by binding the promoters regulated by the ancestral counterpart (E2F), pushing cells to proliferation. This requires the invading SBF to be able to bind regulatory regions controlled by E2F. Using a high-throughput analyses of the DNA-binding properties of the SBF and E2F-family across Eukaryotic lineages I found that E2F and SBF share binding preferences, but that these are not completely overlapping, which could permit the evolutionary conservation of the hybrid E2F/SBF network in Spizellomyces. I then proceeded to test the potential differences \textit{in vivo} in accessibility to E2F and SBF binding sites by coupling in vitro DNA-binding information with nucleosomal and TF-footprints generated from MNase-seq data.
Finally, I developed Agrobacterium-mediated transformation in Spizellomyces, allowing me to describe basic characteristics of its developmental program using live-cell and fluorescence microscopy. By following nuclear dynamics with a fluorescently tagged histone I found that mitosis only initiates after germination, and that nuclei divide synchronously during sporogenesis. Furthermore, by following actin dynamics with LifeAct I showed that zoospores use actin-filled pseudopods to crawl, much like amoeba or animal cells, and that sporangia rely on complex actin dynamics during the formation of zoospores that are reminiscent of animal cellularization processes. This work highlights the importance of non-model systems for finding new solutions to longstanding questions in biology. This is a first step towards establishing Spizellomyces as a model system to study the evolution of key animal and fungal traits, particularly cell cycle regulation and development.
Item Open Access Use of Comparative Genomics for Non-coding Rna Prediction and Investigation of Dna Introgression in Yeast(2008-04-23) Kavanaugh, Laura AnneThe rapid development of large-scale genomic sequencing has dramatically changed the field of genetics, in part through the development of comparative genomics. Fungal comparative genomics is particularly powerful given the large number of genomes currently available, their compact architecture, and their relative ease of genetic manipulation. Fungal comparative genomics was employed in this work to address two related questions. First, it was used along with computational thermodynamic methods to predict non-coding RNA (ncRNA) in Saccharomyces cerevisiae. Sets of positive and negative control genes were evaluated to determine the effect of window sizes and step sizes on the sensitivity of ncRNA identification. The approach was then applied to predict ncRNA genes on chromosome 6 of S. cerevisiae and S. bayanus. Northern blot analysis, rapid amplification of cDNA ends (RACE), and publicly available cDNA library data were used to test the predictions. Strong experimental evidence was accumulated for four new ncRNA genes. Potential structural elements in the 5' and 3' untranslated regions (UTRs) of six annotated protein-coding genes were also identified. This work shows that thermodynamic approaches, coupled with comparative genomics, are powerful tools for predicting structural ncRNA. Second, comparative genomic approaches were employed to identify a non-reciprocal transfer event from Cryptococcus neoformans var. grubii to var. neoformans ~2 million years ago involving a 14 gene (~40 kb) region. The majority of clinical and environmental var. neoformans strains from around the world contain this sequence obtained from var. grubii. The introgression event likely occurred via an incomplete inter-varietal sexual cycle creating a hybrid intermediate where mobile elements common to both lineages mediated the exchange. The subsequent duplication in laboratory strains of a fragment of this same genomic region supports evolutionary theories that instabilities in subtelomeric regions promote adaptive evolution through gene amplification and subsequent adaptation. These data indicate that DNA exchange between closely related sympatric varieties or species may be a recurrent theme in the evolution of fungal species. It further suggests that while evolutionary divergence is the primary force driving speciation, rare introgression events also play a potentially important role.