Arbuscular mycorrhizal (AM) fungi have been living in association with land plants for at least 400 million years. Because they are obligate symbionts, the study of AM symbiosis has focused primarily on its plant host and progress in the molecular biology of AM fungi has been very slow. Using two different approaches, library screening and direct PCR-based assays, genetic information of AM fungi was compared across isolates and species. This allowed the study of novel DNA regions previously unexplored in AM fungi. The following species were investigated: Glomus intraradices, Glomus mosseae, Gigaspora margarita, Scutellospora calospora, Acaulospora scrobiculata and Entrophosphora colombiana. In the first approach, using in vitro grown G. intraradices, a DNA extraction protocol was developed for the construction of a partial genomic library. This library was screened for the presence of microsatellite-containing loci. PCR primers were designed based on five identified loci. Two of these loci were monomorphic for all isolates and species. The second approach used a combination of degenerate and specific primers for fungal chitin synthase genes to explore the variability of this gene family in AM fungi. A total of 21 AM sequences were isolated and sequenced, covering class I and II chitin synthases. RT-PCR with G. intraradices revealed differential expression of chitin synthases in spores and mycelium, as compared to mycorrhized roots. In addition, using primers designed from a highly conserved sequence for plant resistance genes, classical PCR and RT-PCR allowed the detection of a genomic sequence and its cDNA counterpart encoding a putative serine/lysine rich protein in G. intraradices. We have thus investigated genetic variability in AM fungi in functional genes as well as in repetitive DNA regions. Study of gene expression was also possible using in-vitro grown G. intraradices.

Transposons are a diverse collection of mobile genetic elements and are important components of nearly every genome. Because of their mobile and repetitive nature, transposons can have considerable effects on host gene expression, genome organization and evolution. The recent availability of genomic sequence information has expedited the discovery and study of transposons, as exemplified in this thesis by the complete genome analysis of the model plant system Arabidopsis thaliana. Data mining in Arabidopsis has revealed a rich diversity of transposons, of which Basho and Terminal-repeat Retrotransposons In Miniature (TRIM) elements were previously unknown types. The identification of Related to Empty Sites (RESites) provide evidence for past transposition events. Examples of elements contributing to coding regions, acquiring cellular sequences, along with in-depth analysis of the insertions, their target sites and their distribution illustrate the impact of transposons on gene and genome structures. Computer-based searches of genomic sequences has also improved our understanding of previously identified transposon families, such as the origin, classification and mobilization of Tourist elements. In addition, information on transposons gathered from in silico analysis of genomic sequences has served to design in vivo experiments. In a whole genome strategy, Transposon Display was used to investigate transposition and regulation of mobility of Tourist-like elements in A. thaliana and in the nematode Caenorhabditis elegans.

Transposons, also known as transposable elements (TEs), are genetic elements capable of changing their location in the genome and amplifying in number. Because of their ability to cause mutations in the host genome, often with detrimental consequences to the host, yet avoid being eliminated by natural selection, transposons have been labeled selfish elements or genomic parasites. However, the advent of genomics has allowed the identification of numerous instances where transposons have played a crucial role in host genome evolution. In this thesis, I evaluate the extent to which transposons have influenced the genomes of their hosts, with an emphasis on plant genomes. I review the present knowledge of different mechanisms by which this is achieved and provide examples to illustrate them. Next, I tackle the problem of annotating transposons in the completed genomic sequence of domestic rice by comparing RepeatMasker, the standard approach used in transposon annotation, with an alternative approach employing hidden Markov models. In addition, I perform a genome-wide analysis of gene fragment capture by rice Mutator-like transposons. I conclude that, while this is a widespread phenomenon in rice, it is unlikely to represent a major force in generating novel protein-coding genes. Nevertheless, the duplicated gene fragments that are transcribed may playa role in the regulation of host genes they arose from via an RNAi-like mechanism. Finally, I conduct an in silico analysis of a gene family derived from a domesticated Mutator-like transposase, called MUSTANG (MUG), in conjunction with an experimental characterization of the MUG family in Arabidopsis. The results of the study indicate that the MUG family arose in a common ancestor of flowering plants and that the Arabidopsis genes AtMUG1 and/or AtMUG2 may act as global regulators of mitochondrial function. I conclude that our appreciation of the role of transposons in host function and evolution will undoubtedly continue to grow as our understanding of these processes deepens.

Since their discovery, transposable elements (TEs) have been regarded either as useful building blocks of genomes, or as "selfish DNA": genetic parasites that exploit the sexual cycle to spread in copy number within populations to the detriment of their hosts. If the "selfish DNA" hypothesis is correct, TEs are expected to deteriorate and be lost from asexual populations. This thesis tests the predictions of the "selfish DNA" hypothesis in four asexual plant species, focusing on patterns of nucleotide diversity and nucleotide substitution. Sequences bearing strong resemblance to known TE families including Ty1/copia, Ty3/gypsy, and LINE-like elements were successfully isolated from all four plant species, and showed patterns of nucleotide substitution consistent with a long history of purifying selection. Stochastic simulations were also conducted, and suggested that this result is expected if the host species has been asexual for less than tens of thousands of generations.

Despite the ubiquity of transposons in eukaryotic genomes, their evolutionary role remains controversial. The discovery of several domesticated genes has suggested that transposons can gain host functions, and thus contribute to the evolution of their host. Here, I present the results of a genome-wide screen for transposon-derived host genes, which was based on the idea that, once domesticated, the open reading frame of such elements would be maintained, while terminal structures necessary for transposition would be lost. Eight-hundred-and-sixty-three such transposon-dissociated elements were mined from the genome of Arabidopsis thaliana var. Columbia-0, of which less than 10% are associated with expression data. Phylogenetic analysis of Mutator superfamily genes in the genomes of Oryza sativa ssp. japonica (cv Nipponbare) and Arabidopsis, including 121 Mutator-derived transposon-dissociated elements, found that only two gene families are taxonomically widespread. MUSTANG1, a member of one of these families, appears to be under purifying selection. Thus, despite the dearth of taxonomically widespread and/or expressed transposon-dissociated elements, MUSTANG1, as well as three transposon-dissociated elements that may be associated with mutant phenotypes, might be newly discovered transposon-derived host genes.

The sequencing of eukaryotic model organisms has provided us an unprecedented opportunity for a genome-wide characterization of Transposable Elements (TEs) and the study of TE-host relationships. By developing methodologies on database mining, we explored the existence of MUtator (Mu)-Like Elements (MULEs) in Arabidopsis thaliana and Oryza sativa. Mu elements were first discovered in Zea mays; so far, a dozen of the elements have been identified in the genome. We identified a total of 1392 MULEs from the sequenced Arabidopsis genome. They represent one of the most abundant, diversified, yet still mobile DNA transposon families in eukaryotes. The Arabidopsis MULEs are composed of not only the elements showing the typical Mu-family-specific terminal structure (that is the long Terminal Inverted Repeat, TIR), but also a novel type of non-TIR MULEs. Some of this latter type of elements was found to be active both transcriptionally and transpositionally. To understand host-mediated genome-wide regulation(s) on the MULE system in Arabidopsis, we characterized 235 MULE mobility-specific genes (or mudrA-like genes) by mapping them on the sequenced Arabidopsis chromosomes and performing a genome-wide expression assay utilizing Arabidopsis METHYLTRANSFERSE1 (MET1) mutant (met2) plants, we showed that MET1-mediated global CpG methylation can only repress a portion of the gene family; its efficiency depends largely on the gene locations within the context of Arabidopsis chromatin remodeling: stronger in heterochromatic regions but weaker in euchromatic ones. This finding suggests that the Arabidopsis heterochromatic regions are not just a graveyard for the accumulation of defective elements; rather, they may have been playing an important role on the repression of TE activity via, at least in part, exerting MET1-mediated silencing effect. Our expression analysis also suggested that a TIR structure is not necessarily required for the MET1-mediated si

A genome sequence can be conceptualized as a 'book' written with four nucleotide 'letters' in oligonucleotide (oligo) 'words'. These words can be used in genomics, bioinformatics and the development of molecular markers. The whole-genome sequence for rice (Oryza sativa L.) is almost finished and has been assembled into pseudomolecules. For barley ( Hordeum vulgare L.) expressed sequence tags (ESTs) have been assembled into 21,981 tentative consensus sequences (TCs). The availability of such sequence information provides opportunities to investigate oligo usage within and between genomes. For the first of three studies reported in this thesis, a C++ program was written to automatically design oligos that are conserved between two sets of sequence information. In silico mapping between rice coding sequences (CDS) and barley TCs indicated that oligos between 18 and 24 bp provide good specificity and sensitivity (83% and 86%, respectively, for 20mers). Conserved oligos used as PCR primers had a high (91%) success rate on barley lines. Sequencing of PCR products revealed conservation in exon sequence, size and order between barley and rice. Introns were not conserved in sequence but were relatively stable in size. Map locations of eight new markers in barley revealed both genome colinearity and rearrangements between barley and rice. The second study reported in this thesis examined word frequency within the rice genome. A non-random landscape composed of high-frequency and low-frequency zones was observed. Interestingly, high-frequency words seemed to be rice specific while single-copy words were gene specific and conserved across species. As in the first study, oligos of 12 bp or less were not specific, and 18 bp seemed to be a critical length for the specificity of oligos. The third study reported in this thesis involved the development of molecular markers for known genes using public sequence information. Six new polymorphic markers were d

Includes bibliographical references (leaves 201-225). / xiv, 225 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Mitochondrial control region and cytochrome b gene sequence comparisions were used to investigate the molecular systematics of pythons. / Thesis (Ph.D.)--Adelaide University, Dept. of Genetics, 2001

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