Browsing by Subject "Next generation sequencing"

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    B-G gene structure, genetic variation and expression in the turkey (Meleagris gallopavo) major histocompatibility complex
    (2013-02) Bauer, Miranda Marie
    The major histocompatibility complex (MHC) is a genomic region encoding for genes with various immune functions in vertebrates. Although this region has been studied extensively in humans, mice and agriculturally important species such as cattle, horses and chickens, the organization and functional significance of the turkey MHC is relatively unknown. One set of loci located in the avian MHC, designated as B-G genes, have been identified and sequenced within the B-locus of the turkey, chicken and quail, with additional B-G loci identified in the extended MHC of the latter two species. These genes are linked to the class I and class II loci of the MHC and also show regions of extreme polymorphism (Miller, 1984). The total number, function and significance of the B-G genes are yet unknown in any bird species. In turkeys, three B-G genes were previously sequenced within the B-locus (Chaves et al., 2009a), with evidence suggesting additional functional B-G genes located past the 5' ends of the sequenced MHC region. Evidence in the chicken shows differential expression of B-G genes in various immune tissues, which suggests potential immune function. This research used 454 FLX Next Generation Sequencing technology (Roche) for sequencing a bacterial artificial chromosome (BAC) and identified two additional B-G genes located past the 5' end of the core B-locus of the turkey. These genes were annotated using in silico analysis and show organization similar to those in the chicken and quail. Using this information, sequence variation of the B-G genes was compared in different stocks of turkeys. Because each of the three groups, (commercial, heritage and wild), have been selected for different characteristics, variation within these loci was expected. This experiment found variation among loci within the three groups of turkeys. Lastly, B-G gene expression was investigated with quantitative real-time PCR using liver tissue of aflatoxin challenged birds. A low level of expression was observed for three of the four BG genes investigated, with BG5 expression invariant in all individuals across treatment groups. BG4 expression levels fluctuated within and between groups and a higher level of expression was measured in the lactobacillus + aflatoxin group. This work extended the turkey B-locus sequence past the homologous region in the chicken and marks the first examination of sequence variation and gene expression of multiple B-G genes in any species. Sequence variation and expression differences among loci support a hypothesis of distinct functions for these molecules.
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    Genomic analysis of regulatory mechanisms involved in secondary metabolite production.
    (2010-09) Castro-Melchor, Marlene
    Many secondary metabolites have beneficial uses for humans. In addition to their use as antibacterial and antifungal agents, secondary metabolites have been used as immunosuppressants, anti-tumor agents, and antiparasitics. Most of the secondary metabolites known today are produced by filamentous fungi or by members of the Streptomyces genus. Production of secondary metabolites by microorganisms involves a complex, dynamic system, with interconnected elements acting at different levels. Diverse tools were used in this work to explore regulation of secondary metabolite production, mostly in Streptomyces. The tools have a common characteristic: they either generate large amounts of data, or require large amounts of data. Regulation of secondary metabolite production in Streptomyces coelicolor was analyzed at the genome level, by using network modules inferred from a large transcriptome dataset. The upstream sequence of the elements in the network modules was searched for the presence of consensus sequences, and these results combined with information on known interactions, binding sites, and functional relatedness. The combination of this information resulted in a set of twenty networks that have a high likelihood of representing true interactions and represent a starting point for further experimental studies. The characteristics of high productivity were analyzed by comparing the genomes of two strains of the clavulanic acid producer Streptomyces clavuligerus. One of the strains is a high producer of clavulanic acid. Next generation sequence data was used to perform a genome-wide screening to identify all the differences between the two genomes. In addition to mutations in genes involved in β-lactam antibiotic production or their upstream region, structural differences were detected between the two strains. Next generation sequencing technologies were also used to assemble a draft genome for the curdlan producer Agrobacterium sp. ATCC 31749. Curdlan production mimics that of secondary metabolites, it is triggered under starvation conditions. These varied approaches exemplify some of the paths that can lead to a better understanding of secondary metabolism and its regulation.