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Background Wheat ( Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes. Results Using TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry. Conclusions We report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents.

Natural product biosynthesis is heavily influenced by external stimuli; therefore, at any given time the host may contain only a subset of the products that these organisms can biosynthesize. Consequently, compounds isolated from a native source may not be a full collection of the organism's biosynthetic capacity. Genome mining offers the first comprehensive approach to investigating natural product biosynthesis. Genomic sequence provides the ability to obtain, for our purposes, all of the triterpene synthases (oxidosqualene cyclases, OSCs) that exist in a given plant. The origins of triterpenoid skeletal diversity were explored by characterizing OSCs through heterologous expression in the yeast Saccharomyces cerevisiae . The work of this thesis utilized two approaches to increase OSC substrate accumulation and production in Saccharomyces through genetic engineering to increase substrate concentration for heterologous expression of OSCs. Metabolic engineering efforts focused on modifying Saccharomyces to accumulate oxidosqualene (OS) and dioxidosqualene (DOS), the precursors for triterpene biosynthesis. A particularly useful aspect of this work is that several cyclases that showed little to no activity in vitro are quite active in these hosts, which has allowed us to characterize several genes that were previously thought not to encode OSCs. The generated strains facilitated the second aspect of this thesis, which characterized several OSCs from Arabidopsis thaliana, Sorghum bicolor, Lactuca sativa and Lactuca serriola. Genome-mining approaches have uncovered new triterpene skeletons, broadened the metabolic profile of several plants, and illuminated evolutionary relationships between these genes.

Here we provide evidence that LC-MS is useful for the quantification of a transgenic protein that has high identity (89%) with its native homolog. The sequences are merely punctuated by single amino acid differences that we believe are best observed using LC-MS.

Retinoid-related orphan receptor gamma (RORγ) is a lineage-defining transcription factor for T helper 17 (Th17) cells and has long been considered selectively expressed within the immune system. However, accumulating evidence has implicated Th17 cells and other RORγ-expressing immune populations in embryonic brain development and central nervous system (CNS) function. Notably, RORγ-Cre-mediated deletion of the tuberous sclerosis complex (TSC) has been reported to cause spontaneous seizures and premature lethality, leading to the conclusion that RORγ-expressing immune cells directly regulate CNS physiology. Using multiple RORγ-based reporter mouse lines, we identified unexpected and widespread neuronal labeling throughout the forebrain and cerebellum. RORγ-Cre:GFP mice exhibited robust GFP expression in neurons despite the absence of detectable T cells in the brain. Neuronal recombination was evident prenatally and independently validated using the mT/mG reporter line, indicating transient RORγ expression in embryonic neurons. In contrast, a RORγ-GFP reporter showed no detectable RORγ expression in the postnatal brain, either at baseline or following pilocarpine-induced status epilepticus. These findings demonstrate that RORγ expression is not restricted to the immune lineage and reveal previously unrecognized developmental expression in the embryonic brain. Consequently, seizures observed following RORγ-Cre-mediated deletion of TSC1 are unlikely to arise from RORγ-expressing immune cell-dependent mechanisms but instead reflect direct neuronal loss of TSC1. Our results call for careful reinterpretation of prior studies attributing CNS phenotypes to RORγ-expressing immune cells based on RORγ-Cre-driven genetic models.