<i>Pyrenophora teres</i> Drechs. (anamorph: <i>Drechslera teres</i> (Sacc.) Shoem.) is the causal agent of barley net blotch. Net blotch is an economically important disease commonly found throughout the barley producing regions of the world. Significant financial losses result from yield reductions, ranging from 15-35%, and decreased grain quality. Despite its prevalence, it is unclear if the P. teres-barley pathosystem follows a gene-for-gene model, and more generally, little is known about its genetic organization. Three studies were initiated to address these questions.<p>The first study investigated the genetic control of avirulence in <i>P. teres.</i> To establish an appropriate study system, a collection of ten net form (<i>P. teres f. teres</i>) and spot form (<i>P. teres f. maculata</i>) isolates were evaluated on a set of eight differential barley lines to identify two isolates with differential virulence on a specific host line. WRS 1906, exhibiting low virulence on the cultivar Heartland, and WRS 1607, exhibiting high virulence, were mated and 67 progeny were isolated and phenotyped for virulence on Heartland. The population segregated in a 1:1 ratio, 34 avirulent to 33 virulent (Chi-square = 0.0, P = 1.0), indicating single gene control of WRS 1906 avirulence on Heartland. Bulked segregant analysis was used to identify six amplified fragment length polymorphism (AFLP) markers closely linked to the avirulence gene (AvrHeartland). This work provides evidence that the P. teres-barley pathosystem conforms to the gene-for-gene model.<p>In the second study, five isolates of P. teres, representing both net and spot forms, were analyzed by the germ tube burst method (GTBM) and pulsed field gel electrophoresis (PFGE) to determine the species karyotype. Nine chromosomes were observed in all isolates using the GTBM and estimation of chromosome lengths varied from 0.5 to 3.0 µm. PFGE separated 7 to 8 bands depending on isolate, but analysis of bands by densitometry indicated nine chromosomes. Chromosome size ranged from 1.8 to ~6.0 Mb providing a genome size estimate of 32 to 39 Mb. Significant chromosome-length polymorphisms (CLP) were observed between isolates. These CLP did not hinder mating between mating-type compatible net form isolates. No particular CLP or individual chromosome could be associated with differences in disease symptoms observed between pathogen forms. This study provides the first karyotype of both P. teres forms and will assist genetic mapping of this pathogen.<p>A genetic linkage map of <i>P. teres f. teres</i>, was constructed in the third study using the population of 67 progeny derived from the WRS 1906  WRS 1607 cross. The map consists of 138 markers including 114 AFLPs, 21 telomere RFLPs, the mating-type (MAT) locus and an avirulence locus (AvrHeartland) controlling interaction with barley cultivar Heartland. Markers were distributed across 24 linkage groups ranging in length from 2 to 110 cM with an average marker interval of 8.5 cM. The total map length was 797 cM. A telomere-specific probe, consisting of the sequence (TTAGGC)4, was used to map 15 of the 18 telomeres. One of these telomeres mapped to within 3 cM of the AvrHeartland locus. Attempts to consolidate linkage groups by hybridizing markers to the electrophoretically separated chromosomes was unsuccessful because probes bound to multiple chromosomes, likely due to repetitive DNA within the probe. This is the first genetic map reported for this species and it will be a useful genetic tool for map-based cloning of the AvrHeartland gene tagged in this study. <p>This research has provided a number of new insights into the net blotch pathogen and provides a useful research tool in the form of a genetic map. This information lays the foundation for further genetic study of P. teres and will complement studies on barley resistance to net blotch that may potentially lead to more durable resistance.

An increasing body of literature shows that genomes of eukaryotes can contain clusters of functionally related genes. Most approaches to identify gene clusters utilize microarray data or metabolic pathway databases to find groups of genes on chromo- somes that are linked by common attributes. A generalized method that can find gene clusters, regardless of the mechanism of origin, would provide researchers with an unbiased method for finding clusters and studying the evolutionary forces that give rise to them. I present a basis of algorithm to identify gene clusters in eukaryotic genomes that utilizes functional categories defined in graph-based vocabularies such as the Gene Ontology (GO). Clusters identified in this manner need only have a common function and are not constrained by gene expression or other properties. I tested the algorithm by analyzing genomes of a representative set of species. I identified species specific variation in percentage of clustered genes as well as in properties of gene clusters, including size distribution and functional annotation. These properties may be diagnostic of the evolutionary forces that lead to the formation of gene clusters. The approach finds all gene clusters in the data set and ranks them by their likelihood of occurrence by chance. The method successfully identified clusters.

The hsDC2, an unknown gene, was located on chromosome 4q25. Its genetic protein product contains 149 amino acids with the molecular weight of 16.8 kDa approximately. Predicted by bioinformatics, hsDC2 might be a transmembrane protein with three transmembrane helices on the endoplasmic reticulum. Based on the results of reverse transcription-polymerase chain reaction, it revealed that hsDC2 was expressed in many tissues. It was showed that there were more mRNA expression in the cancer tissue. Using real-time quantitative PCR to analyze cancer cell lines, we found that hsDC2 might be related to differentiation status of cells. Among the well-differentiated cells such as nasopharyngeal carcinoma cell line NPC TW01, hepatoma cell line Hep3B and temperature-induced differentiated human fetal osteoblast cell line (hFOB), there were more hsDC2 mRNA expression. We also obtained some useful information from bioinformatics databases. To further elucidate the biological functions of the gene, glutathion S-transferase-hsDC2 fusion protein was used to generate anti-hsDC2 polyclonal antibody. However, we still need further research to clarify the biological function of hsDC2.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 81-84).

Non-viral vectors have been used to deliver therapeutic genes to treat different diseases. There are a variety of non-viral vectors such as liposomes, cationic polymers and peptides. Among all, pH responsive peptides showed excellent DNA transfection efficiency in many types of cell. These peptides are capable of changing their structural conformation as pH decreases, adopting a disordered structure which can destabilize endosomal membrane and therefore enhancing the release of DNA from endosomes into cytosol. Traditional pH responsive histidine-rich peptides showed good DNA transfection efficiency and low toxicity to the cells when compared with other non-viral vectors. However, their low pKa value restricted these peptides to be protonated only at late endosomal stage, in which DNA is extremely susceptible to endosomal degradation. This hindered the DNA to be released to the cytosol efficiently and therefore reduced DNA transfection efficiency. In response to this, it is of great interest to probe into the insertion of either 2,3-diaminopropionic acid (Dap) or methylated-2,3-diaminopropionic acid Dap(Me) to the peptide as alternative pH sensitive components. The pKa values for both Dap and Dap(Me) peptides are higher than that of histidine. It is anticipated that the higher pKa value, the protonation of peptide could be happened at an earlier stage of endosomal maturation. Such protonation of peptide destabilizes the endosome membrane rapidly, causing the release of DNA to the cytosol effectively and hence improving DNA transfection efficiency. In this experiment, LADap(Me)4-L1 peptide was the optimal candidate within the series. It showed good DNA transfection efficiency and cell viability in A549 cells among all Dap and Dap(Me) peptides. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Carotenoids are organic pigments that are mainly found in the chloroplasts and chromoplasts of plants and other photosynthetic organisms. Carotenoid molecules containing oxygen, such as lutein, violaxanthin and zeaxanthin are called xanthophylls and the rest containing un-oxygenated carotenoids are known as carotenes. Carotenoids form the integral part of the photosystem II LHC. Xanthophylls mainly aid in light harvesting and dissipation of harmful excess energy from excited chlorophyll molecules, thereby protecting chlorophyll from photo-degradation. The biosynthesis of carotenoids has been widely studied using plant and algae. However, the regulatory mechanisms involved in carotenoid metabolism need better understanding. This thesis identified novel regulatory mechanisms involved in the carotenoid biosynthetic pathway using activation-tagged Arabidopsis mutants. Two screening methods, red seed coat screening and norflurazon resistance screening, were used in this study. Fourteen mutants were screened using red seed coat screening but a successful mutant characterization could not be performed due to the unavailability of mutants with a single copy T-DNA insertion. Norflurazon screening identified eight mutants, out of which two mutants, KN203 and KN231, were characterized. The KN203 mutant had a defective keto-acyl CoA synthase 19 gene. KN203 mutant had lower carotenoid levels in the leaves and increased carotenoid levels in the mature seeds; the mutant was able to revert back to wild type phenotype after complementation of a functional KCS19 gene copy driven by native promoter. The fatty acid analysis indicated that the mutant KN203 had decreased MGDG and increased lysoPG, lysoPC and lysoPE content. Reduced carotenoid content in KN203 leaves was attributed to changes in fatty acid composition of chloroplast envelope membrane. Mutant KN231 had a T-DNA insertion in a gene encoding a RNA binding protein (RBP47C). KN231 leaf carotenoid levels were similar to wild type but their levels were significantly higher in their seeds. Two allelic mutants were selected to characterize the mutants. Overexpression of functional RBP47C in the mutants reverted to wild type phenotype in some overexpression mutants. A tandem repeat homologue of RBP47C, RBP47C’was identified. In-silico analysis predicted RBP47C to be a potential candidate for chloroplast localization.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 5 uppsatser.

Thesis (Ph.D.)--Baylor University, 2006. / Includes bibliographical references (p. 100-115).

Gene expression quantitative trait loci (eQTL) mapping studies can provide mechanistic insights into the functions of disease-associated variants. However, many eQTLs are cell type and context specific. This is particularly relevant for immune cells, whose cellular function and behaviour can be substantially altered by external cues. Furthermore, understanding mechanisms behind eQTLs is hindered by the difficulty of identifying causal variants. We differentiated macrophages from induced pluripotent stem cells from 86 unrelated, healthy individuals derived as part of the Human Induced Pluripotent Stem Cells Initiative. We generated RNA-seq data from these cells in four experimental conditions: naïve, interferon- gamma (IFNɣ) treatment (18h), Salmonella infection (5h), and IFNγ treatment followed by Salmonella infection. We also measured chromatin accessibility with ATAC-seq in 31-42 individuals in the same four conditions. We detected gene expression QTLs (eQTLs) for 4326 genes, over 900 of which were condition-specific. We also detected a similar number of transcript ratio QTLs (trQTLs) that influenced mRNA processing and alternative splicing. Macrophage eQTLs and trQTLs were enriched for variants associated with Alzheimer’s disease, multiple autoimmune disorders and lipid traits. We also detected chromatin accessibility QTLs (caQTLs) for 14,602 accessible regions, including hundreds of long-range interactions. Joint analysis of eQTLs with caQTLs allowed us to greatly reduce the set of credible causal variants, often pinpointing to a single most likely variant. We found that caQTLs were less condition- specific than eQTLs and ~50% of the stimulation-specific eQTLs manifested on the chromatin level already in the naive cells. These observations might help to explain the discrepancy between strong enrichment of diseases associations in regulatory elements but only modest overlap with current eQTL studies, suggesting that many regulatory elements are in a ‘primed’ state waiting for an appropriate environmental signal before regulating gene expression.

Abstract The progress in recombinant DNA technology has made possible the introduction of exogenous genetic material into cells. Gene therapy aims to correct a defective gene, introduce a therapeutic exogenous gene or a counteracting gene into somatic cells without modification of the germ-cell line. The most important technical interests in the field of gene therapy research have pertained to the development of safe and effective vectors and suitable methods for the delivery of the exogenous gene carrying vectors into the target cells. The aim of this study was to evaluate surgical methods used for gene delivery and to develop an effective gene transfer method for organ-specific gene transfer, primarily into the renal glomeruli. There are genetic and acquired diseases that are candidates for gene therapy. Alport syndrome is an X-chromosome-linked disease caused by a mutation in the type IV collagen α5 chain gene, which causes a defect of the glomerular basement membrane in the kidney, leading to progressive renal failure in males. This manifestation could theoretically be prevented by the transfer of a normal α5 chain gene into the renal glomerular cells. Cystic fibrosis and α1-antitrypsin deficiency are examples of pulmonary diseases and genetic lysosomal storage diseases that are candidates for splenic gene transfer. The gene transfer strategies used so far have proved relatively ineffective. Recombinant adenovirus, retrovirus, adeno-associated virus and liposomes have been previously used as vectors. Direct injection, intra-arterial, intravenous and intratracheal delivery of vectors have been the most extensively studied methods. This preclinical experimental work for marker gene transfer into the kidney, spleen, lung and mammary gland was done by using rabbits, pigs and goats as test animals. The adenoviral vector carrying a β-galactosidase reporter gene was first infused in the renal artery of rabbits and pigs in vivo with or without pharmacological agents. This did not result in any remarkable gene transfer into the kidney. Next, the incubation time between the vector and the target cells was prolonged by ex vivo perfusion of explanted kidneys for 12 hours. Perfusion at room temperature did not improve gene transfer. When the perfusion temperature was raised to 37°C, improved and mostly glomerular gene transfer was observed, with up to 80% of the glomeruli showing β-galactosidase expression in four ex vivo experiments. A closed-circuit organ perfusion method for in vivo gene transfer was developed in this study. The surgical perfusion experiment was tested successfully in ten in vivo perfusions of the kidney, eight of the spleen and eight of the lung in a porcine model. This method led to effective, up to 75% gene transfer into the renal glomeruli as assessed after four days. In the spleen, the perfusion method resulted in relatively effective gene transfer into perifollicular splenic cells, mostly macrophages and endothelial cells. Lung perfusion yielded transgene expression in alveolar epithelial cells, bronchiolar epithelial cells and, to a lesser extent, arteriolar endothelial cells and alveolar macrophages. Perfusion of the goats mammary gland using a retroviral vector in three experiments resulted in growth hormone secretion into the milk. The gene transfer operation was well tolerated by the animals, and no clinical signs of inflammation were observed. No remarkable humoral immunological response against adenovirus or β-galactosidase was elicited in the kidney experiments, but histological signs of inflammation as mononuclear cell clusters in the kidney and lung were seen four and seven days after the experiments. The spleen showed no macroscopic or microscopic pathologic alterations after the perfusion.