Genes encoding ovine insulin like growth factor-I (oIGF-D), -II (oIGF-II) and insulin were cloned, sequenced and characterized. The olGF-I gene contains six exons spanning greater than 30 kilobases. Class 1 and class 2 oIGF-I transcripts contained exons 1 and 2 alternatively-spliced to exon 3, respectively. A novel oIGF-I exon (W) was located upstream of exon 1 and was found alternatively spliced to exon 3. No in-frame methionine codon was found in exon W and therefore translation is proposed to initiate at the methionine codon present in exon 3. Using primer extension, the ovine transcription initiation sites were mapped and found to be well conserved among mammalian and avian IGF-I genes. Expression of exon 1-, 2- and W-specific transcripts was examined in seven tissues from adult or fetal sheep using a reverse transcription-polymerase chain reaction (RT-PCR) assay. Exon 1 transcripts were the most abundant and found in all fetal and adult tissues. Exon 2 transcripts were found in all tissues and in general showed the highest expression in adult liver. Exon W transcripts were expressed at low levels in all tissues examined. To confirm that exon W mRNA produced biologically active IGF-I, an exon W containing cDNA was cloned under the control of a glucocorticoid-inducible MMTV promoter (pMMTV-IGF-IW) and transfected into a bovine mammary epithelial cell line (MAC-T). Stable transfectants were induced with a synthetic glucocorticoid to produce secretable IGF-I. Transcript expression of pMMTV-IGF-IW was confirmed by Northern blot analysis and IGF-I was quantified in the medium of growing cells with RIA. Biological activity of the secreted IGF-I was assayed by measuring the incorporation of [³H]-thymidine into DNA of test MAC-T cells. Media harvested from the pMMTV-IGF-IW transfected clones stimulated labeling of MAC-T cells greater than that of conditioned media from MAC-T cells. Thus, biologically active IGF-I was secreted from pMMTV-IGF-IW cells. The oIGF-II gene is composed of 9 exons that span approximately 25 kilobases. Approximately 750 nucleotides upstream of oJGF-II exon 1 are the three exons of the ovine insulin gene which are transcribed in the same direction as oIGF-II. Four putative promoters direct transcription of six 5’ non-coding exons (1, 3, 4, 5, 6, and 7), which are alternatively spliced to exons 8, 9, and 10. An ovine exon comparable to human exon 2 has not been identified. Multiple transcription initiation sites were identified for exons 1 and 6 by primer extension analysis. Using a RT-PCR assay, exon | and 3 transcripts were shown to be expressed in adult but not fetal liver. In addition, a novel transcript that contained exon 1 spliced directly to exon 8 was detected in adult liver. Exon 4 transcripts were not detected, whereas exons 5, 6 and 7 transcripts were detected in both fetal and adult liver. Like the human and rodent genes, the regulation of expression of the oIGF-I and oIGF-II genes are under complex control. / Ph. D.

Ten winter wheat (Triticum aestivum L.) lines selected from the 1982 International Winter Wheat Mildew and Rust Nurseries were studied to characterize gene number and mode of inheritance of powdery mildew resistance. Two experiments were conducted: 1) each of the lines was crossed to the susceptible cultivar Chancellor, which lacks any known’ gene _ for resistance, and seedlings of the parental lines, F₁, F₂, BC₁ (Chancellor X F₁), and F₃ populations were inoculated with isolate 127 of Blumeria graminis (DC.) E. 0. Speer f. sp. tritici Em. Marchal in the greenhouse and evaluated for powdery mildew reaction; 2) the ten lines were crossed with each other and to each of 13 host differential lines with known genes for powdery mildew resistance, and 300 to 800 F₂ seedlings from each cross were evaluated. All parents were resistant (Infection Type = 1-3), except for ST1-25, which had an intermediate (IT = 4-5) reaction type. Genetic analyses of crosses revealed that the resistance in C39 and SI5 is conferred by three dominant genes (Pm2, 4b, and 6), and resistance in A55-2, R107, and Bulk PV63-6 is governed by one partially dominant gene (Pm4b). Results from F₂, F₃, and BC₁ populations derived from crosses between ‘Armada’ and Chancellor, were inconsistent, but indicated that Armada has at least one dominant gene for resistance, which likely is Pm4b as suggested by others. The resistance gene in OK75R3645 most likely is an allele at the Pm3 locus, and it is probable that the resistance gene in GO4779 is Pm1. Single recessive genes were identified in VPM1 (Pm4b) and ST1-25 (Pm8). / Ph. D.

Past studies of mammary tissue specific expression of transgenes using the murine whey acidic protein (WAP) promoter have shown widely variable, position-dependent and copy number-dependent expression. This study evaluates a series of three WAP transgenes containing the cDNA of human protein C (hPC) for the expression of human protein C in the milk of mice. In two of the transgenes studied, the cDNA of (hPC) was inserted at the translational start site of a 7.8 kbp mouse WAP genomic DNA Eco RI fragment containing 2.6 kbp of 5’ flanking, 3.9 kbp WAP coding (exons and introns), and 1.3 kbp 3’ untranslated region (UTR) and flanking sequences (designated WAPPC1 and WAPPC2). A third transgene consisted of only the 2.6 kbp of WAP 5’ UTR and flanking DNA, 1.4 kbp hPC cDNA, and 1.3 kbp of 3’ WAP UTR and flanking DNA with no linker sequences (designated WAPPC3). The WAPPC1 and WAPPC2 transgenes expressed up to about 10 μg/ml recombinant hPC in mouse milk while WAPPC3 expressed 30-300 (n=10, n=5, n=11, number of founder lines evaluated for each transgene, respectively). In contrast to past studies with WAP-cDNA fusion transgenes where the maximal expression was about 5% of endogenous WAP expression, the WAPPC3 transgene gave maximal expression which was about 30% of endogenous WAP expression. Thus, results from the combination in WAPPC3 of intact 5’ and 3’ WAP UTR with the cDNA of hPC suggests that introns are not necessary to enable high level expression in the mammary gland when using WAP regulatory elements. Relative specific transcript and protein levels in the transgenic animals studied suggest that the rates of translation initiation may be different for the mRNAs of each of the transgenes studied. / Ph. D.

No description available.

Leung Hau Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Thesis Committee --- p.in / Acknowledgement --- p.II / Abstract --- p.III / 摘要 --- p.V / Table of Contents --- p.VII / List of Figures --- p.X / List of Tables --- p.XIII / Appendix Abbreviations --- p.XIV / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Breast Cancer --- p.1 / Chapter 1.2 --- Development of Breast Cancer --- p.2 / Chapter 1.3 --- Risk Factors of Breast Cancer --- p.3 / Chapter 1.3.1 --- Age --- p.3 / Chapter 1.3.2 --- Genetic Factors --- p.4 / Chapter 1.3.3 --- Hormonal Factors --- p.5 / Chapter 1.3.4 --- Lifestyles --- p.6 / Chapter 1.4 --- Drug Metabolism --- p.6 / Chapter 1.5 --- UGT1A1 --- p.7 / Chapter 1.5.1 --- UDP-glucuronosyltransferase --- p.7 / Chapter 1.5.2 --- UGT1A1 --- p.9 / Chapter 1.6 --- Cytochrome P450 I Enzyme Family --- p.10 / Chapter 1.6.1 --- CYP450 subfamily --- p.10 / Chapter 1.6.2 --- CYP1A1 --- p.11 / Chapter 1.6.3 --- CYP1B1 --- p.12 / Chapter 1.7 --- Reasons why UGT1A1 is being studied --- p.13 / Chapter 1.8 --- Outline of this Study --- p.14 / Chapter 1.8.1 --- Effects of Over-expressing UDP-Glucuronpsyltransferase and Cytochrome P450 1A1 Against Xenobiotic Assault in Breast Cancer Cells --- p.14 / Chapter 1.8.2 --- Effects of Genistein and Resveratrol on Phase I and II Enzymes in a Non-cancerous Breast Cell Line --- p.15 / Chapter 1.8.3 --- Effects of UGT1A1 on Cancer Drug Treatment --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.16 / Chapter 2.1 --- Chemicals --- p.16 / Chapter 2.2 --- Cell Culture --- p.16 / Chapter 2.2.1 --- Maintenance --- p.16 / Chapter 2.2.2 --- Preparation of Cell Stock --- p.17 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.17 / Chapter 2.3 --- Cloning and Transfection --- p.18 / Chapter 2.3.1 --- Isolation of RNA from cells and cDNA synthesis --- p.18 / Chapter 2.3.2 --- Amplification of UGTlAl --- p.20 / Chapter 2.3.3 --- Separation and Purification of DNA from Agarose Gel --- p.21 / Chapter 2.3.4 --- Restriction Digestion --- p.22 / Chapter 2.3.5 --- Ligation of DNA Fragment and Vector --- p.22 / Chapter 2.3.6 --- Transformation of DH5a --- p.23 / Chapter 2.3.7 --- Small Scale Plasmid Purification (Miniprep) --- p.24 / Chapter 2.3.8 --- Large Scale Plasmid Purification (Maxiprep) --- p.25 / Chapter 2.3.9 --- Stable Transfection into MCF-7 cells with LipofectAMINE PLUS reagent --- p.26 / Chapter 2.4 --- Analytical Procedures --- p.27 / Chapter 2.4.1 --- Western Blot Analysis --- p.27 / Chapter 2.4.2 --- Measurement of cell proliferation (MTT assay) --- p.28 / Chapter 2.4.3 --- Measurement of DMBA-DNA Adduct Formation --- p.28 / Chapter 2.4.4 --- Comet Assay --- p.29 / Chapter 2.4.5 --- Relative Quantitative Real Time PCR --- p.30 / Chapter 2.4.5.1 --- Real Time PCR Using TaqMan Probe --- p.30 / Chapter 2.4.5.2 --- Statistical Analysis of 2-ΔΔCT Comparative Gene Expression --- p.31 / Chapter 2.4.6 --- Flow Cytometry --- p.31 / Chapter 2.4.7 --- EROD Activity in Intact Cells --- p.31 / Chapter 2.4.8 --- High Performance Liquid Chromatography --- p.32 / Chapter 2.5 --- Statistical Analysis --- p.34 / Chapter Chapter 3 --- Effects of Over-Expressing UDP-GIucuronosyltransferase and Cytochrome P450 1A1 Against Xenobiotic Assault in Breast Cancer Cells --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Results --- p.38 / Chapter 3.2.1 --- Effectiveness of Transfection --- p.38 / Chapter 3.2.2 --- Cell Proliferation Experiments --- p.41 / Chapter 3.2.3 --- Regulation of Estrogen Receptor (ER) Expression --- p.43 / Chapter 3.2.4 --- Formation of DMBA-DNA adduct formation --- p.45 / Chapter 3.2.5 --- Single Cell Gel Electrophoresis (Comet Assay) of DMBA-induced DNA Damage in MCF-7UGT1A1 cells --- p.46 / Chapter 3.2.6 --- HPLC for Estradiol-glucuronidation Analysis --- p.49 / Chapter 3.2.7 --- Single Cell Gel Electrophoresis (Comet Assay) of DMBA or E2-induced DNA Damage in MCF-7cyp1A1 cells --- p.51 / Chapter 3.3 --- Discussion --- p.56 / Chapter Chapter 4 --- Effects of Genistein and Resveratrol on Phase I and II Enzymes in a Non-Cancerous Breast Cell Line --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Results --- p.66 / Chapter 4.2.1 --- "Genistein and Resveratrol Reduced DMBA-induced UGT1A1, CYP1A1 and CYP1B1 Expression" --- p.66 / Chapter 4.2.2 --- Genistein and Resveratrol Reduced the Formation of DMBA-DNA Adduct in MCF-10A Cells --- p.73 / Chapter 4.2.3 --- Genistein and Resveratrol Reduced the Single Strand DNA Damage Generated by DMBA in MCF-10A Cells --- p.76 / Chapter 4.2.4 --- Genistein and Resveratrol Reduced DMBA-induced EROD Activities --- p.81 / Chapter 4.3 --- Discussion --- p.84 / Chapter Chapter 5 --- Effects of Ugtlal on Cancer Drug Treatment --- p.89 / Chapter 5.1 --- Introduction --- p.89 / Chapter 5.2 --- Results --- p.93 / Chapter 5.2.1 --- Cell Proliferation Experiment --- p.93 / Chapter 5.2.2 --- "Expression of Bcl-2 and Bax proteins in Paclitaxel- or VCR-treated MCF-7, MCF-7control and MCF-7UGt1A1 cells" --- p.98 / Chapter 5.2.3 --- Flow Cytometric Analysis of Cell Cycle Phase Distributionin Paclitaxel- or VCR-treated MCF-7 cells --- p.103 / Chapter 5.3 --- Discussion --- p.110 / Chapter Chapter 6 --- Summary --- p.114 / Bibliography --- p.116

Bai Hsing Chen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 89-94). / Abstracts in English and Chinese. / Acknowledgments --- p.ii / Publication --- p.iii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Abstract --- p.vii / 摘要 --- p.x / Table of Contents --- p.xii / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- Molecular Biology in Cancer Development --- p.2 / Chapter 1.1.1 --- Cell Cycle and Cancer --- p.2 / Chapter 1.1.2 --- Oncogenes and Tumor Suppressor Genes --- p.4 / Chapter 1.1.3 --- Epigenetic Alteration in Tumor Cells --- p.6 / Chapter 1.2 --- Colorectal Cancer Development --- p.9 / Chapter 1.2.1 --- Epidemiology of Colorectal Cancer --- p.9 / Chapter 1.2.2 --- Adenoma-Carcinoma Sequence --- p.11 / Chapter 1.2.2.1 --- Hyperplastic (metaplastic) Polyps --- p.11 / Chapter 1.2.2.2 --- Aberrant Crypt Foci (ACF) --- p.13 / Chapter 1.2.2.3 --- Adenomas --- p.13 / Chapter 1.2.2.4 --- Serrated adenomas --- p.15 / Chapter 1.2.2.5 --- Colorectal Carcinomas --- p.16 / Chapter 1.2.3 --- Genetic alterations in CRC --- p.18 / Chapter 1.2.4 --- Epigenetic alterations in CRC --- p.21 / Chapter 1.2.5 --- Staging of Colorectal Cancer --- p.23 / Chapter 1.3 --- Hypothesis --- p.25 / Chapter 1.4 --- Aim of Study --- p.26 / Chapter Chapter 2 --- MATERIALS and METHODES / Chapter 2.1 --- Patient Populations --- p.28 / Chapter 2.2 --- Microdissection and Immunohistochemistry --- p.29 / Chapter 2.3 --- DNA Isolation and Modification --- p.31 / Chapter 2.3.1 --- DNA Extraction from Microdissected Tissues --- p.31 / Chapter 2.3.2 --- DNA Extraction from Frozen Biopsy --- p.31 / Chapter 2.3.3 --- Bisulfite Modification of DNA --- p.32 / Chapter 2.4 --- Detection of K-ras Mutation --- p.33 / Chapter 2.5 --- Methylation-specific PCR (MSP) --- p.36 / Chapter 2.6 --- Bisulfite DNA Sequencing --- p.42 / Chapter 2.7 --- Statistical analysis --- p.44 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- Promoter Hypermethylation of Tumor Related Genes in the Progression of Colorectal Neoplasia --- p.46 / Chapter 3.1.1 --- Clinico-Pathological parameters --- p.46 / Chapter 3.1.2 --- "Frequencies of Promoter Hypermethylation in Colorectal Cancers, Adenomas and Normal Colonic Tissues" --- p.47 / Chapter 3.1.3 --- Promoter Hypermethylation in Multiple Genes --- p.50 / Chapter 3.1.4 --- Promoter Hypermethylation in Advanced vs. Non-advanced Adenoma --- p.50 / Chapter 3.1.5 --- Methylation Patterns in Paired Adjacent Tissues from Cancer Patients --- p.53 / Chapter 3.1.6 --- Immunohistochemistry --- p.55 / Chapter 3.1.7 --- K-ras mutation --- p.61 / Chapter 3.1.8 --- Clinicopathological Correlations with Promoter Hypermethylation --- p.64 / Chapter 3.2 --- DNA Methylation Spread within HLTF CpG Island in Colorectal neoplasia --- p.67 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Methylation is an early event in Colorectal Carcinogenesis --- p.72 / Chapter 4.1.1 --- Methylation is frequently detected in both adenoma and carcinoma --- p.74 / Chapter 4.1.2 --- Concurrent methylation in multiple genes --- p.76 / Chapter 4.1.3 --- Methylation in advanced and non-advanced colorectal adenomas --- p.76 / Chapter 4.1.4 --- Relationship between K-ras mutation and methylation --- p.78 / Chapter 4.1.5 --- Methylation in adjacent tissues --- p.80 / Chapter 4.2 --- DNA Methylation Spread in HLTF gene --- p.81 / Chapter 4.2.1 --- HLTF is Frequently Methylated in Gastrointestinal Neoplasm --- p.82 / Chapter 4.2.2 --- Methylation Spread Patterns in Cancers and Adenomas --- p.83 / Chapter 4.2.3 --- Age Dependent Methylation Spread --- p.85 / Chapter Chapter 5 --- CONCLUSION --- p.87 / References --- p.89

Medulloblastoma (MB) and supratentorial primitive neuroectodermal tumor (stPNET) are pediatric embryonic brain tumors, which arise in a brain that is in the process of growth and development. They differ significantly from adult lesions and may involve unique genetic and epigenetic factors. However, the pathogenesis of these tumors is still elusive. My project consisted of four parts, investigating major genetic and epigenetic alterations of these tumors. / Multiple genetic studies have shown high frequency of loss (30--60%) on chromosome 8p in MBs. Microcell-mediated transfer of chromosome 8 suppressed tumorigenesis or the proliferation of colon and breast cancer cell, indicating that chromosome 8p is likely to include several TSGs in human cancers. In previous studies from our laboratory, results showed the frequency of loss on chromosome 8p is also rather high (66.7%). An overlapping HD region was identified in a 1.8cM interval on 8p22-23.1, between markers D8S520 and D8S1130, in two MBs (Yin et al., 2002), indicating that several candidate TSGs are located within or near this region. PinX1 on 8p23.1, a potential inhibitor of telomerase, is most likely the candidate TSG in MBs due to its location and function. To evaluate the genetic alterations of PinX1 and to investigate its role in MBs, the first part of my study is to perform mutation analysis in a series of 52 primary MBs, 3 MB cell lines and 4 primary stPNETs. Transcript expression of PinX1 was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected tumors and normal cerebellum. Using the telomeric repeat amplification protocol (TRAP) assay, 19 MBs, 2 stPNETs and all 3 MB cell lines were analyzed for telomerase activity. No somatic point mutations and loss of expression of PinX1 were detected in our series, suggesting that PinX1 is not the target gene on 8p23.1 in MBs. Although we did not find a significant association between PinX1 expression and telomerase activity, the presence of telomerase activity in 16 of 22 MBs and 1 of 2 stPNETs indicate that telomerase activation is associated with the development of this malignant disease. Our study represents the largest series of MB examined by telomerase repeat amplification protocol (TRAP) assay. (Abstract shortened by UMI.) / Chang Qing. / "April 2005." / Adviser: Ho-Keung Ng. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0191. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 201-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

[Truncated abstract] This thesis investigates novel methods for the genetic association analysis of haplotype data in samples of unrelated individuals, and applies these methods to the analysis of coronary heart disease and related phenotypes. Determining the inheritance pattern of genetic variants in studies of unrelated individuals can be problematic because family members of the studied individuals are often not available. For the analysis of individual genetic loci, no problem arises because the unit of interest is the observed genotype. When the unit of interest is the linear combination of alleles along one chromosome, inherited together in a haplotype, it is not always possible to determine with certainty the inheritance pattern, and therefore statistical methods to infer these patterns must be adopted. Due to genotypic heterozygosity, mutliple possible haplotype configurations can often resolve an individual's genotype measures at multiple loci. When haplotypes are not known, but are inferred statistically, an element of uncertainty is thus inherent which, if not dealt with appropriately, can result in unreliable estimates of effect sizes in an association setting. The core aim of the research described in this thesis was to develop and implement a general method for haplotype-based association analysis using multiple imputation to appropriately deal with uncertainty haplotype assignment. Regression-based approaches to association analysis provide flexible methods to investigate the influence of a covariate on a response variable, adjusting for the effects of other variables including interaction terms. ... These methods are then applied to models accommodating binary, quantitative, longitudinal and survival data. The performance of the multiple imputation method implemented was assessed using simulated data under a range of haplotypic effect sizes and genetic inheritance patterns. The multiple imputation approach performed better, on average, than ignoring haplotypic uncertainty, and provided estimates that in most cases were similar to those observed when haplotypes were known. The haplotype association methods developed in this thesis were used to investigate the genetic epidemiology of cardiovascular disease, utilising data for the cholesteryl ester transfer protein gene (CETP), the hepatic lipase (LIPC) gene and the 15- lipoxygenase (ALOX15) gene on a total of 6,487 individuals from three Western Australian studies. Results of these analyses suggested single nucleotide polymorphisms (SNPs) and haplotypes in the CETP gene were associated with increased plasma high-density lipoprotein cholesterol (HDL-C). SNPs in the LIPC gene were also associated with increased HDL-C and haplotypes in the ALOX15 gene were associated with risk of carotid plaque among individuals with premature CHD. The research presented in this thesis is both novel and important as it provides methods for the analysis of haplotypic associations with a range of response types, while incorporating information about haplotype uncertainty inherent in populationbased studies. These methods are shown to perform well for a range of simulated and real data situations, and have been written into a statistical analysis package that has been freely released to the research community.

Victoria blight, caused by fungus Cochliobolus victoriae, is a disease originally described on oats and recapitulated on Arabidopsis. Victoria blight is used as a model plant disease that conforms to an inverse gene-for-gene interaction. C. victoriae virulence is dependent upon its production of victorin, a host-specific toxin that induces programmed cell death in sensitive plants. In oats, victorin sensitivity and disease susceptibility is conferred by the Vb gene, which is genetically inseparable from the Pc-2 crown rust resistance gene. In Arabidopsis, victorin sensitivity and disease susceptibility is conferred by the LOCUS ORCHESTRATING VICTORIN EFFECTS 1 (LOV1) gene which encodes a NB-LRR protein, a type of protein commonly associated with disease resistance. LOV1-mediated cell death occurs when victorin binds Thioredoxin-h5 (TRX-h5) and LOV1 appears to "guards" TRX-h5. Together, these results suggest C. victoriae causes disease by inducing a resistance response. The work presented here aimed to determine if the response mediated by LOV1 is functionally related to a resistance response. We genetically characterized the response mediated by LOV1 with virus-induced gene silencing. We determined SUPPRESSOR OF THE G2 ALLELE OF SKP1 (SGT1), a gene required for the function of many resistance genes, is required for victorin sensitivity and involved in LOV1 protein accumulation. We screened a normalized library and identified six genes that suppressed victorin-mediated cell death and cell death induced by expression of the RESISTANCE TO PERONOSPORA PARASITICA PROTEIN 8 (RPP8) resistance gene: a mitochondrial phosphate transporter, glycolate oxidase, glutamine synthetase, glyceraldehyde 3-phosphate dehydrogenase and the P- and T-protein of the glycine decarboxylase complex. Silencing the latter four also inhibited cell death induced by the expression of an autoactive form of the resistance gene PTO, and reduced PTO-mediated resistance to Pseudomonas syringae pv. tabaci. These results provide evidence that victorin-mediated cell death is functionally similar to a resistance response, further supporting the hypothesis that a resistance response is exploited by C. victoriae for pathogenesis in Victoria blight. Resistance function of LOV1 was evaluated by observing Pseudomonas syringae pv. tomato virulence upon LOV1 activation. The LOV1 response pathway in Arabidopsis was adapted to activate upon infection with Pseudomonas syringae pv. tomato expressing the type III-dependent effector protein AvrRpt2, a well-characterized protease. We developed a construct to express a beta-glucuronidase (GUS) and TRX-h5 fusion protein separated by an AvrRpt2 proteolytic cleavage site, in which GUS sterically inhibits TRX-h5 function in LOV1-mediated cell death. The fusion is cleaved upon infection by P. syringae pv. tomato expressing avrRpt2, thereby leading to TRX-h5-mediated activation of LOV1 in the presence of victorin. However, when this strain was inoculated with victorin into transgenic LOV1 trx-h5 plants expressing the GUS/TRX-h5 fusion protein, no decrease in pathogen virulence was observed. Technical shortcomings likely prevented observable LOV1 resistance function. ��� / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Oct. 9, 2012 - Oct. 9, 2013

Les selenoproteïnes constitueixen una família diversa de proteïnes, caracteritzada per la presència del Seleni (Se), en forma de l'amino àcid atípic, la selenocisteïna (Sec). La selenocisteïna, coneguda com l'amino àcid 21, és similar a la cisteïna (Cys) amb un àtom de seleni en lloc de sofre (S). Les selenoproteïnes són els responsables majoritaris dels efectes biològics del seleni i s'ha observat que poden estar implicades en la infertilitat masculina, el càncer, algunes malalties coronàries,l'activació de virus latents i l'envelliment. La selenocisteïna es codifica pel codó UGA, normalment codó de parada (STOP). Per a la recodificació correcta del UGA són necessaris diversos factors. A la part 3' de la regió no traduïda (UTR) dels transcrits dels gens de selenoproteïnes en organismes eucariotes s'hi troba una estructura de "stem-loop" anomenada SECIS. La proteïna SBP2 interactua amb el SECIS, així com amb el ribosoma, i forma un complex amb el factor d'elongació EFsec i el tRNA de la selenocisteïna, el tRNASec. Donat que el codó TGA normalment significa fi de la traducció, les formes tradicionals de cerca de gens no el reconeixen com a codó codificant. Per aquesta raó ha estat necessari desenvolupar una metodologia específica per a la predicció de gens de selenoproteïnes. En els últims anys, hem contribuït a la descripció del selenoproteoma eucariota amb el descobriment de noves famílies (Castellano et al., 2005), amb l'elaboració de nous mètodes (Taskov et al., 2005; Chapple et al., 2009) i l'anotació de diferents genomes (Jaillon et al., 2004; Drosophila 12 genomes Consortium, 2007; Bovine Genome Sequencing and Analysis Consortium, 2009). Finalment, hem identificat el primer animal que no té selenoproteïnes (Drosophila 12 genomes Consortium, 2007; Chapple and Guigó, 2008), un descobriment soprenent donat que, fins el moment, es creia que les selenoproteïnes eren essencials per la vida animal. / Selenoproteins are a diverse family of proteins containing the trace element Selenium (Se)in the form of the non-canonical amino acid selenocysteine (Sec). Selenocysteine, the 21st amino acid, is similar to cysteine (Cys)but with Se replacing Sulphur. In many cases the homologous gene of a known selenoprotein is present with cysteine in the place of Sec in a different genome. Selenoproteins are believed to be the effectors of the biological functions of Selenium and have been implicated in male infertility, cancer and heart diseases, viral expression and ageing. Selenocysteine is coded by the opal STOP codon (TGA). A number of factors combine to achieve the co-translational recoding of TGA to Sec. The 3' Untranslated regions (UTRs) of eukaryotic selenoprotein transcripts contain a stem-loop structure called a Sec Insertion Sequence (SECIS) element. This is recognised by the Secis Binding Protein 2 (SBP2), which binds to both the SECIS element and the ribosome. SBP2, in turn, recruits the Sec-specific Elongation Factor EFsec, and the selenocysteine transfer RNA, tRNASec. The dual meaning of the TGA codon means that selenoprotein genes are often mispredicted by the standard annotation pipelines. The correct prediction of these genes, therefore, requires the development of specific methods. In the past few years we have contributed significally to the description of the eukaryotic selenoproteome2 with the discovery of novel families (Castellano et al., 2005), the elaboration of novel methods (Taskov et al., 2005; Chapple et al., 2009) and the annotation of different genomes (Jaillon et al., 2004; Drosophila 12 genomes Consortium, 2007; Bovine Genome Sequencing and Analysis Consortium, 2009). Finally, and perhaps most importantly, we have identified the first animal to lack selenoprotein genes (Drosophila 12 genomes Consortium, 2007; Chapple and Guigó, 2008). This last finding is particularly surprising because it had previously been believed that selenoproteins were essential for animal life.