Graduation date: 2002

Rat selenoprotein W (SeW) promoter activity was investigated using different concentrations of cadmium, copper, and zinc. Two fragments (404bp and 1265bp) of the SeW promoter, containing a single metal response element (MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter plasmid. The constructs were transfected into cultured rat C6 (glial) and L8 (myoblast) cells and promoter activity measured by means of luciferase reporter gene fused to the SeW promoter fragments in the reporter plasmid. With post-transfection exposure of these cell lines to these metals, copper and zinc, but not cadmium, significantly increased promoter activity of the unmutated 1265bp (not 404bp) construct (p<0.05) only in the C6 cells. Mutation of the MRE sequence abolished promoter response to metal exposure but did not eliminate promoter activity. The results suggest that SeW expression in glial cells can be increased on exposure to copper and zinc and that this response is dependent on the MRE sequence present in the SeW promoter. To understand transcriptional regulation of the SeW gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter, and antibodies, we were able to show that the specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as the MRE. However, the MRE, GRE, AP-1 and LF-A1 did not yield any specific binding. Although, competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore the SeW gene may be a target for Sp1 whose interaction with the SeW promoter may activate or repress the transcription of SeW. / Graduation date: 2003

Molecular tools, coupled with unique germplasm stocks and rigorous phenotyping, are useful for developing a better understanding of qualitative and quantitative disease resistance genes in plants. The identification of molecular markers linked to all types of resistance genes provides opportunities for implementing a range of resistance breeding strategies, ranging from gene pyramiding to gene deployment. This thesis consists of two chapters. The first describes a disease resistance gene mapping effort and the second describes a disease resistance gene introgression effort. The number, location, and effects of genes determining resistance to stripe rust, leaf rust and Barley Yellow Dwarf Virus were determined using a population of doubled haploid (DH) lines from the cross of Shyri x Galena. Resistance to leaf rust was qualitatively inherited, and the locus was mapped to the long arm of chromosome 1. Resistance to stripe rust and BYDV was quantitatively inherited. Multiple QTLs were detected for each type of resistance. The principal stripe rust resistance QTL was on the short arm of chromosome 5 and the principal BYDV resistance QTL was on the long arm of chromosome 1, linked in repulsion phase with the leaf rust resistance gene. Additional QTLs and QTL x QTL interactions were detected. The majority of the qualitative and quantitative resistance loci detected in the Shyri x Galena population coincided with Resistance Gene Analog Polymorphisms (RGAPs) mapped in the same population. These RGAPs were based on degenerate primers derived from cloned resistance gene sequence motifs. These associations should be useful for efficient resistance gene mapping and provide an approach for ultimately isolating and describing quantitative and qualitative resistance genes. The second chapter describes a molecular marker assisted selection (MMAS) effort to introgress stripe rust resistance QTLs on chromosomes 4 and 7 into susceptible germplasm. DH lines were derived form a MMAS backcross-one (BC-1) population, extensively phenotyped for stripe rust resistance, and genotyped for the introgressed QTLs and background genome. The resistance QTLs that were introgressed were significant determinants of resistance in the new genetic background. Additional resistance QTLs were also detected. Together, these chapters describe an integrated approach to disease resistance gene characterization and utilization. / Graduation date: 1999

The level of polymorphisms of many biochemical and DNA markers are low in cultivated sunflower (Helianthus annuus L.). The number of mapped public DNA markers is limited. Molecular markers have not been developed for the most important diseases of sunflower, such as downy mildew. The objectives of this study were (i) to help alleviate the problem of low DNA marker polymorphisms by developing simple sequence repeat (SSR) markers, (ii) to build an integrated AFLP-RFLP linkage map by using previously described probes and newly developed AFLPs, and (iii) to clone and characterize candidate disease resistance genes. Forty-four polymorphic SSR markers were developed from a genomic DNA library. Diversity analysis of these SSRs for variability among 10 public inbred lines produced an average of 1.86 alleles per locus and mean heterozygosity of 0.21. The number of alleles ranged from 1 to 5. Trinucleotide SSRs were less polymorphic than dinucleotide and mononucleotide SSRs. Cluster analysis and multidimensional scaling separated elite inbred lines from wild species. There was more polymorphism in wild species than in elite lines. Three hundred and six AFLP markers were developed using 18 primer combinations. Two sets of previously mapped RFLP markers were tested for segregation in an F��� mapping population. A total of 401 markers were assigned to 17 linkage groups covering 1326 cM with a mean spacing of 3.3 cM between adjacent markers. The RFLP markers were well spaced and well distributed throughout the genome. Some linkage groups are sparsely populated with common markers. There were two gaps of 30 or more cM in two linkage groups. We cloned candidate disease resistance genes for downy mildew resistance based on sequence homology among resistance genes in other species. Eleven unique nucleotide binding sequence (NBS) containing clones were isolated and showed similarity to the corresponding domains of cloned disease resistance genes in other plant species. Seven clones mapped to four linkage groups and identified nine loci. A cleaved amplified polymorphic sequence (CAPS) marker that was 3.7 cM from the Pl1 resistance gene was developed by analysis of NILs. This CAPS marker should facilitate marker-assisted selection in sunflower. / Graduation date: 1999

Currently little is known about the mechanisms and locations of lymphocyte development in teleosts. In this study several aspects of the underlying factors which govern B lymphocyte development in trout were investigated which included: the isolation and characterization of immunoglobulin heavy chain (IgH) genes, the recombination activating genes 1 and 2 (RAG1 and RAG2) and the use of cellular markers to identify tissues harboring precursor B-cells. Immunoglobulin heavy chains are part of the structural components which make up antibody molecules produced by B-cells. We isolated various full-length IgH cDNA clones, some of which contained the secreted while others contained the membrane bound form of IgH. Upon characterization of the membrane bound forms, typical features common to all IgH cDNAs were found including a leader peptide, a variable region and constant domain containing transmembrane (TM) segments as well. Further sequence analysis of this region revealed that the TM domains were spliced directly to the CH3 domains which results in the loss of the entire CH4 region. Our results support previous observations of unusual splicing events in fish IgH genes. RAG1 and -2 in mammals have been shown to be essential for carrying out V (D) J recombination of lymphocyte receptors and are found to be expressed within primary lymphoid tissues and precursor lymphocytes. We isolated the RAG locus from a rainbow trout genomic library and characterized their conservation and expression. Overall the complete amino acid sequences of RAG1 and RAG2 displayed 78% and 75% similarity when compared to RAG genes from higher vertebrates thus demonstrating the highly conserved nature of these genes. Tissue specific expression of both genes was primarily associated with the thymus and pronephros in both juvenile and adult trout. Based upon these observation we conclude that the thymus and pronephros likely serve as the tissue sites for V (D) J recombination in trout and are thus primary lymphoid organs. Finally we addressed the question as to where B-cell lymphopoiesis occurs in trout. Our results using both immunofluorescence and confocal microscopy putatively demonstrate that the thymus harbors precursor B-cells and thus alludes to a dual function for both B and T-cell development in trout. / Graduation date: 1996

Strawbreaker foot-rot is a major limiting factor to cost efficient winter wheat production in the Pacific Northwest. Development of resistant cultivars has been hindered by the lack of adequate levels of genetic resistance and screening techniques which can consistently detect desired genotypes. Studies were conducted to determine if the reported strawbreaker foot-rot resistance of the cultivar "Rendezvous" is effective on isolates of Pseudocercosporella herpotrichoides found in the Pacific Northwest. Protected, naturally infected and artificially inoculated treatments were employed to determine the level of resistance of 10 cultivars including Rendezvous. Different concentrations of inoculum and stages of development were also used to determine if observations on leaf sheath penetration of seedlings obtained in the greenhouse were related to disease severity index readings taken in the field for selected cultivars. In addition, the nature of inheritance of strawbreaker foot-rot was studied in two crosses involving Rendezvous. Experiments were conducted at three locations and over two years at one location. Despite cultivar x treatment interaction, consistent levels of infection were observed in all experiments at each location. Significant differences were found for treatments and cultivars for most attributes. Yield losses, including the components of yield spikes per square meter, 1000 kernel weight, and kernel number per spike were proportional to the severity of the disease. Losses were greater when lodging occurred, which was also associated with disease severity. However, even in the absence of lodging losses were recorded in the naturally and artificially inoculated plots. Traits measured involving Rendezvous and Vpm/Mos 95//*2Hill were only slightly influenced by the treatments. Under greenhouse conditions, it was possible to distinguish the level of resistance of Rendezvous from susceptible cultivars at concentrations of 100 spores/ml, two weeks after inoculation at the seedling stage. Leaf sheath penetration of seedlings was found to be closely associated with the disease severity index obtained under field conditions. Generation means analysis performed in crosses involving Rendezvous indicated that additive and additive x additive gene action were responsible for most of the genetic variability associated with resistance. Narrow-sense heritability estimates also confirmed these fmdings. It would appear that Rendezvous has at least two major genes for resistance to strawbreaker foot-rot. / Graduation date: 1992

Previous work (Thorgaard, G. H. et al., Aquatic Toxicology 46:121-126, 1999) showed triploid rainbow trout (0. mykiss) given embryonic carcinogen bath exposures had significant reduction of induced tumors relative to diploids. In the present study, trout were made triploid by thermal shock after fertilization. At age of 5 months they were given dietary carcinogen: aflatoxin B1 (AFB₁) or 7,12-dimethylbenz[a]anthracene (DMBA) for 30 or 120 days. The dietary exposures were at known tumorigenic levels (100, 200 and 300 ppb AFB₁; 250, 500 and 850 ppm DMBA). At about 16 months after fertilization the fish were sacrificed and tumor incidence and multiplicity were assessed. At all levels of carcinogen and in all tumorous organs tumor incidence was lower in the triploid fish. For DMBA-fed fish it was seen that the diploid:triploid incidence ratios ranged from 2.0 to 9.0 and for AFB₁ from 3.1 to 6.0. Weight class analyses dissociated the tumor incidence effects of growth from the effects of triploidy. Weight classes plotted against logit tumor incidence at all doses and durations showed parallel logistic lines. In every case the triploid curve was substantially lower than the diploid curve, showing the independent suppressive effect of triploidy. Fifteen triploid DMBA liver tumors were examined by direct cycle-sequencing of p53 PCR products across the exons 5, 7 and 8 known to contain nearly all human tumor p53 mutations. There were no p53 mutations seen at, or above, the present threshold of detection, (for radiolabeled manual sequencing, under 5% of mutant in normal). Fluorescent sequencing of 15 stomach tumors, also showed no p53 mutations in the hotspot-containing exons. Mutation detection by sequencing the trout Ki-ras1 gene, ortholog human KRAS2, showed codons 12, and 61 mutations in DMBA-fed trout liver and stomach tumors. The DMBA liver tumor Ki-ras1 mutation incidence showed no change by ploidy. There was a significant reduction in Ki-ras1 exon 1 mutations in triploid stomach tumors (5% in triploids v. 33% in diploids, Fisher's Exact test p<O.O5). AFB₁ liver tumors showed Ki-ras1 mutation incidence of 75% (9/12) in diploids and 90% (9/10) triploids, nearly all in exon 1, this mutation difference with respect to ploidy did not reach significance. / Graduation date: 2001

This study quantified the frequency of simple versus complex races of Puccinia striiformis Westend. in mixtures of wheat cultivars possessing different race-specific resistance genes. A simple race of a pathogen can infect only one component, and a complex race of the pathogen can infect two or more components of an intraspecific plant mixture. The treatments were designed so that the race that was complex changed depending on the host mixture, thus enabling us to observe the influence of pathogen complexity in different host genetic backgrounds. Six cultivar mixtures and one pure stand of winter wheat were inoculated with three races of P. striiformis (CDL 27, CDL 29, and CDL 41) at two locations for two seasons. Potted plants of three winter wheat cultivars (Paha, Tres, and Tyee) that were each susceptible to one of the three races of the pathogen were used to sample the pathogen during the field epidemics. Disease incidence on the differential cultivars was used to calculate the proportion of the three races in each treatment. The specific cultivars included in the mixtures influenced the frequencies of the three races. Increasing the number of virulent races in a mixture reduced the frequency of the complex race relative to the other two races. When two of the races (races 29 and 41) were complex on the same mixture, location had an effect on which of the races was more frequent. When race 29 was the complex race in the mixture, it was more frequent than when race 41 was the complex race. The results suggest that environmental interactions, genetic background of the pathogen race, host composition, and interaction among pathogen races may be as important in determining race frequencies in mixtures as is stabilizing selection sensu Vanderplank (1968). / Graduation date: 1993

The primary purpose of the present study was to investigate a specific area of cognitive functioning to determine if any differences exist between adult children of alcoholics and controls in the processing of emotionally laden word cues. Of secondary importance was the investigation of group differences in self-esteem, extroversion, neuroticism, and attentional control. A modified version of the Stroop Colour Naming Task was used to investigate selective processing of word cues in a sample of 37 adult children of alcoholics (ACOAs) and 37 adult children of non alcoholics (non ACOAs). All subjects were university students who volunteered for the study. The original form of this task required subjects to name the color of ink in which a word was printed while ignoring word content. Modified versions of this task substitute target words and control words for the words standardly used in order to investigate attentional bias for relevant word cues. As predicted, ACOAs were significantly slower than non ACOAs on this task. There was also a significant group x word type interaction. Compared to non ACOAs, ACOAs displayed a significant attentional bias in favor of alcohol and social threat words compared to neutral and positive words as evidenced by increased response times on the Stroop Task. There was also a significant main effect for word type with response time slowest for alcohol words and fastest for positive words. There were no significant group differences in self-esteem, extroversion, neuroticism, or attentional control. The results were discussed in terms of a generalized attentional deficit for the overall slower response time exhibited by the ACOA group. The more specialized Stroop effect of attentional bias for alcohol and social threat words was discussed in terms of the development of danger schemata based on previous life experiences perceived to be threatening. / Graduation date: 1992

IKK[alpha] is a 85KD serine/threonine protein kinase and a subunit of the IKK complex, which contains IKK[alpha], IKK[beta], and IKK[gamma]. IKK[alpha] and IKK[beta] are highly conserved and they contain three functional domains of kinase domain, leucine zipper (LZ), and helix-loop-helix (HLH). Although IKK[alpha] and IKK[beta] can phosphorylate IκB proteins in vitro, IKK[alpha] and IKK[beta] have distinct physiological functions during mouse development. Genetic studies showed that IKK[alpha] is essential for embryonic skin development in mice. Mice deficient in IKK[alpha] display a hyperplastic epidermis that lacks terminal differentiation, resulting a death soon after birth because of the severely impaired skin. Recently, we reported a reduction in IKK[alpha] expression and identified somatic Ikk[alpha] mutations in a high proportion of poorly differentiated human squamous cell carcinomas (SCCs) (Liu et al., 2006). The aim of this study is to investigate the novel role of IKK[alpha] in skin carcinogenesis. We firstly examined IKK[alpha] expression and Ikk[alpha] mutations in human SCCs and found a reduction of IKK[alpha] in poorly differentiated human SCCs and identified somatic Ikk[alpha] mutations in exon 15 of Ikk[alpha] in human SCCs. We then examined the susceptibility of Ikk[alpha] hemizygotes to chemical carcinogeninduced skin carcinogenesis. In this chemical carcinogen-induced skin carcinogenesis setting, 7,12-dimethylbenz[a]anthracene (DMBA) induces Ras mutations and 12-Otetradecanoyl-phorbol-13-acetate (TPA) promotes Ras-initiated cell proliferation. We found two times more papillomas and eleven times more carcinomas in Ikk[alpha superscript +/-] mice than in Ikk [alpha] superscript +/+] mice induced by DMBA/TPA. Ikk[alpha superscript +/-] mice developed larger and earlier tumors than did Ikk[alpha superscript +/+] mice. Poorly differentiated carcinomas expressed low levels of IKK[alpha]. Ninety five percent of the Ikk[alpha superscript +/-] carcinomas and 44% of the Ikk[alpha superscript +/-] papillomas lost the remaining wild type Ikk[alpha] allele. This result indicates that the remaining one wild type Ikk[alpha] allele is important for preventing malignant carcinoma conversion. Also Ikk[alpha] mutations were detected in these skin tumors. Reduced IKK[alpha] was found to enhance TPA-induced mitogenic and angiogenic activities in mouse skin. Taken together, these results suggest that reduction of IKK[alpha] expression provides a selective growth advantage, which cooperates with DMBA-initiated Ras activity to promote skin carcinogenesis. In addition, we observed a small group of FVB female Ikk [alpha superscript +/-] mice for 1.5 years and found that 12/ 24 mice developed various spontaneous tumors including mammary gland carcinomas, uterine and ovary tumors, and dermal fibrosacomas. Somatic Ikk[alpha] mutations, elevated IKK/ NF[subscript -k]B and extracellular signal-regulated kinases (ERK) activities and elevated cyclin D1 levels were detected in these spontaneous tumors. These results suggest that these molecular alterations may contribute to the development of these tumors although the precise role of the down-regulation of IKK in the development of the tumors remains to be determined. Overall, our data and other published results suggest that IKK[alpha] is a new tumor suppressor in men and mice. / text