Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 131-133).

Abstract In tumourigenesis, genetic alterations accumulate in the genes responsible for cell growth, proliferation and DNA repair: proto-oncogenes, tumour suppressor and DNA repair genes. Inactivation of tumour suppressor gene function is commonly recognised as a deletion of one of the two alleles; LOH, loss of heterozygosity. In the present study, LOH of several chromosomal regions was studied in both breast and ovarian cancer. LOH for chromosome region 11q was examined in a large breast cancer consortium cohort (N = 988) and in a Finnish ovarian cancer cohort (N = 78), and the clinical significance of these alterations was evaluated. In breast cancer, LOH of the studied markers at 11q23.1, harbouring approximately 2 Mb of DNA, was seen to be associated with shortened cancer-specific survival. Two candidate genes, ATM (the ataxia telangiectasia disorder gene) and DDX10 (a putative RNA helicase gene) map to this chromosomal region. In ovarian cancer, LOH at 11q23.1–q24 was assigned mainly to two chromosomal regions, A and B, which are proximal and distal to 11q23.2–q23.3, respectively. Only the distal B region was seen to be associated with an aggressive disease course. Therefore, it appears that inactivation of the ATM or DDX10 genes is not crucial for determining the outcome of ovarian cancer. The CHK1 gene at 11q24, encoding a protein kinase required for DNA damage checkpoint function, is a putative target gene at the B region. On the basis of the present results, the main TSG in the studied region involved in the progression of breast cancer maps to 11q23.1 and the corresponding gene for ovarian cancer more distally to 11q23.3-q24. In addition, LOH at 3p, 6q, 8p, 11p, 16q and 17p was examined and their role in the genetic evolution of ovarian cancer was evaluated. Of the studied chromosomal regions, LOH at 17p appeared to be an early event and LOH at 16q24.3, 11q23.3/q24 and 11p appear to occur later in the progression of ovarian cancer.

Aim The aim of this study was to investigate the most commonly used approaches to measure individual genome-wide heterozygosity (IGWH) and to investigate whether IGWH can be considered as a health risk factor or a protective factor in humans. Methods This study was based on two samples from isolated communities of Croatian Adriatic islands, with a total of 1,930 adult examinees from Islands of Vis (N=986) and Korcula (N=944). Examinees were genotyped with a total of 302,662 single nucleotide polymorphisms. Heterozygosity was estimated using five commonly calculated methods. Results Correlation coefficients between different heterozygosity methods were generally in the range of 0.7-0.8. A worsening in some phenotypic traits, including cholesterol and triglycerides as well as increased odds for osteoporosis and metabolic syndrome was recorded in cases of IGWH reduction. Nevertheless, in these cases heterozygosity explained a relatively low amount of variance, generally in range of 0.4-0.6% of total trait variance. Conclusion However, these results were significant in Vis Island sample, while in the replication sample, Korcula Island, most of the associations were not significant, possibly due to the overall lower amount of inbreeding and higher heterozygosity in Korcula Island sample. The results warrant further research in order to provide more information on the extent and importance of individual genome-wide heterozygosity, which might have an important role in communities which experience consanguinity on a greater scale. Two main shortcomings of the study include possible lack of power to detect inbreeding depression and the need to replicate the results in other populations.

Electrophoretic variation in 18 proteins encoded by 20 autosomal loci was used to compare the genetic relationships of 19 natural populations representing three species of the subgenus Neotoma. Of the 20 loci examined nine were monomorphic and fixed for the same allele in all populations. No more than seven loci were polymorphic within a single population. Genetic variability was expressed as the proportion of loci heterozygous in the average individual of a population. Heterozygosity in the three species of Neotoma studied averaged 0.078, a value within the range reported for other rodents. Although the levels of heterozygosity seen in Neotoma could not readily be explained, the variation may be attributed to ecological factors. The three species of Neotoma were compared on the basis of genetic similarity and found to form a close taxonomic unit, probably semispecies. Divergence times were obtained for the three species and found to compare well with divergence times obtained from fossil data. In general, the three species have diverged within the last 112,000 years during the Wisconsin glacial period.

As a prelude to strain selection for domestication and future marker assisted selection, genetic variation revealed by microsatellite DNA was evaluated in yellow perch, Perca flavescens, from four wild North American populations collected in 2003-2004 (Maine, ME; New York, NY; North Carolina, NC; and Pennsylvania, PA), and two captive populations (Michigan, MI; Ohio, OH). For the loci examined, levels of heterozygosity ranged from He = 0.04 to 0.88, genetic differentiation was highly significant among all population pairs, and effective migration ranged from low (Nem = 0.3) to high (Nem = 4.5). Deviation from Hardy-Weinberg 1equilibrium was regularly observed indicating significant departures from random mating. Instantaneous measures of inbreeding within these populations ranged from near zero to moderate (median F = 0.16) and overall inbreeding levels averaged FIS = 0.18. Estimates of genetic diversity, Φ ST and genetic distance were highest between Michigan and all other populations and lowest between New York and Ohio. Genetic differentiation among populations did not correlate with geographic distance. Overall, the patterns of variation exhibited by the captive (Michigan and Ohio) populations were similar to patterns exhibited by the other allegedly wild populations, indicating that the spawning and management practices to date have not significantly reduced levels of genetic variation.

Tsang Wai Kai, Paul. / "July 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 133-157). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The grain amaranths (Amaranthus hypochondriacus L., A. cruentus L., and A. caudatus L.) are important pseudocereals native to the Americas that have received increased attention for their nutritional content, specifically their balance of amino acids. The objective of this project was to produce and characterize a set of highly informative, reproducible microsatellite markers for the grain amaranths. A total of 1457 clones were sequenced from three genomic libraries enriched for the microsatellite motifs AAC, AAT and AC. Of these, 353 (24%) contained unique microsatellites. An additional 29 microsatellite loci were identified among 728 BAC-end sequences of a newly developed amaranth BAC library. Flanking primers were designed for 319 of the microsatellite loci and all were screened on a panel of diverse amaranths, including grain and weedy Amaranthus species. A total of 179 (56%) microsatellites were polymorphic across accessions from the three grain amaranths. Among these polymorphic microsatellite loci, a total of 731 alleles were identified with average of four alleles per locus. Heterozygosity values ranged from 0.14 to 0.83 with a mean value of 0.62. Thirty-seven (21%) of the markers were polymorphic between the parents of a segregating population and were shown to be inherited in a normal Mendelian fashion based on chi-squared analysis, demonstrating the utility of these markers for linkage mapping of the amaranth genome. Phylogenetic analysis using the marker data showed A. hybridus accessions in two of the three major grain amaranth clades, suggesting the polyphyletic evolution of the three cultivated species from different A. hybridus ancestors. The microsatellite markers reported here will be useful for further evaluating the relationships among the grain amaranths and their relatives and are an ideal resource for use in marker-assisted breeding programs, germplasm analysis and varietal identification. The transferability of these markers to A. hybridus, A. powellii, and A. retroflexus as reported here suggests that the markers may be useful to other species with the genus Amaranthus, including economically important weeds, vegetable amaranths, and ornamentals.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A study based in DNA and isozyme markers was carried out to evaluate the diversity and genetic structures of natural populations of Erythrina velutina Willd., aiming monitored prospection of the genetic variability for two populations from the Atlantic Forest (Santana do São Francisco-SE) and Caatinga (Pinhão-SE) Biomes. Young leaves of twenty individuals per population were sampled from each population. Primers of ten arbitrary bases sequence and fifteen enzymatic systems were used. In RAPD analysis, the population from Atlantic Forest Biome originated 100 polymorphic loci and population from Caatinga Biome, 112 loci. It was observed that the genetic structure for population from Caatinga present higher number of observed and effectives alleles, which implicate in a higher heterozigozity. The observed average heterozygozity was higher than the expected heterozigozity by Hardy- Weinberg, which indicate a excess of heterozygotes, and this value might be confirmed by negative value of the Wright´s fixation index (-0.5098). In relation to genetic diversity between populations (Fst and Gst) the values were similar for both markers. The results suggest the use of M1, M7, M11 and M14 individuals from Atlantic Forest Biome; and the M5, M6, M18 and M19 individuals from Caatinga Biome as most divergent for future chemical, biochemical and pharmacological studies. / Um estudo baseado em marcadores de DNA e isoenzimáticos foi realizado para avaliar a diversidade e estrutura genética de populações naturais de Erythrina velutina Willd., visando à prospecção monitorada da variabilidade genética, para fins de seleção das matrizes mais divergentes, em duas populações naturais do estado de Sergipe. Foram amostradas folhas jovens de vinte indivíduos em cada população. Um total de vinte oligonucleotídeos decâmeros de sequência arbitrária e 15 sistemas enzimáticos foram testados. Na análise de RAPD, a população do Baixo São Francisco Sergipano originou 134 locos, sendo 100 polimórficos e a população do município de Pinhão, 143 locos, sendo 112 polimórficos. Observou-se para a estrutura genética da população do município de Pinhão maior número de alelos observados e efetivos, o que implica numa maior heterozigosidade. A heterozigosidade média observada foi maior que a esperada pelo equilíbrio de Hardy- Weinberg, o que indica um excesso de heterozigotos e pode ser confirmado pelo valor negativo do índice de fixação de Wright (-0,2304). Em relação à diversidade genética entre populações (Fst e Gst) os valores foram similares para os dois marcadores. Sugere-se o uso dos indivíduos M1, M7, M11 e M14 do Baixo São Francisco Sergipano; e dos indivíduos M5, M6, M12 e M19 do município de Pinhão como os mais divergentes para futuros estudos químicos, bioquímicos e farmacológicos.

Abstract Prostate cancer is the most frequently diagnosed cancer in men in industrialized countries. Despite the substantial clinical importance of the disease, the mechanisms underlying the development and progression of prostate cancer are poorly understood. In the present study, fragment analysis of chromosome arm 16q was carried out with the aim of searching for sites of consistent chromosomal deletion, possibly uncovering the location of target genes that become inactivated in prostate carcinogenesis. The highest percentage of loss of heterozygosity (LOH) was found at chromosomal region 16q24.1-q24.2, including the gene for 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase (17HSD/KSR) type 2, HSD17B2. The data further indicated an association between loss of the most commonly deleted region and clinically aggressive features of the disease. A fragment analysis performed using sequential primary and locally recurrent prostate cancer specimens suggested the location of the genes related to prostate cancer progression to be at 16q24.3 and, further, gave rise to a hypothesis of the potential role of locus HSD17B2 as a prognostic marker for prostate cancer progression. Quantitative real-time polymerase chain reaction (PCR) revealed a decreased HSD17B2 gene copy number in prostate cancer specimens compared to their normal counterparts. A diminished HSD17B2 gene copy number was significantly associated with poor differentiation of the tumor. The progression of prostate cancer during androgen deprivation is a serious clinical problem, the molecular mechanisms of which largely remain to be clarified. The present data of enzyme activity measurements performed using high-performance liquid chromatography (HPLC) provided evidence of a substantial decrease in oxidative and an increase in reductive 17HSD/KSR activity during the transition of prostate cancer LNCaP cells into an androgen-independent state. Further, the changes detected in the activities largely coincided with the changes in the relative expression levels of genes for the potential 17HSD/KSR isoenzymes; 17HSD/KSR types 2, 5, and 7, as evidenced by relative quantitative reverse transcription PCR (RT-PCR). The data on the expression analysis of mRNA for 17HSD/KSR types 5 and 7 in prostate tissue specimens performed using in situ hybridization showed a moderately low but constitutive level for 17HSD/KSR7 mRNA in tissues of cancerous as well as hyperplastic origin. The expression of mRNA for 17HSD/KSR type 5, instead, varied considerably between different specimens, the highest expressions being strongly associated with aggressive and metastatic prostate cancer. Interestingly, furthermore, the intense expression of 17HSD/KSR5 was significantly associated with the androgen deprivation achieved either surgically or medically. Since 17HSD/KSRs critically contribute to the control of the bioavailability of active sex steroid hormones locally in the prostate, the variation in intraprostatic 17HSD/KSR activity might be crucially involved in the regulation of the growth and function of the organ.

Northern Pike (Esox lucius) is an economically and ecologically valuable species with a circumpolar distribution across the Northern Hemisphere. Northern Pike have been shown to have low levels of genetic variation despite their great capacity to colonize new environments. Here, high-resolution resequencing data from 47 Northern Pike from across North America was used for SNP discovery and population analysis. Our analysis reveals an extraordinary lack of genetic variation among Northern Pike with observed heterozygosity (Ho) of just 0.0835. Our analyses suggest that two major groups of Northern Pike exist in North America that are separated by the North American Continental Divide. Genetic variation associated with the stratification of these two groups resides across the genome particularly in gene regions with multiple copy number variants and functions related to immunity, tissue permeability, and development. Northern Pike from Alaska and the Yukon River harbour about two times more heterozygosity than Northern Pike east of the Continental Divide with an average of one heterozygous SNP every 6,250 bases. Populations east of the Continental Divide possess a remarkable level of genetic homogenization with an average of just one heterozygous SNP every 16,500 bases. For comparison, an average of one heterozygous SNP per 309 bases was reported in herring (Martinez Barrio et al., 2016), one per 500 in Atlantic cod (Star et al., 2011), and one per 750 bases in Coho and chinook salmon (Koop, 2018). This is at least 5 – 10 fold less variation than is seen in humans (the 1000 Genomes Project Consortium, 2015). We observed a recently described master sex-determining gene, amhby, in three western North American populations but not in populations east of the Continental Divide. We could not resolve any signals indicating a genetic sex determination system was present in populations from southern Manitoba or the St. Lawrence River. This may indicate that environmental sex determination is at play in these populations. We found evidence of a possible female-heterozygous, male homozygous ZW-ZZ genetic sex-determination system in New Jersey Northern Pike. With the highest average of 181,268 heterozygous SNPs genome wide and the greatest Ho (0.3228) of all populations, as well as the presence of the sex-determining gene amhby indicate that Northern Pike from our Alaskan population are the oldest in North America. Fewer numbers of heterozygous SNPs (61,073), low Ho (0.0922), and the absence of amhby in Northern Pike east of the Continental Divide suggests that these are relatively young populations and are descended from a small founding population. These results imply that Northern Pike first came to North America through Beringia and colonized its North American range from there, possibly via pro-glacial lake formation and drainage. However, from the data herein it was not possible to trace how re-colonization occurred after the final retreat of glaciers at the end of the last ice age. This thesis provides a genetically high-resolution snapshot of Northern Pike population structure in North America. It demonstrates that organisms with largely homogenous genomes can be incredibly successful and resilient. Finally, it adds to the complex subject of sex determination in fish and provides insight into a sex determination system in transition. / Graduate / 2020-10-15