A majority of methods for identifying sequences in the human genome involve target sequence amplification through PCR. This work presents novel methods for amplifying circularized DNA and presents solutions for some major limitations of PCR. We have developed a novel method to amplify circularized DNA molecules based on a serial rolling-circle replication reaction, called circle to circle amplification (C2CA). Amplified DNA circles can be detected in array-based analyses or in real-time using molecular beacons. The amplification mechanism allows higher precision in quantification than in exponential amplification methods like PCR, and more products can be generated than in PCR. A major limitation of PCR is that amplification artifacts arise when large numbers of specific primer pairs are simultaneously added to a reaction. We have developed a solution to this problem that enables multiplex PCR amplification of specific target sequences without producing amplification artifacts. The procedure is based on oligonucleotide constructs, called selectors. The selectors identify defined target nucleic acid sequences, and they act as ligation templates to direct circularization of these targets. The selectors contain a general primer-pair motif that allows the circularized targets to be amplified in multiplex using a universal PCR primer pair. We also developed a computer program, PieceMaker, that finds an optimal design of selector probes for a given selector application. We demonstrate the method by performing a 96-plex PCR of specific DNA sequences with high success-rate and reproducibility.

Over the last few years, genome-wide association studies (GWAS) have been used to identify numerous obesity associated SNPs in the human genome. By using linkage studies, candidate obesity genes have been identified. When SNPs in the first intron of FTO were found to be associated to BMI, it became the first gene to be linked to common obesity. In order to look for causative explanations behind the associated SNPs, a re-sequencing of FTO had been performed on the SOLiD sequencing platform. In-house candidate gene, SLCX, was also sequenced in order to evaluate a potential obesity association. The purpose of this project was to analyse the sequences and also to evaluate the quality of the SOLiD sequencing. A part of the project consisted in performing PCRs and selecting genomic regions for future sequencing projects. I developed and implemented a sequence analysis strategy to identify obesity associated SNPs. I found 39 obesity-linked SNPs in FTO, a majority of which were located in introns 1 and 8. I also identified 3 associated intronic SNPs in SLCX. I found that the SOLiD sequencing coverage varies between non-repetitive and repetitive genomic regions, and that it is highest near amplicon ends. Interestingly, coverage varies significantly between different amplicons even after repetitive sequences have been removed, which indicates that it is affected by features inherent to the sequence. Still, the observed allele frequencies for known SNPs were highly correlated with the SNP frequencies documented in HapMap. In conclusion, I verify that SNPs in FTO are associated with obesity and also identify a previously unassociated gene, SLCX, as a potential obesity gene. Re-sequencing of genomic regions on the SOLiD platform was proven to be successful for SNP identification, although the difference in sequencing coverage might be problematic.

<p>The fear response of two different captive populations of red jungle fowl (rjf, Gallus gallus) was measured in three different tests, a ground predator test, an aerial predator test and a tonic immobility test. The two populations originated from Copenhagen zoo (Cop) and Götala research station (Got) but had been kept together for four generations. Earlier generations had a confirmed difference in fearfulness where the Cop birds exhibited a higher degree of fear response than Got birds (Håkansson and Jensen, 2005; Håkansson et al., 2007). The most and least fearful birds of each sex and population were identified and used in a gene expression study. The midbrain regions from the candidate birds were collected and RNA was isolated from each brain. The RNA was then reversed transcribed to cDNA which was used in a cDNA microarray experiment. 13 significantly differentially expressed genes were found between the fearful and non-fearful females. Among others were the neuroprotein Axin1, two potential DNA/RNA regulating proteins and an unknown transcript in the Quantitative Trait Locus 1(QTL 1), a well studied QTL on chromosome one with substantial effect on both behaviour and morphology during domestication (Schütz et al., 2002). This thesis succeeds in finding a difference in gene expression between fearful and non fearful female rjf but not between males. It fails in identifying gene expression differences between the two populations. Finally, the found differentiated genes suggest a potential molecular mechanism controlling the fear response in fowl.</p>

The fear response of two different captive populations of red jungle fowl (rjf, Gallus gallus) was measured in three different tests, a ground predator test, an aerial predator test and a tonic immobility test. The two populations originated from Copenhagen zoo (Cop) and Götala research station (Got) but had been kept together for four generations. Earlier generations had a confirmed difference in fearfulness where the Cop birds exhibited a higher degree of fear response than Got birds (Håkansson and Jensen, 2005; Håkansson et al., 2007). The most and least fearful birds of each sex and population were identified and used in a gene expression study. The midbrain regions from the candidate birds were collected and RNA was isolated from each brain. The RNA was then reversed transcribed to cDNA which was used in a cDNA microarray experiment. 13 significantly differentially expressed genes were found between the fearful and non-fearful females. Among others were the neuroprotein Axin1, two potential DNA/RNA regulating proteins and an unknown transcript in the Quantitative Trait Locus 1(QTL 1), a well studied QTL on chromosome one with substantial effect on both behaviour and morphology during domestication (Schütz et al., 2002). This thesis succeeds in finding a difference in gene expression between fearful and non fearful female rjf but not between males. It fails in identifying gene expression differences between the two populations. Finally, the found differentiated genes suggest a potential molecular mechanism controlling the fear response in fowl.

This thesis describes the use of functional genomics screens in yeast to study anticancer drug action and intracellular transport. The yeast Saccharomyces cerevisiae provides a particularly useful model system for global drug screens, due to the availability of knockout mutants for all yeast genes. A complete collection of yeast deletion mutants was screened for sensitivity to monensin, a drug that affects intracellular transport. A total of 63 deletion mutants were recovered, and most of them were in genes involved in transport beyond the Golgi. Surprisingly, none of the V-ATPase subunits were identified. Further analysis showed that a V-ATPase mutant interacts synthetically with many of the monensin-sensitive mutants. This suggests that monensin may act by interfering with the maintenance of an acidic pH in the late secretory pathway. The second part of the thesis concerns identification of the underlying causes for susceptibility and resistance to the anticancer drug 5-fluorouracil (5-FU). In a functional genomics screen for 5-FU sensitivity, 138 mutants were identified. Mutants affecting tRNA modifications were particularly sensitive to 5-FU. The cytotoxic effect of 5-FU is strongly enhanced in these mutants at higher temperature, which suggests that tRNAs are destabilized in the presence of 5-FU. Consistent with this, higher temperatures also potentiate the effect of 5-FU on wild type yeast cells. In a plasmid screen, five genes were found to confer resistance to 5-FU when overexpressed. Two of these genes, CPA1 and CPA2 encode the two subunits of the arginine-specific carbamoyl-phosphate synthase. The three other genes, HMS1, YAE1 and YJL055W are partially dependent on CPA1 and CPA2 for their effects on 5-FU resistance. The specific incorporation of [14C]5-FU into tRNA is diminished in all overexpressor strains, which suggest that they may affect the pyrimidine biosynthetic pathway.

<p>Analysis of genetic relatedness is of great importance in forensic casework such as immigration and identification cases. The conventional methods for relationship testing are not sufficient in the most complicated cases, because more genetic markers are required to obtain results with satisfactory statistical security. This study demonstrates that microarrays, which can be used to genotype thousands of single nucleotide polymorphisms (SNPs), could be a promising solution to this problem. The microarray technique used in this study performed very well on blood samples and also worked well in combination with whole genome amplification, but did not generate any results when used on severely degraded materials.</p><p>Markers suitable for relatedness analysis were selected from the microarray and were successfully tested on families with known genetic relations. Although a maximum of 64 autosomal markers were used, there is a great potential of selecting the hundreds or thousands of markers that may be required in some cases of relatedness investigation.</p>

<p>Small populations are facing a particular risk of extinction due to a lack of appropriate genetic diversity and associated negative effects, factors dealt with in the discipline of conservation genetics. Many orchid species exhibit characteristics that make them a perfect study object in the scope of conservation genetics. The aim with this thesis was to investigate genetic structure at different levels in two orchid species <i>Gymnadenia conopsea</i>, geographically widespread, although diminishing and <i>G. odoratissima</i> with a long history of being rare. Microsatellite markers, developed in and used in studies of <i>G. conopsea</i> were also used in the study of <i>G. odoratissima</i>.</p><p>Populations of <i>G. conopsea</i> expressed high levels of genetic variation and a certain amount of gene flow, although investigated mating pattern in a small population indicated non-random mating among individuals, with the majority of pollen exchange between near neighbours, and noticeable levels of geitonogamous pollinations. Further a pronounced year to year variation in flowering frequency among individuals was found. </p><p>It was also discovered that flowering time variants (early and late) within the species <i>G. conopsea</i> were highly differentiated and seem to have had a more ancient historical separation than the separation between the two different species, <i>G. conopsea</i> and <i>G. odoratissima. </i></p><p>Levels of genetic variation in the rare congener, <i>G. odoratissima</i> differed between island and mainland populations where the more numerous island populations expressed larger levels of genetic variation and were less differentiated compared to the few remaining and genetically depauperated mainland populations.</p><p>Uppsala University Library, Box 510, 75120, Uppsala, Sweden </p>

Small populations are facing a particular risk of extinction due to a lack of appropriate genetic diversity and associated negative effects, factors dealt with in the discipline of conservation genetics. Many orchid species exhibit characteristics that make them a perfect study object in the scope of conservation genetics. The aim with this thesis was to investigate genetic structure at different levels in two orchid species Gymnadenia conopsea, geographically widespread, although diminishing and G. odoratissima with a long history of being rare. Microsatellite markers, developed in and used in studies of G. conopsea were also used in the study of G. odoratissima. Populations of G. conopsea expressed high levels of genetic variation and a certain amount of gene flow, although investigated mating pattern in a small population indicated non-random mating among individuals, with the majority of pollen exchange between near neighbours, and noticeable levels of geitonogamous pollinations. Further a pronounced year to year variation in flowering frequency among individuals was found. It was also discovered that flowering time variants (early and late) within the species G. conopsea were highly differentiated and seem to have had a more ancient historical separation than the separation between the two different species, G. conopsea and G. odoratissima. Levels of genetic variation in the rare congener, G. odoratissima differed between island and mainland populations where the more numerous island populations expressed larger levels of genetic variation and were less differentiated compared to the few remaining and genetically depauperated mainland populations. Uppsala University Library, Box 510, 75120, Uppsala, Sweden

Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods. The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal. This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype. To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank. / QC 20100927

Analysis of genetic relatedness is of great importance in forensic casework such as immigration and identification cases. The conventional methods for relationship testing are not sufficient in the most complicated cases, because more genetic markers are required to obtain results with satisfactory statistical security. This study demonstrates that microarrays, which can be used to genotype thousands of single nucleotide polymorphisms (SNPs), could be a promising solution to this problem. The microarray technique used in this study performed very well on blood samples and also worked well in combination with whole genome amplification, but did not generate any results when used on severely degraded materials. Markers suitable for relatedness analysis were selected from the microarray and were successfully tested on families with known genetic relations. Although a maximum of 64 autosomal markers were used, there is a great potential of selecting the hundreds or thousands of markers that may be required in some cases of relatedness investigation.