Trypanosoma cruzi, the causative agent of Chagas´ disease, is an evolutionarily ancient species with distinct biological and immunological characteristics. A fundamental understanding of the basic biology of the parasite is necessary in order to develop reliable therapeutic and prophylactic agents against T. cruzi. We have, as a part of the T. cruzi genome project launched by the WHO, generated ESTs corresponding to about one third of the functional genes in the parasite. Only about 1/3 of the unique ESTs could be assigned a function upon sequence comparison to all publicly available data. Comparative analysis of the ESTs to functional genes in S. cerevisiae and C. elegans as well as to sequence data from all other kinetoplastids provided primary insights into the evolutionary divergence of T. cruzi. A novel dispersed gene family (DGC3) was identified and shown to be present specifically on chromosome 3 and its homologue. Sequence analysis of ten isolated DGC3 genes revealed a high sequence similarity of almost 98% among copies. The DGC3 genes were transcribed, trans-spliced with the spliced leader and polyadenylated, but did not seem to have any protein-coding property. These data preliminary suggest that it encodes a novel family of functional RNA. In the T. cruzi CL Brener strain, the two alleles of a single copy gene encoding the trypanothione synthetase (TcTRS) enzyme appeared to be highly polymorphic. The divergence of the deduced protein sequence was 4%, almost ten-fold higher than another protein, trypanothione reductase, involved in the same pathway. The observed allelic divergence might influence the TcTRS activity thereby having implications for drug design. Moreover, the TcTRS gene was found to be flanked by a number of genes involved in diverse functions and located to a pair of homologous chromosomes with a size difference of about 2 Mbp. A gene potentially encoding the polypyrimidine-binding protein (TcPTB) was identified and characterised regarding its organisation and function. The deduced amino acid sequence was shown to comprise four RRM domains generally present in other PTBs. Interestingly, the TcPTB gene appeared to be expressed in a stage-specific manner implicating different functions during parasite development.

Domestication has led to genetic changes that affect quantitative traits in farm animals. Both candidate gene analysis using association tests and genome scans based on linkage analysis have been performed to understand the molecular basis underlying quantitative genetic variation in horses, pigs and chickens. To test a possible association of polymorphisms in the PRKAG3 gene, previously found to be associated with excess glycogen content in pig skeletal muscle, with quantitative traits in the horse, the major coding part of the equine PRKAG3 sequence was identified. Bioinformatic characterization of the equine PRKAG3 gene was conducted. A single nucleotide polymorphism (SNP) causing a missense mutation (Pro258Leu) was found. Screening this SNP showed that the Leu258 allele was more frequent in breeds with heavy muscularity. To assess previously reported associations between polymorphisms in the MC4R gene and obesity-related traits further, we conducted linkage analysis between the MC4R locus and fatness-related traits using a Wild BoarxLarge White intercross. No significant association between segregation at the MC4R locus and fatness was detected in this pedigree. A genome scan of quantitative trait loci (QTLs) has been performed in an intercross between chicken lines divergently selected for growth. Divergent parental lines have been established by selecting for high and low 56-day body weight for over 40 generations. The selection has led to approximately a 9-fold difference in 56-day body weight between lines and resulted in correlated responses for a number of traits including appetite, immune response, body composition and metabolic traits. Phenotypic data on growth and other correlated traits were collected from more than 800 F2 individuals. Genome scans using 145 markers on 26 linkage groups have identified QTLs affecting growth and correlated responses to selection for 56-day body weight. No major QTL explaining a large portion of phenotypic variation in growth was revealed in this study.

The population of northern Sweden has previously been shown to be well suited for the mapping of monogenic diseases. In this thesis we have tested the hypothesis that this population could also be used for efficient identification of risk genes for common diseases. In Paper I we have hypothesised that despite the admixture of Swedish, Finnish and Sami, the northern Swedish population consists of sub-populations geographically restricted by the main river valleys running through the region. This geographic isolation, in combination with founder effects and genetic drift, could represent a unique resource for genetic studies. On the other hand, it also underlines the importance of accounting for this e.g. in genetic association studies. To test this hypothesis, we studied the patterns of marriage within and between river valley regions and compared allelic frequencies of genetic markers between these regions. The tendency to find a spouse and live in the river valley where one was born is strong, and allelic frequencies of genetic markers vary significantly between adjacent regions. These data support our hypothesis that the river valleys are home to distinct sub-populations and that this is likely to affect mapping of genetic diseases in these populations. In Paper II, we tested the applicability of the population in mapping HSAN V, a monogenic disease. This disease was identified in only three consanguineous individuals suffering from a severe loss of deep pain perception and an impaired perception of heat. A genome-wide scan combined with sequencing of candidate genes resulted in the identification of a causative point mutation in the nerve growth factor beta (NGFB) gene. In Paper III, a large family with multiple members affected by familial forms of type 1 diabetes mellitus (T1DM) and autoimmune thyroiditis (AITD) was studied. This syndrome was mapped to the IDDM12 region on 2q33, giving positive lodscores when conditioning on HLA haplotype. The linkage to HLA and to the IDDM12 region thus confirmed previous reports of linkage and/or association of T1DM and AITD to these loci and provided evidence that the same genetic factors may be mediating these diseases. This also supported the feasibility of mapping complex diseases in northern Sweden by the use of familial forms of these diseases. In Paper IV, we applied the same approach to study type 2 diabetes mellitus (T2DM). A non-parametric genome-wide scan was carried out on a family material from northern Sweden, and linkage was found to the calpain-10 locus, a previously described T2DM-susceptibility gene on 2q37. Together, these findings demonstrate that selecting for familial forms of even complex diseases, and choosing families from the same geographical region can efficiently reduce the genetic heterogeneity of the disease and facilitate the identification of risk genes for the disease.

With the completion of the human genome sequencing project, attention is currently shifting toward understanding how genetic variation, such as single nucleotide polymorphism (SNP), leads to disease. To identify, understand, and control biological mechanisms of living organisms, the enormous amounts of accumulated sequence information must be coupled to faster, cheaper, and more powerful technologies for DNA, RNA, and protein analysis. One approach is the miniaturization of analytical methods through the application of microfluidics, which involves the manipulation of fluids in micrometer-sized channels. Advances in microfluidic chip technology are expected to play a major role in the development of cost-effective and rapid DNA analysis methods. This thesis presents microfluidic approaches for different DNA genotyping assays. The overall goal is to combine the potential of the microfluidic lab-on-a-chip concept with biochemistry to develop and improve current methods for SNP genotyping. Three genotyping assays using miniaturized microfluidic approaches are addressed. The first two assays are based on primer extension by DNA polymerase. A microfluidic device consisting of a flow-through filter chamber for handling beads with nanoliter liquid volumes was used in these studies. The first assay involved an allelespecific extension strategy. The microfluidic approach took advantage of the different reaction kinetics of matched and mismatched configurations at the 3’-ends of a primer/template complex. The second assay consisted of adapting pyrosequencing technology, a bioluminometric DNA sequencing assay based on sequencing-bysynthesis, to a microfluidic flow-through platform. Base-by-base sequencing was performed in a microfluidic device to obtain accurate SNP scoring data on nanoliter volumes. This thesis also presents the applications of monolayer of beads immobilized by microcontact printing for chip-based DNA analysis. Single-base incorporation could be detected with pyrosequencing chemistry on these monolayers. The third assay developed is based on a hybridization technology termed Dynamic Allele-Specific Hybridization (DASH). In this approach, monolayered beads containing DNA duplexes were randomly immobilized on the surface of a microheater chip. DNA melting-curve analysis was performed by dynamically heating the chip while simultaneously monitoring the DNA denaturation profile to determine the genotype. Multiplexing based on single-bead analysis was achieved at heating rates more than 20 times faster than conventional DASH provides. / QC 20101008

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Sexual conflict is a term that describes the situation where traits can experience opposing selection pressures in the two sexes. Theory suggests this conflict exists in all organisms with separate sexes, and specific chromosome clusters which are possibly sexually antagonistic have been identified in the species Drosophila melanogaster. One of all identified genes is CG3598, which have proved yielding higher fitness for males carrying allele A2 on this gene compared to A1. In this study, factors which contribute to the difference in fitness between these two alleles, with regards to sperm competition and mating success were observed. A double mating design was used, in which males and females were placed in test tubes together in order to examine the number of copulations and the defensive (P1) and offensive (P2) ability of sperm. The relative fitness of males carrying A1 did not differ from males carrying A2, which rejects previous studies, however, A2 had a higher defensive capability compared to A1. On the other hand, A1 instead had better offensive capability and higher amount of rematings. This indicates that the defensive capability of A2 is very strong and opposes the offensive capability of A1, but also the increased rate of rematings in A1. To get a more precise understanding of the fitness relation between A1 and A2 on the gene CG3598, further experiments would need to be performed on the subject. / Sexuell konflikt är ett begrepp som beskriver förhållandet där egenskaper upplever olika selektionstryck beroende på kön. Teorier finns om att den här konflikten existerar i alla organismer med olika kön, och i arten Drosophila melanogaster har det identifierats specifika kromosomkluster som har möjlighet att bidra till sexuell konflikt. En utav alla identifierade gener är CG3598 som har påvisats ge en högre fitness för hanar bärande allel A2 på denna gen jämfört med allel A1. I den här studien undersöks bakomliggande faktorer till skillnaden i fitness mellan dessa två alleler, med avseende på spermiekonkurrens och parningsframgång. Genom en dubbel parningsdesign, där hanar och honor placerades tillsammans i rör undersöktes den defensiva spermieförmågan (P1), den offensiva förmågan (P2) och antal parningar. Den relativa fitnessen hos hanar med A1 skiljde sig inte från A2, vilket motsäger tidigare studier. Däremot var den defensiva förmågan högre för A2 och A1 hade istället högre offensiv förmåga samt en högre andel omparningar. Detta indikerar att A2 har en stark defensiv förmåga som motsätter den offensiva förmågan i A1 men också möjligheten till fler omparningar. För att få en mer precis uppfattning skulle ytterligare experiment behöva utföras då det i denna studie var en väldigt låg andel hanar som parade sig jämfört med vad som förväntas.

RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3′ processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.

There is a basic rule to mammalian neocortical expansion: as it expands, so does it fold. The degree to which it folds, however, cannot strictly be attributed to its expansion. Across species, cortical volume does not keep pace with cortical surface area, but rather folds appear more rapidly than expected. As a result, larger brains quickly become disproportionately more convoluted than smaller brains. Both the absence (lissencephaly) and presence (gyrencephaly) of cortical folds is observed in all mammalian orders and, while there is likely some phylogenetic signature to the evolutionary appearance of gyri and sulci, there are undoubtedly universal trends to the acquisition of folds in an expanding neocortex. Whether these trends are governed by conical expansion of neocortical germinal zones, the distribution of cortical connectivity, or a combination of growth- and connectivity-driven forces remains an open question. But the importance of cortical folding for evolution of the uniquely mammalian neocortex, as well as for the incidence of neuropathologies in humans, is undisputed. In this hypothesis and theory article, we will summarize the development of cortical folds in the neocortex, consider the relative influence of growth- vs. connectivity-driven forces for the acquisition of cortical folds between and within species, assess the genetic, cell-biological, and mechanistic implications for neocortical expansion, and discuss the significance of these implications for human evolution, development, and disease. We will argue that evolutionary increases in the density of neuron production, achieved via maintenance of a basal proliferative niche in the neocortical germinal zones, drive the conical migration of neurons toward the cortical surface and ultimately lead to the establishment of cortical folds in large-brained mammal species.