Helicosporidia are single-celled obligate endoparasites of invertebrates. They have a unique morphology and infection strategy, which make them unlike any other eukaryote. Molecular data were produced to clarify their phylogenetic relationship and to examine the evolution of their cryptic plastid. Phylogenetic analyses of 69 ribosomal proteins identified from an expressed sequence tag (EST) library showed that Helicosporidia are derived green algae and more specifically, are related to the trebouxiophyte algae. An obligate parasitic lifestyle is rare among plant and algal groups, and because Helicosporidium possesses no pigments and no chloroplast-like structure has been identified, photosynthetic ability has presumably been lost in this organism. I sought to examine the role that a relict plastid might play in Helicosporidium. I identified ESTs of 20 putatively plastid-targeted enzymes that are involved in a wide variety of metabolic pathways. As expected, no components of photosynthesis were found, but components of other metabolic pathways including sulfur metabolism and fatty acid, isoprenoid and heme biosynthesis suggest that Helicosporidium retains its plastid for these functions. The complete plastid genome of this species of Helicosporidium was sequenced and revealed only four protein-coding genes not involved in transcription or translation, with two of these confirming the metabolic functions suggested by the nuclear-encoded, plastid-targeted genes identified from the ESTs. In addition, the Helicosporidium plastid genome is one of the smallest known (37.5 kb). Its reduced size results from loss of many genes commonly found in plastids of other plants and algae (including all proteins that function in photosynthesis), elimination of duplicated genes and redundant tRNA isoacceptors, and minimization of intergenic spaces. The Helicosporidium plastid genome is also highly structured, with each half of the circular genome containing nearly all genes on one strand. Both the structure and content of the plastid genome and the deduced function of the organelle show parallels with the relict plastid found in the malaria parasite, Plasmodium falciparum. These unrelated organisms each evolved from photosynthetic ancestors, and the convergence in form and function of their relict plastids suggest that common forces shape plastid evolution, following the switch from autotrophy to parasitism.

Helicosporidia are single-celled obligate endoparasites of invertebrates. They have a unique morphology and infection strategy, which make them unlike any other eukaryote. Molecular data were produced to clarify their phylogenetic relationship and to examine the evolution of their cryptic plastid. Phylogenetic analyses of 69 ribosomal proteins identified from an expressed sequence tag (EST) library showed that Helicosporidia are derived green algae and more specifically, are related to the trebouxiophyte algae. An obligate parasitic lifestyle is rare among plant and algal groups, and because Helicosporidium possesses no pigments and no chloroplast-like structure has been identified, photosynthetic ability has presumably been lost in this organism. I sought to examine the role that a relict plastid might play in Helicosporidium. I identified ESTs of 20 putatively plastid-targeted enzymes that are involved in a wide variety of metabolic pathways. As expected, no components of photosynthesis were found, but components of other metabolic pathways including sulfur metabolism and fatty acid, isoprenoid and heme biosynthesis suggest that Helicosporidium retains its plastid for these functions. The complete plastid genome of this species of Helicosporidium was sequenced and revealed only four protein-coding genes not involved in transcription or translation, with two of these confirming the metabolic functions suggested by the nuclear-encoded, plastid-targeted genes identified from the ESTs. In addition, the Helicosporidium plastid genome is one of the smallest known (37.5 kb). Its reduced size results from loss of many genes commonly found in plastids of other plants and algae (including all proteins that function in photosynthesis), elimination of duplicated genes and redundant tRNA isoacceptors, and minimization of intergenic spaces. The Helicosporidium plastid genome is also highly structured, with each half of the circular genome containing nearly all genes on one strand. Both the structure and content of the plastid genome and the deduced function of the organelle show parallels with the relict plastid found in the malaria parasite, Plasmodium falciparum. These unrelated organisms each evolved from photosynthetic ancestors, and the convergence in form and function of their relict plastids suggest that common forces shape plastid evolution, following the switch from autotrophy to parasitism.

<p>During the development and lifecycle of multicellular organisms, cells have to die, and this occurs by a process called programmed cell death or PCD, which can be separated from necrosis or accidental cell death (Pennell and Lamb, 1997). Senescence is the terminal phase in the development of an organism, organ, tissue or cell, where nutrients are remobilized from the senescing parts of the plant into other parts, and the cells of the senescing organ or tissue undergo PCD if the process is not reversed in time. Leaf senescence involves cessation of photosynthesis, loss of pigments and proteins, nutrient remobilization, and degradation of the plant cells (Smart, 1994). Initiation of leaf senescence is triggered by a wide range of endogenous and environmental factors, that through unknown pathways controls the process, and regulates the expression of senescence-associated genes (SAGs) (Buchanan-Wollaston, 1997). Autumn leaf senescence in deciduous trees is regulated by photoperiod and temperature, and is an attractive experimental system for studies on senescence in perennial plants.</p><p>We have studied the process of autumn senescence in a free-growing aspen (Populus tremula) by following changes in pigment, metabolite and nutrient content, photosynthesis, and cell and organelle integrity. All data were combined in a cellular timetable of autumn senescence in aspen. The senescence process started on September 11 with degradation of pigments and other leaf constituents, and once initiated, progressed steadily without being affected by the environment. Chloroplasts were rapidly degraded, and mitochondria took over energy production after chlorophyll levels had dropped by 50%. At the end of remobilization, around 29th of September, some cells were still metabolically active and had chlorophyll-containing plastids. Over 80% of nitrogen and phosphorus was remobilized, and a sudden change in the 15N of the cellular content on September 29, indicated that volatile compounds may have been released.</p><p>We have also studied gene expression in autumn leaves by analysing EST sequences from two different cDNA libraries, one from autumn leaves of a field-grown aspen and the other from young, but fully expanded leaves of a green-house grown aspen. In the autumn leaf library, ESTs encoding metallothioneins, proteases, stress-related proteins and proteins involved in respiration and breakdown of macromolecules were abundant, while genes coding for photosynthetic proteins were massively downregulated. We have also identified homologues to many known senescence-associated genes in annual plants.</p><p>By using Populus cDNA microarrays, we could follow changes in gene expression during the autumn over four years in the same free-growing aspen tree. We also followed changes in chlorophyll content to monitor the progression of leaf senescence. We observed a major shift in gene expression, occuring at different times the four years, that reflected a metabolic shift from photosynthetic competence to energy generation by mitochondrial respiration. Even though autumn senescence was initiated almost at the same date each year, the transcriptional timetables were different from year to year, especially for 2004, which indicates that there is no strict correlation between the transcriptional and the cellular timetables of leaf senescence.</p>

During the development and lifecycle of multicellular organisms, cells have to die, and this occurs by a process called programmed cell death or PCD, which can be separated from necrosis or accidental cell death (Pennell and Lamb, 1997). Senescence is the terminal phase in the development of an organism, organ, tissue or cell, where nutrients are remobilized from the senescing parts of the plant into other parts, and the cells of the senescing organ or tissue undergo PCD if the process is not reversed in time. Leaf senescence involves cessation of photosynthesis, loss of pigments and proteins, nutrient remobilization, and degradation of the plant cells (Smart, 1994). Initiation of leaf senescence is triggered by a wide range of endogenous and environmental factors, that through unknown pathways controls the process, and regulates the expression of senescence-associated genes (SAGs) (Buchanan-Wollaston, 1997). Autumn leaf senescence in deciduous trees is regulated by photoperiod and temperature, and is an attractive experimental system for studies on senescence in perennial plants. We have studied the process of autumn senescence in a free-growing aspen (Populus tremula) by following changes in pigment, metabolite and nutrient content, photosynthesis, and cell and organelle integrity. All data were combined in a cellular timetable of autumn senescence in aspen. The senescence process started on September 11 with degradation of pigments and other leaf constituents, and once initiated, progressed steadily without being affected by the environment. Chloroplasts were rapidly degraded, and mitochondria took over energy production after chlorophyll levels had dropped by 50%. At the end of remobilization, around 29th of September, some cells were still metabolically active and had chlorophyll-containing plastids. Over 80% of nitrogen and phosphorus was remobilized, and a sudden change in the 15N of the cellular content on September 29, indicated that volatile compounds may have been released. We have also studied gene expression in autumn leaves by analysing EST sequences from two different cDNA libraries, one from autumn leaves of a field-grown aspen and the other from young, but fully expanded leaves of a green-house grown aspen. In the autumn leaf library, ESTs encoding metallothioneins, proteases, stress-related proteins and proteins involved in respiration and breakdown of macromolecules were abundant, while genes coding for photosynthetic proteins were massively downregulated. We have also identified homologues to many known senescence-associated genes in annual plants. By using Populus cDNA microarrays, we could follow changes in gene expression during the autumn over four years in the same free-growing aspen tree. We also followed changes in chlorophyll content to monitor the progression of leaf senescence. We observed a major shift in gene expression, occuring at different times the four years, that reflected a metabolic shift from photosynthetic competence to energy generation by mitochondrial respiration. Even though autumn senescence was initiated almost at the same date each year, the transcriptional timetables were different from year to year, especially for 2004, which indicates that there is no strict correlation between the transcriptional and the cellular timetables of leaf senescence.

The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension. The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results. Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses. Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results. <b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis

<p>The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing.</p><p>Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension.</p><p>The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results.</p><p>Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses.</p><p>Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results.</p><p><b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis</p>