Gene expression in the eukaryotic cell is a fundamental cellular process, which consists of several distinct steps but extensively coupled to each other. From site of transcription in the nucleus to the cytoplasm, both mRNA and rRNA are associated with a proper set of proteins. These proteins influence RNA processing, transport as well as ribosome maturation. We have tried to take advantage of different model systems to understand the process of eukaryotic gene expression at the post-transcription level. To this end, we have focused on identification and characterization of several specific proteins in the context of mRNP and rRNP particles. We have characterized a novel yeast gene MRD1, which encodes a protein with five RNA-binding domains (RBDs) and is essential for viability. Mrd1p is present in the nucleolus and the nucleoplasm. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and 40S ribosomal subunits. Mrd1p associates with the 35S prerRNA and the U3 snoRNA and is required for the initial processing of pre-rRNA at the A0-A2 sites. The presence of five RBDs in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage. Meanwhile, an MRD1 homologue, Ct-RBD-1 with six RBDs, has also been identified and shown to involve in ribosome biogenesis in Chironomus tentans. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In the cytoplasm, Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that Ct-RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes. We have characterized a novel abundant nucleolar protein, p100 in C. tentans. The p100 protein is located in the fibrillar compartment of the nucleolus, and remains in the nucleolus after digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. These results suggest that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus. We have characterized a Dbp5 homologue in C. tentans, Ct-Dbp5. The protein becomes associated with nascent pre-mRNAs at a large number of active genes, including the Balbiani ring (BR) genes. Ct-Dbp5 is bound to nascent BR pre-mRNP particles and accompanies them through the nucleoplasm and the nuclear pore into the cytoplasm. Nuclear accumulation of Ct-Dbp5 takes place when synthesis and/or export of mRNA are inhibited. Our results indicate that most or all of the shuttling Ct-Dbp5 exiting from the nucleus associated with mRNP. Furthermore, Ct-Dbp5 is present along the mRNP fibril extending into the cytoplasm, supporting the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation. We have shown that the export receptor CRM1 in C. tentans is associated with BR pre-mRNP while transcription takes place. We have also shown that the GTPase Ran binds to BR pre-mRNP, but its binding mainly in the interchromatin. Although both CRM1 and Ran accompany BR pre-mRNP through the nuclear pore, Leptomycin B treatment reveals that a NES-CRM1-RanGTP complex is not essential for export of the BR mRNP. Our results suggest that several export receptors associate with BR mRNP and that these receptors might have redundant functions in the nuclear export of BR mRNP. We have analyzed four SR proteins, SC35, ASF/SF2, 9G8 and hrp45, in C. tentans. All four SR proteins genes are expressed in salivary gland cells and in several other tissues in a tissue specific pattern. We found that about 90% of all nascent pre-mRNAs bind all four SR proteins, and that approximately 10% of the pre-mRNAs associate with different subsets of the four SR proteins, suggesting that not all of four SR proteins are needed for processing of pre-mRNA. None of three examined SR proteins leave BR pre-mRNP as splicing is completed. Instead, 9G8 accompanies the mRNP to the cytoplasm, while SC35 and hrp45 leave the BR mRNP at the nuclear side of the nuclear pore complex.

Botryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. As such, this study developed both apparatus and techniques to culture B. braunii for use in the genetic and biochemical characterization. During genetic studies, the genome size was determined of a representative strain of each of the three races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Flow cytometry analysis indicates that the A race, Yamanaka strain, of B. braunii has a genome size of 166.0 +/- 0.4 Mb, which is similar to the B race, Berkeley strain, with a genome size of 166 +/- 2.2 Mb, while the L race, Songkla Nakarin strain, has a substantially larger genome size at 211.3 +/- 1.7 Mb. Phylogenetic analysis with the nuclear small subunit (18S) rRNA and actin genes were used to classify multiple strains of A, B, and L races. These analyses suggest that the evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce. Biochemical studies of B. braunii primarily focused on the B race, because it uniquely produces large amounts of botryococcenes that can be used as a fuel for internal combustion engines. C30 botryococcene is metabolized by methylation to generate intermediates of C31, C32, C33, and C34. Raman spectroscopy was used to characterize the structure of botryococcenes. The spectral region from 1600?1700 cm^-1 showed v(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm^-1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm^-1 was assigned to the backbone C=C bond stretching. Finally, confocal Raman microspectroscopy was used to map the presence and location of methylated botryococcenes within a living colony of B. braunii cells.

Cyanobacteria are an ancient lineage of gram-negative photosynthetic prokaryotes that play an important role in the nitrogen cycle in terrestrial and aquatic systems. Widespread cyanobacterial blooms have prompted numerous studies on the classification of this group, however defining species is problematic due to lack of clarity as to which characters best define the various taxonomic levels. The genera Anabaena, Aphanizomenon and Nostoc form one of the most controversial groups and are typically paraphyletic within phylogenetic trees and share similar morphological characters. This study’s purpose was to determine the taxonomic and phylogenetic relationships among isolates from these three genera using 16S rRNA and bacterial elongation factor P (efp) gene sequences as well as morphological analyses. These data confirmed the non-monophyly of Anabaena and Aphanizomenon and demonstrated that many of the isolates were intermixed among various clades in both gene phylogenies. In addition, the genus Nostoc was clearly not monophyletic and this finding differed from previous studies. The genetic divergence of the genus Nostoc was confirmed based on 16S rRNA gene sequence similarities (≥85.1%), and the isolates of Anabaena were genetically differentiated, contrary to previous studies (16S rRNA gene sequence similarities ≥89.4%). The morphological diversity was larger than the molecular diversity, since the statistical analysis ANOSIM showed that the isolates were morphologically well differentiated; however, the 16S rRNA gene sequence similarities showed some isolates as being related at the species level. Planktonic and benthic strains were not distinguished phylogenetically, although some well-supported clusters were noted. Cellular measurements (length and width of vegetative cells, end cells, heterocysts and akinetes) were noted to be the morphological characters that best supported the differentiation among isolates, more than qualitative characterization. Among the metric parameters, the length of akinetes resulted in better differentiation among isolates. The efp gene sequence analyses did not appear to be useful for the taxonomic differentiation at lower taxonomic levels, but gave well-supported clusters for Aphanizomenon that was supported by the morphological analyses. Both gene regions gave similar trees with the exception of the Aphanizomenon isolates which clustered together in phylogenetic trees based on the efp gene. This differed from the 16S rRNA gene in which this genus was paraphyletic with Anabaena species that were similar in morphology to Aphanizomenon. Hence, the application of multiple taxonomic criteria is required for the successful delineation of cyanobacterial species.

Not many of the abundant freshwater bacterial groups have a representative cultured isolate. In this master thesis project, some abundant bacterioplankton from two lakes (Lake Ekoln and Lake Erken) could be isolated by a dilution-to-extinction approach. Sterilized lake water which was obtained through an ultrafiltration system was used resembling a natural medium. Specific fragments of 16s rRNA of the isolates were amplified by universal bacterial primers (27f and 1492r, 341f and 805r.) for genotyping against a freshwater sequence database and RDP training set (Version 7). A total of 33 isolates from the two lakes were taxonomically classified and revealed the isolation of typical and abundant freshwater bacteria. Original bacterial community of Lake Ekoln was also analyzed by 16S rRNA clone library construction for diversity study. Phylogenetic trees were built through neighbor-joining method by Mega (Version 5) to reveal the evolutionary relationships among database entries, obtained isolates and clones.

Cyanobacteria are an ancient lineage of gram-negative photosynthetic prokaryotes that play an important role in the nitrogen cycle in terrestrial and aquatic systems. Widespread cyanobacterial blooms have prompted numerous studies on the classification of this group, however defining species is problematic due to lack of clarity as to which characters best define the various taxonomic levels. The genera Anabaena, Aphanizomenon and Nostoc form one of the most controversial groups and are typically paraphyletic within phylogenetic trees and share similar morphological characters. This study’s purpose was to determine the taxonomic and phylogenetic relationships among isolates from these three genera using 16S rRNA and bacterial elongation factor P (efp) gene sequences as well as morphological analyses. These data confirmed the non-monophyly of Anabaena and Aphanizomenon and demonstrated that many of the isolates were intermixed among various clades in both gene phylogenies. In addition, the genus Nostoc was clearly not monophyletic and this finding differed from previous studies. The genetic divergence of the genus Nostoc was confirmed based on 16S rRNA gene sequence similarities (≥85.1%), and the isolates of Anabaena were genetically differentiated, contrary to previous studies (16S rRNA gene sequence similarities ≥89.4%). The morphological diversity was larger than the molecular diversity, since the statistical analysis ANOSIM showed that the isolates were morphologically well differentiated; however, the 16S rRNA gene sequence similarities showed some isolates as being related at the species level. Planktonic and benthic strains were not distinguished phylogenetically, although some well-supported clusters were noted. Cellular measurements (length and width of vegetative cells, end cells, heterocysts and akinetes) were noted to be the morphological characters that best supported the differentiation among isolates, more than qualitative characterization. Among the metric parameters, the length of akinetes resulted in better differentiation among isolates. The efp gene sequence analyses did not appear to be useful for the taxonomic differentiation at lower taxonomic levels, but gave well-supported clusters for Aphanizomenon that was supported by the morphological analyses. Both gene regions gave similar trees with the exception of the Aphanizomenon isolates which clustered together in phylogenetic trees based on the efp gene. This differed from the 16S rRNA gene in which this genus was paraphyletic with Anabaena species that were similar in morphology to Aphanizomenon. Hence, the application of multiple taxonomic criteria is required for the successful delineation of cyanobacterial species.

The phylum Chaetognatha is a mysterious group of organisms that has eluded scientists for more than a century because of their unique morphology and developmental characteristics, i.e. protostome (mouth develops from blastopore; e.g. mollusks, annelids, arthropods) versus deuterostome (anus develops from blastopore; e.g. echinoderms and chordates) offer few clues to their evolutionary origins. Some early morphological studies argued that chaetognaths were derived mollusks or nematodes according to gross ultrastructural data, while other studies focused on the coelomic cavity. 33 Although 18S rRNA is widely used in molecular phylogeny studies, it has limits such as long- branch chain attractions and a slow rate of evolutionary change. Long-branch chain attractions are a phenomenon in phylogenetic analyses when rapidly evolving lineages are inferred to be closely related, regardless of their true evolutionary relationships. Hence other genes are used in this study to complement the 18S rRNA such as the cytochrome oxidase genes. The cytochrome oxidase genes are highly conserved throughout all eukaryotic organisms and they are less ambiguous to align as compared to the ribosomal genes, making them better phylogenetic markers as compared to the 18S rRNA gene. This study focuses on using a molecular approach (ARDRA, PCR, phylogenetic tree reconstruction) to determine the phylogeny of pelagic chaetognaths found on Pelican Island, Galveston, Texas. 18S rRNA, Cytochrome Oxidase I and Cytochrome Oxidase II genes were used to help decipher the phylogeny of this group. All analyzed genes in this study (18S rRNA, COI, and COII) grouped the Pelican Island chaetognaths with the protostomes. The maximum parsimony bootstrap tree for the 18S rRNA gene, grouped the samples closest to the arthropods (protostome). For the COI and COII genes, the minimum evolution bootstrap tree grouped the 8 collected samples more closely to two other protostome phyla: the mollusks and annelids (COI) while bootstrapping with the COII grouped the samples with the nematodes (with >66 percent bootstrap). My findings are significant because they reveal phylogenetic results of a protostome lineage for the Chaetognatha using 3 genes, one of which (COII) has not been greatly studied for the Chaetognatha.

Anchialine blue holes found in the interior of the Bahama Islands have distinct fresh and salt water layers, with vertical mixing, and dysoxic to anoxic conditions below the halocline. Scientific cave diving exploration and microbiological investigations of Cherokee Road Extension Blue Hole on Abaco Island have provided detailed information about the water chemistry of the vertically stratified water column. Hydrologic parameters measured suggest that circulation of seawater is occurring deep within the platform. Dense microbial assemblages which occurred as mats on the cave walls below the halocline were investigated through construction of 16S rRNA clone libraries, finding representatives across several bacterial lineages including Chlorobium and OP8. In many blue holes, microbial metabolism of organic matter in the presence of seawater sulfate leads to anoxic and sulfidic conditions at or below halocline. Sunlight penetrating this sulfidic layer allows for in situ primary production to be dominated by bacterial anoxygenic phototrophs. Although water column chemistry and molecular genetic diversity of microbial mats in Cherokee Road Extension Blue Hole were investigated in this study, the full scope of the biogeochemistry of inland blue holes throughout the Bahamas Archipelago is complex and poorly understood. However, these microbial communities are clearly influenced by several factors including solar insolation, terrestrial and marine inputs of oxygen, carbon, and nutrients, water residence times, depth to the halo/chemocline, and cave passage geometry. The biogeochemistry of inland blue holes throughout the Bahamas is so distinctive which makes Abaco Island and the rest of the archipelago valuable as natural experiments, repositories of microbial diversity, and analogs for stratified and sulfidic oceans present early in Earth's history.

Probiotics are viable microorganisms that show beneficial effects on the health of the host by improving their intestinal microflora. The microorganisms applied as probiotics mainly include Lactobacillus and Bifidobacterium species. Probiotics can inhibit the bacterial pathogens, reduce serum cholesterol levels, improve lactose tolerance and stimulate the immune response. They also have other properties such as / tolerance to acid and bile salts, adherence to gastrointestinal cells for colonization, resistance to antibiotics and &beta / -galactosidase acitivity. The properties of probiotic products are determined by the characteristics of the microorganisms they contain. For that reason, isolation and characterization of new strains having probiotic properties is an important issue. New strains are generally isolated from their natural habitats which are fermented dairy products such as kefir. In order to exert beneficial health affects in the digestive system, commercial probiotic products should contain adequate numbers of viable cells. Probiotic microorganisms should protect their viability during their shelf storage. Therefore, the viability of probiotics is especially important for food manufacturers that search for new probiotic strains with good survival and stability properties upon storage. In this study, probiotic microorganisms were isolated from traditional kefir grains known as a &lsquo / complex probiotic&rsquo / . The isolates were firstly identified using biochemical tests, then the putative species belonging to &lsquo / Lactobacillus acidophilus group&rsquo / were identified with 16S rRNA gene sequencing. Analysis of sequencing resulted in differentiation of &ldquo / L. acidophilus group&rdquo / organisms, namely L. amylovorus and L. acidophilus. Moreover, typing of commercial and traditional L. acidophilus strains and L. amylovorus strains were performed with RAPD-PCR by using primer M13. While several L. acidophilus strains showed different RAPD fingerprints most of the L. acidophilus and L. amylovorus strains could not be differentiated due to high similarity of their RAPD fingerprints. Following identification, survival of these isolates in probiotic yogurt preparations were investigated and compared to the survival of commercial probiotics. Consequently, although the survival of kefir grain isolates were less than commercial probiotics, they sustained the minimum recommended level for probiotics (106 cfu/ml) during cold storage. Such level of survival makes them considerably good candidates to be used as commercial probiotic cultures.

The molecular characterization of twenty six Theileria spp. isolates and one C. felis isolate were done on the small subunit ribosomal RNA (SSU rRNA) gene, the 5.8S gene, and the two internal transcribed spacer regions using gDNA. The SSU rRNA gene is increasingly accepted as a widely used marker for characterization, taxonomic classification, and phylogenetic analysis and this gene has been sequenced from a variety of different organisms, resulting in a large database for sequence comparisons (Chae et al. 1998; Chae et al., 1999 a,b,c; Stockham et al., 2000; Cossio-Bayugar et al., 2002; Gubbels et al., 2000). The genomic region consists of the internal transcribed spacer 1 (ITS 1), the 5.8S gene, and internal transcribed spacer 2 (ITS 2) (ITS 1-5.8S-ITS 2 gene region) and separates the SSU rRNA gene from the large subunit ribosomal RNA gene. The 5.8S rRNA gene is highly conserved in size and nucleotide sequence, is relatively constant in molecular weight, and has an average chain length of approximately 160 nucleotides and has proven useful in dividing subgenera of Gyrodactylus ((Nazar, 1984; Zietara et al., 2002). Pairwise comparisons were done between the clones of an individual isolate and among the clones of the different isolates. Phylogenetic trees were made from the resulting sequences. This study shows that different SSU rRNA genes may be associated with ITS 1-5.8S-ITS 2 gene regions of distinct sequence in the same isolate. This study also demonstrates that considerable ITS 1-5.8S-ITS 2 gene region sequence variation may exist within a species. This may be useful for subspeciation designation, or may simply reflect considerable variation within the population. This study shows that the ITS 1-5.8S-ITS 2 gene region may be a useful molecular marker for the taxonomy of Theileria spp.

The ecological niche of the vagina contains a large number of different microbes that are constantly interacting with each other and the host. Culture methods have not been sufficient in order to resolve the complexity of the normal vaginal flora. Further, the methods for delineating normal flora from not normal flora are not easily handled and are traditionally not based on culture but on microscopy of elements of the vaginal fluid. In the work presented in this thesis, an international collaboration was established that pin-pointed some of the difficulties in classifying vaginal floras, including staining, sampling, and discordance when lactobacilli are few in number, and that emphasized the importance of the size of the vision field in microscopes. As lactobacilli are prominent members of the normal vaginal flora they need to be carefully classified if further work towards more robust scoring tools is to be achieved. Phenotypic methods have not been able to separate the closely related Lactobacillus species of the vagina. Progress in molecular biology has provided possibilities to characterize these lactobacilli, which are mainly from the Lactobacillus acidophilus group. In this work a large number of strains collected by true random sampling were subjected to RAPD-PCR, TTGE and multiplex PCR for species identification. The major species found were L. crispatus, L. gasseri and L. jensenii and the recently described L. iners. The presence of L. iners has not been detected in previous studies due to its special nutrient requirements. Development of pyrosequencing technology also made it possible to match signatures of the two variable regions V1 and V3 of the 16S rRNA gene of the vaginal lactobacilli and identify them to the species level in a high throughput manner. The study confirmed that the dominating flora in women with normal vaginal flora comprises the four species mentioned previously. Repetitive sampling during IVF-treatment with highly varying oestrogen levels demonstrates changes that possibly occur during changes in the natural life cycle. Furthermore, L. iners was found to be the first species to be established after spontaneously resolved or treated Bacterial Vaginosis. These findings can be of help in developing new strategies for regaining and retaining the normal vaginal flora.