Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

No description available.

No description available.

Zooxanthellae are very important for the coral reef ecosystem. The diversity of coral hosts is high in the Indo-Pacific, but the diversity of zooxanthellae has not been broadly investigated. Southern Taiwan and Penghu Islands are coral reef and non-reefal communities, respectively. These localities were chosen as the sampling sites for this study to maximize the opportunity of surveying this region in the Indo-Pacific. Zooxanthellae diversity was investigated in 40 host species including 32 species of Scleractinia, 4 species of Actiniaria, 3 species of Milleporina and 1 species of Helioporacea using polymerase chain reaction (PCR) of the ssrRNA gene and restriction fragment length polymorphism (RFLP) patterns. The phylogenetic relationship of partial and complete sequences of the ssrRNA gene were also analysed. Aiptasia puchella harbors clade B; Oulastrea crispata only harbors clade E; while Acropora palifera and Montipora cactus harbor both clades C and E. Zooxanthellae isolated from all except the above 4 host species are identified as "clade C" sensu Rowan and Powers (1991a). Therefore, the clade C is the dominant type in the Indo-Pacific. Phylogenetic analyses based on partial and complete sequences obtained in this study and also from the GenBank data base demonstrate 4 clades (A, B, C and E) in the genus Symbiodinium. Clade E, classed as D3 RFLP type in previous studies, is a distinct clade differing from A, B and C by RFLP and sequencing data. Clade E has only been found in Scleractinia host species collected in shallow-water habitats in the Pacific. The composition of zooxanthellae clades and ecological pattern of polymorphic symbiosis is not consistent with the irradiance adaptation hypothesis in the Caribbean. A literature survey of zooxanthellae in Scleractinian hosts indicates a significant difference between the Caribbean and the Pacific. The documented biogeography of zooxanthellae clades and the ecological pattern of polymorphic symbiosis are also differ between the Caribbean and the Indo-Pacific.

Ribosomal RNA (rRNA) molecules form highly conserved secondary and tertiary structures via rRNA-rRNA and rRNA-protein interactions that collectively comprise the macromolecule that is the ribosome. Because of their cellular universality, rRNA molecules are commonly used for phylogeny estimations spanning all divergences of life. In this dissertation, I elucidate the structure of several rRNAs by analyzing multiply aligned sequences for basepair covariation and conserved higher order structural motifs. Specifically, I predict novel structures for expansion segments D2 and D3 of the nuclear large subunit rRNA (28S) and variable regions V4-V9 of the nuclear small subunit rRNA (18S) from from 249 galerucine leaf beetles (Coleoptera: Chrysomelidae). I describe a novel means for characterizing regions of alignment ambiguity that improves methods for retaining phylogenetic information without violating nucleotide positional homology. In the program PHASE, I explore a variety of RNA maximum likelihood models using the 28S rRNA dataset and discuss the utitilty of these models in light of their performance under Bayesian analysis. I conclude that seven-state models are likely the best models to use for phylogenetic estimation, although I cannot determine with confidence which of the two seven-state models (7A or 7D) is better. Evaluation of the unpaired sites within both rRNAs in Modeltest provided a similar model of evolution for these non-pairing regions (TrN+ I+G). In addition, a sequenced region of the mitochondrial cytochrome oxidase I gene (COI) from the galerucines was evaluated in Modeltest, with each codon position modeled separately (GTR+I+G for positions 1 and 2, GTR+G for position 3). The combined galerucine dataset (28S+18S rRNA helices, 28S+18S rRNA unpaired sites, COI 1st, 2nd and 3rd positions) provided for two mixedmodel Bayesian analysis of five discretely-modeled partitions (using 7A and 7D). The results of these analyses are compared with those obtained from equally weighted parsimony to provide a robust phylogenetic estimate of the Galerucinae and related leaf beetle taxa. Finally, the odd characteristics of strepsipteran 18S rRNA are evaluated through comparison of 12 strepsipterans with 163 structurally-aligned arthropod sequences. Among other interesting results, I identify errors in previously published strepsipteran sequences and predict structures not previously known from metazoan rRNA.

There are two full length variants of the Pokey DNA transposon, PokeyA and PokeyB, and two MITEs, mPok1 and mPok2. Pokey inserts into ribosomal DNA (rDNA) and other genomic locations within the genomes of Daphnia species. I used qPCR to estimate haploid rDNA and Pokey copy number in five Daphnia pulex complex species. In general, rDNA number ranges from ~100 to 500. In four species, low numbers of PokeyA and PokeyB in rDNA and the rest of the genome suggest these elements have low transposition rates, high deletion rates, and/or strong purifying selection against them at the host level. Further, PokeyA may have a higher transposition rate than PokeyB. In these species, mPok1 was not found, and mPok2 is likely inactive. In comparison, the fifth species, D. arenata, which may be a hybrid, has higher Pokey numbers. Higher Pokey numbers could be due to release from epigenetic repression following hybridization. / Ontario Graduate Scholarship in Science and Technology to Shannon H. C. Eagle, Natural Sciences and Engineering Research Council of Canada Discovery Grant to Dr. Teresa J. Crease

Bovine adenovirus (BAdV)-3 is a non-enveloped, icosahedral virus with a double-stranded DNA genome, and is being developed as a vector for vaccination of animals and humans. Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV)-5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. <p> DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported. Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. <p> The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin beta and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin alpha/importin beta pathway for nuclear import. In support of this, a specific interaction between 52K and importin alpha-3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin alpha/importin beta pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. <p> A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells. <p> Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFkB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed <i>in vitro</i> and <i>in vivo</i>. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K.

Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly.

Bovine adenovirus (BAdV)-3 is a non-enveloped, icosahedral virus with a double-stranded DNA genome, and is being developed as a vector for vaccination of animals and humans. Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV)-5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. <p> DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported. Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. <p> The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin beta and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin alpha/importin beta pathway for nuclear import. In support of this, a specific interaction between 52K and importin alpha-3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin alpha/importin beta pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. <p> A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells. <p> Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFkB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed <i>in vitro</i> and <i>in vivo</i>. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K.

Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly.