Polyhydroxyalkanoates (PHAs) comprise a family of macromolecules produced by many bacteria as a carbon and energy reserve and are perceived to have commercial potential as biodegradable thermoplastics. To investigate the biogenesis of PHA inclusion-bodies and the functions of inclusion-body proteins from Bacillus megaterium strain 11561, we identified and cloned a 7.9 kb DNA fragment harboring five genes, phaP, -Q, -R, -B, and -C specifying proteins having known or putative functions in PHA metabolism and/or inclusion-body biogenesis. Sequence similarities to known pha genes identified phaB and -C as specifying acetoacetyl-CoA reductase and PHA synthase, respectively. Putative proteins encoded by phaP, -Q, and -R were not ascribed functions due to lack of significant similarities to known proteins. Both the functionality of the pha gene cluster with respect to PHA accumulation and the transcriptional organization of the genes were determined. Subsequent studies were carried out to further investigate functions of phaP, -Q, and -R. PhaP was established as a major PHA inclusion-body associated protein and was shown to localize to inclusion-bodies in living cells. Further, we demonstrated a phasin-like role for this protein due to its affect on the formation of PHA inclusion-bodies. In addition, our data is consistent with PhaP functioning as a storage protein, implying that the role of PHA inclusion-bodies may be that of a reserve of amino acids in addition to reduced carbon. Regulation of phaP was influenced by PhaQ We showed that PhaQ is a transcriptional repressor of phaP. Moreover, we demonstrated the binding of PhaQ to inclusion-bodies, suggesting that its mode of regulation may involve its localization. Similarly, we showed that PhaR is bound to PHA inclusion-bodies. Our data demonstrated the requirement of phaR for PHA accumulation in vivo and that both PhaC and PhaR were necessary for PHA synthase activity in vitro. Evidence suggests that PHA synthase from strain 11561 can exist in an active or inactive state and that this state is either directly or indirectly influenced by PhaR. A working model is proposed to describe the roles of PhaP, -Q and -R in the metabolism of PHA and biogenesis of PHA inclusion-bodies.

Eukaryotic cells contain scores of small nucleolar RNAs (snoRNAs) that are complexed with proteins in particles known as snoRNPs. SnoRNPs function in processing of ribosomal RNA (rRNA) and nucleotide modification of ribosomal and other RNAs that pass through the nucleolus. The modifications involved are: (1) isomerization of uridine to pseudouridine (Ψ), and; (2) methylation of the 2′-hydroxyl of ribose moieties (Nm). The Ψ and Nm modified nucleotides are the most abundant in rRNA. The purpose of the thesis work is to obtain a better understanding of the synthesis of snoRNPs and the function(s) of the modifications they form. To this end, three lines of research were carried out. Two focus on different snoRNA-associated proteins. The third examines the effect of snoRNP-mediated pseudouridylation of rRNA on ribosome structure and function. One of the snoRNA-associated proteins studied is a conserved helicase previously isolated from a mouse nuclear extract on the basis of association with a conserved snoRNA structure known as the box C/D motif. Here, it was shown that the yeast ortholog of this protein (Rvb2p) is required for production of snoRNAs (both major families) and for localization of snoRNP proteins. The findings are consistent with a role for Rvb2p in the assembly or trafficking of snoRNPs. A second project was aimed at determining if a particular H/ACA snoRNP protein is the enzyme that forms Ψ in rRNA. The protein, Cbf5p, was previously shown to have limited sequence homology with known Ψ synthases (96). Here, in collaboration with the John Carbon laboratory, it was determined that point mutations in Cbf5p in this region of homology can abolish in vivo pseudouridylation of rRNA, arguing that Cbf5p is indeed the global Ψ synthase. Finally, the function of Ψ in the peptidyl transferase center (PTC) was investigated by depleting cells of snoRNPs that modify this domain, and evaluating the under-modified ribosomes. Interestingly, cells lacking Ψs in the PTC are impaired in growth and protein synthesis. Chemical probing revealed changes in the structure of the mutant ribosomes. Enhanced reactivity with dimethylsulfate was observed for nucleotides predicted to influence binding of elongation and initiation factors. The results demonstrate that the structure and activity of yeast ribosomes depend on pseudouridylation of the PTC. The possibility that guide snoRNPs have function(s) other than modification is discussed.

The mitotic apparatus is composed of a complex, seemingly chaotic, but wonderfully organized compilation of cytoskeletal elements and motor proteins. Its task is to assure that each daughter cell receives a single copy of the genome. Microtubules are the biological polymers that make up the bipolar mitotic spindle, serving as tracks to separate sister-chromatids to opposite sides of the dividing cell. Understanding the behavior of microtubules during mitotic entry and exit is extremely important for diagnosing possible defects of atypical or failed mitosis. This research uses the advantages of GFP technology to investigate the dynamics of microtubules throughout the cell cycle. Cells permanently expressing GFP-α-tubulin were generated using lipid transfection and drug selection. We show that GFP-tubulin had little effect on the behavior and role of microtubules; therefore, we conclude that cells expressing GFP-α-tubulin are a reliable method for studying microtubule behavior. Live imaging of an epithelial cell line (LLCPK1) stably expressing GFP-α-tubulin revealed a microtubule bundling phenomenon that stabilizes microtubules during prometaphase. Further analysis shows that these bundles are pulled into the forming spindle in a dynein dependent manner. Our final model is an extension of the search and capture model, where we suggest that centrosomal microtubules also capture peripheral microtubule bundles during spindle assembly. During mitotic exit, the process of moving microtubules inward is reversed, as microtubules are subject to outward forces during anaphase. Three main events are documented following anaphase onset: microtubule elongation, microtubule release, and spindle pole fragmentation. These events serve to populate regions of the cell that were devoid of microtubules during metaphase. Evidence is also presented in support of how microtubules might specify the location of the contractile ring. We show that microtubules at the equator target the cortex more frequently than elsewhere in the cell, suggesting a faster rate of delivering furrow-initiation factors. We use numerous GFP fusion proteins that allowed us to tackle our biological question from several angles, which wouldn't have been possible to answer otherwise. In summary, my work provides new insight into the behavior of microtubules during the assembly and disassembly of mitotic spindles in mammalian somatic cells.

Despite current advances in understanding breast cancer, in 2007 an estimated 178,480 new cases were diagnosed in female patients in the United States, and an estimated 40,640 deaths will occur due to this disease [1]. Numerous risk factors exist for breast cancer including current age, reproductive events, exposure to exogenous hormones or ionizing radiation, and genetic factors [2]. The research presented here is designed to answer questions specifically related to pregnancy and breast cancer risk (parity-induced protection against breast cancer), as well as the molecular pathways involved. We first examined the relationship between p53, a known tumor suppressor gene of breast carcinoma in the mammary gland, and age at first parity in human breast tissue. Using explant cultures from reduction mammoplasty patients, we demonstrated an increase in radiation-induced accumulation of p53 in breast tissue from patients who have undergone an early parity compared to nulliparous patients, or late parous patients. We also demonstrated that p53 accumulation was positively correlated with an increasing number of pregnancies. These are the first studies using cultured human mammary tissue that demonstrate results consistent with experimental rodent models and observational human data. Next, a potential tumor suppressor gene within the breast, cellular retinol binding protein-1 (CRBP1), was found to be significantly up-regulated in response to parity in mice and humans, and that it utilizes transforming growth factor-beta (TGF-β) to confer its signals. Finally, we demonstrated that addition of insulin-like growth factor-I (IGF-I) to mouse mammary gland whole organ cultures (mWOCs) treated with estrogen and progesterone (E+P) could block the normal induction of apoptosis, p53, and its downstream target p21 in response to gamma radiation. In addition, while treatment with E+P resulted in the up-regulation of insulin-like growth factor binding protein-3 (IGFPB3), TGF-β1, and CRBP1, this up-regulation was blocked upon co-treatment with IGF-I. Overall, the research presented here has furthered our knowledge of possible mechanisms by which an early parity can protect against breast cancer. These data suggest that numerous mechanisms can aberrantly alter signaling pathways involved in this protection such that the balance is shifted away from tumor suppression and growth arrest, and towards a phenotype that demonstrates an increased susceptibility toward cancer.

Trypanosoma brucei and related parasites are causative agents of severe diseases that affect global health and economy. T. brucei is responsible for sleeping sickness in humans (African trypanosomiasis) and a wasting disease in livestock. More than 100 years after T. brucei was identified as the etiological agent for sleeping sickness, available treatments remain inadequate, complicated by toxicity, lengthy and expensive administration regiments, and drug-resistance. There is clear need for the development of a new antitrypanosomal drugs. Due to the unique evolutionary position of these early diverging eukaryotes, trypanosomes posses a number of biological properties unparalleled in other organisms, including humans, which could prove valuable for new drug targets. One of the most distinctive properties of trypanosomes is their mitochondrial DNA, called kinetoplast DNA (kDNA). kDNA is composed of over five thousand circular DNA molecules (minicircles and maxicircles) catenated into a topologically complex network. Replication of kDNA requires an elaborate topoisomerase-mediated release and reattachment mechanism for minicircle theta structure replication and at least five DNA polymerases. Three of these (POLIB, POLIC, and POLID) are related to bacterial DNA polymerase I and are required for kDNA maintenance and growth. Each polymerase appears to make a specialized contribution to kDNA replication. The research described in this dissertation is a significant contribution to the field of kDNA replication and the advancement of kDNA replication proteins as putative drug targets for sleeping sickness. Functional characterization of POLIB indicated that it participates in minicircle replication but is likely not the only polymerase contributing to this process. Gene silencing of POLIB partially blocked minicircle replication and led to the production of a previously unidentified free minicircle species, fraction U. Characterization of fraction U confirmed its identity as a population of dimeric minicircles with non-uniform linking numbers. Fraction U was not produced in response to silencing numerous other previously studied kDNA replication proteins but, as we demonstrated here, is also produced in response to POLID silencing. This common phenotype led us to hypothesize that POLIB and POLID both participate in minicircle replication. Simultaneously silencing both polymerases completely blocked minicircle replication, supporting a model of minicircle replication requiring both POLIB and POLID. Finally, we demonstrate that disease-causing trypanosomes require kDNA and the kDNA replication proteins POLIB, POLIC, and POLID. These data provide novel insights into the fascinating mechanism of kDNA replication and support the pursuit of kDNA replication proteins as novel drug targets for combating African trypanosomiasis.

This thesis is the first large-scale molecular phylogenetic study on filarial parasites (family Onchocercidae) which includes 16 species of 6 genera: Brugia malayi, B. pahangi, B. timori, B. patei, B. beaveri, B. buckleyi, Wuchereria bancrofti, W. kalimantani, Mansonella perstans, Loa loa, Onchocerca volvulus, O. ochengi, O. gutturosa, Dirofilaria immitis, Acanthocheilonema viteae and Litomosoides sigmodontis. Two sets of sequence data (5S rDNA spacer and HHa I repeat) were collected by PCR, cloning and dideoxy sequencing. The 5S rDNA gene spacer region sequences were aligned and analyzed by maximum parsimony algorithms, distance methods (neighbor-joining, UPGMA and Fitch-Margoliash methods) and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the robustness of the different phylogenetic reconstructions. The data indicated that both 5S spacer region sequences and Hha I repeat sequences are highly conserved within species yet differ significantly between species. Spliced leader sequences were observed in all of the 5S rDNA spacers sequenced with no sequence variation, although flanking region length heterogeneity was observed even within species. The 5S rDNA spacer sequence data set was used to resolve the branching order of the six genera studied. The various tree-building methods gave very similar results. This study identified four clades which are strongly supported by bootstrap analysis: the Brugia clade; the Wuchereria clade; the Brugia-Wuchereria clade and the Onchocerca clade. Ascaris lumbricoides was used as an outgroup to root the trees generated using 5S sequence data. The analyses indicated that L. sigmodontis and A. viteae may be the most primitive among the 16 species studied. The results of this study do not support the classification which places Loa loa and D. immitis into the Dirofilariinae subfamily. Hha I repeat sequences are 322 nucleotides long, highly repeated and tandemly arranged sequences unique to the nuclear genomes of the genus Brugia. Phylogenetic analyses on the Hha I repeat sequence data set identified at least two clades: the B. pahangi-B. beaveri clade and the B. malayi-B.timori-B. buckleyi clade. Results from the analyses of both data sets were combined and a hypothesis for the phylogeny of the sixteen species of filarial parasites was proposed.

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDE I, CDE II, and CDE III) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wildtype cells shows that some guanines within the centromere DNA are protected from methylation by dimethylsulfate (DMS). These results are consistent with studies demonstrating that yeast contain sequence-specific centromere DNA binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function. We have identified a putative Saccharomyces cerevisiae homologue of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the $\delta$-class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation. plc1-1 cells show an 8-9 fold increase in aberrant chromosome segregation and arrest primarily as large-budded or unbudded cells. Molecular analysis revealed that PLC1 encodes a predicted protein of 94 kD with approximately 50% and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from human, bovine, rat, and Drosophila melanogaster. The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium. Interestingly, the temperature sensitive and chromosome segregation plc1-1 phenotypes are suppressed by exogenous calcium, suggesting a possible relationship between the PI-PLC signaling pathway and control of nuclear events in yeast.

Preparations of unrestricted DNA from Pseudomonas cepacia obtained by lysis of bacteria in agarose plugs contained small amounts of DNA with three different electrophoretic mobilities. The behavior the different species in CHEF gels corresponded to DNA fragments of 3.4, 2.5, and 0.9 Mb. Further analyses were consistent with the notion that the different species represented populations of linear DNAs derived by random double strand breakage of three distinct circular replicons. Treatment of DNA from strain 17616 with SwaI, PacI, and PmeI, restriction enzymes which recognize 8-bp sequences, generated four, six, and three fragments, respectively. Double digestion of the DNA with SwaI and PacI or with SwaI and PmeI generated fragments whose combined molecular weights was 6.8 Mb. Treatment of the DNA with AflII AseI, DraI, SpeI, and XbaI, restriction enzymes which recognize 6-bp sequences, generated about 50 fragments whose combined molecular weight also was about 6.8 Mb. The results indicate that the size of the genome of strain 17616 was 6.8 Mb. The combined molecular weights of the sets of SwaI, PacI, and PmeI fragments was 4.3, 5.9, and 5.9 Mb, respectively, reflecting the fact that the 2.5 Mb replicon contained no SwaI site and that the 0.9 Mb replicon lacked PacI and PmeI sites. The organization of SwaI, PacI, and PmeI fragments comprising each of the replicons was determined by Southern hybridization experiments using various junction fragments as probes and by further restriction analyses. Southern hybridization experiments using r-RNAs and various IS elements from P. cepacia as probes indicated that each of the replicons contained r-RNA genes and IS elements. A SwaI site was introduced into Tn5-751 and the resulting transposon was used to generate and map mutations related to amino acid biosynthesis and the degradation of various carbon sources. Derivatives of strain 17616 were identified in which large segments of the 2.5 and 0.9 Mb replicons had been deleted. The results provide a framework for further genetic analysis of P. cepacia.

No description available.

No description available.