We investigate the regulation of an E.coli iron-sulfur protein DNA damage inducible DNA helicase (DinG) by single-stranded DNA binding protein (SSB). We find that SSB can form a stable protein complex with DinG and stimulate the DinG DNA helicase activity. On the other hand, SSB mutant that retains the single-stranded DNA binding activity but fails to form a protein complex with DinG becomes a potent inhibitor for the DinG DNA helicase. The results indicate SSB stimulates the DinG DNA helicase activity via direct protein-protein interaction. The results from these studies suggest that iron-sulfur proteins are regulated not only by intracellular metal homeostasis but also by specific protein factors in cells. We also describe a novel iron binding protein YrdD in E.coli cell. YrdD is a homolog of the C-terminal zinc-binding region of E.coli topoisomerase I. Purified YrdD contains both zinc and iron. Supplement of exogenous zinc in the growth medium abolishes the iron binding of YrdD in E. coli cells, indicating that iron and zinc may compete for the same metal binding sites in the protein. While the zinc-bound YrdD is able to bind single-stranded (ss) DNA and protect ssDNA from the DNase I digestion in vitro, the iron-bound YrdD has very little or no binding activity for ssDNA, suggesting that the zinc-bound YrdD may have an important role in DNA repair by interacting with ssDNA in cells.

RNA polymerase III (Pol III) transcribes tRNAs as well as other small non-coding RNAs. tRNA genes contain internal promoter sequences (A- and B-boxes) which can be specifically recognized and bound by TFIIIC. Binding of TFIIIC facilitates TFIIIB recruitment, which in turn targets Pol III to be recruited and initiate transcription. In addition to typical tRNA genes, there are other chromosomal regions, referred as extra-TFIIIC (ETC) sites, that are only bound by TFIIIC. Apart from transcription of genes, both complete and partially assembled Pol III complexes perform extra-transcriptional functions such as influencing nearby Pol II transcription, displacement of nearby nucleosomes, as well as chromatin boundary activities. By analyzing transcriptome data from high throughput RNA sequencing, we observed numerous alterations in intergenic transcription in close proximity to tDNAs and other Pol III complex binding sites after TFIIIC binding was globally compromised. Reduction of TFIIIC binding activity was achieved by using a yeast strain containing a mutation in the Reb1p binding site within the TFC6 promoter, which drastically reduces the level of the TFIIIC component Tfc6p. Analysis of loci adjacent to Pol III complex binding sites reveal both 5- and 3-extended transcripts, readthrough transcripts, and increased intergenic cryptic transcription. Many of the effects of 5-UTR extension and de-repression appear to be due to the release of bidirectional activity of neighboring promoters. Translation of affected mRNAs is greatly altered because of the usage of upstream transcriptional start site (TSSs) at both TFC6 and TRM12 loci. The results presented here add another type of boundary activity to the known list of extra-transcriptional functions the blocking activity of transcription from bidirectional promoters. Also, such activities might explain a function of the conserved ETC sites in yeast Saccharomyces cerevisiae. Analysis of the TFC6 locus suggests regulatory effects of assembled Pol III complexes at ETC6 site. Also, Reb1p is confirmed to be the transcriptional factor that binds and activates the TFC6 promoter and it binds several base pairs upstream of the ETC6 site. Analysis of these two divergently transcribed genes (ESC2 and TFC6) reveals that both have 5-UTR extensions after Reb1p is depleted or the Reb1 consensus binding site is mutated, which is consistent with results from our transcriptome data. Similarly, altered TSS results in a significant decrease of the translational level of both TFC6 and ESC2. By assessing nucleosome content within the intergenic region ESC2-TFC6, we find that nucleosome positioning is slightly altered, which could explain the observed 5-extended transcripts of both genes. Taken together, both of these studies demonstrate significant effects of assembled Pol III complexes and Reb1p binding on the transcription of neighboring Pol II promoters and on the translation of their mRNA products.

The capacity to selectively target DNA sequences within a complex mixture is a useful feature for genomic studies. Several methods aimed at undertaking this feat have utilized biotinylated oligonucleotides and streptavidin beads to capture DNA but have low efficiencies, require PCR, or cannot be combined with high-throughput sequencing. The work presented here developed a protocol referred to as selected enrichment through capture (SEC) that uses a biotinylated capture primer to target and concentrate specific DNA sequences at high efficiencies, eliminates the use of PCR, and can be combined with high-throughput sequencing. The effectiveness of SEC was evaluated in a series of studies that determined the efficiency, specificity, and recovery of specific sequences from DNA mixtures. Initially, it was established that SEC could retrieve a single locus as large as 6,200 bases from 3 ng DNA with high yield. This justified attempts to retrieve 16S rRNA genes from DNA from an artificial mock community and DNA extracted from the environment. Combining SEC with high-throughput sequencing allowed for the identification of all 20 species within an artificial community, reconstruction of full-length 16S rRNA genes, and predictions of relative abundances within an order of magnitude of reported values. SEC successful captured 16S rRNA genes from bacteria from seawater, sediment, and in the over abundances of eukaryotic DNA that facilitated the characterization of the microbiome of the Eastern oyster, C. virginica. SEC was further demonstrated to be successful in recovering 16S rRNA genes from the low biomass environment of glacial basal ice with 21 pg/uL of starting DNA. Additionally, SEC was successful in retrieving insertions in the eukaryote Chlamydomonas reinhardtii. SEC has three advantages over other enrichment methods. 1) SEC does not rely on the PCR to enrich samples prior to sequencing. 2) Only a short segment of sequence adjacent to one side of the targeted region needs to be known allowing for adjacent uncharacterized sequence to be recovered. 3) Sequences of interest can be isolated, concentrated, and analyzed without PCR amplification or interference from more abundant DNA found in the sample.

The aim of this dissertation is to investigate stabilization of lipid bilayers during water stress by protectants found in embryos of Artemia franciscana. Two LEA proteins were used: AfrLEA2 and AfrLEA3m. AfrLEA3m was experimentally demonstrated to reside in the matrix. Two detergents were used to differentially solubilize the outer and inner membranes of mitochondria isolated from A. franciscana. Release of AfrLEA3m occurred simultaneously with the release of fumarase, a matrix-resident marker. As a second independent method to corroborate the above findings, I demonstrated that recombinant AfrLEA3m can be imported into mitochondria isolated from rat liver. Molecular modeling of AfrLEA2 and AfrLEA3m revealed structural features that are consistent with amphipathic proteins able to interact with and stabilize cell membranes. The abilities of trehalose and LEA proteins to protect liposomes of various compositions from desiccation-induced damage were evaluated by carboxyfluorescein leakage. AfrLEA2 (cytoplasmic) and AfrLEA3m (mitochondrial) were able to offset damage during drying of liposomes that mimicked the lipid compositions of the inner mitochondrial membrane, outer mitochondrial membrane, and the inner leaflet of the plasma membrane. LEA proteins were more effective than trehalose at preventing desiccation-induced damage when these protectants were confined to one side of the lipid bilayer. When LEA proteins were used in conjunction with trehalose, additive protection was measured in some cases. Little to no additional damage occurred to liposomes dried for one week compared to liposomes dried overnight. The capacity of trehalose and LEA proteins to protect liposomes from freeze-thaw damage was also assessed. Damage to liposomes was less severe after freezing than desiccation. Trehalose provided liposomes with greater protection than LEA proteins from freeze-thaw damage. The greatest stabilization during freezing occurred when trehalose was present on both sides of liposome membranes. Only liposomes mimicking the outer mitochondrial membrane were significantly protected from freeze-thaw damage by LEA proteins. Based on bioenergetic properties assessed by respirometry, the outer membrane of isolated mitochondria (rat liver) remained intact after freezing in 300 mM trehalose solution. Respiratory control ratios were depressed by approximately 30% compared to non-frozen mitochondria, which indicated a limited retention of at least some inner-membrane-dependent properties.

The serotonergic raphe nuclei of the midbrain are principal centers from which serotonin neurons project to innervate cortical and sub-cortical structures. The dorsal raphe nuclei receive light input from the circadian visual system and indirect input from the biological clock nuclei. Dysregulation of serotonin neurotransmission is implicated in neurobehavioral and neurodevelopmental disorders, such as depression, anxiety and autism, and alterations in the serotonergic phenotype of raphe neurons has dramatic effects on affective behaviors in rodents. Here, I demonstrate that day length (photoperiod) during development induces enduring changes in mouse dorsal raphe serotonin neurons - programming their spontaneous neural activity, their responsiveness to noradrenergic stimulation, their intrinsic electrical properties, serotonin and norepinephrine content in the midbrain, gene expression of key serotonergic genes, the ratio of TPH2 positive cells to total cells in the dorsal raphe, as well as depression/anxiety related behavior in a melatonin receptor 1 (MT1) dependent manner. Our results establish mechanisms by which seasonal photoperiods may dramatically and persistently alter the function of serotonin neurons.

Nucleolar stress results when ribosome biogenesis is disrupted. An excellent example is the human Treacher Collins syndrome in which the loss of the nucleolar chaperone, Treacle, leads to p53-dependent apoptosis in embryonic neural crest cells, and ultimately to craniofacial birth defects. We show that depletion of the related nucleolar and Cajal body phospho-protein, Nopp140, in Drosophila melanogaster led to nucleolar stress and eventual lethality when multiple tissues were depleted of Nopp140 by RNAi. We used TEM, immuno-blot analysis, and metabolic protein labeling to show the loss of ribosomes. Targeted loss of Nopp140 only in larval wing discs caused apoptosis, which eventually led to defects in the adult wings. These defects were not rescued by a p53 gene deletion, as the craniofacial defects were in the murine model of TCS, thus suggesting that apoptosis caused by nucleolar stress in Drosophila is induced by a p53-independent mechanism. Loss of Nopp140 in larval polyploid midgut cells induced premature autophagy as marked by the accumulation of mCherry-ATG8a into autophagic vesicles. We also found elevated phenoloxidase A3 levels in whole larval lysates and within the hemolymph of Nopp140-depleted larvae, coincident with the appearance of melanotic tumors. The occurrence of apoptosis, autophagy, and phenoloxidase A3 release to the hemolymph upon nucleolar stress correlated well with the activation of JNK in Nopp140-depleted larvae. Nopp140 is considered a ribosome assembly factor, but its precise functions remain unknown. To approach this problem, we deleted the Nopp140 gene in Drosophila using FLP-FRT recombination. Genomic PCR, RT-PCR, and immunofluorescence microscopy confirmed the loss of Nopp140, its mRNA, and protein products. Nopp140-/- larvae arrested in the second instar stage and died within 8 days. While nucleoli appeared intact in Nopp140-/- cells, the C/D snoRNP methyl-transferase, fibrillarin, redistributed to the nucleoplasm in variable amounts; RT-PCRs showed that 2-O-methylation of rRNA in Nopp140-/- cells was reduced. Ultra structural analysis showed that Nopp140-/- cells were deficient in cytoplasmic ribosomes, but instead contained abnormal electron dense cytoplasmic granules. Furthermore, metabolic labeling showed a significant drop in protein translation. We believe the phenotypes described here define novel intracellular ribosomopathies.

No description available.

In the sixth mass extinction, amphibians are being lost in greater numbers than almost any other taxon. The deadly skin disease, chytridiomycosis, has taken a great toll on amphibian populations around the globe. Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis, appears to have evolved as an obligate pathogen and thus must evade destruction by the amphibian defenses in order to reproduce. Several defenses along the skin mucosa likely inhibit the initial colonization by B. dendrobatidis zoospores, but B. dendrobatidis persists longer in the epithelium where it must survive any onslaught by innate and adaptive immune responses. Batrachochytrium dendrobatidis does this by evading phagocytes inside keratinocytes and impairing lymphocytes that might coordinate a robust immune response. Soluble molecules produced by B. dendrobatidis inhibited B and T cell proliferation and induced lymphocyte apoptosis. These molecules were only produced by cell-walled life-cycle stages that are present in host tissue. Inhibiting cell-wall biosynthesis with a chitin-synthase inhibitor greatly reduced its capacity to inhibit lymphocytes, suggesting that inhibitory factors are located in the cell wall. Treatments of inhibitory factors showed that the soluble molecules are not likely to be proteins or common structural components found in fungal cell walls. Although the B. dendrobatidis cell wall has not been characterized, fungal cell walls are largely composed of carbohydrate suggesting that inhibitory factors may be small molecules and/or carbohydrates. These data suggest that B. dendrobatidis has evolved a survival strategy targeting adaptive immune defenses in the skin of amphibian hosts.

Vesicular transport of proteins is a process essential for cell health and viability. Integral membrane proteins, called phospholipid flippases, play important roles in the formation of transport vesicles at sites of membrane budding. Flippases also establish and maintain membrane asymmetry. This is achieved by using the energy of ATP hydrolysis to unidirectionally translocate specific phospholipid molecules from the lumenal to the cytosolic side of the membrane bilayer. It is hypothesized that the resulting imbalance in phospholipid number between the bilayers causes membrane bending, captured by coat and accessory proteins. This leads to the formation of mature cargo-containing transport vesicles. Drs2p, the founding member of the flippase (P4-ATPase) family, has been implicated in the formation of clathrin-coated vesicles at the trans-Golgi network. The influence of Drs2p flippase activity on membrane curvature is explored in this thesis. When late Golgi membranes were purified, and Drs2p on these membranes were activated by ATP addition, tubulation, elongation, and membrane fusion phenotypes were observed. Furthermore, when purified Drs2p was reconstituted into giant unilamellar vesicles (GUVs), and activated by ATP addition, preliminary data indicates the formation of spindle-like tubes. Due to the presence of purified Drs2p (protein found most abundantly in this system) and dependence of this reaction to ATP addition, it is likely this membrane tubulation is Drs2p-dependent. Thus, these data implicate Drs2p in membrane curvature formation due to the ability to translocate phospholipids across the membrane bilayer. Work is also presented that looks at the role of the C-terminal tail of Drs2p on regulation of flippase activity. The C-terminal tail auto-regulates Drs2p function and loss of auto-inhibition by the tail either by proteolysis or interaction with Drs2p effectors stimulates the flippase activity of Drs2p. Furthermore, phosphatidylinositol 4-phosphate is shown to have a direct influence in binding the C-tail to stimulate Drs2p activity. This is the first evidence, in a P4-ATPase, that the C-tail functions as a domain responsible for regulating flippase activity, and this regulation can be relieved by interaction with a phosphoinositide.

Ku is central to the non-homologous end-joining pathway of DNA double strand break repair, first discovered in eukaryotes and more recently in prokaryotes and archaea. This study concerns the importance of two unique sequence features of Ku protein from Mycobacterium smegmatis that include a lysine-rich extension at the C-terminus and a zinc-binding motif in the DNA-binding bridge-region. The unique C-terminal tail of M. smegmatis Ku contains several lysine-rich low-complexity PAKKA repeats that are absent from homologs encoded by obligate parasitic mycobacteria, but present in other mycobacterial proteins such as histone-like proteins. Removal of the lysine-rich extension from Ku decreased thermal stability and abolished DNA end-joining. The tail contacts the core DNA binding domain of Ku and hinders DNA-protein interaction as evidenced by an increase in DNA binding affinity upon removal of the lysine-rich extension. In contrast to Ku lacking the C-terminus, full-length Ku can directly bind DNA without free ends and form multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer, suggesting that these properties are conferred by its C-terminus. My study suggests that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins and that extensions beyond the shared core domain may have independently evolved to expand Ku function. An in vitro metal binding assay showed zinc binding to a predicted zinc-binding motif in the bridge-region of M. smegmatis Ku, an event that stabilizes the protein and prevents cysteine oxidation, but has little effect on DNA binding. In vivo, zinc induced significant upregulation of the gene encoding Ku as well as a divergently oriented gene encoding a predicted zinc-dependent MarR family transcription factor. In addition, overexpression of Ku conferred zinc tolerance on E. coli. I speculate that zinc binding sites in Ku proteins from M. smegmatis and other mycobacterial species have been evolutionarily retained to provide protection against zinc toxicity. In all, my study identifies novel properties conferred by unique sequences present in M. smegmatis Ku protein, which suggests that the retention and evolution of unique sequences within a protein provides an adaptive advantage to microorganisms against environmental stress.