The SRP and SRP receptor have long been recognized as essential components of the protein translocation machinery in higher eukaryotes. The biochemical studies discussed in this thesis demonstrate that the signal recognition particle (SRP) mediated transport of proteins across the mammalian endoplasmic reticulum requires the participation of guanine ribonucleotides, in a capacity distinct from their role in polypeptide elongation. The requirement for guanine ribonucleotides during translocation was detected by experimentally separating the synthesis and transport phases of the translocation reaction. Here, the initial targeting of ribosomes to the membrane required SRP and an SRP receptor, but not GTP. However, the insertion of the nascent chain into the membrane required the presence of both SRP and SRP receptor, as well as, GTP. Further biochemical characterization of the initially targeted translocation intermediate demonstrated that SRP remains bound to targeted nascent signal sequences, unless GTP is present. The SRP-receptor catalyzed displacement of SRP from ribosomes was GTP-dependent both with intact membranes and with the purified SRP receptor preparations. GTP specific binding localized to the α subunit of the receptor by photoaffinity labeling and by probing nitrocellulose blots of the receptor with GTP. In addition, an analysis of the α subunit primary sequence revealed elements which are similar, yet not identical, to guanine ribonucleotide binding site consensus sequence elements. These results, taken together, indicate that the SRP receptor represents a novel class of GTP binding protein and is responsible for the guanine ribonucleotide mediated displacement of SRP from nascent signal sequences. A more detailed biochemical investigation of the GTP hydrolysis cycle of the SRP receptor demonstrated that the affinity between SRP and the SRP receptor is substantially greater in the presence of bound GTP and that the subsequent hydrolysis of bound GTP by SRα is necessary to recycle SRP to the cytoplasm. Purified SRP receptor was shown to hydrolyze GTP slowly. However, the GTP hydrolysis rate was substantially increased when both the SRP receptor and SRP were present in equimolar quantity. SRP does not hydrolyze GTP under these assay conditions. Moreover, free SRP was found not to compete effectively with SRP-ribosome complexes for the receptor, implying that the conformation of SRP is altered upon binding to a signal sequence. This result suggests that the affinity between SRP and the SRP receptor may be exquisitely regulated in order to prevent futile GTP hydrolysis cycles from occurring in the absence of secretory protein synthesis. Furthermore, the demonstration that the SRP receptor is a GTP binding protein provides fundamental insight into the mechanism of protein translocation. The displacement of SRP appears to be tightly coupled to the membrane insertion of nascent signal sequences. The membrane inserted intermediate in nascent chain translocation can be characterized by i) a resistance to extraction from the membrane with either EDTA or 0.5M KOAc; ii) an insensitivity to protease digestion, even after dissolution of the membrane with nonionic detergent. These results indicate that SRP displacement allows the nascent chain to interact with an additional membrane bound, protein component of the cellular translocation apparatus. Once in contact with this additional component, the nascent chain is shown to be capable to transverse the membrane bilayer in the absence of ribonucleotide hydrolysis or the continued elongation of the polypeptide. Thus, the results are incompatible with postulated mechanisms of protein translocation requiring that energy be derived from the continued elongation of the nascent polypeptide or from the direct interaction of a hydrophobic signal sequence with the lipid bilayer.

The human immunodeficiency virus type-1 matrix (HIV-1 MA) is best described as a multi-functional, structural protein. However, the multitude of functional activities ascribed to this viral component is not nearly as interesting as are its seemingly paradoxical and opposing roles during the viral life cycle. At the time of virus infection, HIV-1 MA remains associated with the reverse transcription complex, in which viral nucleic acids are synthesized, and facilitates its translocation to the host cell nucleus (Bukrinsky, Sharova et al. 1992; Bukrinsky, Sharova et al. 1993). This activity of MA has been proposed to form the basis for the infection of non-dividing cells (Bukrinsky, Haggerty et al. 1993). An interaction between the C-terminally phosphorylated form of MA and HIV-1 integrase, an integral component of the complex, was initially proposed to mediate this association (Gallay, Swingler et al. 1995; Gallay, Swingler et al. 1995). However, conditions which promote dissociation of integrase from the reverse transcription complex do not reduce MA association (Miller, Farnet et al. 1997). The possibility of a direct interaction between MA and the viral genome is discussed in Chapter III. The nucleophilic nature of HIV-1 MA is paradoxical with its reported activity in targeting the viral precursor proteins to the cytoplasmic membrane (Krausslich and Welker 1996), during the particle production phase of the viral life cycle. Furthermore, MA when expressed in the absence of other viral proteins exhibits a cytoplasmic localization (Fouchier, Meyer et al. 1997); a result which does not support a nuclear translocation role for this protein. The work presented here resolves this seemingly controversial issue. We demonstrate that MA exhibits a strong nuclear export activity. This newly discovered activity is designed to effectively counteract the protein's innate nucleophilic nature, thus maintaining a cytoplasmic localization. The nuclear export function of MA is sensitive to changes within the conformation of the protein as C- and N-terminal deletions, as well as point mutations in the protein, abolish the activity. Furthermore, the export activity is mediated by the Crm1 NES receptor (Fornerod, Ohno et al. 1997; Fukuda, Asano et al. 1997; Ossareh-Nazari, Bachelerie et al. 1997) despite the lack of a leucine-rich export signal within the matrix coding region. Therefore, the interaction between matrix protein and Crm1 is most likely to be mediated by another, perhaps cellular, protein. Any changes in matrix structure may lead to the disruption of this protein-protein interaction. We discuss a model implicating a phosphorylation event in the inactivation of this nuclear export signal. An even more fascinating issue regards the role of this nuclear export activity, during the viral life cycle, and is detailed in Chapter II. In short, mutations in MA which impair its nuclear export activity result in nuclear accumulation of the precursor Gag polyprotein (Pr55) and the nucleocapsid-associated viral genomic RNA. As a result, non-infectious virions deficient in genomic viral RNA are produced. Therefore, drugs designed to block this export activity can undermine the carefully orchestrated course of events during HIV replication and can shut down the growth of the virus.

Maternally inherited poly(A)+ RNAs are important for directing early development in many animal species. This thesis investigates the regulation of maternal mRNA in the South African clawed frog, Xenopus laevis. The first portion of this thesis examines an unusual class of maternal RNA, interspersed poly(A)+ RNA, which is composed of co-linear repeat and single copy sequences. A cDNA clone, called pXR, contains the repeat portion of an interspersed RNA that hybridizes to several different oocyte transcripts of diverse size that persist until the neurula stage. DNA sequence analysis of the cDNA and hybrid selection of the oocyte transcripts followed by in vitro translation show that molecules of this repeat family are not translatable. This data, combined with the developmental profile of XR containing RNAs, indicate that members of this repeat family are not likely to be maternal messenger RNAs. The second part of this thesis investigates the expression of a class of maternal mRNAs that are regulated by cytoplasmic polyadenylation during progesterone induced oocyte maturation. One particular mRNA G10, is stored as a polyadenylated RNA in the cytoplasm of stage VI oocytes until maturation when the process of poly(A) elongation stimulates its translation. Injection of mutant and wild-type mRNAs, synthesized in vitro, revealed that two sequence elements, UUUUUUAUAAAG and AAUAAA, were both necessary and sufficient for polyadenylation and polysomal recruitment of G10. Maturation promoting factor and cyclin as well as progesterone can induce polyadenylation but in each case protein synthesis is required. Extracts from oocytes and unfertilized eggs were employed to identify factors that may be responsible for maturation-specific polyadenylation. An 82 kd protein that binds to the UUUUUUAUAAAG in egg, but not oocyte extracts, was identified by UV crosslinking. This data suggests that p82 is a good candidate for a developmentally regulated protein that controls the expression of maternal messenger RNAs in early Xenopus development.

Enterohemorrhagic E. coli (EHEC) has emerged as an important agent of diarrheal disease in the developed countries. Attachment to host cells, an essential step during intestinal colonization by EHEC, is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed attaching and effacing (A/E) lesion, directly beneath bound bacteria. The outer membrane protein, intimin, is required for the formation of this structure, as is Tir, a bacterial protein that is translocated into the host cell and thought to function as a receptor for intimin. In this thesis, we characterized A/E lesion formation by in vivo and in vitro-grown EHEC, aimed at testing whether bacterial adaptation to the mammalian host included up regulation of A/E lesion formation. Our results showed that actin signaling by EHEC was induced upon bacterial growth in vivo, and this induction was likely due to the up regulation of multiple activities by in vivo-grown EHEC. We also focused on the interaction between intimin and the host cell, an interaction that triggers actin condensation of A/E lesion formation. We evaluated the role of β1 integrins, one of the proposed receptors of intimin, in A/E lesion formation, and demonstrated that β1 integrins are not essential for intimin-mediated cell binding and actin condensation. To better understand intimin function, we mapped the functional domains of intimin, showed that the minimal cell binding domain of intimin correlates with the minimal Tir-binding domain. This minimal Tir-binding domain, when purified and coated on latex beads, was sufficient to trigger actin condensation on preinfected mammalian cells, suggesting that Tir-binding by intimin is critical in the final step of A/E lesion formation. To further demonstrate the significance of the interaction between intimin and Tir in A/E lesion formation, we developed a yeast two-hybrid system to identify intimin mutants diminished in Tir-binding, and then characterized those mutants for the ability to trigger actin condensation, the final step of A/E lesion formation. Finally, as a first step to study the downstream actin signaling pathway after Tir-binding, we mapped the domain of Tir involved in intimin-binding, and showed that the N-terminus and C-terminus of Tir are likely to be localized in the host cell cytoplasm, available to interact with downstream effectors in actin signaling.

Resting-state functional magnetic resonance imaging (rs-fMRI) is a non-invasive neuroimaging technique that utilizes spontaneous low-frequency fluctuations of blood-oxygenation-level dependent (BOLD) signals to examine resting-state functional connectivity in the brain. In the past two decades, this technique has been increasingly utilized to investigate properties of large-scale functional neural networks as well as their alterations in various cognitive and disease states. However, much less is known about large-scale functional neural networks of the rodent brain, particularly in the awake state. Therefore, we attempted to unveil local and global functional connectivity in awake rat through a combination of seed-based analysis, independent component analysis and graph-theory analysis. In the current studies, we revealed elementary local networks and their global organization in the awake rat brain. We further systematically compared the functional neural networks in awake and anesthetized states, revealing that the rat brain was locally reorganized while maintaining global topological properties from awake to anesthetized states. Furthermore, specific neural circuitries of the rat brain were examined using resting-state fMRI. First anticorrelated functional connectivity between infralimbic cortex and amygdala were found to be evident with different preprocessing methods (global signal regression, regression of ventricular and white matter signal and no signal regression). Secondly the thalamocortical connectivity was mapped for individual thalamic groups, revealing group-specific functional cortical connections that were generally consistent with known anatomical connections in rat. In conclusion, large-scale neural networks can be robustly and reliably studied using rs-fMRI in awake rat, and with this technique we established a baseline of local and global neural networks in the awake rat brain as well as their alterations in the anesthetized condition.

Autophagy is a cellular process that delivers cytoplasmic materials for degradation by the lysosomes. Autophagy-related (Atg) genes were identified in yeast genetic screens for vehicle formation under stress conditions, and Atg genes are conserved from yeast to human. When cells or animals are under stress, autophagy is induced and Atg8 (LC3 in mammal) is activated by E1 activating enzyme Atg7. Atg8-containing membranes form and surround cargos, close and mature to become the autophagosomes. Autophagosomes fuse with lysosomes, and cargos are degraded by lysosomal enzymes to sustain cell viability. Therefore, autophagy is most frequently considered to function in cell survival. Whether the Atg gene regulatory pathway that was defined in yeast is utilized for all autophagy in animals, as well as if autophagy could function in a cell death scenario, are less understood. The Drosophila larval digestive tissues, such as the midgut of the intestine and the salivary gland, are no longer required for the adult animal and are degraded during the pupal stage of development. Cells stop growing at the end of larval development, and proper cell growth arrest is required for midgut degradation. Ectopic activation of the PI3K/Akt signaling induces cell growth and inhibits autophagy and midgut degradation. Down regulating PI3K/Akt pathway by Pten mis-expression activates autophagy. In addition, mis-expression of autophagy initiator Atg1 inhibits cell growth and knocking down autophagy restore PI3K/Akt activity. Together, these results indicate that autophagy and growth signaling mutually inhibit each other. Midgut destruction relies on the autophagy gene Atg18, but not caspase activation. The intestine length shortens and the cells undergo programmed cell size reduction, a phenomenon that also requires Atg18, before cell death occurs during midgut destruction. To further investigate whether cell size reduction is cell autonomous and requires other Atg genes, we reduced the function of Atg genes in cell clones using either gene mutations or RNAi knockdowns. Indeed, many Atg genes, including Atg8, are required for autophagy and cell size reduction in a cell autonomous manner. Surprisingly, Atg7 is not required for midgut cell size reduction and autophagy even though this gene is essential for stress-induced autophagy. Therefore, we screened for known E1 enzymes that may function in the midgut, and discovered that Uba1 is required for autophagy, size reduction and clearance of mitochondria. Uba1 does not enzymatically substitute for Atg7, and Ubiquitin phenocopies Uba1, suggesting Uba1 functions through ubiquitination of unidentified molecule(s) to regulate autophagy. In conclusion, this thesis describes: First, autophagy participates in midgut degradation and cell death. Second it reveals a previously un-defined role of Uba1 in autophagy regulation. Third it shows that the Atg genes are not functionally conserved and the requirement of some Atg genes can be context dependent.

MicroRNAs play an important role in protecting biological robustness during development. Biological robustness is the ability to maintain a consistent output despite variation in input, such as transcriptional noise or environmental stresses. Here, we show that the conserved microRNAs mir-34 and mir-83 promote the robust migration of the distal tip cells in Caenorhabditis elegans when stressed by changing environmental temperature. Our results show that distal tip cell migration is sensitive to temperature changes occurring within a two hour period during the first larval stage. mir-34 and mir-83 protect distal tip cell migration by regulating potential targets cdc-42, pat-3, and peb-1. cdc-42 and pat-3 are known components of the integrin signaling network controlling pathfinding during migration, while the involvement of peb-1 is a novel finding. Additionally, loss of the two microRNAs leads to a reduction in both fecundity and lifespan, suggesting that the loss of developmental robustness leads to a decrease in fitness. mir-34 and mir-83 are not only conserved in higher organisms, but duplicated. Both have been implicated as tumor suppressor genes in mammalian work. Our work has found a role for both microRNAs in integrin-regulated cell migrations that is potentially conserved in higher organisms. Additionally, our work supports the growing appreciation for the role of microRNAs in both stress response and promoting developmental robustness.

The exocyst is an evolutionarily conserved, hetero-octameric protein complex proposed to serve as a multi-subunit tethering complex for exocytosis, although it remains poorly understood at the molecular level. The classification of the exocyst as a multisubunit tethering complex (MTC) stems from its known interacting partners, polarized localization at the plasma membrane, and structural homology to other putative MTCs. The presence of 8 subunits begs the questions: why are so many subunits required for vesicle tethering and what are the contributions of each of these subunits to the overall structure of the complex? Additionally, are subunit or subcomplex dynamics a required feature of exocyst function? We purified endogenous exocyst complexes from Saccharomyces cerevisiae, and showed that the purified complexes are stable and consist of all eight subunits with equal stoichiometry. This conclusion contrasts starkly with current models suggesting that the yeast exocyst tethers vesicles by transient assembly of subcomplexes at sites of exocytosis. Using a combination of biochemical and auxininduced degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer, and demonstrated that several known exocyst binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex using negative stain electron microscopy; our results indicate that exocyst exists predominantly as an octameric complex in yeast with a stably assembled, elongated structure. This is the first complete structure of a CATCHR family MTC and it differs greatly from the EM structures available for the partial COG and Dsl1 complexes. Future work will be necessary to determine whether exocyst conformational changes are a required feature of vesicle tethering and how such changes are regulated. These architectural insights are now informing the design of the first in vitro functional assay for the exocyst complex. We developed methodology for attaching fluorescently-labeled exocyst complexes to glass slides and monitoring the capture of purified, endogenous secretory vesicles by single molecule TIRF microscopy. By this approach, we can monitor tethering events in real time and determine the required factors and kinetics of exocytic vesicle tethering.

A comprehensive understanding about how genetic variants and mutations contribute to phenotypic variations and alterations entails experimental technologies and analytical methodologies that are able to detect genetic variants/mutations from various biological samples in a timely and accurate manner. High-throughput sequencing technology represents the latest achievement in a series of efforts to facilitate genetic variants discovery and genotyping and promises to transform the way we tackle healthcare and biomedical problems. The tremendous amount of data generated by this new technology, however, needs to be processed and analyzed in an accurate and efficient way in order to fully harness its potential. Structural variation (SV) encompasses a wide range of genetic variations with different sizes and generated by diverse mechanisms. Due to the technical difficulties of reliably detecting SVs, their characterization lags behind that of SNPs and indels. In this dissertation I presented two novel computational methods: one for detecting transposable element (TE) transpositions and the other for detecting SVs in general using a local assembly approach. Both methods are able to pinpoint breakpoint junctions at single-nucleotide resolution and estimate variant allele frequencies in the sample. I also applied those methods to study the impact of TE transpositions on the genomic stability, the inheritance patterns of TE insertions in the population and the molecular mechanisms and potential functional consequences of somatic SVs in cancer genomes.

Herpesvirus ateles is an oncogenic tumor virus of New World primates, which has sequence homology and biological properties in common with Herpesvirus saimiri. Each causes acute T-cell lymphomas in susceptible species of New World primates, while establishing a latent infection of the T lymphocytes of its normal host. The thesis research consists of characterization of the viral genome, cloning of viral DNA, in vitro immortalization of T cells with the virus, and mapping of viral transcripts within immortalized cells. Additional experiments performed to transfect cloned DNA into immortalized cells were unsuccessful. Fragments of H. ateles virion DNA were cloned into the vector pHyg, which is selectable in both prokaryotic and mammalian cells. Overlapping clones of >95% of the viral genome were characterized by restriction mapping, and were used to determine restriction maps of H. ateles strains 73 and 810. Peripheral blood T lymphocytes from several species of New World primate were expanded in medium containing IL-2. T-cells from cottontopped tamarins were immortalized with H. ateles 73, H. saimiri 11, and H. saimiri 484-77, becoming IL-2-independent and growing continuously in culture. Immortalization was highly efficient, occurring reproducibly in cultures of 104-105 cells. Immortalization of IL-2-expanded T-cells of red-bellied tamarins, spider monkeys, and squirrel monkeys was unsuccessful with all strains of virus used. The right end of H. saimiri DNA is deleted in nononcogenic mutants. It has been found to produce four small RNAs in immortalized cells. Similarly, two small viral RNAs were found to be transcribed in cells immortalized by H. ateles 73. The RNAs, of 115 and 119 nuc1eotides, were mapped in the right end of the viral genome. The RNAs contain two regions of high conservation with H. saimiri RNAs. The genes for the small RNAs contain promoter, internal, and terminator sequences characteristic of cellular U RNAs.