No description available.

No description available.

The development of somatic nuclear transfer procedures and the factors that are likely to affect the success of nuclear transplantation were discussed in the first chapter. In chapter 2, the effects of cell cycle stage and age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages and the in vitro life span of these cell lines were analyzed. The cell cycle characteristics of both fetal and adult cells were analyzed as a population of cells in culture. To study the individual cells from a population, a “shake off” method was developed to isolate G1 cells and evaluate progression through cell cycle. Irrespective of the age of the cell donor, the mean cell cycle length in isolated cells was shorter than what was observed for cells cultured as a population. In vitro development was analyzed after fusing confluent and isolated G1 cells to enucleated metaphase II oocytes. These results indicate that there were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells and confluent cells. Because previous studies do not clearly demonstrate the cell cycle stage of the donor cell that resulted in development to term, Chapter 2 evaluates the methods of producing populations of G0 and G1 cells and the cell cycle characteristics of G0 and G1 cells. Furthermore, in vitro and in vivo development of embryos derived from high confluent, G0 cells and “shake off” G1 cells were evaluated. These results indicate that the isolated G1 cells supported development to term better than confluent cells. Finally, in Chapter 3, pregnancy establishment, pregnancy loss and health of newborn calves were evaluated for nuclear transfer embryos derived from cells of different genotypes from dairy and beef breeds. In addition, the effect of overnight shipping of cloned embryos and the synchrony between the age of the embryo and the estrus cycle of the recipient female on development were assessed. The results indicate that genotype has an effect on development of nuclear transfer embryos.

The dissertation is focused on the role of chemotaxis receptor played in the transmembrane signaling. The first project accomplished was to study the interaction between serine receptor and methyltransferase. As an effort to probe the regulation mechanism of methyltransferase CheR, Isothermal Titration Calorimetry (ITC) was applied to characterize any possible regulation associated allosteric effect between methyltransferase CheR's multiple interactions. It was found that methyltransferase CheR binds to its receptor docking site and substrate SAH independently, further experiments are proposed to explore the interaction between the receptor methylation region and methyltransferase, CheR. It is speculated that conformational change in the receptor upon ligand binding controls the accessibility of the receptor methylation region to methyltransferase. The second project was carried out as a continued effort to determine the receptor ligand binding activity in the ternary complex with the adaptor protein CheW and the histidine kinase CheA, and it was expected that some cooperativity between receptor dimers would be observed. Surprisingly, in the absence of the cytoplasmic signaling proteins, CheW and CheA, serine receptors exhibited negative cooperativity in ligand binding between dimers, and the negative cooperavtivity disappeared in detergent solution. For the first time, direct evidence was found as a strong support the transmembrane signaling mechanism that involves receptor clusters. Chemoreceptors form patches at the poles of bacteria in the presence and absence of signaling proteins, and the active interaction between receptor dimers could play a significant role in information integration when passing through the bacterial membrane. Experiments are also designed for further clarify the function of receptor cluster in the future.

Centromeres are the site on a chromosome that allows for proper chromosome segregation during cell division. The kinetochore, a DNA-protein complex that forms at the centromere, attaches the chromosome to the spindle fiber and therefore ensures faithful separation of the chromosomes. Cse4p was identified in the budding yeast S. cerevisiae and is protein that resembles histone H3. Due to its centromere specific localization led to the proposal of the centromere specific nucleosome(s). Further identification of Cse4p homologues in many diverse organisms, including mammalian systems, supported the idea that the centromere specific nucleosome is an evolutionarily conserved structure. The localization of the centromere specific histone H3-like proteins (CenH3) to the centromere is one of the remaining questions in centromere biology. It is known that the CenH3 proteins have two domains, the conserved Histone Fold Domain (HFD), and a unique N-terminus, and that each domain has a distinct function. I analyzed the function of the Cse4 N-terminus and HFD in centromere localization and showed that the N-terminus has no function in centromere localization, but rather is involved in regulating Cse4 protein turnover. The HFD, therefore, contains all the centromere targeting information and is necessary and sufficient for Cse4 function. Further analysis revealed that the specific DNA contacts of the Cse4 HFD are important for localization to the centromere. In the budding yeast, S. cerevisiae, the centromere DNA is defined by a specific DNA sequence that is divided into three distinct regions, CDEI, II, and III. Mutations of these regions affect centromere function to varying degrees. Analysis of Cse4p localization to mutant centromere constructs revealed that CDEII is necessary for Cse4 centromere localization, but that the intrinsic bend of this element is not involved in Cse4p centromere localization. Cse4p localization to two other centromere mutants, cen3-X35 and cen3-X50, was analyzed. Cse4p could recognize cen3-X35, a construct with a CDEII deletion, but failed to recognize cen3-X50, a CDEII and CDEIII mutant centromere construct. Furthermore, the CBF3 complex was present at both of these centromere constructs, indicating that the CDEIII by deleted in cen3-X50 directly affects Cse4p centromere localization.

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.

Nonenveloped viruses must navigate a number of host cell membrane barriers during entry and after replication for release. Simian Virus 40 (SV40) pirates the endocytic pathway of the host cell for transport from the cell surface until it reaches the endoplasmic reticulum (ER). Within the ER lumen the capsid is thought to be disassembled releasing the viral genome which then must be transported across the ER and nuclear membrane barriers to initiate viral replication. Following replication the SV40 viral progeny must cross the nuclear plasma membrane without becoming membrane encapsulated. To overcome these obstacles during release, viruses such as SV40 utilize a lytic infectious cycle that results in the permeabilization of the host cell membranes through an unknown mechanism. The research presented here utilized numerous techniques to investigate the roles of the minor structural proteins in the SV40 life cycle. VP2 and VP3 were both shown to perform essential functions with VP2 contributing to cell binding while VP3 functioned downstream of this event. Both VP3 and VP2 showed the ability to post-translationally integrate into the ER membrane where VP3 attained a multi-membrane spanning topology. These findings combined with the propensity of VP2 and VP3 to oligomerize and permeabilize bacterial membranes provide the premise for the following hypothesis. We propose that upon disassembly in the ER lumen these minor structural oligomerize and integrate into the ER membrane where they act as a viroporin to aid in the transfer of the viral genome across the ER membrane. The SV40 mutant lacking both the VP2 and VP3 initiation codons revealed that in addition to VP3, another viral protein was synthesized from the VP2 reading frame. This protein we termed VP4 is expressed at the onset of lysis, it is not sequestered by VP1 and its removal inhibited the lytic property of VP3 towards E. coli and caused a significant 2 day delay in cell lysis during infection. In summary, my work provides new insight into how nonenveloped viruses have evolved to traverse the host cell membrane barriers during entry, when they must remain intact, and during release when they become dispensible.

The earliest steps of nascent protein folding are critical to the overall folding efficiency. Folding events start as soon as the protein is translocated into the endoplasmic reticulum lumen, where the co-translational machinery ensures the fidelity of protein folding by coupling molecular chaperones, foldases and folding sensors. We seek to investigate the co-translational maturation events of disease-related glycoproteins containing different membrane topology to determine the generality and substrate specificity of this process, to hopefully provide new insight into therapeutic methods by targeting protein maturation at early stages. A variety of cell biological, biochemical, and molecular biological approaches using cell-free assays, isolated organelles and live cells have been applied in this study. The work on co-translational maturation of a type I membrane protein human tyrosinase has shown that Hsp70 family member BiP handed off tyrosinase to the lectin chaperones calnexin/calreticulin as glycans were added. The maturation pathway of the albino mutation of tyrosinase (C71R) diverges from that of the wild type co-translationally through its recognition by the oxidoreductase ERp57. The work on a type II membrane protein influenza neuraminidase (NA) subtype N9 has shown that calnexin co-translationally interacted with NA prior to calreticulin. This sequential manner was found to be a common feature of the ER assembly line determined by the membrane localization and soluble characteristics of calnexin/calreticulin, respectively. These interactions were required for the proper maturation of NA as NA aggregated if calnexin/calreticulin interaction was abolished by glycosylation inhibition or removal of specific glycans. Surprisingly, a subset of NA molecules can form intermolecular disulfides co-translationally supporting NA homodimerization. NA co-translational dimerization also occurs for a NA mutant lacking the critical large loop disulfide bonds, indicating that the dimerization of the stem domain does not require proper folding of the top globular domain of NA. This represents an exception to the general rule that protein oligomerization happens after the folding of individual domains. Future work on a variety of other substrates will help illuminate a global pathway of glycoprotein co-translational maturation.

Viruses have evolved to exploit cellular pathways and machinery in order to deliver their genome to the cell, replicate, and produce viral progeny. Nonenveloped viruses must overcome membrane barriers to infect host cells and trigger lysis for virion release. The model nonenveloped virus, Simian Virus 40 (SV40), is bound at the cell surface and eventually delivered to the endoplasmic reticulum (ER) where penetration occurs resulting in delivery of the viral genome to the nucleus by an unknown mechanism. During the later stages of infection viral progeny are assembled in the nucleus and are liberated from the host cell through a cytolytic process. SV40 appears to initiate cell lysis by expressing the late viral protein VP4 at the end of infection for virus release. Bacterially expressed and purified VP4 forms size selective pores in membranes. To investigate the role of VP4 in host cell lysis an inducible expression system was used to produce VP4 in mammalian cells. The viral protein was mainly localized along the nuclear envelope and correlated with the mislocalization of nuclear proteins and was associated with cell death. These results indicate that VP4 acts as a viroporin in the nuclear membrane to promote virus release. Previous results indicated that the two minor structural proteins, VP2 and VP3, may act as membrane proteins during viral infection. Studies using purified proteins, bioinformatics, a cell-free membrane insertion assay and a thorough examination of viral propagation, assembly and infection processes have provided new insights into the role of the minor structural proteins during infection. Targeted disruption of the viroporin activity of VP2 and VP3 inhibited viral infection. Together, these results support that the late viral proteins VP2, VP3 and VP4 each act as viroporins and serve as critical triggers for the progression of the viral life cycle. This investigation provides new insight into how the viroporin activity of the late viral proteins is utilized in viral infection and release.

Nur77, an orphan nuclear receptor, plays a key role in T cell apoptosis. As a transcription factor, Nur77 is assumed to exert its functions by driving the expression of target genes. However, Nur77 targets in T cell apoptosis are unknown. In cancer cell lines, Nur77 can induce apoptosis through the intrinsic apoptotic pathway but it remains controversial how Nur77 kills T cells. In this study, we provide biochemical, pharmacological and genetic evidence demonstrating that Nur77 induces apoptosis through the activation of the intrinsic pathway in T cells. We also show that Nur77 is a physiological substrate of the MEK-ERK-RSK-cascade. Specifically, we demonstrate that RSK phosphorylate Nur77 at serine 354 and this modulates Nur77 nuclear export and intracellular translocation during T cell death. Our data reveal that Nur77 phosphorylation and mitochondrial targeting, regulated by RSK, may define a role for the MEK1/2-ERK1/2 cascade in T cell apoptosis.