Bibliography: leaves 71-79. Almost 80 percent of plant taxa develop vesicular-arbuscular mycorrhizae (VAM) which are symbiotic associations between plant roots and soil fungi. The fungi are biotropic-obligate symbionts. Identification of VAM fungi is currently based on spore characteristics. Molecular techniques provide tools for better and more accurate identification of species, as well as for the examination of genetic variability occuring between individual spores of a single species.

Bibliography: leaves 153-185. Vesicular-arbuscular (VA) mycorrhiza formation is a complex process which is under the genetic control of both plant and fungus. This project aims to develop a model infection system in Hordeum vulgare L. (barley) suitable for molecular analysis; to identify host plant genes differentially expressed during the early stages of the infection process; and to screen a mutant barley population for phenotypes which form abnormal mycorrhizas.

Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor (GEF) that converts inactive eIF2 GDP-bound binary complexes into active eIF2 GTP-bound complexes that can bind initiator t-RNA molecules and ribosomes to begin translation. eIF2B is functionally divided into two subcomplexes: the catalytic core comprised of eIF2B epsilon and eIF2B gamma, and the regulatory core comprised of eIF2B alpha, eIF2B beta and eIF2B delta. While the catalytic subunits are responsible for exerting GEF activity, the regulatory subunits recognize eIF2 and respond to eIF2 alpha phosphorylation. Cellular stress, such as virus infection, inhibits host protein synthesis by activating specific kinases that are capable of phosphorylating the alpha subunit of eIF2, which can then sequester eIF2B to stall guanine nucleotide exchange by a currently unresolved mechanism. Importantly, we demonstrate that loss of eIF2B alpha or expression of a variant of the human eIF2B alpha subunit harboring a single point mutation (T41A) is sufficient to neutralize the consequences of eIF2 alpha phosphorylation, and render primary MEFs significantly more susceptible to vesicular stomatitis virus infection. To extend this analysis, we further exhibit the vital function of eIF2B alpha in protein synthesis through phenotypic studies in yeast. Here, we report that this subunit can sufficiently substitute for its yeast counterpart, GCN3, and reproduce similar growth phenotypes under normal and amino acid deprived conditions. In addition, the human eIF2B alpha-T41A variant was unable derepress GCN4 translation in response to an inhibitor of amino acid biosynthesis in yeast, an activity that requires sensitivity to phosphorylation of the yeast eIF2 alpha homolog, SUI2. Previously, we have demonstrated that vesicular stomatitis virus can infect and replicate to high levels in tumor cells. Moreover, these cells appear to contain defects in eIF2 alpha-mediated translational control, plausibly due to disregulation of eIF2B activity, which overcomes the inhibitory effects of eIF2 alpha phosphorylation. Our data suggest a role for eIF2B, specifically eIF2B alpha, in suppression of translation following virus infection, and imply that this complex may contribute to oncogenic transformation. These results emphasize the importance of eIF2B alpha in mediating eIF2 kinase translation inhibitory activity and may provide insight into the complex nature of viral oncolysis and cellular transformation.

Oncolytic virotherapy is emerging as a new treatment option for cancer patients. At present, there are relatively few oncolytic virus clinical trials that are underway or have been conducted, however one virus that shows promise in pre-clinical models is Vesicular Stomatitis Virus (VSV). VSV is a naturally occurring oncolytic rhabdovirus that has the ability to preferentially replicate in and kill malignant versus normal cells. VSV also has a low seroprevalence, minimal associated morbidity and mortality in humans, and simple non-integrating genome that can be genetically manipulated, making it an optimal oncolytic vector. Currently, many labs are using a variety of different strategies including inserting trans genes that can modulate the innate and adaptive immune response. VSV can also be retargeted by altering its surface glycoprotein (G) or be made replication incompetent by deleting the G protein. Currently, our lab has engineered a series of new recombinant VSVs, incorporating either the murine p53 (mp53), IPS-1, or TRIF transgene. mp53, IPS-1 and TRIF were incorporated into the normal VSV-XN2 genome and mp53 was also incorporated into the mutated VSV-ΔM vector generating VSV-mp53, VSV-IPS-1, VSV-TRIF and VSV-ΔM-mp53. Our data using these new viruses indicate that these viruses preferentially replicate in and kill transformed versus non-transformed cells and efficiently express the transgene. However, despite the ability for VSV-IPS-1 and VSV-TRIF to induce a robust type 1 IFN response, VSV-ΔM-mp53 was the only construct that had reduced toxicity and elicited an increased anti-tumor response against a syngeneic metastatic mammary tumor model. VSV- ΔM-mp53 treatment lead to a reduction in IL-6 and IP-10 production, an increase in tumor specific CD8+ T cells, and immunologic memory against the tumor. Collectively these studies highlight the necessity for additional VSV construct development and the generation of new clinically relevant treatment schema.

Graduation date: 1991

Graduation date: 1991

Vesicular stomatitis virus (VSV) infection causes inhibition of host protein synthesis, in part by sequestering initiation factors required for mRNA cap recognition. The viral mRNAs share a common mRNA structure to those of the host cell, with a 5' cap and 3' polyadenylate tail, but continue to be efficiently translated despite host translational shutoff. This observation suggests that a non-canonical translation pathway is utilized for viral protein synthesis. To investigate this pathway, we performed an RNA interference screen to identify genes required for VSV replication. In contrast to bulk cellular translation, viral translation is hypersensitive to knockdown of a protein constituent of the 60S ribosomal subunit, rpL40. Depletion of rpL40 diminishes VSV protein synthesis by >90% and is restored through complementation with an siRNA-resistant mutant of rpL40. To delineate the mechanism by which rpL40 is required for viral protein synthesis, we reconstituted translation of VSV mRNA in yeast extracts in vitro. In the absence of rpL40, we show that the two ribosomal subunits fail to associate on VSV mRNA, and the small subunit does not scan to the initiation codon. Regulation by rpL40 occurs in context of the large subunit, providing direct evidence for translational control by the ribosome itself. This rpL40- dependent mechanism of translation initiation is broadly conserved within eukaryotes, governed solely through an RNA determinant, and is utilized by several viruses within the order Mononegavirales. To determine whether a subset of cellular transcripts also require rpL40 for translation, we identified polysome-associated mRNAs in yeast by deep sequencing. We demonstrate that in vitro and in vivo translation of candidate mRNAs, including factors involved in stress responses, are inhibited in the absence of rpL40. This finding suggests that rpL40 plays a critical role in transcript-specific translation during cellular stress. Collectively, our work identifies an alternative translation pathway that is specifically dependent on rpL40, revealing a previously unappreciated mechanism of protein synthesis regulation by the ribosome.

Arbuscular mycorrhizal (AM) fungi have been living in association with land plants for at least 400 million years. Because they are obligate symbionts, the study of AM symbiosis has focused primarily on its plant host and progress in the molecular biology of AM fungi has been very slow. Using two different approaches, library screening and direct PCR-based assays, genetic information of AM fungi was compared across isolates and species. This allowed the study of novel DNA regions previously unexplored in AM fungi. The following species were investigated: Glomus intraradices, Glomus mosseae, Gigaspora margarita, Scutellospora calospora, Acaulospora scrobiculata and Entrophosphora colombiana. In the first approach, using in vitro grown G. intraradices, a DNA extraction protocol was developed for the construction of a partial genomic library. This library was screened for the presence of microsatellite-containing loci. PCR primers were designed based on five identified loci. Two of these loci were monomorphic for all isolates and species. The second approach used a combination of degenerate and specific primers for fungal chitin synthase genes to explore the variability of this gene family in AM fungi. A total of 21 AM sequences were isolated and sequenced, covering class I and II chitin synthases. RT-PCR with G. intraradices revealed differential expression of chitin synthases in spores and mycelium, as compared to mycorrhized roots. In addition, using primers designed from a highly conserved sequence for plant resistance genes, classical PCR and RT-PCR allowed the detection of a genomic sequence and its cDNA counterpart encoding a putative serine/lysine rich protein in G. intraradices. We have thus investigated genetic variability in AM fungi in functional genes as well as in repetitive DNA regions. Study of gene expression was also possible using in-vitro grown G. intraradices.

Organelles within the endomembrane system of all eukaryotic cells exchange membrane lipids and proteins using membrane-bound transport vesicles. This highly conserved vesicular transport process is essential for life and is highly regulated. Much of this regulation is provided by small monomeric GTP-binding proteins such as Arf and Arl that act as molecular switches, cycling between inactive GDP-bound and active GTP-bound states. This cycle of GTP binding and hydrolysis is controlled by guanine-nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. I have investigated regulatory interactions involving two ArfGAPs, Age1 and Gcs1, involved in post-Golgi vesicular transport in the budding yeast Saccharomyces cerevisiae. In yeast, the Age2 + Gcs1 ArfGAP pair is essential and facilitates post-Golgi transport. I found that overexpression of either the poorly characterized ArfGAP Age1 or the Sfh2 phosphatidylinositol-transfer protein can bypass the requirement for Age2 and Gcs1. Indeed, endogenous Age1 is required for efficient Sfh2-bypass. Moreover, the yeast phospholipase D protein, Spo14, which is activated by Sfh2 and regulates membrane lipid composition, is required for Age1 to effectively alleviate the deleterious effects of defective Age2 + Gcs1 function. My findings suggest that Age1 is regulated by membrane lipid composition and can provide ArfGAP function for post-Golgi transport. Gcs1 is involved in multiple vesicular transport stages, is a dual-specificity GAP for both Arf and Arl1 proteins and, as shown here, also has functions independent of its GAP activity. The absence of Gcs1 causes cold sensitivity for growth and endocytic transport. The cold sensitivity of cells lacking Gcs1 is alleviated by the elimination of either the Arl1 or Ypt6 vesicle-tethering pathway at the trans-Golgi, or by overexpression of Imh1, an effector of the Arl1 pathway. I found elimination of the Ypt6 pathway also prevents Arl1 activation and membrane localization, that Arl1 binding by Imh1 is necessary and sufficient for alleviation, and that the Gcs1 function required for growth and transport in the cold is independent of any GAP activity. My findings suggest that in the absence of this GAP-independent function of Gcs1 the resulting dysregulated Arl1 causes the gcs1? defects through the sequestration of a yet-to-be-determined cellular factor.

Oxidative stress is one of the major characteristics in Alzheimer’s disease, and converging evidence indicates that cysteine S-nitrosylation might be related in AD pathology. My results demonstrated exogenous S-nitrosoglutathione was able to S-nitrosylate vAChT, vMAT2, vGluT1 and vGluT2. S-nitrosylation of these vesicular transporters inhibited the uptake of [3H]acetylcholine, [3H]dopamine and [3H]glutamate respectively. APP/PS1 transgenic mice were used to investigate neurotransmission dysfunctions of Alzheimer’s disease. Global protein S-nitrosylation was increased in the 9 and 12 month APP/PS1 mice. Further investigation demonstrated an increase of vAChT and vGluT1 S-nitrosylation in frontal cortex of 6, 9 and 12 month APP/PS1 mice and an increased vAChT and vGluT1 S-nitrosylation was found in hippocampus of 3 month APP/PS1 mice. These findings together suggest that S-nitrosylation of vesicular transporters inhibits the uptake of neurotransmitters, and S-nitrosylation of vAChT and might be associated with the neurotransmission dysfunction of acetylcholine and glutamate in Alzheimer’s disease.