The effect of altering gap junction activity on Drosophila melanogaster survival after bacterial infection
Nicole Guito, 2014 (Advisor: Dr. Brad Hersh)
The intercellular channels that connect one cell to another are termed gap junctions, and they are responsible for direct communication between neighboring cells. Such cell communication is also important during the immune response, and, therefore, gap junctions are hypothesized to play a role in the immune system. To further investigate this role, we studied viral innexins or vinnexins, which are proteins that are encoded by a Polydnavirus carried by some ichneumonid parasitoid wasps. Vinnexin proteins can be overexpressed in a desired location using the UAS-GAL4 system in order to see their effect on the organism. We first looked at the effects of vinnexin overexpression in the Drosophila eye and throughout the body. We hypothesized that vinnexins disrupt host physiology, and we found that while vinnexins do not disrupt proper development, they are lethal when expressed throughout the body. We also looked at the effects of vinnexin overexpression in the gut on Drosophila survival after bacterial infection with S. marcescens, and we hypothesized that vinnexins disrupt the immune response. We found that vinnexinG overexpression was lethal, but that the parental lines from the original vials had a lower survival rate than the offspring. Therefore, we need to further investigate the levels of gene expression in the offspring in order to interpret these results. This project was beneficial because we have a better understanding of the location in which vinnexin overexpression causes disruption in Drosophila.
An Investigation of the Promoter Region of Arabidopsis thaliana Peroxidase Gene 32
Gabriella Izzo, 2014 (Advisor: Dr. Ann Kleinschmidt)
Arabidopsis thaliana has 73 different full length class III heme peroxidase genes, some of which are expressed throughout each life stage of the plant, and have a variety of functions such as in pathogen defense, and cell wall synthesis. Though overall changes in peroxidase protein activity have been characterized, the functions of the majority of individual peroxidase proteins have not yet been identified. Sequence conservation of the mRNA’s and post-translational modifications of the protein make linking proteins to the mRNA sequences which encode them difficult. One way to gain more information about specific peroxidase proteins is through localization of the activity of the promoter, with the aim of using this information about important regulatory sequences to make predictions about what the protein does. This study used the promoter region of Arabidopsis thaliana peroxidase gene 32 to gain more information about the function of the protein the gene encodes, as well as about important regulatory sequences within the promoter. As little has been done to map the peroxidase promoters, different lengths of the promoter were amplified so that conclusions could be made about where important regulatory sequences are located within the promoter region. While the appropriate clones were made, analysis within plants was not completed due to difficulties with the transformation of the plasmid into Agrobacterium tumefaciens. In silico analysis of the promoter was performed, however, and based upon this it could be hypothesized that A. thaliana peroxidase gene 32 is inhibits floral organ development and is involved in stress response.
Further Investigation of the Cell Proportioning and Spore Formation Phenotype of the fbiA– mutant in Dictyostelium discoideum
Rachel Kloecker, 2014 (Advisor: Dr. Margaret Nelson)
Dictyostelium discoideum is a unicellular slime mold used as a model for cell differentiation in more complex organisms due to the role that cell fate and proportioning play in its life cycle. Dictyostelium transition from unicellularity to a multicellular fruiting body under conditions of starvation and release spores into an environment with a more abundant food source. FbiA is one protein that may influence cell fate in this process and may be a target of ubiquitination by a previously characterized protein, FbxA. In previous studies, the fbiA– mutant has produced more prestalk cells, fewer prespore cells, and fewer mature spores than the wildtype. However, the wildtype used in those studies was lost in a freezer failure, and follow-up experiments suggested that the mutant phenotype was less marked than originally described. This study aimed to clarify the fbiA– phenotype by quantifying the percent prespore and percent spore using Mud1 immunofluorescence and detergent treatment, respectively. Synergy experiments via actin15-lacz transformation were also conducted to determine any mutant preference in proportioning and its degree of autonomy. Reliable conclusions could not be drawn from the percent prespore and spore data due to inconsistencies between trials. However, the synergy experiment images suggest that fbiA– cells tend towards the PstO and PstB fate, especially later in development. Additional trials of all aspects of the experiment need to be conducted to achieve greater consistency and more reliable conclusions.
The role of innexins 2 and 5 and vinnexins D and Q1 in the innate immune response of Drosophila melanogaster
Kelsey Sadlek, 2014 (Advisor: Dr. Brad Hersh)
Innexins are transmembrane proteins that comprise gap junctions, communication channels between cells, in Drosophila melanogaster and are speculated to play a vital role in the organism’s cellular immune response. When invaded by the parasitoid wasp, the Drosophila’s immune defense system is often compromised. The female parasitoid injects virus particles into the host, which are often associated with a change in hemocyte cell morphology and immune suppression. The polydnavirus, a type of dsDNA virus present in many parasitoid wasps, contains vinnexins, viral homologs of innexins that disrupt the normal immune response of the Drosophila by a mechanism that is not completely understood. Though these vinnexins have been found to interact with innexin 2, specifically, in addition to all other vinnexins in previous studies, the role of this interaction and its relation to immune suppression is not clear. Using the UAS-GAL4 express ion system, we knocked-out innexins 2 and 5 to elucidate their role in the Drosophila cellular immune response. In addition, we used the yeast 2-hybrid method to investigate specific interactions between innexin 2 and vinnexin D and vinnexin Q1. We were unable to observe a trend between encapsulation and RNAi knock-out genotypes, and we were unable to observe any interactions between innexin 2 and vinnexins, though we gained insight into the molecular techniques that we used.
Characterization of UBX binding of Cpr47Ee edge in the Drosophila melanogaster haltere
Rachel Stegemann, 2014 (Advisor: Dr. Brad Hersh)
Hox genes are a highly conserved group of genes that encode transcription factors that regulate various direct and indirect target genes. These genes are essential for anterior to posterior patterning in all bilaterally symmetrical metazoans. Although the mechanism by which they choose their targets is not well understood, Hox proteins bind preferentially to a core DNA sequence of ATTA, which will occur approximately every 256 base pairs in an average genome. We have hypothesized that other DNA sequences, both upstream and downstream of this core site, increase specificity of Hox protein binding. In this experiment, we used the Hox protein Ultrabithorax (UBX) and one of its target binding sites, Cpr47Ee edge cis-regulatory element to identify additional sequences important for Hox function. This direct UBX target contains two core ATTA sites, one of which is essential for proper expression. UBXIa protein was induced in BL21(DE3)pLysS E. coli bacteria and then purified on a Ni-NTA column. The purified protein was used in electromobility shift assays (EMSA) to determine binding affinity with wild-type and mutated sequences of the Cpr47Ee edge regulatory DNA.
Isolation and Characterization of the Extracellular Polymeric Substances of Haemophilus ducreyi
Josh Taylor, 2014 (Advisor: Dr. Tricia Humphreys)
Previous research has suggested that the bacterium Haemophilus ducreyi, the etiological agent of the sexually transmitted genital ulcerative disease chancroid, is capable of biofilm formation. Biofilms are surface-adherent communities of microorganisms, oftentimes consisting of multiple species of bacteria. These formations have been specifically noted to augment the virulence and overall pathogenicity of infectious microorganisms. In this way, the possibility of biofilm formation in H. ducreyi poses major implications to diagnosis and treatment. This study sought to confirm the secretion of extracellular polymeric substances (EPS) required for successful biofilm formation by H. ducreyi. These substances include extracellular DNA (eDNA), extracellular polysaccharides, as well as extracellular amino acids and protein. Observation of eDNA was made through the use of DAPI staining of green fluorescent protein expressing H. ducreyi grown on positively charged slides, which were then examined through the use of fluorescent microscopy. The amount of eDNA noted during fluorescent microscopy was relatively extensive in proportion to the number of cells present, indicative of a large amount of extracellular matrix. This matrix was isolated through the use of sonication at various amplitudes, followed by ultracentrifugation and filtration in order to remove large molecule contaminants. The resulting supernatant was tested for nucleic acid, protein, and polysaccharide content via various spectrophotometric methodologies. In this way the presence of DNA, protein, and polysaccharides was observed in the supernatant and their respective concentrations quantified. The fact that H. ducreyi possessed all major EPS components further indicates that this microorganism is properly equipped for biofilm formation, a potentially critical factor in the overall pathology, transmission, and persistence of chancroid infection.