2015 Select Projects

Matt, University of Scranton
Mentor: Haixin Sui

Glycosylphosphatidylinositol-anchored proteins are ideal sites for expression of heterologous proteins in the methylotrophic yeast Pichia pastoris. Previous studies fused Candida antarctica lipase B (CALB) and hydrophobins to the known GPI-modified cell wall proteins Gcw51p and Gcw61p, respectively. However, both the growth behavior and cell-surface morphology remain unclear in these new, genetically-modified yeast. In this study, spectrophotometry, cell counts, and viability tests were performed to construct a growth profile of the yeast. Subsequently, high-pressure freezing, freeze resubstitution, and ultramicrotomy were performed to prepare specimens for electron microscopy (EM). The growth profile indicated viable cell density peaked after 96 hours in methanol-containing media, and EM results revealed the structures of cell surfaces with expression of GPI-anchored proteins. These results provided insight into optimizing Pichia pastoris growth for industrial applications.

Several eukaryotic cells rely on flagellar undulation for motility, and the structure of the axonemal shaft, constituting the cytoskeletal core of the flagellum, has been partially elucidated.  Of particular interest, the axoneme contains nine microtubule doublets arranged in a ring around two microtubule singlets.  Previous work on sea urchin sperm has indicated that microtubule doublets have accessory proteins, possibly related to their structural integrity, known as intralumenal microtubule associated proteins (iMAPs), but the identity and structural role of iMAPs remain unclear.  Following common microtubule doublet isolation techniques of Chlamydomonas, as well as dialysis and sucrose gradient ultracentrifugation, transmission electron microscopy was conducted on negatively stained samples to assess purity.  Following achievement of sufficient purity, SDS-PAGE was conducted to separate proteins.  SDS-PAGE results indicated that at least 20 proteins remained in the sample.  Molecular weight estimates and staining intensity indicated three protein bands as potential iMAPs.  This work lays the foundation for future studies to conclusively identify several iMAPs, and it bears further implications for understanding microtubule doublet composition.

Mauricio, Dutchess Community College
Mentor: Joseph Wade

Bacterial systems employ Rho-dependent termination to terminate transcripts that are not translated. However, several genes have been shown to have intragenic Rho termination, though the functionality of this mechanism remains unclear. It has been proposed that Rho termination is regulated by a process known as antitermination, which requires an array of proteins: NusB, NusE (also known as ribosomal protein S10), NusG, and NusA. Although these antitermination factors are highly conserved, their only described target in bacteria is rRNA. rRNA contains BoxA elements that serve as binding sites for a NusB:S10 complex. NusG and NusA act as elongation factors that associate with RNAP in the transcriptional complex. The mechanism of antitermination is not fully understood, but structural data support a model in which S10 competitively inhibits Rho by associating with NusG, a key contact site for Rho during termination. The inositol monophosphatase, SuhB, has a functional connection to the antitermination complex. suhB, a Rho terminated gene, also contains the BoxA consensus in its 5’ UTR. To determine the mechanism of BoxA-mediated regulation of suhB, we sought to identify and characterize sequence features upstream of, and within the suhB gene that are required for regulation. Random mutations were introduced into suhB and its upstream regulatory sequences. We then screened for mutants with up-regulated expression of suhB. We saw that mutation of boxA significantly increased suhB transcriptional levels, strongly suggesting that NusB directly regulates suhB by binding the BoxA. It is hypothesized that due to BoxA residing only 6 nucleotides upstream from the Shine-Dalgarno sequence, the binding of NusB:S10 to BoxA blocks the binding of the ribosome preventing translation, thus allowing Rho termination of the non-translated mRNA. Though several mutants were identified outside of boxA that resulted in an increase of suhB expression, there was no obvious connection between their motifs. This suggests that antitermination proteins are functioning as negative regulators at suhB in contrast to their positive regulatory role at rRNA.

Rinaldys, Dickinson College
Mentor: Joan Curcio, Randall Morse

Nearly all eukaryotic and prokaryotic genomes carry mobile genetic elements that play a significant role in genome structure and gene regulation. The 5.9 kb Ty1 long terminal repeat (LTR) retrotransposon of the budding yeast Saccharomyces cerevisiae was the first LTR retrotransposon demonstrated to mobilize through an RNA intermediate, and not surprisingly, it is the best studied—which makes it a perfect model for investigating the mechanisms of retrotransposition. As an evolutionary progenitor of retroviruses, Ty1 generates a complement of its RNA genome, known as complementary DNA (cDNA), through reverse transcription in cytoplasmic nucleocapsids. This cDNA is imported into the nucleus of the cell and transposed into the host’s genome, which renews the life cycle of Ty1 with the expression of the newly inserted cDNA. This process, however, does not occur at a constant frequency in all organisms, and can be modulated by numerous genetic and environmental factors that either stimulate or inhibit the progression of retrotransposition. Retrosomes, the cytoplasmic sites of assembly of Ty1 RNA and protein into nucleocapsids, are associated with spindle pole bodies (SPBs), the yeast functional analogues to the centrosomes. This study aimed to understand the role of essential genes, specifically the genes involved in SPB duplication, in the retrotransposition cycle of Ty1 through doxycycline-controlled transcriptional regulation of SPB duplication genes, and to understand the importance of SPB duplication in retrosome formation. When cells are arrested at an early stage in SPB duplication, retrotransposition is strongly repressed, but when cells are arrested directly after SPB duplication by depletion of CDC53, retrotransposition increases significantly. These data suggest that retrotransposition is linked to a specific step in the duplication of SPBs.

Simey, Sewanee – The University of the South
Mentors: Randall Morse, Joan Curcio

Mediator is a large multisubunit complex that is critical for the expression of RNA Polymerase II regulated genes. Even though Mediator is well known for its role in eukaryotic transcription, its function in retrotransposition of Pol II regulated retroviruses is not yet understood. Ty1 retrotransposition in yeast serves as a model for studying retroviral life cycles. Previous studies in Saccharomyces cerevisiae characterized gene mutations (including some Mediator mutants) that affect Ty1 retrotransposition posttranscriptionally. When Ty1 transcription was driven by the wild type Ty1 promoter, mutant yeast strains showed altered Ty1 mobility. Because the mutant yeast strains showed normal Ty1 transcript levels, we expected that the promoter used to drive Ty1 retrotransposition should not affect the Ty1 mobility observed in mutant yeast. However, when Ty1 retrotransposition was driven by the pTEF1 promoter, the mutant yeast strains showed normal levels of Ty1 mobility. These preliminary results suggest that normal progression through later steps in the Ty1 retrotransposon life cycle is promoter dependent.  Here we analyze 68 mutant yeast strains and use the Ty1-his3AI reporter to examine whether differences in Ty1 mobility are seen compared to when Ty1 transcription is driven by the pTEF1 promoter.

Mediator complex is known to play a role in transcription of Polymerase II regulated genes, however, its presence at Polymerase III genes has not been thoroughly studied. Unpublished data using Chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) suggests that Mediator subunits associate with Polymerase III genes. However, proteins that do not serve a biological function in yeast (such as green fluorescent proteins (GFP)) also associate with Pol III genes, making it difficult to know whether the observed association of Mediator with Pol III genes is physiologically meaningful. To examine this problem further, we used an “anchor away” yeast strain that allowed Mediator to be evicted from the nucleus. We then epitope tagged a Mediator subunit and used chromatin immunoprecipitation (ChIP) to test Mediator and GFP association with Pol III genes before and after Mediator eviction from the nucleus.

Yannick, Schenectady County Community College
Mentors: Magdia De Jesus, Nicholas Mantis

The gut-associated lymphoid tissues (GALT) such as Peyer’s patches (PPs) are sites of continuous antigen sampling and presentation in the intestinal mucosa. PPs are considered the immunological frontline providing immunity against potentially harmful antigens and microbes. We are particularly interested in PP uptake of the fungal organisms Candida albicans and Candida tropicalis as these organisms have been found to play an inflammatory role in patients with inflammatory bowel diseases (IBD). We have found that the sampling of C. albicans and C. tropicalis is not indiscriminate, as these are transcytosed by specialized M-cells within the follicle-associated epithelium (FAE) of PPs. Once transported, C.albicans and C.tropicalis are delivered to the subepithelial dome (SED) where they are sampled by the underlying network of dendritic cells (DCs). Although the mechanism (s) of selective fungal sampling remains unknown, our results begin to elucidate the immune cells that play a role in this process. Future experiments are geared towards understanding the immunological fate of C. albicans, C. tropicalis as well as the DCs that sample them.

Chelsea, Stockton University
Mentor: Nicholas Mantis

Ricin is a naturally occurring toxic protein derived from the beans of the castor oil plant. It can cause fatality in an animal if even very small amounts are ingested, inhaled, or injected. Specifically, ricin is a eukaryotic ribosome-inactivating protein, which means it damages the ribosomes that are responsible for protein synthesis in a cell and eventually results in cell death. There are several different types of cell death pathways that ricin may trigger, including apoptosis and necroptosis. Both of these are programmed forms of cell death that share similar initial signaling molecules. Human macrophage-like cells were challenged in vitro with various concentrations of ricin to establish a significant dose response curve. Cell viability was determined after ricin exposure both with and without inhibitor. Results were analyzed using cytotoxicity assays and flow cytometry and suggest that ricin-induced cell death is apoptotic. These results lay a good foundation in determining the cell death pathway, but additional experiments are needed to identify the specific caspases involved.

Jan, Stony Brook University
Mentors: Janice Pata, Nilesh Banavali

DNA replication is an essential process of all living organisms and is dependent on the coordination of multiple proteins and enzymes. A key enzyme involved in this process is DNA polymerase. This enzyme is responsible for adding deoxyribonucleotide triphosphates (dNTPs) to the daughter strand based on the DNA sequence of the parent strand. However, the fidelity of nucleotide incorporation can vary dramatically for different types of polymerases. This project involved developing an assay to determine and quantify errors made by various types of DNA Polymerases using Next Generation Sequencing. This assay used an engineered plasmid that had all the possible combinations of five nucleotides as the template, which allowed errors based on sequence context to be determined. The polymerases that were tested were Taq DNA Polymerase and Dpo4 Trans-Lesion Synthesis Polymerase. The validity of this assay was confirmed through Sanger Sequencing of the polymerase extension products. The results showed that the designed specific primer sequence was inserted.