Tag: Molecular Biology

Determining roles of Wnt signaling in mammalian heart valve development

Presenter: Andrew Mckay, Biology

Poster: C-2

Mentor: Kryn Stankunas, Institute of Molecular Biology

Heart defects occur in 2% of live births, and of these, valve defects are the most common. By studying normal heart valve development we hope to find the genetic causes of these defects. Our primary question is: what signals direct the remodeling of embryonic heart valves into thin, elongated leaflets? We hypothesize that local Wnt signals direct heart valve remodeling by regulating cell proliferation and morphogenesis through activation of the NFATc1 transcription factor. To accomplish this, we use transgenic mice that allow us to block Wnt signaling during narrow time windows of embryogenesis. We inject transgenic mice with doxycycline, a molecule that causes the transgenes to temporarily express the Wnt antagonist Dkk-1 and inhibit Wnt signaling in particular cell types. I helped determined which of three transgenic lines best inhibits Wnt signaling and produces the most consistent and robust phenotype for valve defects. This line will be used for future experiments in which we will stain the sections for various cell type markers, indicators of proliferation, and localization of the transcription factor NFATc1. We will then use these results to determine if Wnt signaling affects cell proliferation and NFATc1 localization in targeted cell types in the developing valve.

Investigating an Unusually Bright Variant of the Red Fluorescent Protein mKeima

Presenter: A.J. Risenmay, Biology

Poster: C-4

Mentor: Jim Remington, Physics, Institute of Molecular Biology

mKeima is a monomeric red fluorescent protein (λem¬¬max ~620 nm) that is maximally excited in the blue (λex¬¬max ~440 nm). This extraordinarily large stokes shift can be significantly reduced following chromophore deprotonation under acidic conditions. By designing mutants to exploit the varying excitation species of mKeima, our lab was able to develop a redox-sensitive red fluorescent protein to be used as a quantitative reporter of the thiol/disulfide status in reducing subcellular compartments. This ratiometric variant was identified as mKeima M159K_TDCC and was found to be unusually fluorescent under green light (580 nm). Further examination revealed that mKeima M159K_TDCC is colorless when expressed in the dark, but irreversibly becomes pink when exposed to blue light. Here we combine x-ray crystallography and fluorescence/absorption spectroscopic techniques to investigate the fascinating chemistry behind this mKeima mutant.

Determining the degree to which chloroplast genome copy number limits the expression of chloroplast genes

Presenter: Dylan Udy, Biology

Poster: D-1

Mentor: Alice Barkan, Institute of Molecular Biology

The relationship that exists between chloroplasts and the plant cells they occupy is very complex. Chloroplasts evolved from a cyanobacterial endosymbiont, and throughout evolution many of the ancestral bacterial genes have been transferred to the plant nuclear genome. The proteins from many such nuclear genes are sent back to the chloroplast where they perform a variety of functions. We identified a non-photosynthetic maize mutant that accumulates reduced levels of several chloroplast mRNAs. We showed that the causal mutation is a transposon insertion in a nuclear gene encoding a protein that is closely-related to bacterial DNA polymerase I. Angiosperm genomes include two closely related paralogs encoding this protein. These have been studied in Arabidopsis (a dicot plant), where they are dual-targeted to both the mitochondria and chloroplast and have redundant functions. I have shown that our maize mutant has a 10-fold reduction in chloroplast DNA but normal levels of mitochondrial DNA, suggesting that the two paralogs have become specialized for either chloroplast or mitochondrial DNA replication in maize (a monocot plant). I am using the maize mutant to investigate the degree to which the abundance of chloroplast DNA limits chloroplast gene expression. I have found that the abundance of some chloroplast mRNAs decreases in parallel with the abundance of chloroplast DNA, whereas the abundance of other mRNAs does not. These results show that different factors limit the expression of different genes in the chloroplast.

Investigating Early Effects Following Glia Cell Ablation in Medulloblastoma

Presenter: Kelsey Wahl, Chemistry

PosterPoster: D-2

Mentor: Hui Zong, Institute of Molecular Biology

Medulloblastoma is the most common type of malignant brain tumor in children. During cerebellar development, granule neuron precursor cells (GNPs) proliferate along the external germinal layer in response to the sonic hedgehog signaling pathway. In our lab, medulloblastoma is modeled in mice by inducing heterozygous mutations in both the sonic hedgehog signaling receptor patched (Ptc) and the tumor suppressor gene p53. These mutations lead to a brain tumor in the cerebellum through over-proliferation of GNPs. From previous research, it is shown that unipotent GNPs in a tumor can somehow give rise to glia cells. In order to determine the role of glia cells within the tumor, they were selectively ablated through thymidine kinase (TK)-mediated cell ablation with administration of Ganciclovir (GCV). Amazingly, the proper dosing regime of GCV leads to complete tumor regression. To further understand the ablation process, we studied early time points during GCV injections to observe cellular processes within the tumor.

Utilizing a Fusion Protein for Sequence Specific Nucleosome Shifting in Chromatin

Presenter(s): William Reed-dustin − Biology, Human Physiology

Faculty Mentor(s): Jeffrey McKnight

Poster 81

Research Area: Natural/Physical Science (Molecular Biology)

Chromatin refers to the organization of DNA in eukaryotic organisms. Chromatin is organized such that DNA wraps around protein groups called histones. The units of histones wrapped in DNA are called nucleosomes, nucleosomes are connected by short stretches of linker DNA. DNA in nucleosomes is relatively inaccessible to RNA polymerase and transcription factors and thus, is effectively turned off. The goal of this research was to move nucleosomes onto specific DNA sequences by producing a fusion protein that would combine the binding domain from a specific transcription factor, XBP1, and the active domain from a known chromatin remodeler protein, CHD1. A procedure originally developed by Dr. Jeffrey McKnight was used to produce a plasmid that coded for a protein with the binding domain of XBP1 and the active domain of CHD1. This plasmid was then transformed into yeast. The cells’ DNA was then digested into mono-nucleosomes which were sequenced and compared to yeast without the plasmid inserted. This was done to see if the fusion protein had altered the nucleosomes’ locations.
The goal of this research is to show that the strategy for fusion protein production can be applied to diverse transcription factors across the yeast genome. Ultimately, this strategy could be useful in cancer treatment, silencing oncogenes by moving nucleosomes onto their binding sites.

Determining Scrib binding partners relevant to its spindle orienting function

Presenter(s): Hussein Al-Zubieri – Biochemistry

Faculty Mentor(s): Ken Prehoda, Nicole Paterson

Poster 82

Research Area: Molecular Biology

Asymmetric stem cell division requires a mitotic spindle oriented relative to its axis of polarity. Spindle orientation determines where the cleavage furrow is positioned, thereby determining the location of cell division. Without a correctly oriented spindle, tumorigenesis can occur. Indeed, many of the proteins in the pathway are tumor suppressors. Two pathways have been found to position the spindle, Mud pathway and Dlg pathway. Both are required for spindle orientation. Our research focuses on a Dlg pathway protein member called Scrib which is a tumor suppressor protein that has been shown to be required for spindle orientation. The function of Scrib is not yet fully understood, and our research is focused on discovering the mechanism of its function in this process. A starting point for identifying function is to test the requirement of the functional domains of Scrib in vivo, and determine the specific function of these domains in vitro. My project is to determine the Scrib binding partners relevant to its spindle orienting function.