Hughes Scholars 2006 >>next previous<<

Alex Barash
	Research Advisor & Department John Schimenti - Biomedical Sciences Name of Project: Transcribing Fertility: Transcription Factor A-MYB Abstract: A-MYB is a transcription factor that binds DNA in a sequence-specific manner, with MYB protein binding sites usually located immediately adjacent to binding sites for other transcription factors. MYB proteins coordinate with other transcription factors to cooperatively activate the promoter. Homozygous null A-myb female mutants are unable to nurse pups due to mammary underdevelopment and growth defects, while homozygous null A-myb males have defects in growth and infertility due to a block in spermatogenesis (a pachytene arrest). The Repro9 mutation, which I am studying, potentially encodes a hypomorphic missense allele of A-myb, manifesting itself as an arrest in spermatogenesis but doesn’t exhibit growth defects. In previous semesters, I used bioinformatics to pinpoint genes which were being directly affected by A-myb using microarray data from 17 day wild-type and mutant testes. I found that a large number of affected genes related to spermatogenesis had a MYB site roughly 300bp upstream of their transcription sites. In addition, antibody staining for A-MYB protein was much lower in mutant mice, suggesting protein destabilization in mutants. Several differences in histone composition were observed through antibody staining. Over the summer, I will look at the expression of Myb genes using real-time PCR to determine if they are likely to have overlapping roles in spermatogenesis. Further bioinformatics comparing 14 and 17 day Myb microarrays, as well as Mei1, a similar meiotic mutation will also be done. Finally, gel assays and potentially ChIP will allow us to find genes whose promoters are directly regulated by MYB binding.
Anna Beavis
	Research Advisor & Department Andrew Clark - Molecular Biology and Genetics Name of Project: Sexual Selection in Female Drosophila Melanogaster: An Association Test Between Female Genes Triggered by Mating and Observed Phenotypes Abstract: Female Drosophila melanogaster tend to mate twice and store the sperm of both males in their sperm-storage organs. This evolutionarily advantageous adaptation protects the female’s fitness by increasing genetic variability in her offspring and guarding against the possibility of infertility or sterility in one of her mates. Because of the physical juxtaposition of the sperm, sperm competition occurs. Thus, males have adapted and evolved defensive and offensive methods to increase their chances of siring, or producing, the most offspring. Mating, sperm, and seminal proteins activate a variety of genes in the female that affect various female phenotypes. These include her propensity to re-mate, her fecundity, egg-laying rates, and sperm storage patterns. However, they do so in variably across different female genotypes, indicating that the female plays a role in the determination of which male sires more offspring. Candidate genes have been identified in the female and it is now possible to identify associations between the up- or down-regulation of these genes post-copulation and the phenotypes (re-mating rate, etc) observed in different lines of the female Drosophila melanogaster. This summer, I will perform an association test that compares polymorphisms found in the candidate genes on the second chromosome of female fruit flies to their respective mating-related phenotypes. I will look at three phenotypes: re-mating rate, how long the female waits to mate with a another male; fecundity, how many offspring she produces; and lifespan, how long the female survives post copulation. I will use two male lines of Drosophilae that have different color eyes: brown dominant and wild-type red; these will be mated in succession in both directions to female wild-type flies. The number of each type of progeny from each will be counted and the time the female takes to remate will be recorded. This experiment will be performed on 94 different lines of 2nd chromosome females (females whose 1st, 3rd, and 4th chromosomes are homozygous), and each line will have 20 reps; the entire experiment will be performed twice. I will also continue to sequence the candidate genes in each line, looking for polymorphisms such as single nucleotide polymorphisms (SNPs), microsatellites, insertions and deletions that may affect the gene’s translation, and compare the polymorphisms to the phenotypic data. Then we can identify associations between the polymorphisms on a certain gene and an observed phenotype, offering insight into female defense and control of sexual selection in Drosophila melanogaster.
Cassie Bigelow
	Research Advisor & Department Drew Noden - Biomedical Sciences Name of Project: Myogenic Capabilities of Limb Bud Mesoderm in Avian Embryos Abstract: Craniofacial myogenesis has been heavily studied in the Noden lab for many years. Viral infection and transplant studies have led to detailed fate maps for all muscles of the head and neck region in chick embryos. More recently, transplant experiments have been used to analyze the myogenic capabilities of specific tissues placed in a different embryonic environment. The use of quail-chick chimeric embryos (generated through microsurgeries) allows us to locate and analyze the behavior of quail tissue transplanted into chick embryos by recognizing quail-specific markers using antibodies such as QCPN and QH1 in immunocytochemistry assays. The goal of this project is to transplant quail limb mesoderm into the head region of a chick embryo (stage 9-10) in the location known to give rise to extraocular muscles (specifically the lateral rectus and dorsal oblique muscles). Two experiments will be performed. The first, which is already in progress, is a transplant of distal limb mesoderm. This region is not normally myogenic and will form blood vessels and connective tissue in the fully-formed limb of the embryo. The second region will be a transplant of proximal limb mesoderm from the quail, which will contain myoblasts migrating in from the sclerotomal regions of paraxial mesoderm in the trunk. This mesoderm normally forms limb muscle. By transplanting these limb tissues into the head region of a chick embryo, we hope to see that this mesoderm receives myogenic signals from the head and forms the normal head muscles expected (lateral rectus and dorsal oblique) as opposed to the normal tissues generated if left in the limb. This can be detected by whole mount immunocytochemistry using QCPN or QH1 to detect the presence of quail cells in the chimeric embryo. In situ hybridization of these embryos can be used to determine the types of tissues that have formed based upon their expression patterns. We will specifically look for expression of muscle-specific genes such as myf5 and myoD. By the end of the project, we hope to be able to draw conclusions about the level of responsiveness of avian limb mesoderm in myogenesis and the strength of myogenic signaling pathways in the head region of avian embryos.
Megan Blanchard
	Research Advisor & Department Antonio DiTommaso - Crop and Soil Sciences Name of Project: Does Polyembryony Confer Greater Competitive Ability in the Non-Native Invasive Vine, Pale Swallow-wort? Abstract: Pale swallow-wort (Vincetoxicum rossicum) is an invasive non-native plant that is threatening natural systems in many northeastern states and in Quebec and Ontario, Canada. It is a twining herbaceous perennial vine that spreads vegetatively, but primarily reproduces by seeds, some of which exhibit polyembryony (i.e. individual seeds that have multiple seedlings). Polyembryony has been found to occur in a significant percentage of V. rossicum seeds, and may allow the plant to invade new habitats quicker because of the ability of fewer seeds to produce more seedlings. This experiment will be the first to study the competitive effects of V. rossicum polyembryony . In order to test the hypothesis that polyembryony confers an advantage to V. rossicum in both intra- and inter-specific competition, an experiment has been set up under controlled greenhouse conditions. V. rossicum of three polyembryonic classes, singles, doubles, and triples (one, two, and three seedlings per seed, respectively) are competing with themselves in all combinations and with Canada goldenrod (Solidago canadensis) and common milkweed (Asclepias syriaca). The competition with S. canadensis, an ecologically related species, and A. syriaca, a phylogenetically and ecologically related species, will allow comparisons of the competitive effects of polyembryony in V. rossicum. Growth rates have been measured and will be taken until the plants are harvested, dried, and the above- and below-ground biomass measured. The main effects of polyembryonic class and competing species (and their interactions) on V. rossicum growth rate and total height, and shoot, root, and total biomass will be analyzed and mean separations will be determined.
Anna Bottar
	Research Advisor & Department Stephen Bloom - Microbiology and Immunology Name of Project: Investigation of mechanisms involved in bypass of Bcl-2-mediated resistance to drug-induced apoptosis in Burkitt’s lymphoma cell lines Abstract: Sensitivity of lymphoid and other cell types to drug-induced apoptotic cell death is regulated in large part by the levels of Bcl-2 and related proteins which are expressed. Bcl-2 is found to be expressed at high levels in many cases of Burkitt’s lymphoma (BL), a B-cell lymphoma. This high Bcl-2 expression is considered to be responsible, in large part, for the resistance of BL cells to anti-cancer drug-induced apoptosis. Two cell lines will be compared, one which is resistant to multiple drugs (REW) and one variant which has similar high levels of Bcl-2 proteins, but shows more sensitivity to high-density conditions and some drugs (SEW). The REW and SEW cell lines will be treated with three different classes of drugs as follows: topoisomerase I inhibitor (camptothecin), topoisomerase II inhibitor (etoposide), and anti-microtubule inhibitor (vincristine). Multiple concentrations will be used, and differences in the extent of apoptosis will help to characterize the cell lines for specific versus general differences in drug sensitivity and to help identify particular drug targets and associated signaling pathways that determine that drug sensitivity. Through the use of a Coulter counter, it will also be noted if differences exist in the effects of the drugs on cell proliferation and cell cycle progression. This could be due to differential activation of the p53 pathway or of cell cycle checkpoint proteins. By phenotyping the two cell lines’ responses to drugs, I will better understand the candidate protein-receptors and associated signaling pathways that can circumvent Bcl-2 based resistance in Burkitt’s lymphoma cell lines.
Christine Cheng
	Research Advisor & Department William Kraus - Molecular Biology and Genetics Name of Project: Effects of Cofactor Activity on ERα-Regulated Transcription Abstract: Transcription induced by estrogen signaling occurs through at least two pathways, one of which involves the ligand-bound estrogen receptor α homodimer (ERα) binding directly to specific DNA segments called estrogen-response elements (EREs) and recruiting numerous cofactors. These cofactors play an important role in the histone modifications and recruitment of basal transcription machinery that must occur for transcription to take place. Modifications such as displacing nucleosomes occurs in part through acetylation of histones by the coactivators p300 and CBP acetyltransferase, which adds an acetyl group to the substrate. However, p300 and CBP also directly acetylates ERα and increases both transcriptional regulatory and DNA-binding activities of ERα. Like many other forms of post-translational modifications, the acetylation of ERα is reversible. Previous studies from the Kraus lab have shown that SIRT1, a nuclear NAD⁺-dependent protein deacetylase, can deacetylate ERα in vitro. The activity of ERα may therefore be regulated by the competition between acetyltransferases and deacetylases. The first question is What is the role of SIRT1, p300, and CBP in the acetylation of ERα, and ultimately ERE-dependent transcription in vivo? To address this question, I have completed the knockdown of endogenous SIRT1 and p300 using short hairpin RNA (shRNA) constructs introduced by retroviral infection into 231 cells, an ERα negative breast cancer cell line. Wild type and mutant ERα constructs are to be introduced into 231 cells via retroviral expression constructs. The two mutants are both double mutants: 1) 266/268K replaced with 266/268Q, which mimics acetylated ERα; 2) 266/268K replaced with 266/268R, which mimics deacetylated ERα. Acetylation assays will be used to evaluate the in vivo effect of both SIRT1 deacetylase activity and p300 acetyltransferase activity on ERα function. The second question is the effect of SIRT1 and p300, CBP underexpression on the ERα cofactor complex of which the three proteins are part of. Characterization of cofactor-binding effects will be achieved using antibody against ERα to immunoprecipitate (IP) both wildtype and double mutant ERα and its bound cofactors in both SIRT1 and p300 RNA-silenced 231 cells. Then, by separating the bound factors and treating with antibody targeting different cofactors, I can look at whether the binding interaction of other cofactors, such as SRC with ERα is affected by knockdown of SIRT1 or p300/CBP RNA. Running a Western to blot for targeted cofactors in parallel with the immunoprecipitation with ERα will compare overall cofactor presence in cell with cofactor bound to ERα. Furthermore, immunoprecipitation of ERα-bound cofactors can also reconfirm knockdown of SIRT1 and p300. PLAN: Confirm knockdown of p300 and CBP through Western Blot screening. Combine knockdown of both p300 and CBP and confirm through Western Blot again. Overexpress wild type or mutant ERα in 231 cells underexpressing SIRT1 or p300/CBP and confirm by Western Blot. Perform in vivo acetylation assays. Parallel with immunoprecipitation (IP) of wild type and mutant ERα and its corresponding bound cofactors to characterize effect of SIRT1 or p300/CBP RNA silencing on cofactor-binding. Run Western Blot for targeted IP cofactors to compare with IP results. Immunoprecipitation of ERα with antibody against SIRT1 and p300/CBP is another way of confirming RNA-silencing.
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