Jennifer Burg
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Research Advisor & Department
Volker Vogt - Molecular Biology & Genetics
Name of Project:
Cleavage and Activation of Rous-sarcoma Reverse Transcriptase in vivo.
Abstract:
The gene Gag encodes the main structural proteins of the Rous-sarcoma virus (RSV). In order to incorporate the enzymes reverse transcriptase (RT) and integrase (IN) into the virion, a frame shift occurs in approximately 5% of Gag transcripts forming the polyprotein Gag/Pol. The integration of Gag/Pol into the virion followed by the proper cleavage of RT and IN gives the virion functional enzymes. The cleavage and activation of RT is difficult to study because, in vivo, variations in the genetic sequence affect not only RT cleavage but also the assembly of the virion, and, in vitro, the N-terminus of RT is refractory to cleavage. To overcome the problem of a lack of in vitro cleavage I will construct a HIV Vpr and RSV RT fusion protein which, when incorporated into virus-like particles containing RSV Gag/p6, will allow for the study of RT cleavage out of Pol in vivo. Along with experiments to study the cleavage of RT from Pol, I will also use the Vpr-RT system, by altering the amino acid sequences upstream of RT, to assess the effects amino acid changes have on the activity and functionality of RT. To use this system I will first transfect cells with the Vpr-RT DNA, then radioactively label the transfected cells with radioactive amino acid. Virus-like particles, which bud from these cells, will also be radioactive and can be collected and analyzed. The results of these experiments will lead to a more detailed understanding of the cleavage and activation of RT in viruses. Better knowledge of these processes is essential to the understanding of retrovirus activity.
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Christopher Chandler
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Research Advisor & Department
Kelly Zamudio - Ecology & Evolutionary Biology
Name of Project:
Effect of body size on male reproductive success in Ambystoma maculatum
Abstract:
Little research has been done on the mating systems of aggregate breeders like the spotted salamander (Ambystoma maculatum). In this species, hundreds of individuals congregate in early spring to breed for several days. During mating, males deposit spermatophores on the ground, which females pick up with their cloacas. Because so many individuals take part in these short-lived breeding aggregations and because males always outnumber females, multiple mating and multiple paternity within clutches is common in the wild. Furthermore, multiple fathers do not always sire equal proportions of the offspring within a clutch, suggesting there may be sexual selection acting in this species. In one field experiment, males placed first in breeding chambers with previously unmated females sired significantly higher numbers of offspring than later males, suggesting sexual selection for early arrival time to breeding ponds. Another trait on which sexual selection may act is body size. In other species such as the red-backed salamander (Plethodon cinereus), larger males often receive more attention from females; however, this species is territorial and does not mate in aggregations, so the determinants of reproductive success might not be the same in the spotted salamander. To see if body size affects male reproductive success in the spotted salamander, I conducted breeding experiments in the spring of 2002 in which an unmated female was placed in a chamber with three males of different sizes. I preserved the spermatophores and offspring that resulted from the matings and took DNA samples from all the potential parents. By genotyping the larvae and parents at several microsatellite loci, I will determine which male fathered each of the larvae to see if larger males have higher reproductive success. In addition, I will determine which male each of the spermatophores came from to see if the most successful males are the ones that deposit the most spermatophores.
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Michael Chao
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Research Advisor & Department
Marci Scidmore - Vet Microbiology & Immunology
Name of Project:
The Role of Rab6 in Chlamydia trachomatis Infection
Abstract:
The genus Chlamydia encompasses gram-negative obligate intracellular bacterial eukaryotic parasites that are associated with infectious blindness, pneumonia and sexually transmitted disease. A key aspect of the chlamydial infection cycle involves the endocytosis of the infectious elementary body and rapid formation of a host derived parasitophorous vacuole, also called an inclusion. The inclusion is vital to chlamydial infection because it creates an isolated niche separate from cell components. It is within this inclusion that Chlamydia replicates without harm from host lysosomes and degradative enzymes as well as evades immune system detection. After internalization, Chlamydia express inclusion membrane proteins (Incs) on the cytoplasmic face of the endocytic vesicle, which theorized to interact with host trafficking proteins such as Rab6, a GTPase found to be involved in exocytosis. This interaction may help direct the inclusion away from the lysomal pathway and avoid digestion in the host cell, but it is not well defined. The proposed research project involves trying to determine the interaction between the host cell protein Rab6 and the chlamydial inclusion. By transfecting HeLa cells with mutated recessive dominant or constitutively active Rab6 encoding vectors, and then infecting with C. trachomatis, we observe what functions Rab6 may serve in vesicle trafficking during infection. We will look for possible inhibition of infection, changes in bacterial replication rate and changes in chlamydial growth rate. Trafficking of Golgi-derived vesicles will also be examined as well as acquisition of cholesterol by the inclusion. Secondary effects such as structural changes in the inclusion membrane morphology may also be observed. These observations may elucidate how host cell proteins interact with Incs to produce the inclusion, and can open up new avenues for drugs and treatments. Additionally, such interactions can enhance understanding of the intricate nature of cell trafficking systems.
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Clara Cheong
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Research Advisor & Department
Klaas van Wijk - Plant Biology
Name of Project:
A Reverse Genetics Approach to Identify the Role of the Trigger Factor Protein in Plastids of Arabidopsis thaliana
Abstract:
Chloroplasts are essential for the proper function of higher plants, carrying out photosynthesis as well as biosynthesis. The chloroplast is evolutionarily related to bacteria, and the two share similar machinery for transcription, translation and transport, among others. Although the chloroplast contains a circular genome of its own, not all chloroplast proteins are encoded for by this, and many proteins are encoded by nuclear genes then post-translationally imported into the chloroplast. As such, while mechanistic similarities exist between bacteria and chloroplasts, the chloroplast also contains several proteins without homologues in bacteria. Recently, a homologue of the bacteria Trigger Factor protein (Tig) has been found to be nuclear encoded in Arabidopsis thaliana. Additionally, this homologue carries a likely chloroplast targeting signal on its N-terminal domain. In E.coli, Tig has been characterized, and has a potential role as a protein chaperone. Tig is located at the exit tunnel of a 70S ribosome, and contains a peptidyl-prolyl isomerase domain, which may serve to stabilize nascent proteins for proper folding. However, Tig mutants have no phenotypic differences from wild type bacteria, though a Tig/DnaK double deletion mutant exhibits significant developmental differences, suggesting a complementary role for the two in protein synthesis. To determine if a similar role exists for Tig in Arabidopsis, my project has employed biochemical techniques to identify true Tig mutants from Salk T-DNA insertion lines. Thereafter, I will verify the expression and localization of Tig in Arabidopsis at both RNA and protein levels. To understand the role of Tig in Arabidopsis, the expression of other plastid chaperones (DnaK, GrpE, Cpn60, cpSRP54) will be concurrently observed, to determine if Tig mutants are able to partially compensate for the deletion via these chaperones. A double KO of Tig and cpSRP54 will also be generated and assayed biochemically, since cpSRP54 may share an overlapping function with Tig, based upon the known role for cpSRP54 in post-translational targeting.
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Esther Chong
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Research Advisor & Department
Lee Kraus - Molecular Biology & Genetics
Name of Project:
The Effects of Mediator Complex on the Expression of Estrogen-Regulated Genes
Abstract:
Hormonal signaling by estrogens plays an important role in many physiological processes, such as reproduction, development, growth, and metabolism. Abnormalities in estrogen signaling can lead to diseased states, such as breast cancers (overstimulation of cell growth by estrogens) and osteoporosis (loss of bone density due to estrogen insufficiency). Estrogens exert their cellular actions via nuclear estrogen receptor (ER) proteins that function as transcriptional regulatory proteins. ERs act in conjunction with coactivator proteins that enhance the transcriptional activity of ER. Coactivators include Mediator, a protein complex that contains about 20 polypeptides including an ER binding subunit called Med220. The Mediator complex binds directly to ER through Med220 in the presence of estradiol (an estrogen). The activated ER then facilitates transcription of ER-regulated genes, such as pS2 and CatD. It has been shown that inactivation of the Mediator complex reduces ER-dependent transcription in vitro. My long term goal is to establish a system to reduce Med220 expression in vivo using RNA interference (RNAi) techniques. In RNAi, small fragments of double stranded RNA target homologous mRNA sequences for degradation, inhibiting expression of the corresponding gene. I plan to prepare dsRNA that will target endogenous Mediator mRNA, thus reducing the levels of Med220 protein in cells. As an initial trial of this approach with the cell lines that we use in the Kraus lab, I will first target estrogen receptor alpha (ERa), a well characterized factor for which we have many useful reagents to characterize the RNAi-induced gene silencing. Ultimately, I will examine the effects of reduced levels of Med220 and other related Mediator proteins on the expression of ER-regulated genes. The expression of these genes will be quantified by measuring their transcription using Northern blots and RT-PCR. It is expected that by reducing expression of the Med220 protein, the transcriptional activity of ER will be reduced and the ER-regulated genes will be expressed to a lesser extent. The techniques used in this project will include cloning, cell culture and transfection methods, Western blots, immunoprecipitations, Northern blots and RT-PCR.
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Jesse Colangelo-Lillis
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Research Advisor & Department
Alice Churchill - Plant Protection
Name of Project:
Chemical and Genetic Analyses of Anthraquinone Production in Mycosphaerella Pathogens of Banana and Plantain
Abstract:
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Summer Day
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Research Advisor & Department
Tom Brenna - Nutritional Sciences
Name of Project:
Comparison of the ability of 1 month and 9 month-old ACOX mice to synthesize Docosahexaenoic acid (DHA) in liver, heart, let muscle, brian and mitochondria of these tissues.
Abstract:
Homozygous (ACOX -/-) mice lack the expression of the acyl-CoA oxidase (ACOX) protein believed to be the first, rate-limiting step of peroxisomal b-oxidation of long chain polyunsaturated fatty acids. These knockout mice were used as a tool to test the peroxisomal b-oxidation involvement in the polyunsaturated fatty acid synthesis. We believe according to prior investigation that there may be an alternative pathway taking place in the mitochondria. We tested the ACOX -/- mice against age-matched controls (+/+) to determine the legitimacy of this alternative pathway. Mitochondria were isolated under nitrogen from the tissues through homogenization and a series of centrifugations to isolate a pellet. The mitochondrial isolate was tested for membrane integrity by observing the uptake of a cationic carbocyanine dye JC-1. We then fed the mitochondria (in vitro) isotopically labeled 18:3n-3. The mitochondria were extracted by a modified Bligh and Dyer procedure, and methylated to yield fatty acid methyl esters (FAMEs). The FAMEs were analyzed using a gas chromatograph (GC) with flame ionization detector (FID). FAMEs were analyzed for double-bond position by an acetonitrile chemical ionization tandem mass spectrometer (CIMS/MS) and they were analyzed isotopically with a gas chromatography-combustion isotope ratio mass spectrometer (GCC-IRMS). These analyses were done to determine if labeled DHA (22:6n-3) was produced by the mitochondria from labeled 18:3n-3. If significant amounts of labeled DHA were produced by the mitochondria, we would conclude that an alternative pathway in the mitochondria may be occurring.
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