Ligia Acosta
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Research Advisor & Department
Maureen Hanson - Molecular Biology & Genetics / Plant Biology
Name of Project:
Analysis of RNA Editing by Chloroplast Transformation
Abstract:
RNA editing converts cytidine to uridine in the chloroplast. In tobacco, 32 editing sites have been described in 15 different genes. Chateigner-Boutin and Hanson (1) found that the overexpression of rpoB-2 in the chloroplast decreased editing of other sites in vivo, particularly the sites rps14-1 and psbL-1. Upon visual inspection, they found that the rpoB-2, rps14-1, and psbL genes share similar sequences surrounding the edited C. I hypothesize that there could be a reciprocal relationship, that is, the overexpression of an rps14-1 transgene might have a significant effect on the percentage of editing in the endogenous rpoB and psbL genes. In the first half of this project, which was performed during the spring semester, I introduced the rps14 transgene into chloroplasts in icotiana tabacum var. Petit Havana through a chloroplast transformation vector via the gene gun. Transgenic tissue was identified by resistance to spectinomycin. The second half of the project, which will be performed during the summer, will concentrate on ensuring homoplasmy in the transgenic tissue through cyclical tissue culture on media with spectinomycin. Homoplasmy will be confirmed by DNA blot analysis of genomic DNA. Once most of the chloroplast genomes are found to contain the transgene, the percentage of editing will be analyzed by poisoned primer extension (PPE) to determine the effects of rps14 overexpression on the editing extent of the sites in rpoB and pbsL. If rps14 overexpression affects editing extent of other cluster members, then one can infer that these genes share a mechanism for editing. While waiting for transgenics, I will analyze transgenic plants generated by other researchers and collect data for my lab. In addition, I will develop a new transformation construct carrying an affinity tag. It will be used to make transgenic plants that will be useful to isolate proteins involved in RNA editing.
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Georgina Aldridge
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Research Advisor & Department
James Giovannoni - Plant Biology
Name of Project:
Characterization and Mapping of the Leutescent Mutant of Tomato
Abstract:
During ripening, fruit undergo many alterations such as changes in color, texture, and compounds that affect flavor and aroma. Mutations that disrupt or alter this process can be useful in the discovery of genes related to ripening. Our laboratory uses tomato as a model system because, among other reasons, it has an obvious ripening phenotype that can aid in screening for useful mutants. Two such mutations, lutescent 1 (l-1) and lutescent 2 (l-2), show a 9-10 day delay in ripening. In addition, the immature fruit of both mutants usually lack the normal green pigment when compared to wild type fruit of the same age, suggesting that the defect may affect the chloroplast. The leaves of both mutants tend to lose pigment and senesce at a higher rate than the wild type plants. The two mutations are located on separate chromosomes, and during previous semesters I refined the map of l-2 using RFLP mapping of an F2 population generated from a cross between L. esculentum and L. cheesemannii. A cosmid clone containing the l-2 gene was isolated by my lab. We sub-cloned this into a bluescript vector and sequenced, designing new primers as needed. The finished sequence was 38.5kb and contained five open reading frames. A candidate was identified by genetic mapping. The candidate gene was cloned and sequenced from wild type and l-2 and compared for base pair differences. l-2 was found to have a single base pair substitution resulting in a stop codon in the open reading frame instead of the amino acid leucine. This mutation causes a 23 amino acid truncation. Using tissue culture methods we will now test the candidate by introducing the wild type gene into the mutant plants and looking for complementation. We will also introduce the mutant gene into the wild type to test for protein interference, as well as creating a knock-out using RNAi.
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Michelle Armsby
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Research Advisor & Department
Nancy Tisch - Biometrics
Name of Project:
Community Structure of Subtidal Amphipods: A Look At Jassa marmorata and Corophium sp.
Abstract:
Two coexisting amphipod species, Jassa marmorata and Apocorophium acutum, have similar lifestyles within subtidal algal communities. Both species build tubes on algae and hard substrata, and are thought to rely heavily on filter feeding. However, recent studies suggest that intraguild predation (the killing and eating of a species that is a potential competitor) and cannibalism are also important amphipod feeding strategies (Tisch, pers.comm.; Macneil et al., 1997; Dick et al. 1996) and may influence the relative abundances of these two amphipods. Amphipods are also likely to be a food source for other decapod crustaceans such as Carcinus meanus, Cancer borealis, juvenile Homarus americanus (Tisch, pers.comm), and the invasive Hemigrapsus sanguineus (O’Connor, in prep), as well as commercially important fish species within the subtidal (Macneil et al., 1999). The construction of tubes may provide protection from all types of predation, as well as a stationary structure to filter feed. Amphipods that build tubes on many types of algae and at a faster rate may be able to escape predation more efficiently. The aim of this research is to gain a better understanding of the subtidal amphipod community and its role in the food web dynamics of the Gulf of Maine. First, monthly field sampling will be performed in order to document seasonal patterns of abundance of these two species. Next, a series of laboratory experiments will be performed to assess the role of cannibalism, intraguild, fish and crab predation on the abundance of these two species. Tube building experiments will also be conducted to compare rates of shelter construction. Gut contents of available predators, as well as algal sampling will be obtained from field sites to evaluate the diversity of predators and habitat of this amphipod community. These studies will provide insight on a community that has largely been overlooked in the past, but has the potential to have a large impact on trophic level dynamics.
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Caren Armstrong
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Research Advisor & Department
Thomas Cleland - Neurobiology & Behavior
Name of Project:
Task-Specific Modulation of Chemosensory Function of the Vomeronasal Organ in Anosmic Mice
Abstract:
The vomeronasal organ (VNO) has long been known to sense non-volatile and semiochemicals related to mating and aggressive behaviors. However, the role of the VNO as a more typical sensory modality has not yet been investigated. Previous studies have indicated several chemicals that are able to stimulate the VNO without being urinary extracts or other known semiochemicals (Wong et al., 2000; Trinh & Storm, 2003). Other studies have developed means by which mice may be rendered anosmic-- including the intraperitoneal injection of 3-methylindole to destroy the cells of the olfactory epithelium and the use of adenylyl cyclase III knockouts who lack olfactory epithelial function-without disrupting VNO function. During the course of the Hughes project, we plan to first ensure that the 3-MI experiments can be replicated using the CD-1 strain of mice and to then use the technique to render trained mice anosmic in order to test their ability to use their VNO to perform blocking-type behavioral tasks (Giannaris, et al., 2001). In addition, we will begin to establish a colony of adenylyl cyclase-III knockout mice here at Cornell which we can then use to perform the same types of experiments in order to increase the likelihood that the VNO is indeed the sensory modality mediating these task-specific blocking tasks.
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Rebecca Baker
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Research Advisor & Department
Gregory Martin - Boyce Thompson Institute
Name of Project:
A Study of the Relationship Between Structure and Virulence Function in the acrPto Protein of the Plant Pathogen Pseudomonas syringae Bacterium.
Abstract:
The gene product of the tomato resistance gene Pto recognizes the gene product of the bacterial plant pathogen Pseudomonas syringae avirulence gene avrPto in a gene for gene interaction, and triggers a signal transduction cascade that results in the hypersensitive response and cell death. The three-dimensional structure of AvrPto comprises a folded central core region (D29-I33, 105 residues) flanked by flexible N-terminal and C-terminal domains. The N-terminus contains a site required for myristoylation of AvrPto inside the tomato cell. Along with avirulence function within the tomato recognition system, AvrPto has a virulence function within the tomato recognition system, AvrPto has a virulence function that contributes to Pseudomonas pathogenicity. Mutational analysis of AvrPto introduced to susceptible tomato plants will determine the structural basis of AvrPto virulence function. Clones containing a binary vector of avrPto within a sequence of potato virus X was sequenced in E. coli and introduced to plants via inoculation with transformant Agrobacterium tumifaceans. Upon introduction to a susceptible tomato plant, Agrobacterium will remain localized at the site of innoculation, but the PVX viral vector will be conveyed to the tomato cell and spread systemically, conveying the avrPto sequence to all infected tissue. Susceptible tomatoes infected with AvrPto will display severe necrosis and massive cell death, while susceptible tomato plants infected with an AvrPto mutant with diminished virulence function will display reduced disease symptoms by comparison. Preliminary results show certain mutations cause a delay in recognition of AvrPto as well as reduced virulence function. The removal of the myristoylation site, rather than abolish AvrPto virulence function, may alter its phenotype. More clones will be created for further information in plant assays, and cytological techniques will be used to interpret these results.
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Anne Bjorkman
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Research Advisor & Department
Barbara Bedford - Natural Resources
Name of Project:
The Role of Dispersal in the Species Diversity of Fens
Abstract:
Wetland ecosystems include some of the most biologically diverse habitats in the world (Bedford and Godwin 2003). In particular, wetlands called rich fens support an unusually large number of rare and endangered plant species. Previous studies have shown the effects of water and soil chemistry on species diversity in fens. One aspect that has not previously been studied in these fens is the relationship between diversity and the distance between fens. Given what we know about island biogeography (MacArthur and Wilson 1967, Gotelli and Ellison 2002), we would expect that fen area, the distance between fens, and the dispersal ability of seeds to affect the number of species found at a given site (Nekola 1999, Soons and Heil 2002). In my project, I explored this relationship in order to determine the significance of its role in fen diversity. Using information about plant species composition, seed dispersal type, and distance between fens, I calculated the similarity decay over distance (Nekola and White 1999) for each type of seed dispersal mechanism. These data help to reveal the relationship between dispersal ability and species diversity in fens. The results of this research are important in helping to understand the factors that affect species diversity in fens. Without a complete understanding of all the factors at play, it is impossible to develop a plan to protect that diversity.
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Rebecca Brown
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Research Advisor & Department
Bik-Kwoon Tye - Molecular Biology & Genetics
Name of Project:
Generation of a True Null Mutant for Drosophila melanogaster MCM 10 Gene
Abstract:
This summer I will be working with true null mutant Drosophila melanogaster for the MCM 10 gene. The MCM 10 gene is a member of the minichromosome maintenance gene family that codes for proteins involved in the initiation of DNA synthesis. Studies of the MCM 10 gene family include studies on MCM 2 - 7 as they code for six conserved proteins that form a hexameric complex that is involved in the initiation of DNA replication in all eukaryotes. MCM 10 has become a gene of interest because studies have shown that the Mcm 10 protein interacts with the proteins of the MCM 2 - 7 hexameric complex, and that these interactions mediate the binding and disassociation of the MCM 2 - 7 complex at the replication origin of DNA. Father studies may reveal the full role and necessity of the interaction of the Mcm 10 protein with the MCM 2 -7 hexameric complex and how important it is in the processes of DNA replication. This summer, I will attempt to successfully generate a true null mutant Drosophila for the MCM 10 gene through directed matings, using a P1 element transposon insertion just upstream of the MCM 10 gene, located on the second chromosome of Drosophila melanogaster. If the inserted transposon makes an imprecise excision, successfully cleaving out part of the gene (there by deactivating it), the Drosophila with the excision will be true null mutants for the MCM 10 gene. Of the flies that are lacking in functional copies of the MCM 10 gene, we may begin to explore the viability of the Mcm 10 protein in DNA replication based on the survivorship of the flies unable to produce the Mcm 10 protein. In the event that true null mutants of MCM 10 are unable to survive, we may say that the Mcm 10 protein is necessary for the successful initiation of DNA replication. In the event that true null mutants of MCM 10 survive, this summer we will begin to study true and alternate roles of the MCM 10 gene through additional genetic analysis.
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