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Research Advisor & Department
Irby Lovette - Ecology and Evolutionary Biology
Name of Project:
Quantification of Genetic Variation to Determine Evolutionary History of House Finch (Carpodacus mexicanus) Populations Abstract:
The house finch (Carpodacus mexicanus) is a native western North American bird species that has populations across most of the mainland U.S., Mexico, Hawaii, and Guadalupe Island. My goal is to determine the extent of genetic variation between the different extant populations of house finches. There are distinct phenotypic differences between the Guadalupe house finch, mainland western house finch, and eastern introduced populations. Although the phenotypic differences are well documented, no study has examined genetic differences among mainland and island house finch populations. I will test whether genetic changes underlie the dramatic phenotypic differences that we see, particularly for house finches on Guadalupe Island, which may in fact be a distinct species. During the summer I will be sequencing the mitochondrial marker ND2. I will be doing this for approximately twenty samples from each of six separate mainland populations. I will also do this for the few samples I have from Hawaii, Mexico, and Guadalupe Island. Once I have all of the samples sequenced I will be able to compare them in order to find single base pair differences. These genetic differences can then be used to construct a phylogenetic tree for the house finch populations.
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Research Advisor & Department
Richard Harrison - Ecology and Evolutionary Biology
Name of Project:
Investigation
of Fatty Acid Reductase as a Candidate Speciation Gene Responsible for
Pheromone Blend Specificity in the European Corn Borer Abstract:
The European Corn Borer (ECB) (Ostrinia nubilalis) is an excellent model system for studying the genetic basis of pre-zygotic barriers to gene exchange. Two strains of ECB are known to exist, and are characterized by different pheromone blends of the E and Z isomers of 11-tetradecenyl acetate. In one strain females produce a 3:97 blend of E/Z, and in the other strain females produce a 99:1 E/Z blend. Z males respond only to the characteristic 3:97 blend. E males respond mostly to the 99:1 E/Z blend, however a significant number respond to intermediate blends, and rarely to the Z blend. Work in the Harrison lab has been dedicated to understanding the genetic architecture of the ECB mating system. Through simple genetic crosses (Roelofs et al. 1987), it was discovered that pheromone production (Pher) and male behavioral response (Resp) are both controlled by single major genes. Resp is sex linked and located on the Z chromosome, while Pher is autosomal and located on chromosome 12. Dopman et al. (2004) have since placed these genes on a linkage map for ECB. My efforts in the Harrison lab will center on identifying the gene responsible for producing the different pheromone blends. It is theorized that a fatty acid reductase (FAR) produces the characteristic pheromone blends, and as a result such a gene is a candidate "speciation gene" (Zhu et al. 1996). A FAR homologue has recently been identified and sequenced in the silkworm moth Bombyx mori (Moto et al. 2002). Reductase activity and substrate specificity were confirmed when the gene was expressed in Yeast. Using this known FAR sequence, as well as other homologous FAR sequences identified via GenBank, I will design PCR primer pairs in an attempt to identify the homologous FAR gene in the European Corn Borer. These primer pairs will first be used to probe cDNA from the pheromone gland, and if successful, genomic DNA as well. I will then sequence the FAR gene in a large number of individuals from both the E and Z strains. If FAR is responsible for pheromone blend specificity, we expect that it will map to the same location as Pher identified in the genetic map constructed by the Harrison lab. Furthermore, we also expect both strains to be exclusive groups at the FAR locus.
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Research Advisor & Department
David McCobb - Neurobiology and Behavior
Name of Project:
Adrenal
Dysfunction in Mice and its Affect on Social Behavior: Use of Mice With
Impaired Adrenal Function in Testing Virtual Dominance Theory Abstract:
The specific aims of this project are to 1) determine how dominance is established between a pair of mice, 2) determine whether adrenal function contributes to establishing dominance, and how pair-wise interactions are affected by altered adrenal function, and 3) test predictions of virtual dominance theory based on what is learned about dominance and adrenal function. The ability of an animal to mount a vigorous adrenaline-secretory response may be an essential determinant of dominance status and territory establishment. In some animals such as lizards and baboons, a faster adrenaline response or higher adrenaline levels can set apart a dominant individual over a subordinate. McCobb et al showed that stress due to chronic subordination changes adrenomedullary function at the level of BK channel properties in adrenaline-secreting chromaffin cells. BK channels have also been shown to be a major determinant of intrinsic excitability of these cells; BK activation promotes rapid repetitive firing, which enables rapid secretion of adrenaline. Adrenaline appears to be a key player in provoking and modulating social interactions, and the BK channel plays an important role in adrenaline release, so investigating the affects of the BK channel on social interactions is very appealing. Normal mice do show establishment of dominance over one another. When two males are placed together, they will usually fight until one is clearly dominant over the other. Dominant mice are observed to police and attack their subordinates. Dominance is being measured by observing which mouse provokes attacks, how long attacks last, and how long a mouse adopts a defensive posture. An individual might also be considered subordinate if it is excluded from friendly huddling. Wild-type mice will be observed in pairs for indications of dominance. Dominance might be affected by size, excitability, activity levels, age, sex or some other factor. It is important to determine which factors contribute most strongly to dominance and subordinance. Differences in adrenal function will then be introduced into the experiment by testing experimental mice against wild-type and other experimental mice. We hypothesize that mice with impaired adrenaline secretion will be handicapped against normal mice in establishing themselves as dominant. The decreased ability to secrete adrenaline may even outweigh all the other variables contributing to dominance. If adrenaline secretion is extremely important to establishing dominance, and there is little or no compensatory mechanism to counteract their dysfunction, experimental mice might not even be able to establish dominance over each another. After investigating the pair-wise behavior of the experimental mice, I would like to use what I learned to investigate virtual dominance theory proposed by Kern Reeve, a faculty member involved with this project. The most powerful prediction the virtual dominance model makes is who will be dominant in a group of related individuals. For example, in unrelated mice the larger or stronger mouse would be expected to dominate over the other mice. When related mice interact, the mouse that is most related to all the members of the group would be dominant, even if it is a smaller mouse or one that is prone to being subordinate in other situations (Reeve & Jeanne). If the experimental mice do indeed show impaired dominance assertion, they would an excellent tool in performing robust tests of the virtual dominance model. A mouse that would otherwise have little or no chance of being dominant over another mouse may end up being dominant over many mice if it is the most related to others in a group. Thus this project is unique in that it relates predictions based on evolutionary theory to the underlying physiological and molecular mechanisms of behavior. References: (1) McCobb, David P., Yuko Hara, Guey-Jen Lai, Sahar F. Mahmoud, Gabriele Flugge. 2002. Subordination stress alters alternative splicing of the Slo gene in tree shrew adrenals. Hormones and Behavior. 43, 180-186. (2) Reeve, H.K., & Robert L. Jeanne. 2003. From individual control to majority rule: extending transactional models of reproductive skew in animal societies. Published Online. The Royal Society. 1041-1045.
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 | Research Advisor & Department
David - Molecular Biology and Genetics
Name of Project:
Search for mutations that activate PTPa in human breast cancer cells Abstract:
c-Src was the first proto-oncoprotein to be identified. It is the prototype of a family of protein-tyrosine kinases (PTKs) that are bound to perinuclear membranes, endosomes, secretory vesicles, and at the cytoplasmic face of the plasma membrane (ref: 1, 2, 3). c-Src is transiently activated during the cell cycle, and is also activated in 70% of human cancers, especially those of the breast and colon. However, the mechanism of c-Src activation in human cancer is unknown. Dephosphorylation at Tyr527 plays a key role in c-Src activation. Dephosphorylation can be accomplished by protein tyrosine phosphatases like PTPa. The Shalloway laboratory has developed a model of how PTPa activates c-Src, and is interested in PTPa because its over-expression activates c-Src in vivo (ref: 4, 5). Zheng et al showed that over-expression of PTPa in rat embryo fibroblasts activates c-Src and causes cell transformation, suggesting that PTPa may be a potential proto-oncogene (ref: 6). There are several possible mechanisms that may activate PTPa. One of the mechanisms may be via mutations. Therefore, the goal of my project is to see whether there are PTPa mutations that activate PTPa, which in turn, activates c-Src. To search for possible mutations, the total RNA was prepared from 15 cell lines and used to make and to amplify PTPa cDNA. This cDNA will then be cloned, sequenced, and then compared to the known, placental PTPa sequence. If mutations are found, then the cDNA will be subcloned and placed into expression plasmids. These expression plasmids will then be transfected into NIH 3T3 to express the mutated protein; the effects will then be observed. References: (1) Thomas, S.M. and Brugge, J.S. (1997) Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol., 13, 513-609. (2) Bjorge, J.D., Jakymiw, A. and Fujita, D.J. (2000) Selected glimpses into the activation and function of Src kinase. Oncogene, 19, 5620-5635. (3) Zheng, X.M., Resnick, R., and Shalloway, D. (2000) A phosphotyrosine displacement mechanism for activation of Src by EMBO, 19 No. 5, 964-978. (4) Zheng, X.M., Wang, Y. and Pallen, C.J. (1992) Cell transformation and activation of pp60 (c-Src) by overexpression of a protein tyrosine phosphatase. Nature, 359, 336-339. (5) den Hertog, J., Pals, C.E.G.M., Peppelenbosch, M.P., Tertoolen, L.G.J., de Laat, S.W. and Kruijer, W. (1993) receptor protein tyrosine phosphatase a activates pp60(c-Src) and is involved in neuronal differentiation. EMBO J., 12, 3789-3798. (6) Taylor, S.J. and Shalloway, D. (1996) Src and the control of cell division. BioEssays, 18, 9-11.
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 | Research Advisor & Department
Kelly Zamudio - Ecology and Evolutionary Biology
Name of Project:
Phylogeography of the Green Frog (Rana clamitans): Glacial Refugia and Postglacial Colonization Abstract:
The climatic fluctuations of the past 2.4 million years have had a significant influence on the genetic structure of contemporary species. Oscillations between glacial and interglacial periods caused corresponding cyclic changes in the geographical distribution of organisms. Advancing ice sheets reduced the range of species to isolated patches of suitable habitat known as refugia. After the ice sheets retreated, these isolated populations dispersed and colonized previously inhospitable regions. Though the effect of these repeated range constrictions and expansions on the present-day genetic structure of a number of European species has been examined, little research has been done with eastern North American taxa. My project is concerned with reconstructing the post-glacial evolutionary history of such a taxa — Rana clamitans, or the green frog. Specifically, I hope to accomplish three tasks: (1) determine the refugial dynamics of R. clamitans during the last glacial advance, (2) understand the extent and routes of postglacial colonization by divergent lineages, and (3) compare the refugia and colonization characteristics of R. clamitans to those of the other eastern North American species that have been studied. In order to fulfill these three tasks, I will be employing a number of molecular techniques. I will first use phenol chloroform methods to extract DNA from tissue samples that have been collected from 145 individuals that represent 56 localities across the range of R. clamitans. I will then amplify and sequence a segment of cytochrome b, a protein-encoding mitochondrial gene. Phylogeographic analysis of the sequence variations between populations will allow me to examine the role of the most recent glacial cycle on the genetic structure of R. clamitans today. In doing so, I will be contributing to the present body of knowledge about the complex relationship between earth history and biotic diversification, especially the biogeographic patterns and origins of genetic diversity in eastern North America. I will also be providing important insights into the impact habitat reduction and fragmentation can have on the genetic diversity of a species.
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 | Research Advisor & Department
Ruth Collins - Molecular Medicine
Name of Project:
Analysis of Conservation of Elp1p Function from Yeast to Human Abstract:
Vesicular trafficking is regulated by Rab proteins. Rabs are members of the superfamily of Ras small GTPases. Like all GTPases, Rab functions as nucleotide dependent molecular switches. They cycle between GTP and GDP bound states, a process which is regulated by guanine nucleotide exchange factor (GEFs) and GTPase activating proteins (GAPs). Sec4 is a Rab protein functioning in the regulation of polarized exocytosis in yeast from the Golgi to the plasma membrane. Sec2p, GEF specific for Sec4p, is recruited to the sites of exocytosis to activate Sec4p. Elp1p is a member of the six-subunit Elongator complex. It was recently found that Elp1p is responsible for the correct localization of Sec2p and negatively regulates polarized exocytosis. The Elongator complex is composed of 2 subcomplexes. Elp1p, Elp2p, and Elp3p form one complex and Elp4p, Elp5p, and Elp6p form another. Elp1p is the yeast homolog of the human gene lKAP. Mutations in IKAP have been shown to cause the human neurodegenerative disease Familial dysautonomia (FD.) Because of this we are interested in studying if the function of Elp1p in yeast is conserved in IKAP. Our lab has previously shown that the C-terminal 185 residues of Elp1p interact with Sec2p and are critical for Elp1p function. The truncation mutant Elp1D185 can not function as Elp1p. We will construct a chimera protein combining yeast Elp1D185 with the IKAP equivalent C-terminus. Then we can test if this yeast-human chimera can function in yeast. We will also create FD mutation, truncation further up the C-terminus, in yeast Elp1p and design a chimera with IKAP C-terminus and see if it will complement elp1D. The results could be compared to full length IKAP cloned in expression vector in yeast. These will give clues to whether or not the Elp1p Sec2p interacting domain is conserved in humans, shedding light on the mechanisms of FD and Elp1p function.
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