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Research Advisor & Department
Mariana Wolfner - Molecular Biology and Genetics
Name of Project:
The study of the wispy protein/protein interactions in Drosophila melanogaster using the Yeast-Two-Hybrid system
Abstract:
Drosophila melanogaster is an animal model used in the study of early embryo development. In particular, these studies are focused on the activation of the mature oocyte and the initiation of development by the fertilized egg. One of the genes needed to start this early phase of development is the wispy gene, which is important in the formation of normally shaped spindles, the transition to embryo mitosis, and the localization and polyadenylation of maternal mRNAs. To further study the wispy gene, I examined the interaction of its protein product with the other proteins in Drosophila using the Yeast-Two-Hybrid system. In this system, I used the GAL4 transcription activator which is composed of two domains, the transcription-Activation Domain (AD) and the DNA-binding Domain (BD), which binds to the promoter UAS. Together, these two domains activate the transcription of a down-stream target gene, such as UAS-His+. Because AD and BD have to be in a protein complex to activate transcription, fusing the wispy protein to BD and another protein from a cDNA library to AD allows us to test for the interaction between wispy and the other protein: if they interact there will be transcription of UAS-His+, resulting in the production of Histidine and the growth of the yeast colonies on a Histidine-free plate. Using this test, 30 different genes whose products interacted with the wispy protein were identified. Among these, CG11486 and CG10042 are of particular interest because of their nucleic acid binding activity.
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Research Advisor & Department
Mary Lou Zeeman - Neurobiology and Behavior
Name of Project:
The effects of the stress hormone corticosterone on the release of luteinizing hormone from the anterior pituitary
Abstract:
The anterior pituitary releases luteinizing hormone (LH) in response to gonadotropin releasing hormone (GnRH) from the hypothalamus. A surge in LH levels causes ovulation in females, and constant pulses of GnRH will yield an LH surge. We seek to observe how the mouse pituitary integrates information independently of the hypothalamus, particularly in response to the stress hormone corticosterone (cort). We perfused ringers solution over the pituitary gland with pulses of GnRH or a pulse of GnRH and cort every hour. We collected the runoff in five or ten minute samples, and tested for LH levels using an Enzyme Linked ImmunoabSorbent Assay. We found that cort did not inhibit the release of LH unless GnRH was constantly dripped over the pituitary glands. In some cases LH release was increased during or shortly after the cort pulses. Our results conflict with those in the literature, and further investigation is necessary to determine by what mechanism cort is affecting the pituitary gland.
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Research Advisor & Department
Irby Lovette - Ecology and Evolutionary Biology
Name of Project:
Population genetics of a recent colonization event of Barn Swallows in Argentina
Abstract:
In 1980, six pairs of Barn Swallow (Hirundo rustica) were discovered breeding in Argentina, within the species' wintering range (Mart’nez, 1983). Today thousands of pairs breed along the coast between Mar del Plata and Bah’a Blanca in Buenos Aires province. We are exploring the genetic history of this population to determine whether the current large population experienced a population bottleneck (indicating a small founding population with little or no ongoing recruitment). We surveyed allele diversity of eight microsatellite (Kleven, 2005; Tsyusko, 2007) loci from five populations, three from Argentina and two from North America (California and New York). Although there was significant population differentiation, Fst values were small. In addition, there was no evidence for a population bottleneck in Argentina. These patterns raise the possibility of ongoing gene flow between North America and Argentina and suggest that a substantial number of individual Barn Swallows have been recruited into the Argentinean breeding population, which would require a breeding cycle shift of six months.
References:
Hawley, D.M., Hanley, D, Dhondt, A.A., Lovette, I.J. Molecular evidence for a founder effect in invasive house finch (Carpodacus mexicanus) populations experiencing an emergent disease epidemic. Molecular Ecology. 15, 263-275 (2006)
Kleven, O, Jacobsen, F, Robertson R.J., Lifjeld, J.T. Extrapair mating between relatives in the barn swallow: a role for kin selection?. Biological Letters. 1, 389-392. (2005)
Mart’nez, M. M. Nidificaci—n de Hirundo rustica erythrogaster (Boddaert) en la Argentina (Aves, Hirundinidae). Neotr—pica 29, 83-86 (1983).
Tsyusko, O.V., Peters, M.B., Hagen, C., Tuberville, T.D., Mousseau T.A., M¿ller, A.P., Glenn, T.C. Microsatellite markers isolated from barn swallows (Hirundo rustica). Molecular Ecology Notes (2007)
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Research Advisor & Department
Patricia Cassano - Nutritional Sciences
Name of Project:
Comparing the Accuracy of FEV6 and FVC in Measuring Lung Function and Predicting the Presence of Chronic Obstructive Pulmonary Disease (COPD)
Abstract:
Current standards used to define disease in epidemiologic studies of Chronic Obstructive Pulmonary Disease (COPD) are derived from spirometric lung function testing. COPD is considered present if a subject's Forced Expiratory Volume in One second (FEV1) is less than 70% of his or her Forced Vital Capacity (FVC) and FEV1 is less than 80% of a predicted value. However, recent studies suggest that substituting Forced Expiratory Volume in 6 seconds (FEV6) for FVC in the diagnostic ratio yields a more reliable and accurate classification. This theory was investigated using spirometric data from the Health, Aging, and Body Composition (Health ABC) cohort. Subjects were classified as diseased or non-diseased at baseline using both criteria and then the distribution of subjects was compared. 314 of 2,095 subjects were classified with COPD by either criterion, with 21% of those cases being disputed between the ratios. These disputed cases showed lower prevalence of non-respiratory conditions (e.g. cancer) than either non-diseased or confirmed diseased subjects. Results also suggested an implausibly high rate of reversion among COPD cases to non-diseased status after four years (80%). Further analysis is needed to clarify if this results from improper data collection or from characteristics inherent to the classification groups.
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Research Advisor & Department
Jun (Kelly) Liu - Molecular Biology and Genetics
Name of Project:
Dissecting the mechanisms of striated muscle fate specification in the C. elegans postembryonic mesoderm
Abstract:
Understanding how different cell fates are specified is fundamental to the study of various developmental processes. This project investigates how three transcription factors, FOZI-1, HLH-1, and MAB-5 function redundantly to specify fourteen body wall muscles in C. elegans that are derived from the M lineage, a postembryonic mesodermal lineage that the Liu Lab uses to study mesodermal development. The yeast two-hybrid system was used to determine if the functions of FOZI-1, HLH-1, and MAB-5 in specifying body wall muscle are mediated through direct physical interaction. In addition, previous studies in the Liu Lab (Amin et al., 2007) show that the FOZI-1 protein contains an evolutionarily conserved FH2 domain that serves to bind actin and mediate self-dimerization. Thus, the yeast two-hybrid system was also used to determine whether FOZI-1 self-dimerizes and if so, whether the FH2 domain mediates this dimerization. Preliminary data from the yeast two-hybrid assay indicate that HLH-1, a transcription factor known to self-dimerize, binds to itself. However, no interaction among the three proteins was detected. Furthermore, no self-interaction was detected for FOZI-1. Future experiments will use Western Blotting to determine whether each of the tested proteins is being stably expressed in the two-hybrid system.
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Research Advisor & Department
Mariana Wolfner - Molecular Biology and Genetics
Name of Project:
More Than She Bargained for: An Exploration into How Drosophila Males Influence Their Mates
Abstract:
Unbeknownst to most females, the seminal fluid of males from fruit flies to humans contains more than just sperm and nutrients, but an entourage of proteins that vary in function from hormones to proteases and have a wide range of effects on females.1 The accessory glands of male Drosophila melanogaster produce one such protein called ovulin that induces ovulation in females for a period of ~1.5-6 hours post-mating. 2 Ovulin is of particular interest because it has been shown to be one of the most rapidly evolving genes in the D. melanogaster genome.
Previous work has shown that ovulin self-interacts to form a dimer within the female reproductive tract and residues conserved across multiple Drosophila species support a coiled-coil motif as the method of interaction. 3 My work focuses on the effects of the diverged regions of ovulin on maintaining its self-interaction and function in ovulation induction. Using the yeast two-hybrid system I have shown that only three closely related species of Drosophila ovulin can interact with that of D. melanogaster even though the coiled-coil residues are conserved in much more distant relatives. I am now testing the ability of ovulin from other species to induce ovulation in female D. melanogaster by ectopically expressing them through the use of germ-line transformation. These studies could help to shed light on other important regions of the ovulin gene and how its entrance into the female reproductive tract is linked to the ovulation phenotype.
1Wolfner MF. The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila. Heredity. 2002 Feb;88(2):85-93. Review.
2Heifetz Y, Lung O, Frongillo EA Jr, Wolfner MF. The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary. Curr Biol. 2000 Jan 27;10(2):99-102.
3Wong A, Albright SN, Wolfner MF. Evidence for structural constraint on ovulin, a rapidly evolving Drosophila melanogaster seminal protein. Proc Natl Acad Sci U S A. 2006 Dec 5;103(49):18644-9. Epub 2006 Nov 27.
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Research Advisor & Department
Bik Tye - Molecular Biology and Genetics
Name of Project:
Inter-Species Functional Analysis Reveals Novel Origins of Replication
Abstract:
The sequence conservation and location of replication origins in the Saccharomyces cerevisiae genome have been extensively analyzed. There are about 350 replication origins in the budding yeast, each with a specific DNA sequence of about 200 bp known as Autonomously Replicating Sequences (ARSs). However, only a small fraction of ARSs is activated in any given S phase, suggesting functional redundancy. A comparison of ARSs identified an 11-17 bp AT-rich consensus sequence (ACS) that is necessary, but not sufficient for ARS activity. To identify other sequence motifs essential for ARS function, we have embarked on a large-scale isolation of genomic ARSs from five other Saccharomyces species using S. cerevisiae, and have isolated hundreds of such ARSs to date. Though ARSs could be readily isolated from the more closely related S. mikatae, S. kudriavzevii, and S. bayanus strains, many are not conserved in sequence or synteny with endogenous S. cerevisiae origins, and some do not contain an obvious ACS. Genomic ARSs from the more distantly related S. castellii and S. kluyveri strains are more difficult to isolate, but AT-rich mitochrondrial ARSs can be isolated readily. We have also isolated numerous kluyveri ARSs using an auxotrophic S. kluyveri strain. Interestingly, we have identified two classes of ARSs: Inter-species ARSs that function in multiple species, and species-specific ARS that function exclusively in one yeast species. This finding suggests that there are both conserved and divergent mechanisms for origin function among the related yeasts. Testing ARSs for function in multiple yeast species will allow us to understand the evolutionary dynamics of replication origins, and to build a phylogenetic tree based on origin function that is independent of sequence similarity.
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