CHARACTERIZATION OF OTK18 DURING DEVELOPMENT USING DROSOPHILA MELANOGASTER

University Of Nebraska Medical Center, Omaha NE

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The research interests of Dr. Kimberly A. Carlson focus on understanding the role of a novel transcriptional suppressor, OTK18, in regulation of developmental processes, innate immunity and neuronal fate (demise or growth) utilizing Drosophila melanogaster and Sprague-Dawley (SD) rat cortical neurons (RCNs) as model systems. It is assumed that this transcriptional regulator plays multiple roles and multiple functions within human development and the innate immune response. In two recent publications by the PI, it was demonstrated that OTK18 can act to suppress HIV-1 viral replication in monocyte-derived macrophages, and is up-regulated in mononuclear phagocytes of human brain tissue with severe HIV encephalitis (HIVE). Also, it was found that OTK18 is ubiquitously expressed in all normal human tissues, increases in expression in aging and dying cells regardless of HIV-1 infection and is produced in response to neuronal demise. At this time, the normal homeostatic function of OTK18 has not yet been characterized, the relationship of OTK18 to development has not been looked at and the relationship of OTK18 to neuronal state remains to be determined. My research has three specific aims: 1) to characterize the homeostatic, developmental, and immune functions of OTK18, 2) to determine the mechanism underlying the neuropathological (or anti-neuropathological) effects of OTK18, and 3) to utilize D. melanogaster and RCNs as model systems for investigation. My research utilizes transgenic D. melanogaster containing the human OTK18 gene to determine developmental and immune roles of this gene. Currently, we have constructed stable OTK18 transgenic lines. These transgenic lines will be crossed with various D. melanogaster developmental mutants to dissect the role OTK18 has in each developmental process or in homeostatic function. This transgenic system in combination with molecular techniques such as conventional and quantitative polymerase chain reaction (PCR), reverse-transcription (RT)-PCR, Adapter Fragment Length Polymorphism (AFLP), and microarray analysis, will be used to determine the genes that are being differentially regulated due to increased OTK18 expression at each stage of development. This data, when combined with proteomic techniques such as Western blotting and enzyme-linked immunosorbant assays (ELISAs), will assist in developing a rational mechanism for OTK18 regulation. Concurrently, the role of OTK18 in neuronal development or demise will be studied utilizing SD fetal RCNs, tissue culture techniques, standard neurotoxicity assays and molecular biology techniques. Because OTK18 is known to be upregulated in the brain of the most severe HIVE cases in which neuronal demise is rampant, it is important to determine whether OTK18 is acting as a neuroprotectant or neurotoxin. At this time, there are four research projects that are ongoing. The first is the creation, stabilization, and characterization of the OTK18 transgenic D. melanogaster lines. We have successfully defined the p-element insertion to the X, the 2nd and the 3rd chromosome. In addition, we have just finished stabilized the X chromosome insertion stock and will start characterizing the role of the transgene after the first of the year (2005). This project is being done by me and Darby Carlson. The second project is being done by Cole Spresser. Cole has successfully transfect a Drosophila embryonic cell line with the human OTK18. The RNA was collected from these cells and sent to UNMC for microarray analysis. At this time, we have done the initial 24 hours time point. Data will be back soon for us to do 2 other time points to determine OTK18 gene regulation. We did have some problems, due to old equipment. We could not maintain the temperature of the cells and contamination has been a problem. We are using an incubator that is 30 years old, but Jim Turpen is purchasing us a new one. Therefore, these problems should be fixed. The third project is to clone the OTK18 into a bacterial periplasmic expression vector. Using conventional Cytoplasmic expression vectors, we found that the protein was toxic to the cells. With this new system, we should be able to express, purify, and produce antibodies against OTK18. This is a project being worked on by Anjeza Pashaj. She has successfully cloned the gene into the vector and we are awaiting sequence data for confirmation. The last project is the RCN culture and neuropathogenic analysis. This is being done by my graduate student, Stephanie Bogers. Stephanie has successfully isolated rat cortical neurons, transfected them with human OTK18, extracted RNA and optimized RT-PCR parameters. She has shown that there is no significant cell death due to OTK18 expression in these neurons. One huge problem has be the lack of a good fluorescent camera. Jim Turpen is purchasing this for us, so this problem should be resolved.