The goals of this research are to identify, isolated, and characterize inducible genes from livers of female rats treated with pregnenolone 16 alphacarbonitrile ( a compound with antiglucocorticoid properties, PCN). We propose to achieve this goal by identifying differentially expressed mRNA species by an improved state of the art display technique commonly known as Differential Display Reverse Transcription-PCR(DDRT-PCR). This method is based in the assumption that every cell expresses some 15,000 genes and that every mRNA molecule could be transcribed and amplified by the polymerase chain reaction. By comparing the patterns of expressed mRNA from two cell types one is able to detect both quantitative and qualitative changes. This kind of method would allow not only to identify new genes but also the detection of any changes in gene expression involved in a particular cellular process such as drug treatment such as Dexamethasone or xenobiotic exposure (PCN). We have demonstrated that our approach would be successful since preliminary data has been generated using this technique. We have identified, isolated and obtained clones from true positive differentially displayed cDNA fragments. Some of these fragments have been shown to originate from PCN induced mRNA while others are from PCN inhibited mRNA. We have also been able to demonstrate that some of these amplified cDNA fragments are concurrently induced by DEX. We will continue to expand or data on the number of differentially displayed hands by augmenting the number of AP primers tested in treated animals. Cloning and sequencing of true positive cDNA fragments of uncharacterized or unidentified mRNAs obtained by our preliminary research, will continue until we obtain complete cDNA clones for the amplified mRNAs and the characterization of their cognate genes. Future work will concentrate on the identifying the regulatory sequences for the steroid and or anti-steroid gene induction. This research will continue to develop as a strong collaboration between the University of Colorado (Health Sciences Center) and the University of Puerto Rico. We have been training and working with Dr. Philip Guzelian who has an R01 grant from NIEHS, to study the regulation of cytochrome P4503A family and specially with CYP4503A1 (a known DEX and PCN inducible gene). This research has been and will continue to be an essential and key element for the training of graduate and undergraduate students in a relatively new biomedical research area (Toxicology) in the U.S. and certainly a new area that need to be developed in Puerto Rico. This collaboration with the University of Colorado will serve as a minority student bridge between the mainland and Puerto Rico. Evidence of this is the acceptance of a graduate student of mine to their biomedical graduate program after a summer experience at the Medical Toxicology lab. Many undergraduate students are now being exposed to hands on molecular toxicology techniques completely new to our School and to the Medical Sciences Campus.
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