ISOLATION/CHARACTERIZATION OF PREGNENOLONE 16 ALPHA CARBONITRILE INDUCED GENE

本研究的目标是识别、分离和表征 雌鼠肝脏中的诱导基因 三甲腈（一种具有抗糖皮质激素特性的化合物，PCN）。 我们建议通过鉴定差异表达的 通过改进的现有技术展示技术，mRNA种类通常 称为差异显示逆转录-PCR（DDRT-PCR）。 这 一种方法是基于这样的假设，即每个细胞表达大约15，000个 基因，每个mRNA分子都可以被转录和扩增， 聚合酶链反应。通过比较mRNA表达模式， 从两种细胞类型中，能够定量和定性地检测 变化 这种方法不仅可以识别新的基因， 而且还可以检测到参与免疫应答的基因表达的任何变化， 特定的细胞过程，如药物治疗，如地塞米松或 异生物质暴露（PCN）。 我们已经证明，我们的方法将是 成功的原因是使用这种技术生成了初步数据。 我们从真阳性克隆中鉴定、分离并获得了克隆， 差异显示的cDNA片段。 其中一些碎片 结果表明，有10种基因来源于PCN诱导的mRNA，而其他基因则来源于PCN 抑制mRNA。 我们也已经能够证明，其中一些 扩增的cDNA片段同时被DEX诱导。 我们将继续扩大或数据的数量差异 通过增加治疗组中检测的AP引物的数量来显示手 动物 大肠杆菌真阳性cDNA片段的克隆及序列分析 通过我们的初步研究获得的未表征或未鉴定的mRNA， 直到我们获得扩增mRNA的完整cDNA克隆 以及它们同源基因的特征。 未来的工作将 集中精力鉴定类固醇的调节序列， 或抗类固醇基因诱导。 这项研究将继续发展为一个强大的合作， 科罗拉多大学（健康科学中心）和 波多黎各。 我们一直在培训和菲利普·古泽利安博士 他拥有NIEHS的R 01资助，研究细胞色素的调节， P4503 A家族，特别是与CYP 4503 A1（一种已知的DEX和PCN诱导的 基因）。 这项研究已经并将继续是一个重要的和关键的 研究生和本科生的培训要素， 美国相对较新的生物医学研究领域（毒理学）， 这肯定是波多黎各需要开发一个新领域。 这 与科罗拉多大学的合作将成为少数 美国大陆和波多黎各之间的学生桥梁。 证据是 我的一个研究生被他们的生物医学研究生录取 在医学毒理学实验室的夏季经验后，计划。 许多 本科生现在接触到的分子 毒理学技术完全新的我们的学校和医学 科学校园。

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.