ISOLATION/CHARACTERIZATION OF PREGNENOLONE 16 ALPHA CARBONITRILE INDUCED GENE

这项研究的目标是识别、隔离和表征 孕烯醇酮16处理雌性大鼠肝脏的诱导基因 碳腈(一种具有抗糖皮质激素特性的化合物，PCN)。 我们建议通过识别差异表达来实现这一目标 用改进的最先进的展示技术共同进行mRNA的筛选 称为差异显示反转录-聚合酶链式反应(DDRT-PCR)。这 方法是基于这样的假设：每个单元格表示大约15,000个 基因和每一个信使核糖核酸分子可以被转录和扩增 聚合酶链式反应。通过比较表达的mRNA的模式 从两种细胞类型中，我们能够同时检测到定量和定性 改变。这种方法不仅可以识别新的基因 还可以检测涉及到的基因表达的任何变化 特殊的细胞过程，如药物治疗，如地塞米松或 异种生物暴露(PCN)。我们已经证明，我们的方法将是 成功，因为已经使用该技术生成了初步数据。 我们已经从真阳性中鉴定、分离并获得了克隆 差异显示的cDNA片段。其中一些碎片已经被 显示来自PCN诱导的信使核糖核酸，而其他来自PCN 抑制了mRNA的表达。我们还能够证明其中一些 扩增出的cDNA片段同时被地塞米松诱导。 我们将继续扩大或提供不同数量的数据 通过增加在处理中检测的AP引物的数量来展示手 动物。猪瘟病毒真阳性基因片段的克隆及序列分析 通过我们的初步研究获得的未知或未识别的mRNAs， 将一直持续到我们获得扩增的mRNAs的完整cdna克隆。 以及它们的同源基因的特征。未来的工作将是 专注于确定类固醇和类固醇的调节序列 或抗类固醇基因诱导。 这项研究将继续发展为两国之间的强有力的合作 科罗拉多大学(健康科学中心)和加州大学 波多黎各。我们一直在培训菲利普·古泽利安博士并与之合作 世卫组织获得了NIEHS的R01拨款，用于研究细胞色素的调节 P4503A家族，特别是CYP4503A1(一种已知的地塞米松和PCN诱导物 基因)。这项研究一直是，并将继续是一个必要的和关键的 培养研究生和本科生的要素 美国相对较新的生物医学研究领域(毒理学)和 当然，这是一个需要在波多黎各开发的新领域。这 与科罗拉多大学的合作将成为少数 大陆和波多黎各之间的学生桥。这方面的证据是 我的一名研究生被他们的生物医学毕业生录取 在医学毒理实验室的一次暑期体验之后。许多 本科生现在接触到了亲手操作的分子 毒理学技术对我校和医学界来说都是全新的 理工学院校园。

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.