REGULATION OF EXPRESSION OF OPIOID PEPTIDES & THEIR REEP

阿片肽存在于神经和中枢神经系统的许多不同部位， 内分泌系统中，它们介导不同类型的反应。 所有 已经证明，许多这种大家族的肽（超过15种） 衍生自3种不同的前体，称为阿黑皮素原， 脑啡肽原和强啡肽原。 阿片肽结合的受体 在性质上也是多种多样的。 它们可以分为3个主要的 基于它们结合的阿片类药物的差异的类型。 每种受体 阿片肽在大脑中的分布有些不同， 可以调节不同的反应。 这项研究的主要目的是进一步了解 调节阿片肽和阿片样物质表达的机制 受体。 这一目标将通过多层次的方法来实现 采用分子生物学、免疫学和蛋白质化学的方法。 阿片肽的合成位点将在大脑中被定位， 使用每种阿片样物质特异性DNA探针的原位杂交技术 先驱 阿片样物质基因中的调控位点将由以下定义： 基因转移技术 这将通过研究以下因素的影响来实现： 阿片样物质基因的特异性改变（体外位点特异性 诱变）对转录活性和对蛋白质加工的影响。 两种不同类型的受体细胞（哺乳动物细胞系）中的前体 和青蛙卵母细胞）。 阿片受体研究的第一步将是确定 通过重组DNA技术构建一种类型的受体。 一 一种称为杂交选择程序的相对较新的方法将 用于纯化编码受体的mRNA种类。 青蛙 卵母细胞表达系统将用于测定mRNA的能力， 指导受体蛋白的合成。 cDNA将从以下制备： 纯化mRNA并克隆。 从cDNA的序列来看， 可以确定受体中亚基的数量， 每个子单元的结构。 下一步将是使用各种受体 cDNA探针用于确定阿片受体基因的数量以及 这些基因彼此相似。

Opioid peptides are found in many different sites in the nervous and endocrine systems where they mediate diverse types of responses. All numbers of this large family of peptides (more than 15) have been shown to be derived from 3 different precursors known as pro-opiomelanocortin, proenkephalin and prodynorphin. The receptors that opioid peptides bind to are also diverse in nature. They can be classified into 3 predominant types based on the differences in opioids they bind. Each type of receptor and opioid peptide has a somewhat different distribution in the brain and may mediate different kinds of responses. The major goal of this research is to further our understanding of the mechanisms that regulate expression of opioid peptides and opioid receptors. This goal will be accomplished by a multilevel approach employing methods of molecular biology, immunology and protein chemistry. The sites of synthesis of opioid peptides will be mapped in the brain by in situ hybridization techniques using DNA probes specific for each opioid precursor. The regulatory sites in the opioid genes will be defined by gene transfer techniques. This will be done by studing the effects of specific alterations of the opioid genes (site-specific in vitro mutagenesis) on transcriptional activity and on processing of the precursors in two different types of recipient cells (mammalian cell lines and frog oocytes). The first step in the study of opioid receptors will be to determine the structure of one type of receptor by recombinant DNA techniques. A relatively new approach called the hybridization selection procedure will be used to purify the mRNA species that code for the receptor. The frog oocyte expression system will be used to assay the mRNA for its capacity to direct the synthesis of the receptor proteins. cDNA will be prepared from the purified mRNA and cloned. From the sequence of the cDNA it will be possible to determine the number of subunits in the receptor and the structure of each subunit. The next step will be to use various receptor cDNA probes to determine the number of opioid receptor genes and how similar these genes are to one another.