REGULATION OF EXPRESSION OF OPIOID PEPTIDES & THEIR REEP

阿片肽存在于神经和大脑的许多不同部位。 在内分泌系统中，它们调节不同类型的反应。全 这一大家族多肽(超过15个)的数量已经被证明是 来自3种不同的前体，称为前阿片黑素皮质素， 前脑啡肽和前强啡肽。阿片肽结合的受体 在性质上也是多样的。它们可分为三大类 根据它们所结合的阿片类药物的差异而确定的类型。每种类型的受体 阿片肽在大脑中的分布略有不同 可能会调节不同类型的反应。 这项研究的主要目的是加深我们对 阿片肽和阿片样物质表达的调控机制 感受器。这一目标将通过多层次的方法实现。 采用分子生物学、免疫学、蛋白质化学等方法。 阿片肽的合成位置将通过in在大脑中进行定位 使用针对每种阿片类药物的DNA探针的原位杂交技术 前身。阿片类药物基因中的调控位点将由 基因转移技术。要做到这一点，就需要研究 阿片类基因的特异性改变(体外定位 突变)对转录活性的影响以及对 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)。 研究阿片受体的第一步将是确定 用重组DNA技术研究一种受体的结构。一个 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信使核糖核酸。青蛙 卵母细胞表达系统将被用来检测mRNA的能力 指导受体蛋白的合成。将从以下来源制备cDNA 纯化后的mRNAs进行克隆。从cDNA的序列来看，它将是 有可能确定受体中的亚基数量和 每个亚基的结构。下一步将使用各种受体 用于确定阿片受体基因数量和方式的cDNA探针 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.