QTL AND MICROARRAY MAPPING LEAD SENSITIVITY GENES

描述(申请人提供)：人类活动导致了许多有毒物质在环境中的分布，其中包括遍布我们生物圈的重金属。除了铅对人类的急性毒性影响外(即在含铅涂料或受污染的饮用水中)，长期接触对所有生物体的发育也有更多的潜在影响。暴露在低水平铅中的儿童会改变发育过程，这些儿童会出现多动、感觉功能改变和认知能力(智商)改变等症状。果蝇是一种很有前途的模式生物，可以用来研究发育过程中铅暴露的影响，因为(1)对它的遗传学有深入的了解，而且很容易操纵它的基因组；(2)有对极低剂量铅的微小影响敏感的行为和形态分析。据认为，铅暴露的敏感性存在很大的变异性，人类和果蝇细胞都被认为在暴露于铅时诱导“保护基因”的表达。这一提议的假设是，人们可以利用数量性状基因座(QTL)定位技术，结合微阵列和复杂的遗传分析，识别使有机体对铅毒性的行为和发育影响产生抵抗力的一些“保护基因”。为了验证这一假说，目标1是通过将90个重组近交系果蝇品系的幼虫暴露于环境相关剂量的铅，并分析受铅影响的几个行为和突触连接方面，如幼虫运动、成虫学习、成虫运动、成虫运动和乙醇蒸气暴露，以及幼虫神经肌肉连接的结构，来定位对铅敏感的QTL。目的2是利用目标1中确定的在铅诱导的行为变化方面表现出最大差异的重组近交系的整个幼虫和成虫头部的标记mRNA进行DNA微阵列分析。目的3是通过条件性过度表达或通过现有的突变和RNA干扰技术下调在目标1和2中进行的分析中重叠的基因，并确定铅对这些基因改变的果蝇的认知、运动和突触功能的影响。这些研究的结果将确定在人类铅暴露期间发生变化的最重要基因的候选基因，并很可能导致生物检测或治疗重金属暴露在NOAEL值或LOAEL值或以下。

DESCRIPTION (provided by applicant): Human activity has resulted in the environmental distribution of many toxic substances, among them the heavy metals that are spread throughout our biosphere. In addition to the acute toxic effects to humans exposed to lead (i.e. in lead paint or in contaminated drinking water) there are more insidious effects of chronic exposure on the development of all organisms. Children exposed to low levels of lead have altered developmental processes, and these children develop symptoms such as hyperactivity, changes in sensory function, and changes in cognitive abilities ("IQ"). Drosophila is a promising model organism to study the effects of lead exposure during development because of (1) the sophisticated understanding of its genetics, and the ease of manipulating its genome; (2) the availability of behavioral and morphological assays sensitive to small effects of very low doses of lead. There is thought to be a great deal of variability in the sensitivity of lead exposure, and both human and Drosophila cells are thought to induce expression of "protective genes" upon exposure to lead. The hypothesis for this proposal is that one can identify some of the "protective genes" that make an organism resistant to the behavioral and developmental effects of lead toxicity using quantitative trait loci (QTL) mapping techniques combined with microarray and sophisticated genetic analyses. To test this hypothesis, Aim 1 is to map lead sensitive QTL by exposing larvae of 90 recombinant inbred Drosophila strains to environmentally relevant doses of lead, and assaying several behavioral and synaptic connection aspects that are affected by lead - such as larval locomotion, adult learning, adult locomotion, adult locomotion combined with ethanol vapor exposure, and the structure of the larval neuromuscular junction. Aim 2 is to perform DNA microarray analyses with labeled mRNA from whole larvae and adult heads from the recombinant inbred lines that show the greatest differences in lead-induced behavioral alterations, as determined in Aim 1. Aim 3 is to up-regulate, by conditional over-expression, or down-regulate, by using existing mutations and RNA interference techniques, the genes that overlap in the assays performed in Aims 1 and 2, and to determine the effects of lead on cognition, locomotion and synaptic function of these genetically altered flies. Results of these studies will identify candidates for the most important genes that are altered during lead exposure in humans, and could well lead to bioassays or treatments for heavy metal exposure at or below NOAEL and LOAEL values.