MOLECULAR BASIS OF SUPPRESSION IN DROSOPHILA

描述：维克多·科尔斯博士的长期目标是获得一些 对高级染色质组织在细胞周期中的作用的认识 真核生物中基因表达的调控。为此，他提议 继续他对吉普赛逆转座子诱导的性质的研究 突变及其抑制。其中一些突变是由 对开放阅读框架的直接改变；兴趣的变化 由于吉普赛人被插入到监管机构 该基因的区域。在这些情况下，元素存在于 启动子和一些转录增强子阻止 这些增强剂的功能。使用突变，首先是黄色和 后来，在果蝇的分叉基因中，科尔塞斯博士确定 那个吉普赛人含有12个毛翼抑制因子的结合位点 蛋白质和这种蛋白质结合形成的结构是一种 一类称为绝缘体的染色质元素的例子。绝缘子 防止活跃或抑制的染色质的影响扩大 在他们的位置上。正如预期的那样，基因的功能缺失突变 SU(HW)逆转了吉普赛插入的表型效应。科尔斯博士 还确定了su(HW)蛋白有12个锌指， 介导其与DNA和两个酸性和一个亮氨酸拉链区域的结合 这是它抑制基因表达所必需的。其他人则有 发现了MDG4的修饰性基因，其突变会影响 吉普赛人诱发突变的表型。双变异体的遗传分析 在考克斯的实验室里完成。表明MOD(MDG4)蛋白是 吉普赛效应在增强子上的方向性所必需的 功能：在缺乏这种蛋白质的情况下，上游和下游的增强剂 吉普赛插入点的下游会受到影响。科尔斯博士 相信他已经克隆了mod(MDG4)基因，尽管这还没有 已经被变种人营救证实。为了确定哪些额外的 蛋白质参与了su(HW)和mod(MDG4)的相互作用，Dr。 CORCES已经筛选出MOD的显性抑制因子(MDG4)，并已经 确定了3个他称之为SU(Mod)的基因；这些蛋白质似乎 允许SU(HW)在没有方向性的情况下形成绝缘体 MOD(MDG4)基因产物。其他人已经发现，在一种压力下 携带女性不育突变OVO[D]，插入吉普赛人导致 女性生育能力的频繁恢复。在这个品系里，吉普赛人 生殖系中的易位是由母系效应突变引起的 叫做弗拉门戈。通过与这些工作人员的合作，科尔塞斯博士 确定弗拉门戈雌性的卵巢有一种新的吉普赛人 编码所需类型的包膜蛋白的转录本 在其他逆转录病毒中形成具有感染性的颗粒。他已经进行了 一些分馏、EM和进料实验，其结果表明 火龙雌虫提取物中含有感染性吉普赛颗粒。 在目前的应用中，科尔塞斯博士专注于这一机制 SU(HW)蛋白形成染色质边界的基础。 首先，他建议继续分子遗传特征 包括MOD(MDG4)的三种蛋白的相互作用，它 编码su(HW)蛋白及其DNA结合部位。下一步他会 表征参与形成和形成的其他组件 SU(HW)绝缘子的功能。他将从每一个基因中克隆一个基因 调节基因突变效应的两类突变 SU(HW)蛋白：三个e(Y)基因之一，其突变增强 Y[2]等位基因及其三个SU(Mod)基因之一的表型 突变逆转了MOD(MDG4)突变的影响。一旦这些基因 一旦被克隆，他将决定编码蛋白的能力 与吉普赛DNA以及su(HW)和mod(MDG4)蛋白相互作用。 最后，他建议研究染色质结构的变化 由于SU(HW)绝缘体的形成而产生的 对其对上游方向干扰的影响负责 转录增强剂。为此，他将分析 在核小体的定位和在 特定转录因子与调控序列的结合 两个基因中专门为此目的组装的镶嵌基因。

DESCRIPTION: Dr. Victor Corces' long-term goal is to gain some understanding of the role of higher order chromatin organization in the control of gene expression in eukaryotes. To this end he proposes to continue his studies on the nature of gypsy retrotransposon-induced mutations and their suppression. Some of these mutations are caused by a direct alteration of the open reading frame; the mutations of interest to Dr. Corces result from the insertion of gypsy in the regulatory region of the gene. In these cases, the presence of the element between the promoter and some of the transcriptional enhancers prevents the function of these enhancers. Using mutations, first in the yellow and later also in the forked genes of Drosophila, Dr. Corces has determined that gypsy contains 12 binding sites for the suppressor of Hairy-wing protein and that binding of this protein creates a structure that is an example of a class of chromatin elements called insulators. Insulators prevent the influence of active or repressed chromatin from extending across their location. As expected, loss-of-function mutations in the su(Hw) reverse the phenotypic effects of gypsy insertions. Dr. Corces has also determined that the su(Hw) protein has twelve zinc fingers that mediate its binding to DNA and two acidic and a leucine zipper regions that are necessary for its repression of gene expression. Others had discovered a gene, modifier of mdg4, whose mutation affects the phenotype of gypsy-induced mutations. Genetic analysis of double mutants performed in Corces' lab. indicates that the mod(mdg4) protein is necessary for the directionality of the gypsy effect on enhancer function: in absence of this protein, enhancers both upstream and downstream of the point of gypsy insertion are affected. Dr. Corces believes that he has cloned the mod(mdg4) gene although this has not been confirmed by mutant rescue. In order to determine what additional proteins are involved in the su(Hw) and mod(mdg4) interaction, Dr. Corces has screened for dominant suppressors of mod(mdg4) and has identified 3 genes that he has called Su(mod); the proteins appear to allow su(Hw) to form an insulator without directionality in the absence of the mod(mdg4) gene product. Others had discovered that, in a strain carrying the female-sterile mutation ovo[D], insertion of gypsy caused the frequent restoration of female fertility. In this strain, gypsy transposition in the germ line is caused by a maternal-effect mutation called flamenco. In collaboration with these workers, Dr. Corces has determined that the ovaries of flamenco females have a novel gypsy transcript that encodes an envelope protein of the type that is needed in other retroviruses to form infectious particles. He has carried out some fractionation, EM and feeding experiments whose results indicate the presence of infective gypsy particles in extracts of flam females. In the current application, Dr. Corces concentrates on the mechanism underlying the formation of chromatin boundaries by the su(Hw) protein. To begin, he proposes to continue the molecular genetic characterization of mod(mdg4) including the interaction of the three proteins that it encodes with the su(Hw) protein and its DNA binding site. Next he will characterize additional components that mediate the formation and function of the su(Hw) insulator. He will clone one gene from each of the two classes of mutations that modulate the mutagenic effect of the su(Hw) protein: one of the three e(y) genes whose mutations enhance the phenotype of the y[2] allele and one of the three Su(mod) genes whose mutations reverse the effect of mod(mdg4) mutations. Once these genes are cloned, he will determine the ability of the encoded proteins to interact with gypsy DNA and with the su(Hw) and mod(mdg4) proteins. Finally, he proposes to study the changes in chromatin structure that result from the formation of the su(Hw) insulator and that may be responsible for its directional interference with the effect of upstream enhancers on transcription. To this end, he will analyze changes in hypersensitive sites, in the positioning of nucleosomes and in the binding of specific transcription factors to the regulatory sequences of two genes mosaic genes especially assembled for this purpose.