Analysis of the Inverse Effect in Drosophila

拟议研究的总体目标是表征一类反式作用调控基因，这些基因在白色眼睛颜色位点上产生反向剂量反应。饱和诱变包括杂交发育不良，甲磺酸乙酯和x射线，将进行恢复转座子插入，点突变和染色体重排在反向调控有效的白色。其次，常染色体将被筛选为片段三组，改变白色的表达。以前分离的突变体在一个反向调节白色将用于确定隐性重组的组织。在眼睛中表达的其他基因将被测试，以确定这些基因是否受到对白色起作用的反向调节因子的影响。一套精致的白色启动子融合将被构建并转化到果蝇的种系中。这些将被反向调节测试，以确定所需的白色基因的序列作出反应。将克隆影响白色的P元素插入突变的逆转录调节因子，并通过DNA测序和蛋白质融合生产来分析基因产物。最后，将通过确定白色基因座调节突变体是否在超雌性中表现出剂量补偿，并通过对white- ivory（一种不响应反向调节因子的非补偿等位基因）进行逆转分析，来检查X染色体剂量补偿中反向效应的参与。建立对逆调节器的补偿和响应是否相关。了解基因表达是如何被调控的，是洞察遗传机制和发育的核心。该提案包含创新实验，这将导致增加有关果蝇基因剂量补偿和反式调控位点机制的知识。

The overall objective of the proposed research is to characterize a class of trans-acting regulatory genes that produce an inverse dosage response on the white eye color locus. A saturation mutagenesis involving hybrid dysgenesis, ethyl methanesulfonate and X-rays will be conducted to recover transposon insertions, point mutants and chromosomal rearrangements in inverse regulators effective upon white. Secondly, the autosomes will be screened for segmental trisomics that alter the expression of white. A previously isolated mutant in an inverse regulator of white will be used to determine the tissues in which the recessive recombination. Other genes expressed in the eye will be tested to determine whether these are affected by the inverse regulator that works on white. A refined set of white promoter fusions will be constructed and transformed into the Drosophila germline. These will be tested with the inverse regulator to determine the sequences of the white gene required to respond. Selected examples of inverse regulators that are P element insertion mutants affecting white will be cloned and used to analyze the gene products via DNA sequencing and protein fusion productions. Finally, the involvement of the inverse effect in X chromosomal dosage compensation will be examined by determining whether white locus regulatory mutants exhibit dosage compensation in metafemales and by conducting a reversion analysis on white- ivory, a non-compensating allele that does not respond to inverse regulators, to establish whether compensation and response to inverse regulators is correlated. An understanding of how gene expression is regulated is central to insight into genetic mechanisms and development. This proposal contains innovative experiments which should result in increased knowledge relating to mechanisms involved in gene dosage compensation and trans-acting regulatory loci in Drosophila.