Analysis of the Prune/Killer of Prune Lethal Interaction

黑腹果蝇的异常翼盘(AWD)基因是在干扰想象盘正常发育的致命性突变的杂交异常基因筛查中被鉴定出来的。虽然纯合子为零等位基因的幼虫存活到第三龄幼虫的末期，但想象组织如成像盘、淋巴腺和大脑视觉中心的增殖严重减少。这些突变的想象盘在移植到变态幼虫体内时不能区分成虫结构。AWD是AWD基因的多肽产物，是具有核苷二磷酸激酶(NDP Kinase)活性的六聚体蛋白的亚基。修剪杀手(KPN)是一种AWD基因突变，它将Pro 97改变为丝氨酸。虽然，KPN突变纯合子只有30%的野生型NDP激酶特异性活性，但它们是活的，没有可检测到的形态表型。在野生型AWD基因背景中，X染色体修剪(Pn)基因的零突变导致眼睛颜色表型，但不影响生存能力。然而，在缺乏pn基因功能的情况下，Kpn是一种显性致死突变。几年前，对pn/kpn致死相互作用的非连锁抑制子进行了大规模的突变筛选，但未能产生任何抑制子。为了解释未能恢复抑制子的原因，假设即使在KPN突变杂合子中积累的突变蛋白水平也太高，任何潜在的抑制子突变都无法克服。因此，设计了一种改进的突变方案，以KPN转基因作为突变蛋白的来源来筛选未连锁的显性抑制基因。从这个改良的筛查中发现了三个抑制子突变。令人难以置信的是，这三个都是单个第三条染色体基因的隐性致死等位基因。该基因命名为su(KPn)，通过基因重组定位在3R近端，通过缺失定位确定了其与84C的细胞遗传学定位。本研究的目的之一是克隆SU(KPN)基因，确定其转录本的序列，鉴定其蛋白产物的定位，并检测直接蛋白：AWD与SU(KPN)和/或Pn与SU(KPn)之间的蛋白质相互作用。另一个目标是利用详细的镶嵌分析来检测su(Kpn)致死表型，以帮助阐明su(Kpn)基因产物的功能。了解抑制基因产物的同源性，将有助于了解核桃与核桃杀手之间的致死相互作用以及AWD基因的正常功能。这些实验的结果可以让我们深入了解发育信号通路。到目前为止在该系统中收集的数据表明，表型缺陷是缺乏想象中的盘增殖所需的扩散物质或存在抑制想象中的盘增殖的扩散物质的次要结果。对主要缺陷的分析有望为幼虫和成象细胞之间的信号传递提供新的见解。

The abnormal wing discs (awd) gene of Drosophila melanogaster was identified in a hybrid dysgenic screen for lethal mutations that interfere with the normal development of imaginal discs. Although larvae homozygous for a null allele survive until the end of the third larval instar, the proliferation of imaginal tissues such as, imaginal discs, lymph glands and optic centers of the brain is severely reduced. These mutant imaginal discs are not capable of differentiating adult structures when transplanted into metamorphosing larvae. AWD, the polypeptide product of the awd gene, is the subunit of a hexameric protein that has nucleoside diphosphate kinase (NDP kinase) activity. Killer of prune (Kpn) is an awd gene mutation which changes proline 97 to serine. Although, Kpn mutant homozygotes have only 30% of wild-type NDP kinase specific activity, they are viable and have no detectable morphological phenotype. In a wild-type awd gene background, null mutations in the X chromosome prune (pn) gene cause an eye color phenotype but do not affect viability. Nevertheless, in the absence of pn gene function, Kpn acts as a dominant larval lethal mutation. Several years ago, a large scale mutagensis screen for unlinked suppressors of the pn/Kpn lethal interaction, failed to yield any suppressors. To account for the failure to recover suppressors, it was hypothesized that the level of mutant protein that accumulates even in Kpn mutant heterozygotes was too great for any potential suppressor mutation to overcome. So, a modified mutagenesis protocal was designed to screen for unlinked dominant suppressors using a Kpn transgene as the source of mutant protein. Three suppressor mutations were recovered from this modified screen. Incredibly, all three are recessive lethal alleles of a single third chromosome gene. This gene was named su(Kpn) and localized by genetic recombination to proximal 3R; its cytogenetic location with 84C was determined by deletion mapping. One objective of this research is to clone the su(Kpn) gene, determine the sequence of its transcript, identify the localization of its protein product, and assay for direct protein:protein interactions between AWD and SU(KPN) and/or between PN and SU(KPN). Another objective is to examine the su(Kpn) lethal phenotype utilizing a detailed mosaic analysis to help elucidate the function of the su(Kpn) gene product. Learning the identity of the suppressor gene product will help to understand the lethal interaction between prune and Killer of prune and the normal function of the awd gene. The results of these experiments can give insight into developmental signaling pathways. The data thus far collected in this system suggests that the phenotypic defects are the secondary consequence either of the absence of a diffusible substance that is required for imaginal disc proliferation or to the presence of a diffusible substance that inhibits imaginal disc proliferation. Analysis of the primary defect promises to provide new insight into the signaling that occurs between larval and imaginal cells.