REGULATION OF CELL AND ORGAN SIZE IN VIVO

动物最终达到的大小取决于细胞总数和单个细胞的大小。总细胞数由细胞增殖和细胞死亡决定。 近年来，我们对这两个过程的调控的理解取得了重大进展。 相比之下，调节单个细胞大小的机制还不清楚。本提案中详细描述的实验旨在了解体内调节细胞大小的机制。 使用FLP-重组酶诱导的果蝇眼睛中的有丝分裂重组的遗传筛选用于鉴定导致细胞大小增加的突变。 迄今为止分离出的突变映射到六个不同的遗传位点。 其中一个基因座对应于gigas基因，该基因已被其他基因座表征并导致大的多倍体细胞。 gigas基因是TSC 2基因的果蝇同源物，TSC 2基因在大约70%的结节性硬化症患者中发生突变。在筛选中确定的基因座之一，洛基的表型与吉加斯的表型极其相似，并映射到TSC 1基因的位置，该基因在30%的结节性硬化症患者中突变。 另一种突变，增加细胞大小而不改变倍性，映射到PTEN基因附近，该基因在许多不同的人类癌症中发生突变。 另一个，扩大，似乎以非自主的方式行事。 另外两个尚未绘制。该提案的具体目标1描述了完成全面的遗传筛选，以确定导致细胞大小增加的突变。 突变将被分类到互补组中，基因将被定位。 具体目标2描述了一个详细的表型分析吉加斯，岩石，狼和扩大。实验将解决这些突变增加细胞大小的确切机制。 在每种情况下，将确定突变对倍性、生长和周期调节的影响，并在已知的细胞大小调节剂的背景下研究基因的作用。 具体目标3描述了用于表征剩余突变的表型以及随着筛选接近完成而将被鉴定的那些突变的表型的一般策略。 还描述了克隆基因的方法。对筛选中鉴定的突变的分析将有助于我们对体内细胞大小调节的理解。

The eventual size reached by an animal is determined both by the total number of cells as well as the size of individual cells. Total cell number is determined by cell proliferation and cell death. Recent years have witnessed substantial progress our understanding of the regulation of these two processes. In contrast, the mechanisms that regulate the size of individual cells are not well understood. The experiments detailed in this proposal are aimed at understanding the mechanisms that regulate cell size in vivo. A genetic screen using FLP-recombinase induced mitotic recombination in the Drosophila eye was used to identify mutations that resulted in increased cell size. The mutations isolated to date map to six different genetic loci. One of the loci corresponds to the gigas gene which has been characterized by others and results in large polyploid cells. The gigas gene is the Drosophila homologue of the TSC2 gene which is mutated in approximately 70 percent of patients with tuberous sclerosis. One of the loci identified in the screen, rocky has a phenotype that is extremely similar to that of gigas and maps to the location of the TSC1 gene which is mutated in 30 percent of patients with tuberous sclerosis. Another mutation, which increases cell size without altering ploidy, maps to the vicinity of PTEN gene which is mutated in a number of different human cancers. Another, expanded, appears to act in a non-autonomous manner. Two others have not yet been mapped. Specific Aim 1 of this proposal describes the completion of a comprehensive genetic screen to identify mutations that lead to an increase in cell size. Mutations will be sorted into complementation groups and the genes will be mapped. Specific Aim 2 describes a detailed phenotypic analysis of gigas, rocky, wolf and expanded. Experiments will address the precise mechanism by which these mutations increase cell size. In each case the effect of the mutation on ploidy, growth and cycle regulation will be determined and the role of the gene will be studied in the context of known regulators of cell size. Specific Aim 3 describes a general strategy for characterizing the phenotype of the remaining mutations and those that will be identified as the screen moves to completion. An approach to cloning the genes is also described. Analysis of the mutations identified in the screen will contribute to our understanding of cell size regulation in vivo.