REGULATION OF CELL AND ORGAN SIZE IN VIVO

动物最终达到的大小既取决于细胞的总数，也取决于单个细胞的大小。细胞总数由细胞增殖和细胞死亡决定。近年来，我们对这两个过程调控的理解取得了实质性进展。相比之下，调节单个细胞大小的机制尚不清楚。在本建议中详细的实验旨在了解调节体内细胞大小的机制。使用flp -重组酶诱导果蝇眼有丝分裂重组的基因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与六个不同的基因位点相对应。其中一个位点对应于gigas基因，该基因已被其他基因所表征，并导致大型多倍体细胞。gigas基因是TSC2基因的果蝇同源物，TSC2基因在大约70%的结节性硬化症患者中发生突变。在屏幕上发现的一个基因座，rocky的表型与gigas非常相似，并且定位于TSC1基因的位置，该基因在30%的结节性硬化症患者中发生突变。另一种突变，在不改变倍性的情况下增加细胞大小，与PTEN基因的附近相对应，PTEN基因在许多不同的人类癌症中发生突变。另一个，扩大后，似乎以一种非自主的方式行动。另外两个尚未绘制地图。本提案的具体目标1描述了一个全面的基因筛选的完成，以确定导致细胞大小增加的突变。突变将被分类到互补组，基因将被绘制出来。具体目标2描述了详细的表型分析gigas，岩，狼和扩大。实验将解决这些突变增加细胞大小的精确机制。在每种情况下，突变对倍性、生长和周期调节的影响将被确定，基因的作用将在已知的细胞大小调节的背景下进行研究。特异性目标3描述了描述剩余突变和那些将在筛选完成时被识别的突变表型的一般策略。还描述了一种克隆基因的方法。分析筛选中发现的突变将有助于我们了解体内细胞大小调节。

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.