RegA, Chloroplast Biogenesis, and Germ-Soma Differentiation in Volvox

柯克博士试图了解一个完整的细胞分化程序是如何在基因组中编码的，以及它是如何在细胞和分子水平上执行的。正在研究的模型系统是Volvox carteri，这是一种简单的球形绿藻，在2000个最终分化的体细胞（在提供运动性后）和16个可能不朽的生殖细胞之间进行分工。regA是调节胚体分工的一个关键基因，它在体细胞中阻断生殖发育。RegA蛋白具有转录因子的特征，其许多假定的靶标已被克隆、测序并证明是编码叶绿体蛋白（“NGECPs”）的核基因。他将验证RegA在体细胞中作为NGECPs的活性抑制因子在胚体分化中发挥关键作用的假设，从而阻止叶绿体的生物发生、生长和繁殖。他将测试可诱导regA转基因对这些NGECPs在生殖腺和衣藻细胞中的转录的影响。然后使用重组RegA和抗RegA抗体来检测RegA与克隆的假定靶基因的上游调控区域的结合，并免疫沉淀（随后克隆DNA）结合RegA的染色质片段。这些结合位点的意义将通过报告基因构建来检验。与RegA相互作用的蛋白质也将通过抑制筛选和至少双杂交分析来寻找。

Dr Kirk seeks to understand how a complete program for cellular differentiation is encoded in a genome, and how it is executed at the cellular and molecular levels. The model system being studied is Volvox carteri, a simple spherical green alga with a division of labor between 2,000 terminally differentiated somatic cells that undergo programmed cell death (after having provided motility), and 16 germ cells that are potentially immortal. A key gene regulating this germ-soma division of labor is regA , which acts in somatic cells to block reproductive development. The RegA protein has features of a transcription factor, and many of its putative targets have been cloned, sequenced and shown to be nuclear genes encoding chloroplast proteins ("NGECPs"). He will test the hypothesis that RegA plays its key role in germ-soma differentiation by acting in somatic cells as an active repressor of NGECPs, thereby preventing chloroplast biogenesis, growth, and reproduction. Hewill test the effects of inducible regA transgenes on transcription of these NGECPs in both Volvox gonidia and Chlamydomonas cells. Then recombinant RegA and anti-RegA antibodies will be used to test for binding of RegA to upstream regulatory regions of its cloned putative target genes, and to immunoprecipitate(and subsequently clone DNA of) chromatin fragments that bind RegA. The significance of such binding sites will be tested with reporter constructs. Proteins interacting with RegA will also be sought via a suppresser screen and ayeast two-hybrid analysis.