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，以及它如何在细胞和分子水平上执行。 被研究的模型系统是团藻（Volvox carteri），一种简单的球形绿色藻，在2,000个经历程序性细胞死亡（在提供运动性之后）的终末分化体细胞和16个潜在永生的生殖细胞之间进行分工。 调节这种生殖体分工的关键基因是regA，它在体细胞中起作用，阻止生殖发育。 RegA蛋白具有转录因子的特征，并且其许多推定的靶标已被克隆、测序并显示为编码叶绿体蛋白（“NGECP”）的核基因。 他将测试这一假设，即RegA通过在体细胞中作为NGECP的活性阻遏物，从而阻止叶绿体生物发生，生长和繁殖，在生殖体分化中发挥关键作用。 他将在团藻和衣原体细胞中检测诱导型regA转基因对这些NGECP转录的影响。 然后，重组RegA和抗RegA抗体将用于测试RegA与其克隆的推定靶基因的上游调控区的结合，并用于免疫沉淀结合RegA的染色质片段（并随后克隆其DNA）。 这些结合位点的显著性将用报告构建体进行测试。 与RegA相互作用的蛋白质也将通过抑制筛选和酵母双杂交分析来寻找。

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.