RUI: Trans-Acting Factors in Large-Scale Genome Rearrangement

Coyne9806573这项建议的重点是了解在纤毛原生动物嗜热四膜虫中发现的基因组重组过程，这是正常发育过程的一部分。为了协调和进行这种基因组重排，必须需要大量的反式作用因子，但到目前为止，只鉴定了两个，命名为Pdd1p和Pdd2p。Pdd1p属于染色质蛋白家族，通常与浓缩的染色质有关。它被发现与基因组DNA在加工时被消除的部分密切相关，这表明它在DNA消除机制中起直接作用。亲本、体细胞PDD1基因的中断会导致DNA消除和染色体断裂的缺陷，以及无法完成成功的接合。异构体不能分裂，最终会死亡。这种蛋白质的活性将通过在四膜虫的种系和体细胞核中破坏编码它的基因，以及通过在该基因中产生定点突变来研究。这些突变将通过基因替换技术重新引入四膜虫基因组，并分析它们对特定DNA重排、核分化事件的进展以及Pdd1的适当核靶向的影响。基因组的稳定性通常是发育和遗传的标志。一些众所周知的例外包括脊椎动物免疫球蛋白基因的重排和酵母交配型基因座。然而，有趣的是，作为正常发育计划的一部分，许多生物都经历了大规模的基因组重组。拟议的实验将有助于确定这种DNA重排所涉及的机制。他们还将阐明染色质结构形成和基因组重排之间的联系。这些实验特别适合培养本科生对实验生物学研究的严谨和兴奋。

Coyne9806573The focus of this proposal is on understanding the process of genome reorganization found in the ciliated protozoan, Tetrahymena thermophila, that occurs as part of the normal developmental process. To coordinate and carry out this genome rearrangement, a large number of trans-acting factors must be required, but thus far, only two, named Pdd1p and Pdd2p have been identified. Pdd1p, the more well characterized of the two, belongs to the chromo family of proteins, which are usually associated with condensed chromatin. It is found in close association with that fraction of genomic DNA that is eliminated, at the time of its processing, suggesting a direct role in the DNA elimination mechanism. Disruption of the parental, somatic PDD1 gene causes a defect in DNA elimination and chromosome breakage and a failure to complete successful conjugation. Exconjugants are unable to divide and eventually die. The activities of this protein will be studied by disrupting the gene encoding it in both the germline and somatic nuclei of Tetrahymena, as well as by generating site-directed mutations in the gene. These mutations will be reintroduced into the Tetrahymena genome by gene replacement technology and analyzed for their effects on specific DNA rearrangements, the progression of nuclear differentiation events, and proper nuclear targeting of Pdd1p.Stability of the genome is normally a hallmark of development and inheritance. Some well-known exceptions include the rearrangement of vertebrate immunoglobulin genes and the yeast mating type loci. Intriguingly, however, many organisms undergo massive genome reorganization as part of their normal developmental program. The experiments proposed will help define the mechanisms involved in such DNA rearrangements. They will also clarify the connection between chromatin structure formation and genome rearrangement. The experiments are particularly suited to the training of undergraduate students in the rigor and excitement of experimental biological research.