Regulation of Trypanosome DNA Replication

这项拟议的研究涉及两个相关问题：(1)调控的分子机制是什么 S期基因在布氏锥虫中的表达以及(2)特定的线粒体是如何表达的 DNA连接酶在细胞周期中的调节及其表达如何对动质粒结构和 组织？ 在动质体内，蛋白质编码基因似乎很少或没有显著的转录调节。 相反，基因表达的调节主要发生在转录后水平。在早期的工作中， 我们鉴定了几个基因在S阶段表达所需的八聚体共有序列 以及两个与八聚体序列高度特异性结合的蛋白质复合体。编码这些的基因 布鲁氏毛滴虫中存在蛋白质，以及其他尚未被研究的相关基因。我们有 成功同步化布氏毛滴虫，并将利用同步培养研究S期基因表达 并鉴定和鉴定特异性结合蛋白。这些蛋白质的亚基将用 免疫学方法和质谱学。蛋白质复合体也将被提纯和表征。 采用串联亲和纯化。将使用RNA干扰方法来敲除每个基因的表达 结合蛋白并确定它们在序列特异性结合八聚体序列中的重要性 在成绩单上。我们还将使用免疫沉淀和RT-PCR直接显示在体内的结合 这些蛋白质复合体转变为含有八聚体序列的转录本。 束枝藻DNA连接酶ka(LIG Ka)是一种不稳定的蛋白质，具有周期性表达和周期性表达的特点。 定位到动质粒。我们将研究八聚体相关序列在循环表达中的作用 通过单独的八聚体相关序列的点突变获得LIG ka及其邻近的LIG k？基因 并结合在一起。这些研究将解决每个基因转录本的循环对此的可能影响 另一个人的。我们还将调查LIG ka在调节修复仍然存在的缺口中的可能作用 小圈复制起源于它们关闭之前，作为网络分裂的前奏。KDNA的结构 在表达LIG ka？的布鲁氏毛滴虫细胞中将确定网络和网络微环的数量。在不变的情况下 和整个细胞周期的调控水平。自从DNA连接酶被发现与DNA相互作用以来 缺口修复反应中的聚合酶我们将确定特定的线粒体DNA聚合酶 与LIG ka一起发挥作用？并将检测连接酶-聚合酶复合体的能力 修复复制原点处的模型微圆间隙。

The proposed research addresses two related questions: (1) What are the molecular mechanisms that regulate S-phase gene expression in Trypanosoma brucei? and (2) How is the expression of a specific mitochondrial DNA ligase regulated during the cell cycle and how does its expression contribute to kinetoplast structure and division? There appears to be little or no significant transcriptional regulation of protein coding genes in kinetoplastids. Rather, regulation of gene expression occurs largely at a post-transcriptional level. In earlier work with C. fasciculata we identified an octamer consensus sequence required for S-phase expression of several genes and also two protein complexes that bind to the octamer sequence with high specificity. Genes encoding these proteins are present in T. brucei as well as additional related genes that have not been investigated. We have successfully synchronized T. brucei and will use synchronized cultures to investigate S-phase gene expression and to identify and characterize the specific binding proteins. Subunits of these proteins will be identified using immunological methods and mass spectrometry. Protein complexes will also be purified and characterized using tandem affinity purification. RNA interference methods will be used to knock down expression of each of the binding proteins and to determine their importance in sequence-specific binding to the octamer sequence in transcripts. We will also use immunoprecipitation and RT-PCR to directly demonstrate the binding in vivo of these protein complexes to transcripts containing the octamer sequences. The C. fasciculata DNA ligase ka (LIG ka) is an unstable protein that shows cyclic expression and cyclic localization to the kinetoplast. We will examine the roles of octamer-related sequences in the cyclic expression of LIG ka and the adjacent LIG k¿ gene through point mutation of individual octamer-related sequences alone and in combination. These studies will address the possible effect of the cycling of each gene transcript on that of the other. We will also investigate the possible role of LIG ka in the regulated repair of gaps that remain at minicircle replication origins prior to their closure as a prelude to network division. The structure of the kDNA networks and of the network minicircles will be determined in T. brucei cells expressing LIG ka? at a constant and regulated level throughout the cell cycle. Since DNA ligases have been found to interact with DNA polymerases in gap repair reactions we will identify the specific mitochondrial DNA polymerase that might function together with LIG ka? in gap repair and will examine the ability of the ligase-polymerase complex to repair a model minicircle gap at the replication origin.