Gene Amplification-Sciarid DNA Puffs

超过空格_ROMIDED。在唾液腺巨大染色体中发生的程序性发育事件--Sciara DNA泡芙扩增的独特系统，为我们提供了一个研究再复制控制的机会，这是我们的长期目标。为了实现这一目标，我们必须首先确定扩增的起源，将其与在发育早期每个细胞周期仅激活一次的同一基因座的起源进行比较，并研究顺式作用的调控元件。最近，我们发展了复制起始点(RIP)定位的方法；将该技术应用于酵母ARS1和Sciara DNA Puff 11/9A，再加上定位延伸到真核生物的起源识别复合体(ORC)结合部位，以及DNA合成的起始点与启动蛋白的结合部位相邻的病毒范例，在这项拨款申请中，我们建议：(1)鉴定顺Actinq元件。我们将把我们的研究扩展到Sciara 11/9A起源的未被研究的右半部分，它似乎有另一个复制起点。此外，我们还将定位DNA puff 11/2B的起始点和ORC结合位点，以便与DNA puff 11/9A进行序列比较，以揭示保守的顺式作用序列。(2)顺式肌动蛋白元件的功能测试。我们已经证明ORC在体外与来自11/9A的160bp的片段中的80bp结合。诱变将鉴定ORC结合所需的160bp片段中的序列，包括对在Sciara 11/9A起始点的左侧和右侧复制起始点发现的14BP共识序列进行功能测试。P元件的转化将为顺式作用调节元件提供体内功能分析。此外，混合匹配实验将给出西亚拉人和果蝇起源的顺式作用序列的功能比较。一种新的体内试验将使用聚酰胺来阻断假定的顺式作用元件，例如直接靠近Sciara 11/9A起源中ORC结合部位的蜕皮激素反应元件，并分析其对重新复制的影响。(3)起爆区的规定。我们发现，Sciara 11/9A复制起始区(IZ)在发育过程中大小发生变化。染色质免疫沉淀(ChIP)将指示在DNA扩增过程中是否也存在正常复制机制的蛋白质，并将指示它们在起始区的分布。决定IZ边界的因素将通过插入hsp26启动子的含源克隆的P元件转换来研究，其中对结合RNA聚合酶、转录因子(热休克因子)或核小体定位至关重要的序列将依次突变，以分析它们对IZ边界的影响。从独特的SCHARA系统获得的信息应该有助于理解后生动物用来确定来源和调节其激活的机制。表演网站========================================Section End===========================================

EXCEED THE SPACE _ROMIDED. The unique system of Sciara DNA puff amplification which occurs as a programmed developmental event in salivary gland giant chromosomes, offers an opportunity to investigate re-replication control, which is our long term goal. To approach this goal, we must first identify the origin of amplification, compare it the origin of the same locus that is activated just once per cell-cycle earlier in development, and study the cis- acting regulatory elements. Recently we developed the method of Replication Initiation Point (RIP) mapping; application of this technique to yeast ARS1 and to Sciara DNA puff 11/9A,coupled with mapping the Origin Recognition Complex (ORC) binding site extended to eukaryotes the viral paradigm that the start site for DNA synthesis is adjacent to the binding site for the initiator protein, in this grant application we propose: (1) Identification of Cis-actinq Elements. We will extend our research to the unstudied right half of the Sciara 11/9Aorigin that appears to have another replication start site. In addition, we will map the origin and ORC binding site in DNA puff 11/2B,for sequence comparison with DNA puff 11/9Ato reveal conserved cis-acting sequences. (2) Functional Tests of Cis-actinq Elements. We have shown that ORC binds in vitro to 80 bp in a 160 bp fragment from the 11/9Aorigin. Mutagenesis will identify sequences in the 160 bp fragment required for ORC binding, including a functional test of a 14 bp consensus sequence found both at the left and right replication start sites in the Sciara 11/9A origin. P-element transformation will provide an in vivo functional assay for putative cis-acting regulatory elements. Furthermore, mix-and-match experiments will give a functional comparison of cis-acting sequences in Sciara and Drosophila origins. A novel in vivo assay will use polyamides to block putative cis-acting elements, such as the ecdysone response element directly adjacent to the ORC binding site in the Sciara 11/9Aorigin, with analysis of the effect on re-replication. (3) Specification of the Initiation Zone. We have found that the Sciara 11/9A replication initiation zone (IZ) changes size during development. Chromatin ImmunoPrecipitation (CHIP) will indicate if proteins of the normal replication machinery are also present during DNA amplification, and will indicate their distribution in the initiation zone. Factors that may determine the IZ boundary will be studied by P-element transformation of an origin-containing clone with an insertion of the hsp26 promoter, where sequences important for binding RNA polymerase, a transcription factor (heat shock factor), or for nucleosome placement will be mutated in turn to analyze their effect on the IZ boundary. Information gained from the unique Sciara system should be helpful in understanding the mechanisms used by metazoa to specify an origin and to regulate its activation. PERFORMANCE SITE ========================================Section End===========================================