Gene Amplification-Sciarid DNA Puffs

描述（由申请人提供）：Sciara DNA puff扩增的独特系统作为唾液腺巨染色体中的程序化发育事件发生，为研究再复制控制提供了机会，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，将其与发育早期每个细胞周期仅激活一次的同一位点的起源进行比较，并研究顺式作用调控元件。最近，我们开发了复制起始点（RIP）作图的方法;将该技术应用于酵母ARS 1和Sciara DNA puff 11/9A，再加上将起源识别复合物（ORC）结合位点作图扩展到真核生物，即DNA合成的起始位点与起始蛋白的结合位点相邻的病毒范例，在本授权申请中，我们提出：（1）顺式肌动蛋白元件的鉴定。我们将把我们的研究扩展到未研究的Sciara 11/9A起点的右半部分，它似乎有另一个复制起始位点。此外，我们将绘制DNA puff 11/2B的起源和ORC结合位点，与DNA puff 11/9A进行序列比较，以揭示保守的顺式作用序列。(2)顺式作用元件的功能测试。我们已经证明，ORC在体外结合到来自11/9A起源的160 bp片段中的80 bp。诱变将鉴定ORC结合所需的160 bp片段中的序列，包括在Sciara 11/9A起点的左右复制起始位点发现的14 bp共有序列的功能测试。P-元件转化将为推定的顺式作用调节元件提供体内功能测定。此外，混合和匹配的实验将给出一个功能比较的顺式作用序列在Sciara和果蝇的起源。一种新的体内试验将使用聚酰胺阻断推定的顺式作用元件，例如与Sciara 11/9A起点中的ORC结合位点直接相邻的蜕皮激素反应元件，并分析对再复制的影响。(3)起爆区的规格。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）将指示正常复制机制的蛋白质是否也存在于DNA扩增过程中，并将指示它们在起始区中的分布。可能决定IZ边界的因素将通过插入hsp 26启动子的含源克隆的P元件转化来研究，其中对结合RNA聚合酶、转录因子（热休克因子）或核小体放置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Sciara系统中获得的信息应该有助于理解后生动物用于指定起源和调节其激活的机制。

DESCRIPTION (provided by applicant): 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 cisacting 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 11/9A origin 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-acting 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 11/9A origin, 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 behelpful in understanding the mechanisms used by metazoa to specify an origin and to regulate its activation.