CHROMOSOME STRUCTURE DURING DROSOPHILA DEVELOPMENT

描述：本申请建议继续和扩展 异染色质和真异染色质边界的研究 开始是艾伦·斯普拉德林实验室的博士后研究员。 的 研究人员正在使用Dp（1;f）1187微型染色体系统， 在过去的几年里，斯普拉特林实验室。 这个1.3Mb的微型染色体 显示出典型高等真核生物的所有特征 染色体，但它提供了不寻常的实验机会。 在工作 作为一名博士后研究员，PI发现， 常染色质-异染色质边界具有不寻常的特征， 应用程序，他描述了他的计划，以进一步表征这些 序列和异染色质。 该提案包含三个具体目标。 首先，PI将 继续描述异染色质限制性片段 Dp 1187在不同组织中的表达 在以前的实验中， PI发现，例如，唾液 腺体和卵巢。 PI将进行映射实验，以进一步 描述这些染色体的结构和限制 片段在卵巢DNA的实验中，他将专注于了解 Dp 1187片段在Southern杂交中表达不足的两个原因是 污点。 PI发现这些片段不会转移 典型的效率，以及有缩短的Dp 1187分子 在这个样本中。 PI还将检查唾液腺Dp 1187。 在 该组织中存在Dp 1187序列的真实表达， 这不是因为转移问题。 分子的性质 引起的代表性不足将以2D为特征 杂交分析 PI还建议检查Dp 1187， 二倍体细胞（胚胎和成虫盘/脑）。 PI已经 有证据表明，在胚胎DNA中，Dp 1187序列的代表性不足， 由于转移不良，DNA印迹中的Dp 1187片段 在胚胎DNA中发现了增加的大小。 他一直在开发 从单倍体精子中分离DNA的技术， 我们也检测了Dp 1187在这些细胞中的结构。 第二个具体目标是检查结果的一般性， dp 1187通过检查其他真异染色质边界。 其中一 他将研究的是sc 8倒位， 染色体为Dp 1187。 在其他实验中，他将研究欧盟- 由于插入P元素而产生的异色边界 变成常染色体异染色质。 第三个具体目标是确定和描述物理 Dp 1187异染色质导致选择性转移的性质 抑制作用 他已经在溶液中分离出了抗转移DNA 他会用不同的方法来处理这些DNA，比如用碱， 变性，然后以各种方式检查效果，例如 通过检测其在蔗糖梯度中的沉降， 构象

DESCRIPTION: This application proposes to continue and extend the research on heterochromatin and the eu-heterochromatin boundary that he started as a Postdoctoral Fellow in Allan Spradling's laboratory. The investigator is using the Dp(1;f)1187 minichromosome system developed in the Spradling lab over the last few years. This 1.3 Mb minichromosome displays all of the characteristics of a typical higher eukaryotic chromosome, but it affords unusual experimental opportunities. In work done as a postdoctoral fellow the PI found that DNA sequences at the euchromatic-heterochromatic boundary have unusual features and in this application he describes his plans to further characterize these sequences and heterochromatin in general. The proposal contains three specific aims. In the first the PI will continue his characterization of heterochromatic restriction fragments of Dp1187 in a number of different tissues. In previous experiments the PI found that there were differences between, for example, salivary gland and ovary. The PI will carry out mapping experiments to further characterize the structure of these chromosomes and restriction fragments. In the experiments on ovary DNA he will focus on understanding the two reasons that Dp1187 fragments are under represented on Southern blots. The PI has found that these fragments do not transfer with typical efficiency and as well that there are shortened Dp1187 molecules in this sample. The PI will also examine salivary gland Dp1187. In this tissue there is a true under representation of Dp1187 sequences that is it is not due to transfer problems. The nature of the molecules that give rise to the under representation will be characterized by 2D hybridization analysis. The PI also proposes to examine Dp1187 in diploid cells (in embryos and in imaginal discs/brains). The PI already has evidence that in embryo DNA Dp1187 sequences are under represented on Southern blots due to poor transfer and that Dp1187 fragments of increased size are found in embryo DNA. He has been developing techniques for the isolation of DNA from haploid sperm and he will examine the structure of Dp1187 in these cells as well. The second specific aim is to examine the generality of the results on Dp1187 by examining other eu-heterochromatin boundaries. One of those that he will examine is the sc8 inversion which is the parental chromosome for Dp1187. In other experiments he will examine eu- heterochromatic boundaries that result from the insertion of a P element into autosomal heterochromatin. The third specific aim is to identify and characterize the physical property of Dp1187 heterochromatin that leads to selective transfer inhibition. He has already isolated transfer resistant DNA in solution and he will treat this DNA in a variety of ways, such as alkaline denaturation and then examine the effects in a variety of ways such as by examining its sedimentation in sucrose gradients to look for altered conformation.