BACTERIOPHAGE CLONING SYSTEM--HIGH MOLECULAR WEIGHT DNA

在过去的两年半里，我们付出了很大的努力， 改进噬菌体P1克隆系统。 目前， 被认为是粘粒和YAC系统的合理替代品， 克隆高分子量（HMW）DNA。 我们构建了Pl小鼠 和人类图书馆，并支持了 其他人则构建大鼠和果蝇库。 在本提案中，我们 我们希望在四个方向扩展我们的研究： 1.我们想评估PI克隆过程的可靠性， 新的P1小鼠和人文库具有代表性。 为此我们 将确定PI系统是否能够克隆基因组的区域， 在粘粒载体中不可克隆或不稳定的DNA。 我们也 将开始在小鼠的两个区域中构建重叠群 Gasser博士和Nadeau博士合作， PI系统可以有效地生成远程物理地图。 2.我们希望提高Pl克隆的效率和Pl的纯度。 包装和回收克隆DNA所需的反应。 这将允许我们 在低于最佳条件下克隆HMW DNA（例如，用DNA 来源于排序的染色体），并将避免与 在粗包装提取物中的核酸酶活性， 这些提取物的功效。 3.我们希望发现PI系统如何包装小于头部大小的 (110 kb）大头部的DNA。 希望我们能利用这些信息 抑制反应。 4.我们希望继续努力开发转座子系统， 允许我们以可控的方式将任何克隆的插入片段片段化。 这 将有助于定位物理和功能决定因素， 这些插入物并将允许它们重新引入哺乳动物细胞。

During the past two and one-half years we have devoted much effort to improving the phage Pl cloning system. At the present time it is accepted as a reasonable alternative to cosmid and YAC systems for the cloning of high molecular weight (HMW) DNA. We have constructed Pl mouse and human libraries in our laboratory and have supported the efforts of others to construct rat and Drosophila libraries. In this proposal we wish to extend our studies in four directions: 1. We want to evaluate how faithful the Pl cloning process is and how representative the new Pl mouse and human libraries are. To do this we will determine whether the Pl system is able to clone regions of genomic DNA that are not clonable or are unstable in cosmid vectors. We also will initiate efforts to construct contigs in two regions of the mouse genome in collaboration with Drs. Gasser and Nadeau to determine how efficiently the Pl system can generate a long range physical map. 2. We want to increase the efficiency of Pl cloning and the purity of the reaction needed to package and recover cloned DNA. This will permit us to clone HMW DNA under less than optimal conditions (e.g. with DNA derived from sorted chromosomes) and will avoid problems associated with nuclease activity in the crude packaging extracts that compromise the efficiency of those extracts. 3. We wish to discover how the Pl system packages less than headful-sized (110 kb) DNA in large heads. Hopefully we can use that information to inhibit that reaction. 4. We want to continue efforts to develop transposon systems that will permit us to fragment any cloned insert in a controlled manner. This will be useful in localizing physical and functional determinants on those inserts and will permit their re-introduction into mammalian cells.