CELL REGULATION: BIOCHEMICALLY ISOLATED DNA SEGMENTS

将为脉冲场凝胶开发新的仪器 电泳，使用轮廓夹紧均匀电 场，用于分析和制备分离非常大 DNA分子（多达1000万个碱基对）。 二维的- 一种独特分离大DNA分子的装置， 还规划拓扑约束。 这是通过 通过E. coliRecA反应。 由D-引起的拓扑约束 循环将在所提出的二维中引起独特的移动性 脉冲场凝胶仪 这将允许特定人类的基因 需要隔离的疾病，包括囊性纤维化。 技术 高分子量DNA的独特切割将是 开发 这利用了将EDTA分子拴系到 小的单链DNA 与亚铁离子络合， 在氧的存在下，双链体DNA可以被切割， DNA探针通过一个连接到一个大的DNA分子上的超分子链， 或由E. coliRecA反应。 它 将允许在人类的身体 和遗传RFLP图谱。 将开发一种新的酵母载体， 这些非常大的DNA分子的特异性克隆。 这 载体将允许外源DNA序列，包括人类， 作为人工染色体保存在酵母细胞中。 是 预计分子大小可达100万个碱基对， 直接在酵母中分离。 将开发一种快速的方法 绘制大片段DNA中的单碱基对变化。 这 技术依赖于分支迁移的热力学 允许分支的DNA分子驻留在错配的 在DNA中的定位和单链在分支点的切割 核酸酶 此外，还将开发一种新的载体， 克隆编码DNA蛋白质的基因。 它将用于 分离与酵母着丝粒结合的DNA结合蛋白 以及参与细胞周期和DNA损伤的序列 酵母核糖核苷酸还原酶的调节。 DNA序列 已经被分离出来，保护那些序列参与 维持酵母染色体，包括着丝粒和 从转录。 一种灵敏的定量菌落颜色 已经开发了用于直接测量 这种保护序列的有效性，从而使其 详细调查。

New instrumentation will be developed for pulse field gel electrophoresis, using a contour clamped homogeneous electric field, for the analytical and preparative separation of very large DNA molecules (up to 10 million base pairs). A two dimensional- apparatus for the unique separation of large DNA molecules with a topological constraint is also planned. This is accomplished by tagging a specific DNA molecule by a D-loop promoted by the E. coli RecA reaction. The topological constraint caused by the D- loop will cause a unique mobility in the proposed two dimensional pulse field gel apparatus. This will allow genes for specific human diseases to be isolated, including cystic fibrosis. Techniques for the unique cleavage of high molecular weight DNA will be developed. This makes use of tethering EDTA molecules to a small single-stranded DNA. Complexed with ferrous ion and in the presence of oxygen, duplex DNA can be cleaved when the DNA probe is bound to a large DNA molecule through a paranemic or plectonemic joint catalyzed by the E. coli RecA reaction. It will allow a correlation to be drawn between the human physical and genetic RFLP map. A new yeast vector will be developed for the specific cloning of these very large DNA molecules. This vector will allow a foreign DNA sequence, including human, to be maintained in a yeast cell as an artificial chromosome. It is anticipated that molecules as large as 1 million base pairs can be directly isolated in yeast. A method will be developed for rapidly mapping single base pair changes in large stretches of DNA. This technique relies on the thermodynamics of branch migration allowing a branched DNA molecule to reside at a mismatched location in DNA and cleavage at the branch point by single strand nucleases. Also, a new vector will be developed for the direct cloning of a gene coding for a DNA protein. It will be used to isolate the DNA binding proteins that bind to a yeast centromere and the sequences involved in the cell cycle and DNA damage regulation of yeast ribonucleotide reductase. DNA sequences have been isolated that protect those sequences involved in the maintenance of yeast chromosomes, including centromeres and ARS from transcription. A sensitive, quantitative colony color assay has been developed for the direct measurement of the effectiveness of this protective sequence, thus allowing its detailed investigation.