AN IMPROVED IN VIVO DNA FOOT PRINTING BY A NON-RADIOISOTOPE DNA

随着几种生物基因组测序的完成，有必要从细胞核内细胞功能的角度来研究基因组组织。其中一个重要的信息是蛋白质结合位点在特定基因组区域的位置。我们利用非放射性同位素DNA测序仪开发了一种体内DNA足迹，直接研究染色体上DNA-蛋白质复合物的模式。为了建立该方法，我们以酿酒酵母基因组的复制起始序列为模型，得到如下结果：1. 基于紫外辐射依赖性嘧啶二聚体的形成，通过标记DNA的核苷酸，建立了从每个基因组的单拷贝序列获得体内足迹的最具重复性的方法。2. 在放射性同位素足迹中产生可探测波段的DNA量估计为0.1-0.5 fmol。这种灵敏度可以通过DNA测序仪（ALF express, Pharmacia inc.）和一种发红色的花青素染料（Cy-5）标记引物DNA获得。通过将紫外光足迹与检测系统（Cy-5标记DNA和ALF表达）相结合，我们可以在单倍体酵母细胞中检测到与传统放射性同位素足迹相似的染色体复制起点特异性蛋白质结合模式。4. 由于数据分析的性能更高，自动DNA测序仪的体内足迹比放射性同位素更有用和有利。

By completion of genome sequencing of several organisms, it will be necessary to study genome organization in terms of cellular functions in nuclei. One of the important information is locations of protein binding sites at a particular genomic region. We have developed an in vivo DNA footprinting with a non-radioisotope DNA sequencer to study directly a mode of DNA-protein complex on chromosome. To establish the method, we have taken the replication origin sequence of S.cerevisiaegenome as a model and obtained the following results. 1. The most reproducible method to obtain in vivo footprinting from a single copy sequence per genome was established by marking nucleotides of DNA based on UV irradiation dependent pyrimidine dimer formation. 2. The amount of DNA to produce a detectable band in the radiosotope footprinting was estimated as 0.1-0.5 fmol. This sensitivity could be obtained using a DNA sequencer (ALF express, Pharmacia inc.) and a red-emitting cyanine dye (Cy-5) labeled primer DNA.3. By combination of UV-photofootprinting and the detection system (Cy-5 labeled DNA and ALF express), we could detecta specific protein binding pattern at a chromosomal replication origin in haploid yeast cells similarly as that by a conventional radioisotope footprinting. 4. Due to the higher performance of data analysis, the in vivo footprinting by auto DNA sequencer is more useful and advantageous than that with radioisotope.