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

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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.