Cloning of Large Segments from the Centromere Region of Human Chromosome

点击翻译按钮获取中文摘要

To examine the structure and function of the centromere of human chromosome, we have been trying to isolate large DNA fragments from the centromere region. On the other hand, another approach to investigate DNA-protein interaction revealed the specific recognition of a centromeric alphoid DNA by a centromere antigenie protein.1. Cloning of the centromere region. Alphoid DNA family, a highly repeated DNA sequence, is located at the centromere region of human chromosome. The array of 170 bp long alphoid repeats seems to extend more than several hundreds kb. Digestion of chromosomal DNA with the restriction enzyme BglII yielded about 200 kb long alphoid DNA fragments, which can be separated from smaller DNA fragments derived from other regions of chromosome by centrifugation in a sucrose density gradient. To clone long alphoid DNA and then to examine its biological activity in human cultured cell, a derivative of pYAC plasmid of S. cerevisiae was constructed by inserting the hygromycine-resistant gene and an ARS (autonomously replicating sequence). Yeast transformants of pYAC library are under investigation for the presence of long alphoid repeats by using pulse field gel electrophoresis and southern hybridization.2. DNA-Protein interaction in the centromere region. We have shown one of the human centromere antigenic proteins that are detected by antisera of autoimmune disease patients specifically interacts with alphoid DNA. Immunoprecipitation using centromere antibody or gel-shift assay revealed that the 80 kD CENP-B protein binds to 17 bp long DNA motif present in alphoid repeats. Analysis of DNA sequence of the cloned alphoid sequence shows that the 17 bp motif (CENP-B box) is present in all the chromosome except for Y- chromosome. Therefore, it is likely that the interaction between CENP-B protein and the CENP-B box participates in the function of the centromere in the segregation of chromosome.