Analysis of molecular structure of nucleosomes consisting of repetitive DNA sequences and their function in chromatin

我们研究了单核细胞体重建的DNA片段含有（GGA/TCC）n重复的DNA-DNA复合物的形成和检查的功能方面的GGA-重复分离的基因组DNA片段含有GGA-重复，并通过分析它们的染色体定位。主要研究结果如下：1.采用三重亲和捕获法从小鼠基因组中分离到19个含有（GGA /TCC）n重复序列的DNA片段.其中19个克隆定位于13条染色体的不同位置.几乎一半（9/19）的分离的DNA片段含有（GGA/TCC）n重复富集异染色质组分，其余的均匀分布于异染色质和常染色质组分。所检测的两个克隆均未富集常染色质组分。凝胶迁移率变动分析和DNase I保护图谱的结果表明，含有GGA重复序列的基因组DNA片段以序列依赖的方式形成DNA- DNA复合物.对含有不同重复数的GGA重复序列或在GGA重复序列的一个或两个位点上携带不同侧翼序列的质粒DNA片段重组的单核细胞的DNA-DNA复合物形成的分析结果表明，通过GGA重复序列形成复合物是GGA重复序列和GGA重复序列两个位点上同源侧翼序列所必需的。

We investigated the ability of mononucleosomes reconstituted on DNA fragments containing (GGA/TCC)n repeats for DNA-DNA complex formation and examined the functional aspect of GGA-repeats by isolating genomic DNA fragments containing GGA-repeats and by analyzing their chromosomal localization. The results obtained were as follows.1. We isolated 19 DNA fragments containing (GGA /TCC)n repeats from mouse genome by triplex affinity capture.2. 19 clones of them were mapped to different locations of 13 chromosomes.3. Almost half (9/19) of the isolated DNA fragments containing (GGA/TCC)n repeats were enriched to heterochromatin fraction and the remains were equally distributed to hetero- and eu-chromatin fractions. Neither of the clones examined was enriched to euchromatin fraction.4. The results from gel mobility shift assay and DNase I protection mapping indicated that genomic DNA fragments containing GGA-repeats formed DNA- DNA complexes in a sequence-dependent manner.5. The results from the analysis of DNA-DNA complex formation of mononucleosomes reconstituted on various fragments of plasmid DNAs which contained different repeat-number of GGA-repeats or different flanking sequences carrying at either or both sites of GGA-repeats indicated that their complexes formations though GGA-repeats were required for both GGA-repeat number and homologous flanking sequences at both sites of GGA-repeats.