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

我们研究了含有(GGA/TCC)n个重复序列的DNA片段上重组的单核小体形成DNA-DNA复合体的能力，并通过分离含有GGA-重复序列的基因组DNA片段并通过分析它们的染色体定位来研究GGA-重复序列的功能。研究结果如下：1.研究结果。通过三链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)含有(GGA/TCC)n重复序列的DNA片段被富集成异染色质组分，其余部分平均分布在异染色质组分和欧盟染色质组分中。所检测的两个克隆均未富含常染色质。凝胶迁移率改变分析和DNase I保护图谱的结果表明，含有GGA-重复序列的基因组DNA片段以一种序列依赖的方式形成DNA-DNA复合体。对含有不同重复数的GGA-重复序列或在GGA-重复序列的任一个或两个位点携带不同侧翼序列的不同DNA片段上重组的单核小体DNA-DNA复合体的分析结果表明，尽管GGA-重复序列的两个位点都需要GGA-重复序列，但它们的复合体的形成仍然需要GGA-重复序列。

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.