Mandrioli M, Bizzaro D, Manicardi G C, Gionghi D, Bassoli L, Bianchi U
Dipartimento di Biologia Animale, Università di Modena, Italy.
Chromosoma. 1999 Dec;108(7):436-42. doi: 10.1007/s004120050395.
Electrophoresis following digestion of Myzus persicae genomic DNA with HindIII showed the presence of a prominent band of approximately 200 bp whereas a faint electrophoretic band corresponding to DNA fragments of about 3000 bp was observed after digestion with ApaI. In situ digestion with restriction enzymes, followed by in situ nick translation, showed that ApaI targets are localized at the nucleolus organizer-bearing X telomeric region, whereas HindIII restriction sites are clustered in intercalary C-positive areas on the same X chromosome. Fluorescent in situ hybridization (FISH) carried out by using digoxygenin-labeled HindIII repeats as probe fully confirmed overlapping between the hybridization sites of this probe and the AT-rich intercalary heterochromatic bands on the X chromosome. These findings, together with published data, allow us to conclude that the M. persicae genome possesses three classes of C-positive heterochromatin: (i) a GC-rich argentophilic band located on one telomere of the X chromosome that contains ApaI targets; (ii) AT-rich intercalary bands located on the X chromosome containing clustered HindIII fragments; (iii) AT-rich telomeric bands located on autosomes, consisting of HaeIII repeats. Molecular analysis has shown that the length of the HindIII repeat consensus sequence is 189 bp with an AT content of 67%. Southern blotting with HindIII monomers revealed a regular ladder of bands composed of multimers of basic length that are characteristic of satellite DNAs. The HindIII repeat displays other features typical of eukaryotic satellite arrays such as overlapping with heterochromatic bands and a high degree of sequence similarity among monomers (84%-94%). A similarity plot showed that sequences were particularly variable in the 50-100 bp region whereas they proved to be highly conservative in the first 50 bp, thus suggesting that this portion of the repeat might be functionally important.
用 HindIII 消化桃蚜基因组 DNA 后的电泳显示存在一条约 200 bp 的明显条带，而用 ApaI 消化后观察到一条对应于约 3000 bp DNA 片段的微弱电泳条带。用限制性内切酶进行原位消化，随后进行原位缺口平移，结果表明 ApaI 的靶位点位于携带核仁组织区的 X 染色体端粒区域，而 HindIII 限制性位点聚集在同一 X 染色体的居间 C 阳性区域。使用地高辛标记的 HindIII 重复序列作为探针进行的荧光原位杂交（FISH）充分证实了该探针的杂交位点与 X 染色体上富含 AT 的居间异染色质带之间存在重叠。这些发现与已发表的数据一起，使我们能够得出结论，桃蚜基因组具有三类 C 阳性异染色质：（i）位于 X 染色体一个端粒上的富含 GC 的嗜银带，其中含有 ApaI 靶位点；（ii）位于 X 染色体上的富含 AT 的居间带，其中含有聚集的 HindIII 片段；（iii）位于常染色体上的富含 AT 的端粒带，由 HaeIII 重复序列组成。分子分析表明，HindIII 重复共有序列的长度为 189 bp，AT 含量为 67%。用 HindIII 单体进行的 Southern 印迹显示出由基本长度的多聚体组成的规则条带阶梯，这是卫星 DNA 的特征。HindIII 重复序列显示出真核卫星阵列的其他典型特征，如与异染色质带重叠以及单体之间的高度序列相似性（84%-94%）。相似性图显示，序列在 50-100 bp 区域特别可变，而在前 50 bp 中被证明高度保守，因此表明该重复序列的这一部分可能在功能上很重要。
