人类α卫星DNA的序列异质性及错配α卫星DNA双链体的热稳定性

Sequence heterogeneity of the human alphoid satellite DNA and thermal stability of mismatched alphoid DNA duplexes.

Bachvarov D R, Markov G G, Ivanov I G

Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia.

Int J Biochem. 1987;19(10):963-71. doi: 10.1016/0020-711x(87)90179-0.

Abstract

340 bp (dimer) and 680 bp (tetramer) fractions of the human alphoid satellite DNA (h alpha RI DNA) were isolated after complete cleavage of total human DNA with EcoR I and cloned in pBR 32.5. 2. Ten clones containing 340 bp inserts and one clone containing 680 bp insert were sequenced in order to investigate the sequence heterogeneity of this satellite DNA and the sequence data were compared with the consensus h alpha RI DNA sequence of Wu and Manuelidis (1980). 3. It was shown that in all clones studied the mutations are nonrandomly distributed along the human alphoid monomers forming distinct conservative and variable regions. 4. This mutation distribution pattern was compared with the nucleotide variations between the consensus sequences of different primate alphoid DNAs and it was found that the interspecies nucleotide divergency of this satellite DNA is quite similar to the intragenomic one. 5. The sequenced h alpha RI DNA clones were used for preparation of DNA-DNA hybrids with a known percentage of base pair mismatching. 6. These hybrids were melted on hydroxyapatite (HAP) and the results obtained were used to determine the relationship between the thermal stability (Tm) and the extent of base pair mismatching for naturally diverged DNA sequences. 7. A value of 0.7 degrees C decrease in Tm per 1% base pair mismatching was found.

摘要

用人α卫星DNA（hαRI DNA）的340 bp（二聚体）和680 bp（四聚体）片段，在人总DNA用EcoR I完全切割后分离得到，并克隆于pBR 32.5中。
对10个含340 bp插入片段的克隆和1个含680 bp插入片段的克隆进行测序，以研究该卫星DNA的序列异质性，并将序列数据与Wu和Manuelidis（1980）的hαRI DNA共有序列进行比较。
结果表明，在所有研究的克隆中，突变沿着人α卫星单体非随机分布，形成明显的保守区和可变区。
将这种突变分布模式与不同灵长类α卫星DNA共有序列之间的核苷酸变异进行比较，发现该卫星DNA的种间核苷酸差异与基因组内差异非常相似。
对已测序的hαRI DNA克隆用于制备具有已知碱基对错配百分比的DNA-DNA杂交体。
这些杂交体在羟基磷灰石（HAP）上解链，所得结果用于确定自然分化的DNA序列的热稳定性（Tm）与碱基对错配程度之间的关系。
发现每1%碱基对错配，Tm值降低0.7℃。