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

Abstract

1. 1. 340 bp (dimer) and 680 bp (tetramer) fractions of the human alphoid satellite DNA (hαRI DNA) were isolated after complete cleavage of total human DNA with EcoR I and cloned in pBR 32.5. 2. 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 hαRI DNA sequence of Wu and Manuelidis (1980). 3. 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. 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. 5. The sequenced hαRI DNA clones were used for preparation of DNA-DNA hybrids with a known percentage of base pair mismatching. 6. 6. These hybrids were melted on hydroxyapatite (HAP) and the results obtained were used to determine the relationship between the thermal stability ( T m) and the extent of base pair mismatching for naturally diverged DNA sequences. 7. 7. A value of 0.7°C decrease in T m per 1% base pair mismatching was found.