SV40 Replication core origin에 존재하는 early palindrome의 구조와 기능에 대해 알아보기 위해 SV40 origin을 포함하는 0.8kb DNA fragment를 filamentous phage M13 DNA에 cloning시킨 후 oligonucleotide-directed site-specific mutagenesis에 의해 early palindrome이 이룰 수 있는 hairpin 구조 중 stem 부분이 파괴된 mutant들과 base의 치환은 있으나 stem 부분은 다시 회복된 mutant들을 만들었다 in vivo replication assay를 위해 mutation이 일어난 origin 부위를 pAT153 vector로 subcloning하여 COS-1 cell에 transfection함으로써 replication level을 측정하였다. 그 결과 early domain의 inverted repeats는 fuctional cruciform structure를 형성함으로써 SV40 replication에 관여하는 것은 아니라는 것을 알 수 있었다.
To define the roles of the early palindrome domain within the SV40 replication origin region involved in initiation of DNA synthesis, 800-bp DNA fragment containing the simian virus 40(SV40) origin of replication was inserted into filamentous phage M13 DNA. Four kinds of mutants were introduced into this recombinant phage DNA, M13SV-2, by oligonucleotide-directed site-specific mutagenesis. Then, the origin fragments containing one or two base substitutions were subcloned into pAT153 vector, a derivative of pBR322. In two mutants (pATSV-A,-B) the stems were destroyed by single base substitutions. In the other two mutants(pATSV-C,-D) the normal stems were restored by additional base substitutions which complements the bases altered initially. While pATSV-A and pATSV-C could tolerate base substitutions, pATSV-B and pATSV-D showed dramatic decrease in DNA replication.
