Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide–deoxyadenosine adducts into M13mp7L2

number of these plaques to analyze their properties and found incorporated at the underlined site into four oligonucleothat the majority lacked the oligonucleotide insert. In a few tides, 5-CAGATTTAGAGTCTGC-3 ,5 -CAGTGCAGcases, the hairpin that should have been cut out by the ATTTAGAG-3 ,5 -GTGCAGATTTAGA-3 and 5-TGCrestriction enzyme, EcoRI, was still present. The high number AGATTTA-3. The oligonucleotides were inserted into of plaques lacking the insert has proved to be a considerable M13mp7L2 and the vector transfected into SOS-induced inconvenience in screening by differential hybridization. In Escherichia coli SMH77 which were then plated on agar some cases, the majority of the plaques could not be assigned. plates. The experiments reported here were designed to This vastly increased the number of plates that had to be test the effect of the lesion position (the underlined A in screened in order to obtain reliable numbers for comparing the sequences above) on the ligation efficiency of the insert mutation frequencies from different adducts in different conand the frequency of failed constructs, as well as any texts. We were interested in investigating the reason for the possible effects on the mutagenic consequences of the lesion. failure to insert the oligonucleotide into the constructed vectors. The construct survival was estimated from the number of Therefore, we constructed oligonucleotides with sequences plaques formed following transformation, and mutation based on context II(A), 5-CAGATTTAGAGTCTGC-3 frequencies were estimated from sequencing of randomly