3.1 Mutagenic properties of AFB1-FapyGua in
different sequence contexts
The single-stranded pSBL vectors containing AFB1-FapyGua
in the AXC, CXC, TXC, and CXA (X = AFB1-FapyGua)
sequence contexts were constructed and replicated in COS7 cells. The CGC
sequence has been identified as a hot-spot for
AFB1-induced G > T transversions, while the
AGC, TGC sequences and especially the CGA sequence were less prone to
these substitutions (Fedeles and Essigmann 2018). Following isolation of
the progeny DNAs, individual clones were obtained by transformation ofE. coli DH5α cells and subjected to Sanger sequencing. The
experiments were conducted in triplicate, beginning from transfection of
COS7 cells. The sample size was fixed in advance at the level of ten
clones per DNA isolate, excluding clones that either did not contain the
insert sequences (~ 20%) or had alterations others than
point mutations (Table 1 ).
These data revealed that regardless of the sequence contexts,
AFB1-FapyGua was highly mutagenic, inducing base
substitutions at frequencies of ~ 80-90%
(Figure 2 ). The difference between sequence contexts with
regard to non-mutagenic replication was insignificant. Similar to
results of the prior study, in which the TXA sequence was utilized (Lin
et al. 2014), mutational spectra were dominated by G > T
transversions. In addition, G > A transitions and G
> C transversions were observed in each of the four
sequences tested. The levels of G > A transitions were
higher in the AXC and CXA sequences relative to the CXC and TXC
sequences, with difference observed for the AXC versus CXC sequence
approaching significant level (p = 0.057). The level of G
> C transversions was elevated in the CXA sequence;
relative to the AXC sequence, the difference was significant (p =
0.047).