Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2.
Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2.
复制标题
加合物位置和序列长度对含有苯并[c]菲二醇环氧化物-脱氧腺苷加合物的寡核苷酸连接至M13mp7L2的影响。
DOI：
10.1093/mutage/16.1.65
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发表时间：
2001
期刊：
影响因子：
2.7
通讯作者：
Jerina,DM
中科院分区：
文献类型：
--
作者：
Ponten,I;Waters,LS;Sayer,JM;Pilcher,AS;Dipple,A;Jerina,DM
由（+）-的顺式开环产生的加合物（1 S，2 R，3R，4S）-3，4-二羟基-1，2-环氧苯并[c]菲（苯并[c]菲二醇环氧化物-2，其中苄基羟基和环氧化物氧是反式的）通过脱氧腺苷的外环N6-氨基在下划线位点掺入到四种寡核苷酸中，5′-CAGATTTAGAGTCTGC-3′，5′-CAGTGCAGATTTAGAG-3′，5′-GTGCAGATTTAGA-3′和5′-TGCAGATTTA-3′。将寡核苷酸插入到M13 mp 7 L2中，并将载体转染到SOS诱导的大肠杆菌SMH 77中，然后将其接种在琼脂平板上。本文报道的实验被设计成测试损伤位置（上述序列中下划线的A）对插入物的连接效率和失败构建体的频率的影响，以及对损伤的诱变后果的任何可能的影响。从转化后形成的噬斑数量估计构建体存活率，并从随机挑选的噬斑的测序估计突变频率。将加合物位点移动到序列的中间显著增加了连接效率，而不管插入的寡核苷酸的长度如何，并且改变插入长度或加合物位置不会显著影响观察到的突变的频率（40-58.6%）或分布。因此，只要局部序列（加合物周围的五个或六个碱基）保持恒定，就可以调节寡核苷酸插入物的大小和加合物在其中的位置，以得到最佳的连接效率，而不改变损伤的诱变结果。
The adduct that would arise fromcisopening of (+)-(1S,2R,3R,4S)-3,4-dihydroxy-1,2-epoxy-benzo[c]phenan-threne (benzo[c]phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen aretrans) by the exocyclicN6-amino group of deoxyadenosine was incorporated at the underlined site into four oligonucleotides, 5′-CAGATTTAGAGTCTGC-3′, 5′-CAGTGCAGATTTAGAG-3′, 5′-GTGCAGATTTAGA-3′ and 5′-TGCAGATTTA-3′. The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-inducedEscherichia coliSMH77 which were then plated on agar plates. The experiments reported here were designed to test the effect of the lesion position (the underlined A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion. The construct survival was estimated from the number of plaques formed following transformation, and mutation frequencies were estimated from sequencing of randomly picked plaques. Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40–58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.