プラスミド・pHT114mutの段階でＡＢＣの能力を持ち、
大腸菌に組み込んで、取り出して
pHT014に組み込んでpHT016になると
ＡＣの能力しか発現しないという流れだと思います。
大腸菌に入れて、出す間に何があるのですか？
（論文の該当部分）
A HindIII-BamHI fragment containing a region corresponding to the remaining single type III module was prepared from plasmid pHT014.
The fragment was subcloned into BamHI- and HindIlldigested vector plasmid pUC119 to give plasmid pHT114mut.
Oligonucleotide-directed mutagenesis was performed by the method of Kunkel with the Mutan-K mutagenesis kit.
Plasmid pHT114mut was introduced into E. coli BW313, and uracil-containingsingle-stranded DNA was prepared.
This DNA was used as a template and hybridized with a synthetic oligonucleotide, 5'-ACCGGAGGTACAGTGACGACAAATCCTGGT-3'.
The left half of the oligonucleotide is complementary to the DNA region encoding the C-terminal end of the large N-terminal domain (catalytic domain), and the other half is complementary to the region encoding the N-terminal part of the Cterminal domain.
Mutant clones were selected after sequencing by the dideoxy chain termination method of Sanger et al.
The HindIII-BamHI segment in plasmid pHT014 was replaced by the HindIII-BamHI fragment prepared from pHT114mut with the desired deletion, giving plasmid pHT016.
＞Plasmid pHT114mut was introduced into E. coli BW313, and uracil-containingsingle-stranded DNA was prepared.
の部分ですね。ここにかかれているように、uracilを含む単鎖DNAを調製するためですね。
下記のURLに原理がかかれています。
時間に余裕があれば、
http://www.kochi-u.ac.jp/~tatataa/tech2003/gene/uracil.html
にかかれている参考文献を読んでみると、更に理解が深まるでしょう。
