Rand K N, Gait M J
EMBO J. 1984 Feb;3(2):397-402. doi: 10.1002/j.1460-2075.1984.tb01819.x.
The sequence of gene 63 of bacteriophage T4 was determined by a shotgun approach. Small DNA fragments, derived by sonication of a restriction fragment that encompasses the region of gene 63, were cloned in M13 vectors and sequenced by the 'dideoxy' method. The position of the gene was established by comparison with the sequence of a gene 63 amber mutant. Knowledge of the DNA sequence of gene 63 and surrounding regions has allowed the construction of a clone of gene 63 in which RNA ligase production is under the control of the lac promoter of bacteriophage M13mp8. Infected E. coli cells can be induced to produce a protein indistinguishable from commercially available RNA ligase.
噬菌体T4基因63的序列是通过鸟枪法测定的。通过对包含基因63区域的限制性片段进行超声处理得到的小DNA片段,被克隆到M13载体中,并采用“双脱氧”法进行测序。通过与基因63琥珀突变体的序列进行比较确定了该基因的位置。基因63及其周围区域的DNA序列信息使得构建出了一个基因63克隆,其中RNA连接酶的产生受噬菌体M13mp8的lac启动子控制。被感染的大肠杆菌细胞可被诱导产生一种与市售RNA连接酶无法区分的蛋白质。