Iatrou K, Meidinger R G
University of Calgary, Department of Medical Biochemistry, Alberta, Canada.
Gene. 1989 Jan 30;75(1):59-71. doi: 10.1016/0378-1119(89)90383-1.
The polyhedrin gene of the nuclear polyhedrosis virus of the silkmoth Bombyx mori (BmNPV) has been subjected to deletion mutagenesis. A number of clones containing partially deleted polyhedrin genes were characterized and four clones containing limited deletions of the 5'-untranslated or 5'-flanking sequences of the gene were further analyzed with respect to polyhedrin promoter activity. The functional characterization of the deletion mutants was achieved through the insertion of a chloramphenicol acetyl transferase (CAT) gene (cat) into each deletion junction. The resultant cat constructs were introduced into the genome of BmNPV through homologous recombination and the effect of each deletion on the activity of the polyhedrin promoter was evaluated by measurements of CAT enzymatic activity in extracts of tissue culture cells infected with the corresponding recombinant BmNPVs as well as by primer extension assays. Removal of the entire leader region and eleven adjacent residues of the 5'-flanking sequences of the polyhedrin gene results in a dramatic decrease in promoter activity, which, however, remains detectable through CAT activity measurements. Elimination of an additional 30 nucleotides (nt) of the upstream sequences results in the complete inactivation of the polyhedrin promoter. The functional characterization of a deletion mutant lacking 41 nt of the 5'-flanking sequences has demonstrated that no functions necessary for viral infectivity, replication or assembly are disrupted by this deletion, since the corresponding recombinant viruses propagate in the cells with the same kinetics and to the same extent as wild-type BmNPV. As a result of the deletion mutagenesis, two classes of transfer vectors have become available. The first class can be used for introducing into the viral genome foreign nucleotide sequences under polyhedrin promoter control, while the second one can be used for obtaining recombinant viruses harboring foreign genetic material in an environment which is devoid of polyhedrin promoter activity.
家蚕核型多角体病毒（BmNPV）的多角体蛋白基因已进行缺失诱变。对多个含有部分缺失多角体蛋白基因的克隆进行了表征，并对四个含有该基因5'-非翻译区或5'-侧翼序列有限缺失的克隆进行了多角体蛋白启动子活性的进一步分析。通过将氯霉素乙酰转移酶（CAT）基因（cat）插入每个缺失连接处，实现了缺失突变体的功能表征。通过同源重组将所得的cat构建体引入BmNPV基因组，并通过测量感染相应重组BmNPV的组织培养细胞提取物中的CAT酶活性以及引物延伸试验，评估每个缺失对多角体蛋白启动子活性的影响。去除多角体蛋白基因5'-侧翼序列的整个前导区和11个相邻残基会导致启动子活性急剧下降，不过，通过CAT活性测量仍可检测到。上游序列再额外消除30个核苷酸（nt）会导致多角体蛋白启动子完全失活。对一个缺失5'-侧翼序列41 nt的缺失突变体的功能表征表明，该缺失不会破坏病毒感染性、复制或组装所需的任何功能，因为相应的重组病毒在细胞中的增殖动力学和程度与野生型BmNPV相同。由于缺失诱变，现已获得两类转移载体。第一类可用于在多角体蛋白启动子控制下将外源核苷酸序列引入病毒基因组，而第二类可用于在缺乏多角体蛋白启动子活性的环境中获得携带外源遗传物质的重组病毒。
