ISOLATION OF GENOME-ENZYME COMPLEX FROM CYTOPLASMIC POLYHEDROSIS VIRUS OF SILKWORM BOMBYX MORI

The genome-enzyme complex is isolated from the silkworm cytoplasmic polyhedrosis viruswith DEAE-Sephadex column chromatography after ultraviolet irradiation. The genome-enzyme complex shows both RNA-polymerase and methyltransferase activities while using ~3H-UTP and [~3H-methyl]-S-adenosyle-L-methionine as substrates. Like the double-stranded RNAgenome of CPV, the genome-enzyme complex eould be separated into nine segments on pol-yacrylamide gel electrophoresis. It is demonstrated that the RNA po1ymerase and methyl-transferase are tightly bound to the double-stranded RNA genome and that the individualsegments of the genome-enzyme complex all possess RNA-polymerase and methyltransferaseactivities. It appears that each segment of the double-stranded RNA genome is transcribedindependently.     

CODING ASSIGNMENTS OF EACH mRNA SYNTHESIZED IN VITRO AND PROTEIN COMPONENTS OF CPV OF SILKWORM BOMBYX MORI

The coding propertics of each mRNA synthesized in vitro of CPV have been examined. The mRNAs were separated by polyacrylamide gel electrophoresis and isolated from the gel slices. The individual mRNA was then translated into the wheat germ cell-free protein synthesizing system. The products of translation were analyzed by the immuno-reaction with the ~(125)I-IgG of each of the five electrophoretic protein components of CPV. The coding assignments were deduced on the basis of the immuuo-reaction as follows: mRNA_1, mRNA_3 and mRNA_9 encode group P_1 of viral proteins, mRNA_2 and mRNA_6 encode group P_2 and P_3, respectively, mRNA_7 and mRNA_6 encode group P_4 while mRNA_9 encode group P_5.There axe more than 10 polypeptides in the CPV virion as demonstrated by two-dimensional electrophoresis.     

体外合成的家蚕细胞质多角体病毒mRNA于麦胚无细胞系统中翻译其结构蛋白

家蚕细胞质多角体病毒(Cytoplasmic polyhedrosis virus以下简称CPV)颗粒中含有以双链RNA为模板的RNA多聚酶,于体外合成系统中可以获得毫克量的CPV单链RNA.体外合成反应液经DEAE-Sephadex A-25柱层析分离、纯化、浓缩,再经2.5％聚丙烯酰胺凝胶电泳分离,表明体外合成的单链RNA含有九条区带.体外合成的单链RNA,于小麦胚无细胞制剂翻译系统中给予同位素标记的氨基酸,翻译的蛋白与家蚕CPV抗血清产生免疫沉淀线,说明体外系统转录的单链RNA含有足够的基因信息,指导家蚕CPV的结构蛋白的合成.     

[3H-甲基]-甲硫氨酸可以作为体外合成家蚕细胞质多角体病毒mRNA 5′-端的甲基供体

本文报道了用[³H-甲基]-甲硫氨酸代替S-腺苷-L甲硫氨酸作为甲基供体,在家蚕细胞质多角体病毒(简称CPV)颗粒所带的RNA多聚酶催化下于体外合成³H-mRNA。体外合成的³H-CPV mRNA经 DEAE-Sephadex A-25柱层析分离纯化。用核糖核酸酶P₁和蛇毒磷酸二酯酶降解、纸电泳分离,结果表明甲硫氨酸的[³H-甲基]已参入CPV-RNA的5-末端。即使在S-腺苷-L-甲硫氨酸的存在下,[³H-甲基]-甲硫氨酸对于CPV-mRNA的5’-末端罩式结构的形成仍具有甲基供体的功能。     

家蚕细胞质多角体病毒基因-酶复合物的分离

本文报道了家蚕细胞质多角体病毒(简称CPV)颗粒经紫外光照射后,通过DEAE-Sephadex A-25 柱层析,用氯化钠溶液分步洗脱分离得到基因-酶复合物。以³H-UTP和[³H-甲基]-S-腺苷-L-甲硫氨酸(SAM)作为底物时,该复合物均呈现RNA多聚酶和甲基转移酶活力。与CPV的RNA基因相同,基因-酶复合物能由聚丙烯酰胺凝胶电泳分成9条片段,各个片段也均呈现RNA多聚酶和甲基转移酶的酶活力。说明CPV颗粒里的RNA多聚酶和甲基转移酶紧密结合于双链RNA基因上,在复制过程中,CPV的双链RNA基因的每一片段各自独立地进行转录。     

家蚕细胞质多角体病毒的体外合成的mRNA编码信息及病毒蛋白组成

本文报道了体外合成的家蚕细胞质多角体病毒(CPV)mRNA的编码性质。该mRNA经聚丙烯酰胺凝胶电泳分离后,各mRNA片段分别从切下的凝胶中抽提得到,并且分别置于麦胚无细胞体外翻译系统进行翻译。翻译生成的产物与CPV 5种蛋白组份的~(125)I标记的抗体(IgG)进行免疫沉淀反应。所得结果表明,家蚕CPVmRNA的编码如下:mRNA_1,mRNA_3和mRNA_9编码病毒蛋白P_1组;mRNA_2和mRNA_6分别编码P_2和P_3组蛋白;mRNA_7和mRNA_8共同编码P_4组蛋白,而mRNA_9编码P_5组蛋白。经双向电泳分离,观察到CPV颗粒蛋白组份至少有10种以上。