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.     

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。     

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.     

家蚕细胞质多角体病毒(CPV)双链RNA为模板的RNA多聚酶和转甲基酶的蛋白亚基组成

本文以家蚕CPV的基因与酶复合物为材料进一步研究了家蚕CPV-的RNA多聚酶和转甲基酶的蛋白亚基组成,一种方法是直接用~(125)Ⅰ-标记的基因与复合物中的蛋白质分析。其中含有三种不同分子量的蛋白质即结构蛋白P1(分子量33,000),P2(分子量67,000)和P4(分子量142,000),这三种结构蛋白在SDS-等电聚焦电泳中都表现了二种以上分子量相同而电荷不同的蛋白。第二种方法是分别应用CPV的5种结构蛋白的抗体抑制CPV RNA多聚酶和转甲基酶的能力进行判断,结果表明CPV RNA多聚酶合有结构蛋白P1(33,000),P2(67,000)和P4(142,000),而转甲基酶主要由结构蛋白P1组成。     

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。     

THE PROTEIN SUBUNITS OF THE DOUBLESTRANDED RNA DEPENDENT RNA POLYMERASE AND METHYLTRANSFERASE OF THE CYTOPLASMIC POLYHEDROSIS VIRUS OF SILKWORM, Bombyx mori

The particles of CPV of silkworm contain double-stranded RNA polymerase and methyltransferase. It was reported in a previous paper that the genome-enzyme complex could be isolated. The genome-enzyme complex shows high enzyme activity of RNA polymerase and methyltransferase in spite of the fact that it consists of only 5 percent of the protein. In order to clarify the protein subunits of the RNA polymerase and methyltransferase, two methods were adopted. The SDS-polyacrylamide gel electrophoretogram showed that the ~(125)I-labeled genome-enzyme complex of CPV contained three protein components in molccular weight of 33 K, 67 K and 142 K daltons respectively and each protein component of them consisted of more than two protein subunits with different isoelectric points in 2-dimensional electrophoretogram. The antibody to the five protein components (P1, P2, P3, P4, P5) was prepared and used to inhibit the enzyme activities of RNA polymerase and mthyltransferase. It showed that the RNA polymerase was inhib