Modulation of the cellular phenotype by integrated adeno-associated virus.
Modulation of the cellular phenotype by integrated adeno-associated virus.
复制标题
整合腺相关病毒对细胞表型的调节。
DOI：
10.1016/0042-6822(92)91218-j
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发表时间：
1992
期刊：
影响因子：
3.7
通讯作者：
Lavi,S
中科院分区：
文献类型：
--
作者：
Winocour,E;Puzis,L;Etkin,S;Koch,T;Danovitch,B;Mendelson,E;Shaulian,E;Karby,S;Lavi,S
腺相关病毒 (AAV)rep 基因编码一系列重叠、多功能、非结构蛋白 (Rep 蛋白)，这些蛋白调节病毒生命周期，也能够反式调节非病毒基因表达 (Berns, 1990, Microbiol. Rev.54, 316–329 综述)。为了研究 AAVrep 基因在细胞染色体环境中的表达，用 AAV/新杂交病毒感染 SV40 转化的中国仓鼠胚胎 (OD4) 细胞，并选择和扩增对抗生素 G418 具有抗性的后代。 AAV 和neoDNA 插入片段的染色体整合和RNA 转录通过Southern 和Northern 印迹程序得到证实。其中一个 G418R 细胞系稳定表达一种蛋白质，该蛋白质在蛋白质免疫印迹中与 AAV 抗 Rep 抗血清发生特异性反应。 AAVrepDNA 的稳定整合在正常生长条件下不会干扰细胞增殖，与细胞表型的两个变化相关：9 个细胞系中有 8 个细胞系对紫外线 (254 nm) 明显比亲本 OD4 细胞更敏感；与亲本 OD4 细胞相比，9 个细胞系中的 7 个细胞系失去了在体外促进 SV40 起源 DNA 扩增的能力。通过删除了rep基因的AAV/neoDNA构建体或通过缺乏AAV DNA的aneoDNA构建体转化为G418R的OD4细胞没有表现出这些表型变化。有人认为，AAVrepgene 的稳定整合会干扰与 DNA 修复和基因扩增相关的细胞过程。
The adeno-associated virus (AAV)repgene encodes a series of overlapping, multifunctional, nonstructural proteins (Rep proteins) which regulate the viral life cycle and which are also capable of trans-regulating nonviral gene expressions (reviewed in Berns, 1990,Microbiol. Rev.54, 316–329). To investigate the expression of the AAVrepgene in a cellular chromosomal context, SV40-transformed Chinese hamster embryo (OD4) cells were infected with an AAV/neohybrid virus and progeny resistant to the antibiotic G418 were selected and amplified. Chromosomal integration and RNA transcription of the AAV andneoDNA inserts were confirmed by Southern and Northern blotting procedures. One of the G418Rcell lines stably expressed a protein which reacted specifically with AAV anti-Rep antiserum in Western immunoblots. The stable integration of AAVrepDNA, which did not interfere with cell proliferation under normal growth conditions, was associated with two changes in cellular phenotype: eight of nine lines were markedly more sensitive to UV light (254 nm) than were the parental OD4 cells; and seven of the nine lines had lost the capacity to promote SV40 origin DNA amplificationin vitro, in contrast to the parental OD4 cells. OD4 cells transformed to G418Rby AAV/neoDNA constructs with a deletedrepgene, or by aneoDNA construct lacking AAV DNA, did not display these phenotypic changes. It is suggested that stable integration of the AAVrepgene interferes with cellular processes connected with DNA repair and gene amplification.