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首页> 外文期刊>Molecular and Cellular Biology >Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids.
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Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids.

机译：在嵌合质粒稳定转化的细胞中从多瘤晚期启动子转录。

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We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.
机译：我们已经检查了嵌合质粒的表达，该质粒包含单纯疱疹病毒胸苷激酶（tk）基因或新霉素抗性（neo）的Tn5基因的编码序列，该基因与多瘤DNA的晚期启动子相连。尽管多瘤病毒晚期基因通常在仅包含整合的病毒DNA分子的转化细胞中不表达，但是用tk或neo质粒转染的大鼠tk或野生型细胞能够在含有次黄嘌呤-氨基蝶呤-胸腺嘧啶核苷或G418分别处于不允许染色体外DNA复制的条件下，表明tk或neo基因已完全表达。此外，即使在没有直接选择该活性的情况下，细胞也能在次黄嘌呤-氨基蝶呤-胸腺嘧啶核苷或G418中生长。 Northern分析表明tk或neo转录本的稳态水平接近多瘤早期转录本的水平。 S1分析表明，这些转录本在多瘤病毒的晚期启动子内启动，并且其5'端定位在与晚期溶菌感染期间使用的那些相似或相同的位置。检查了聚腺苷酸化信号的取代作用。尽管包含多瘤早期聚腺苷酸化信号的质粒在赋予细胞稳定的G418抗性表型方面比使用晚期信号的类似构建体更为有效，但发现两种信号均得到有效利用。这表明在没有游离病毒DNA产生的情况下无法检测到多瘤转化的细胞中的晚期转录本不是无效的mRNA裂解或聚腺苷酸化的影响。我们的结果表明，整合多瘤基因组中的晚期基因表达不受消息起始水平的调节，而最有可能是通过转录后事件调控的。

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