REGULATION OF VIRAL GENE EXPRESSION IN INSECT CELLS

基于病毒基因的模型系统是其中最有用和最重要的 可用于动物细胞基因调控研究的系统。 杆状病毒是大的DNA病毒，其在细胞内转录和复制。 昆虫细胞的细胞核，因此，提供了一个极好的模型， 昆虫细胞中基因调控的研究。 进入细胞后， 一些杆状病毒早期启动子被立即识别并转录 被宿主细胞机制所控制 杆状病毒早期启动子的研究将 在我们对启动子识别的基本理解中很重要， 在昆虫细胞中的调节。 杆状病毒主要包膜糖蛋白 （gp 64）基因进入细胞后立即被识别并转录 细胞和gp 64被认为在病毒感染中起重要作用 周期 此外，转录和翻译调控， GP 64看起来很复杂。 Gp 64由不同的启动子转录 在感染周期的早期和晚期， 可以以不同的效率进行翻译。 因此，杆状病毒 gp 64基因为研究提供了一个方便的多用途模型系统 昆虫细胞和病毒-细胞相互作用的几个方面。 在 在这些研究中，我们将研究昆虫中的转录调控， 细胞和识别早期病毒启动子，调节和选择性 病毒晚期启动子在昆虫中的应用及翻译调控 细胞 此外，阐明调控表达的机制， 这一重要的包膜糖蛋白将提供深入了解 这组病毒使用复杂的复制策略， 对其他大型DNA病毒的影响。 gp 64表达的调节将通过多重免疫组化检测。 补充方法。 立即早期gp 64启动子的识别 将以下列方式检查昆虫细胞的细胞毒性：缺失， 将生成并使用连接子扫描和饱和点突变 用于功能分析（CAT和引物延伸试验），以鉴定顺式 决定转录速率和准确性的作用元件 入会仪式 细胞蛋白质的DNA结合位点将通过凝胶定位 迁移率变化和DNA酶足迹。 功能重要的细胞 DNA结合蛋白将被分离、克隆和测序 frugiperda和/或舞毒蛾细胞。 转录调控 gp 64晚期启动子将通过类似的方法和病毒基因进行检测 与晚期启动子直接相互作用的编码蛋白将被 克隆和序列测定 从早期到晚期的gp 64表达调控 晚期gp 64 mRNA将通过确定转录起始来检测 早期和晚期启动子的比率，早期和晚期mRNA的稳定性， 和晚期mRNA的翻译效率。

Model systems based on viral genes are among the most useful and important systems available for studies of gene regulation in animal cells. Baculoviruses are large DNA viruses that are transcribed and replicate in the nuclei of insect cells and as such, provide an excellent model for studies of gene regulation in insect cells. After entry into the cell, some baculovirus early promoters are immediately recognized and transcribed by the host cell machinery. Studies of baculovirus early promoters will be important in our basic understanding of promoter recognition and regulation in insect cells. The baculovirus major envelope glycoprotein (gp64)gene is recognized and transcribed immediately upon entry into the cell and gp64 is believed to play an important role in the viral infection cycle. Additionally, the transcriptional and translational regulation of gp64 appear to be complex. Gp64 is transcribed from different promoters at early and late times in the infection cycle, resulting in mRNAs which may be translated with different efficiencies. As such, the baculovirus gp64 gene provides a convenient multipurpose model system for the study of several aspects of the insect cell and the viral-cell interactions. In these studies, we will examine transcriptional regulation in the insect cell and recognition of early viral promoters, regulation and selective utilization of viral late promoters, and translational regulation in insect cells. In addition, elucidation of the mechanisms regulating expression of this important envelope glycoprotein will provide insight into the complex replication strategy utilized by this group of viruses and may have implications for other large DNA viruses. The regulation of gp64 expression will be examined by multiple complementary methods. Recognition of the immediate early gp64 promoter by the insect cell will be examined in the following manner: Deletion, linker scanning, and saturating point mutations will be generated and used for functional analyses (CAT and primer extension assays) to identify cis acting elements which determine the rate and accuracy of transcription initiation. DNA binding sites for cellular proteins will be located by gel mobility shift and DNAase footprinting. Functionally important cellular DNA binding proteins will be isolated, cloned and sequenced from Spodoptera frugiperda and/or Lymantria dispar cells. Transcriptional regulation of the gp64 late promoter will be examined by similar methods and viral genes encoding proteins which directly interact with late promoters will be cloned and sequenced. Regulation of gp64 expression from the early and late gp64 mRNAs will be examined by determining transcription initiation rates of early and late promoters, stabilities of the early and late mRNAs, and translational efficiencies of earl and late mRNAs.