REGULATION OF VIRAL GENE EXPRESSION IN INSECT CELLS

基于病毒基因的模型系统是最有用和最重要的系统之一 可用于研究动物细胞中的基因调控的系统。 杆状病毒是一种大型DNA病毒，可在 昆虫细胞的细胞核，因此提供了一个很好的模型 昆虫细胞中基因调控的研究。进入单元格后， 一些杆状病毒早期启动子立即被识别和转录 被宿主细胞机器。杆状病毒早期启动子的研究将 在我们对启动子识别和 昆虫细胞的调节。杆状病毒主要包膜糖蛋白 (Gp64)基因进入后立即被识别和转录 细胞和gp64被认为在病毒感染中起重要作用。 周而复始。此外，对转录和翻译的调节 Gp64似乎很复杂。Gp64由不同的启动子转录而成 在感染周期的早期和晚期，导致mRNA 可能会以不同的效率翻译。因此，杆状病毒 Gp64基因为肿瘤的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……里面 在这些研究中，我们将研究昆虫的转录调控 细胞与早期病毒启动子的识别、调节和选择性 昆虫病毒晚期启动子的利用及其翻译调控 细胞。此外，还阐明了基因表达的调控机制 这一重要的包膜糖蛋白将提供对 这组病毒使用的复杂复制策略，可能具有 对其他大型DNA病毒的影响。 对gp64表达的调节将通过多个 互补的方法。早期gp64启动子的识别 将以以下方式检查昆虫细胞：删除， 链接器扫描和饱和点突变将生成并使用 用于功能分析(CAT和引物延伸分析)以识别顺式连接蛋白 决定转录速度和准确性的作用元件 入会仪式。细胞蛋白质的DNA结合部位将通过凝胶定位 流动性转移和DNA酶足迹。具有重要功能的蜂窝 拟从夜蛾中分离、克隆和测序DNA结合蛋白 果蝇和/或舞毒蛾细胞。转录调控基因 Gp64晚期启动子将通过类似的方法和病毒基因进行检测。 编码与后期启动子直接相互作用的蛋白质将是 克隆并测序。早期和晚期对gp64表达的调控 将通过确定转录起始来检查晚期gp64 mRNAs 早期和晚期启动子的速率，早期和晚期mRNAs的稳定性， 以及Earl和Late mRNAs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.