GENETIC AND BIOCHEMICAL ANALYSIS OF AD 5 REGION 2

其目的是了解控制机制。 用腺病毒调控真核基因表达 类型5(Ad5)早期区域2A作为模型系统。的基因产物 早期区域2A是一个72,000道尔顿的单链DNA结合蛋白 (DBP)参与调节病毒基因表达的至少有四个 途径：(1)DNA合成的起始和延伸步骤；(2)DNA合成的起始和延伸步骤 几个Ad5早期基因的转录调控，(3) 早期mRNAs的稳定性，以及(4)病毒的宿主范围。 近期目标是在DBP内准确定义 参与蛋白质的每一种功能。为了实现这一目标，我们 将通过定点突变技术在DBP中产生突变 并分析突变的DBP与单链结合的能力 DNA，并帮助腺相关病毒在人类细胞中复制。 深入探索蛋白质的功能特性将是 在突变基因重新导入病毒后执行。

The objective is to gain an understanding of the control mechanisms operative in the regulation of eukaryotic gene expression, using adenovirus type 5 (Ad5) early region 2A as the model system. The gene product of early region 2A is a 72,000 dalton single-stranded DNA-binding protein (DBP) that is involved in regulating viral gene expression in at least four ways: (1) the initiation and elongation steps of DNA synthesis, (2) the regulation of transcription of several of the Ad5 early genes, (3) stability of early mRNAs, and (4) the host-range of the virus. The immediate goal is to precisely define within the DBP domains that are involved in each of the functions of the protein. To accomplish this, we will generate mutations in the DBP by site-specific mutagenesis techniques and analyze the mutant DBPs for their ability to bind to single-stranded DNA, and to help adeno-associated virus replicate in human cells. In-depth probing of the functional properties of the protein will be performed following re-introduction of the mutated gene back into the virus.