Mammalian L1 retrotransposon replication

最新发现： ORF 1 p的结构与功能研究-ORF 1 p是L1基因编码的两种蛋白质之一。其他人对小鼠ORF 1 p的早期研究表明，它结合核酸，作为核酸伴侣，并通过高度保守的卷曲螺旋结构域形成三聚体。然而，ORF 1 p在逆转录转座中的功能在很大程度上是未知的。我们正在使用几种方法来研究这个问题，包括分析适应性进化的结构、生物化学和生物学效应，其主要涉及卷曲螺旋结构域（Boissinot等人，Mol.生物进化18：2186）。为此，我们从已灭绝的L1家族L1 Pa 5中恢复了一个预适应的ORF 1 p，该家族是目前活跃的人类L1 Pa 1家族适应的ORF 1 p的祖先。我们还创建了包含现代和祖先区域的ORF 1 p的马赛克版本，以及删除各种结构域的其他变体。我们广泛的特点，这些蛋白质的反转录转座和相互作用与几种哺乳动物宿主蛋白在体内。此外，我们纯化了mg量的各种ORF 1 ps至均一。我们在体外研究了它们的各种生物物理和生物化学特性，包括反映它们的核酸伴侣活性的几种测定。其中一些研究报告目前正在准备出版。 （本年度PI校内研究预算的百分比）：

RECENT FINDINGS: STRUCTURAL AND FUNCTIONAL STUDIES OF ORF1p - ORF1p is one of two L1 encoded proteins. Earlier studies by others of mouse ORF1p showed that it binds nucleic acids, acts as a nucleic acid chaperone, and forms trimers via a highly conserved coiled coil domain. However, the function of ORF1p in retrotransposition is largely unknown. We are using several approaches to examine this problem including analysis of the structural, biochemical, and biological effects of adaptive evolution, which involved mainly the coiled coil domain (Boissinot, et al, Mol. Biol. Evol. 18: 2186). To do so we resuscitated a pre-adapted ORF1p from an extinct L1 family, L1Pa5, which is ancestral to the adapted ORF1p of the currently active human L1Pa1 family. We also created mosaic versions of ORF1p that contain modern and ancestral regions, and other variants that were deleted of various domains. We extensively characterized these proteins with respect to retrotransposition and interaction with several mammalian host protein in vivo. In addition, we purified mg amounts of some the various of ORF1ps to homogeneity. We examined their various biophysical and biochemical properties in vitro including several assays that reflect their nucleic acid chaperone activity. Some of these studies are now being prepared for publication. (percentage of PI's Intramural Research Budget for this project for this year):