Ilves H, Kahre O, Speek M
Laboratory of Molecular Genetics, Institute of Chemical Physics and Biophysics, Tartu, Estonia.
Mol Cell Biol. 1992 Sep;12(9):4242-8. doi: 10.1128/mcb.12.9.4242-4248.1992.
The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.
大鼠长散在核元件（L1Rn）DNA元件的基因组结构包含两个重叠的开放阅读框（ORF），显然有可能编码一种DNA/RNA结合蛋白（在ORF1中）和一种逆转录酶（在ORF2中）。我们鉴定了一个1630 bp的L1Rn cDNA克隆，其包含重叠的ORF，以及一个600 bp的基因组片段，该片段源自一个全长L1Rn成员并包含ORF1的起始部分。这些DNA在SP6聚合酶启动子的控制下克隆到pSP65载体中后，被用于部分恢复L1Rn的ORF1-ORF2结构。为了测试L1Rn的ORF1和ORF2是否作为融合蛋白表达，制备了一系列含有一个或两个ORF且有渐进性截短的加帽RNA，并在兔网织红细胞裂解物中进行翻译。我们的分析表明，体外推定的由逆转录酶编码的L1Rn ORF2的表达受翻译重新起始或内部起始的调控，而非核糖体移码的调控。
