Studies on the Arabidopsis LINE, ATLNs : Their expression and the function of ORF proteins encoded.

拟南芥基因组具有LINE，称为ATLN，其表达受DNA甲基化负调控。对5' UTR中缺失的分析揭示，5' UTR具有两个启动子，在此指定为P1和P2。通过5' RACE分析转录物表明，它们的5'末端位于P1区的上游或P2区的下游。这一观察结果和P1区不含TATA序列的事实表明P1是一个内部启动子，它从启动子上游的位点启动转录。ATLN成员有两个连续的开放阅读框架（orf 1和orf 2），这是逆转录转座所必需的。ORF 1蛋白的C-末端区域具有核定位信号（NLS）样序列KRKR和KKGRK。在烟草BY-2和洋葱皮细胞中表达的GFP-ORF 1融合蛋白定位于细胞核，而NLS样序列突变的GFP-ORF 1融合蛋白则不定位于细胞核。由于ORF 1蛋白的N端区域具有逆转录病毒锌指基序，我们还通过在酵母和烟草BY2细胞中使用双杂交系统来检测蛋白质-蛋白质相互作用。我们发现ORF 1可以相互作用，而在锌指基序以及亮氨酸拉链基序的取代突变的ORF 1不能。这些结果表明，ORF 1由两种功能结构域组成，参与核定位和蛋白质-蛋白质相互作用。

The Arabidopsis thaliana genome has LINEs, called ATLNs, whose expression is negatively regulated by DNA methylation. Analysis of deletion in the 5' UTR revealed that the 5' UTR has two promoters, designated here as P1 and P2. Analysis of transcripts by 5' RACE showed that their 5' ends were located at sites immediately upstream of the P1 region or at sites downstream of the P2 region. This observation and the fact that the P1 region contains no TATA sequence indicate that P1 is an internal promoter that initiates transcription from sites upstream of the promoter.ATLN members have two consecutive open reading frames (orf1 and orf2) that are essential for retrotransposition. The C-terminal region of the ORF1 protein has nuclear localization signal (NLS) -like sequences, KRKR and KKGRK. The GFP-ORF1 fusion protein, which was expressed in tobacco BY-2 and onion skin cells, was localized to the nuclei of their cells, but the GFP-ORF1-fusion protein with mutations in NLS-like sequences was not. Because the N-terminal region of the ORF1 protein has a retroviral zinc-finger motif, we also examined protein-protein interactions by using two hybrid system in yeast as well as in tobacco BY2 cells. We found that ORF1 can interact with each other, whereas ORF1 with substitution mutations in the zinc-finger motif as well as in the leucine-zipper motif cannot. These results show that ORF1 consists of two kinds of functional domains involved in nuclear localization and protein-protein interaction.