MODEL SYSTEMS TO EVALUATE TRANSPOSITION IN EUKARYOTIC CELLS

英文名：Drosophila melanogaster 通过尖端特异性转位过程维持端粒长度。 已知两种非LTR反转录转座子HeT-A和TART， 负责这种生物的端粒延长。基于in 原位杂交，HeT-A在活跃分裂的细胞中表达， 尤其是在生殖细胞系中。在标准的实验室菌株中， 每有性世代有1%的染色体末端延伸， 这表明转录不是转座的限制步骤。 少数菌株的末端HeT-A阵列比正常的长得多。 3号染色体上的一个遗传因子已经被确定， 终端HeT-A阵列生长。与其他逆转录酶不同，HeT-A 有吗t编码其自身的逆转录酶（RT），RT是 反转录转位为了研究HeT-A RT的来源，我们 已经采用了一种生物化学测定法来测量细胞中的RT活性， 提取物和原位。初步数据表明，RT活动是 在端粒较长的菌株中更高。探讨 RT基因的来源，并显示该元件转座 通过从其序列预期的RNA中间体，我们得到 制备具有HeT-A序列和两个 报告基因白眼基因，被一个反向的 内含子，仅在HeT-A转座后表达。胚胎 生产转化体的注射正在进行中。一旦我们能够 量化换位，这些元素将被用来表征 刺激HeT-A转座的遗传因素。的基因 绿色荧光蛋白（GFP）也将用作报告基因， 转座使表达HeT-A mRNA的细胞与那些 允许换位。类似的结构正在使用L1， 人非LTR反转录转座子。一些稳定转化的 正在使用细胞系。其中两个细胞系是人类结肠癌细胞系， 错配修复基因hMSH6突变的癌细胞系 hMLH1。测量换位的实验正在进行中 突变细胞系中的频率。另一种p53突变细胞系 正在接受调查

Summary of Work: Drosophila melanogaster maintains telomere length by a process of tip specific transposition. Two non-LTR retrotransposons, HeT-A and TART, are known to be responsible for telomere elongation in this organism. Based on in situ hybridizations, HeT-A is expressed in actively dividing cells, especially in the germ line. In standard laboratory strains only about 1% of chromosome ends are extended per sexual generation, suggesting that transcription is not the limiting step in transposition. A few strains have terminal HeT-A arrays much longer than normal. A genetic factor on chromosome 3 has been identified that induces terminal HeT-A arrays to grow. Unlike other retroposons, HeT-A doesn?t encode its own reverse transcriptase (RT), a key enzyme in retrotransposition. To investigate the source of the HeT-A RT, we have adapted a biochemical assay to measure RT activity in cell extracts and in situ. Preliminary data suggest that the RT activity is higher in the strains with longer telomeres. To investigate the source of RT genetically and to show that this element transposes via an RNA intermediate as expected from its sequence, we have prepared plasmid constructs with HeT-A sequence and two reporter genes. The white-eye gene, interrupted with a reversed intron, will be expressed only after HeT-A transposition. Embryo injections to produce transformants are in progress. Once we can quantify transposition, these elements will be used to characterize the genetic factors that stimulate HeT-A transposition. A gene for Green Fluorescent Protein (GFP) will also be used as a reporter for transposition to relate cells expressing HeT-A mRNA to those that allow transposition. Similar constructs are being made using L1, a human non-LTR retrotransposon. A number of stably transformed cell lines are being used. Two of these cell lines are human colon cancer cell lines that are mutant for mismatch repair genes, hMSH6 and hMLH1. Experiments are underway to measure transposition frequency in the mutant cell lines. Another cell line mutant for p53 is under investigation.