MODEL SYSTEMS TO EVALUATE TRANSPOSITION IN EUKARYOTIC CELLS

工作总结：果蝇通过顶端特异性转位来维持端粒长度。两个非LTR反转录转座子，HET-A和START，已知在这种生物中负责端粒的延长。虽然HET-A在活跃分裂的细胞中表达，特别是在生殖系中，但在野生型菌株中，每一有性世代只有大约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HET-A末端排列比正常长得多。3号染色体上的一个遗传因素已被确定，它可以诱导末端HET-A阵列生长，但不能诱导非端粒反转录转座子的转座。在有丝分裂时，长端粒以端粒-端粒结合的形式出现，频率很高。与其他逆转录酶不同，HET-A不编码自己的逆转录酶(RT)，这是逆转录转座子中的一个关键酶。端粒较长的菌株RT活性较高。为了从遗传学上探讨RT的来源，并证明该元件如预期的那样通过ITS序列的RNA中间体转座，我们准备了含有HET-A序列和报告基因的载体，只有在HET-A转座后才能表达。用于产生转化子的胚胎注射正在进行中。一旦我们能够量化转座，这些元素将被用来描述刺激HET-A转座的遗传因素。绿色荧光蛋白(GFP)基因也将被用作转座的报告基因，将表达HET-A mRNA的细胞与允许转座的细胞联系起来。类似的构建也在使用L1，一种人类非LTR反转录转座子。目前正在使用一些稳定转化的细胞系。其中两个细胞系是错配修复基因突变的人类结肠癌细胞系，hMSH6和hMLH1。另一种p53基因突变的细胞系正在研究中。在p53细胞中，转座发生的频率很高，反转座子活跃的细胞中有很高的凋亡率。这个项目得到了爱尔兰共和军的支持。-染色质、DNA损伤、DNA修复、异染色质、果蝇、黑腹果蝇、卵子、端粒

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. Although HeT-A is expressed in actively dividing cells, especially in the germ line, in wild type 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, but does not induce that transposition of non-telomeric retrotransposons. The long telomeres are found in telomere-telomere associations at a high frequency at mitosis. Unlike other retroposons, HeT-A doesn?t encode its own reverse transcriptase (RT), a key enzyme in retrotransposition. 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 constructs with HeT-A sequence and a reporter gene that 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. Another cell line mutant for p53 is under investigation. In p53 cells transposition occurs at a high frequency and there is a high incidence of apoptosis in cells with the active retrotransposon. This project is supported by an IRA. - Chromatin, DNA Damage, DNA Repair, Heterochromatin, Drosophila Melanogaster, Ooycte, Telomere