DROSOPHILA TELOMERES--GENE, TRANSCRIPTS, AND CHROMATIN

端粒序列及其调控被认为与细胞衰老和癌症的发展有关；然而，很明显，这些关系并不像最初想象的那么简单。果蝇是研究端粒的有价值的模型，因为这种有机体通过与大多数其他物种使用的机制不同来维持其端粒。果蝇端粒是由端粒特异的非LTR反转录转座子HET-A和START的多个重复序列组成，而不是由端粒酶产生的简单重复序列。比较这两种机制的异同为确定端粒活性的基本特征提供了一个强有力的途径，端粒活性是我们工作的长期目标。这些实验的一个主要目标是了解这些反转录转座子的调节。一系列实验将集中在最近发现的HET-A启动子上。这个启动子很有趣，不仅因为它在端粒维持中的作用，而且因为它是第一个在分子水平上研究的异色启动子。此外，它是非LtR元件和LtR元件的典型启动子之间意想不到的中间产物，这一发现对逆转录病毒具有进化意义。在培养细胞中的研究已经检测到启动子序列中的几个功能元件。这项工作将扩展到清楚地定义每个元件，通过在完整的果蝇中使用报告转基因来测试启动子，识别启动子中的任何组织特异性元件，并探索与异染色质和常染色质染色体环境的相互作用。Start同时产生正义和反义转录本。对反义启动子转录本进行了鉴定，并对其和正义启动子进行了定义。两者都是异色的，其特征与HET-A启动子一样。HET-A在老年果蝇和一些细胞培养物中的表达显著增加。我们假设这是由于年龄相关的异染色质变化，并将测试这一点以及影响位置效应差异的因素对HET-A和START启动子表达的影响。一种新的相关逆转录酶的来源将被确定。有证据表明，这种RNA要么是TART的加工产物，要么是来自“独立的”细胞基因的信使核糖核酸。这个mRNA很有趣，因为它可能与端粒酶的催化亚单位有进化关系。转座子维持端粒机制的系统发育研究将扩大到包括果蝇属以外的昆虫，以拓宽对该机制基本特征的认识。

Telomere sequences and their regulation have been implicated in- both cellular aging and the development of cancer; however, it is clear that these relationships are not as simple as first thought. Drosophila is a valuable model for the study of telomeres because this organism maintains its telomeres by a variation of the mechanism used by most other species. Drosophila telomeres are composed of multiple repeats of the telomere specific non-LTR retrotransposons, HeT-A and TART, rather than the simple repeats generated by telomerase. Comparison of similarities and differences between these two mechanisms offers a powerful approach for determining the essential features of telomere activity, a long term goal of our work. A primary goal of these experiments is to understand the regulation of these retrotransposons. One line of experiments will focus on the recently identified HeT-A promoter. This promoter is interesting not only for its role in telomere maintenance, but because it is the first heterochromatic promoter studied at the molecular level. Additionally, it is an unexpected intermediate between the typical promoters for non-LTR and LTR elements, a finding with evolutionary implications vis a vis retroviruses. Studies in cultured cells have detected several functional elements within the promoter sequence. This work will be extended to clearly define each element, to test the promoter by using reporter transgenes in intact flies, to identify any tissue-specific elements in the promoter, and to explore interactions with both heterochromatic and euchromatic chromosomal environments. TART yields both sense and antisense transcripts. The antisense promoter transcript was identified; it and the sense promoter will be defined. Both are heterochromatic and will be characterized as was the HeT-A promoter. HeT-A expression is dramatically increased in older flies and some cell cultures. We hypothesize that this is due to age-related changes in heterochromatin and will test this and the effects of factors affecting position effect variegation on the expression of the HeT-A and TART promoters. The origin of a novel related reverse transcriptase mRNA will be determined. Evidence suggests that this RNA is either a processed product of TART or the mRNA from a "free-standing" cellular gene. This mRNA is interesting because of a possible evolutionary relationship to the catalytic subunit of telomerase. The phylogenetic study of the transposon mechanism for telomere maintenance will be extended to include insects outside the genus Drosophila to broaden the view of its essential features of this mechanism.