MECHANISM OF MEIOTIC PAIRING IN DROSOPHILA MALES

雄性果蝇减数分裂配对机制

• 批准号: 2734606
• 负责人: Bruce D. McKEE
• 金额: $ 21.69万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734606
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA topoisomerases; Drosophilidae; chromosome movement; cytogenetics; fungal genetics; genetic mapping; genetic promoter element; genetic transcription; histones; male; meiosis; nucleic acid repetitive sequence; oligonucleotides; radionuclides; sex chromosomes; site directed mutagenesis; tissue /cell culture; transfection; yeasts

The major goals of this study are to identify meiotic sites in Drosophila and to use them to gain insight into the mechanisms of meiotic pairing. We have utilized cytogenetic methods to show that pairing capacity is widely distributed throughout the euchromatin of chromosome 2, a major autosome, but that a strong pairing site maps to the base of chromosome arm 2L in the same region occupied by the repeated structural genes of the histone. We have also shown that X-Y pairing is mediated by 240bp intergenic spacer repeats from the rDNA loci on the X and Y. X chromosomal insertions of these repeats can restore pairing capacity to X chromosomes deleted for the native pairing region in the heterochromatin. By contrast, fragments of the rDNA transcription unit without promoters or spacers have no pairing ability. Each of the spacer repeats has a functional copy of the pre-rRNA promoter and we have shown by in vitro mutagenesis and functional testing that disabling the "spacer promoters" in arrays of these repeats also prevents them from stimulating X-Y pairing. We propose that pairing sites in Drosophila consist of transcribed sequences or the promoters thereof. The proposed experiments involve development of a novel mini- chromosome assay that can be used to test any candidate sequence for pairing capacity. It will be used to test transgenic insertions of histone repeats and a variety of other candidate sequences, including arrays of promoter sequences from both Drosophila and yeast. The idea that transcription is required for pairing will be tested by attempting to "activated" sequences that lack autonomous pairing capacity by transcription from heterologous promoters. The possibility that pairing involves formation of joint molecules will be investigated by assessing sensitivity of pairing to mismatches between potential partners, with special attention to whether mismatches upstream or downstream of the initiation site are especially disruptive. This latter test is aimed at discovering evidence for or against direct involvement of nascent transcripts in the pairing process. We will also search for sites outside the promoter region of the 240bp spacer repeats that are essential for pairing function of the repeats.

本研究的主要目标是确定果蝇减数分裂位点 并利用它们来深入了解减数分裂配对的机制。我们 利用细胞遗传学方法表明配对能力广泛存在 分布在2号染色体（主要常染色体）的常染色质中， 但强配对位点映射到染色体臂 2L 的碱基 组蛋白的重复结构基因占据的同一区域。 我们还表明 X-Y 配对是由 240bp 基因间间隔介导的 来自 X 和 Y 上的 rDNA 位点的重复。X 染色体插入 这些重复序列可以恢复删除的 X 染色体的配对能力 异染色质中的天然配对区域。相比之下，碎片 没有启动子或间隔区的 rDNA 转录单位没有 配对能力。每个间隔重复序列都有一个功能性副本 pre-rRNA 启动子，我们通过体外诱变和功能证明 测试禁用这些重复序列中的“间隔启动子” 也阻止它们刺激 X-Y 配对。我们建议配对 果蝇中的位点由转录序列或启动子组成 其中。拟议的实验涉及开发一种新型迷你 染色体分析可用于测试任何候选序列 配对能力。它将用于测试组蛋白的转基因插入 重复序列和各种其他候选序列，包括数组 来自果蝇和酵母的启动子序列。这个想法是 配对所需的转录将通过尝试进行测试 缺乏自主配对能力的“激活”序列 从异源启动子转录。配对的可能性 涉及联合分子的形成将通过评估进行研究 配对对潜在伴侣之间不匹配的敏感性， 特别注意上游或下游是否不匹配 起始位点尤其具有破坏性。后一个测试的目的是 发现支持或反对新生直接参与的证据 配对过程中的转录本。我们还将搜索外部网站 240bp 间隔重复序列的启动子区域对于 重复的配对功能。