MECHANISM OF MEIOTIC PAIRING IN DROSOPHILA MALES

雄性果蝇减数分裂配对机制

基本信息

批准号：
2396055
负责人：
Bruce D. McKEE
金额：
$ 21.64万
依托单位：
UNIVERSITY OF TENNESSEE KNOXVILLE
依托单位国家：
美国
项目类别：
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

项目摘要

DESCRIPTION: Although the segregation of homologous chromosomes during the first meiotic division is usually ensured by the formation of recombinational chiasmata that physically join the homologs, nonexchange chromosomes without chiasmata can also segregate properly. This is abundantly clear during meiosis I in Drosophila males, where homologous chromosomes pair with each other to form bivalents in the complete absence of recombination. Dr. McKee's laboratory has been instrumental in defining which sequences act as pairing sites to allow recognition of homologs in Drosophila primary spermatocytes. Their results to date indicate that autosomal heterochromatin plays little if any role in pairing. There appears to be a widespread distribution of weak pairing sites throughout the autosomal euchromatin, but in addition, there are some pairing sites that are highly preferred. One of these is at the base of chromosome arm 2L, and may correspond to the histone gene repeats. On the X chromosome, most euchromatic sequences and most heterochromatic sequences are inactive in male meiotic pairing (even in the presence of X chromosomal DNA elsewhere in the genome with which it could potentially pair). Instead, pairing between the X and Y chromosomes is restricted to the array of rRNA genes within the centric heterochromatin. Recent results from the McKee lab indicate that the responsible sequences are located within the intergenic spacers that separate adjacent rDNA transcriptional units. These spacers consist of several repeats of a 240 bp sequence that contain transcription-competent promoters. Mutations in these "spacer promoters" abolish both transcription and the ability to mediate pairing. This data has suggested the working hypothesis that pairing sites consist of actively transcribed sequences or to active promoters, and that particularly strong pairing sites are those that contain a high density of transcribed genes, such as is the case for rDNA and histone gene clusters. The first major aim of the research described in this proposal is to determine in more detail what kinds of sequences can serve as pairing sites. Most of the work under this specific aim will utilize a mini-chromosome assay, in which sequences to be tested for pairing activity will be "jumped" onto the free X duplication Dp(1;f)1187. Two copies of the unaltered Dp segregate randomly from each other or from an attached X-Y in the same spermatocytes, so there are no functional male-meiotic pairing sites in this minichromosome. Sequences added to the Dp will be tested for their ability to promote either proper disjunction of two minichromosomes or the regular segregation of minichromosomes from related DNA elsewhere in the genome. Sequences to be tested by this assay include: (1) rDNA, focusing on the intergenic spacer repeats; (2) histone gene repeat units; (3) random fragments of autosomal DNA; (4) Stellate sequences; (5) arrays of promoter sequences, particularly those whose activity can be controlled; and (6) arrays of binding sites for proteins (such as those in the Polycomb complex) that are associated with promoters. In a second approach, Dr. McKee will exploit X-4 translocations and terminal 4th chromosome deletions to identify pairing sites on the small 4th chromosome. The second specific aim is to understand some of the rules governing the usage of pairing sites. First, rDNA sequences that lack pairing capacity will be placed downstream of strong promoters that themselves are also incapable of promoting pairing. These constructions will be transformed into the Drosophila genome and their pairing activity checked using both the current assay (where the transgene is placed on an X chromosome lacking the rDNA repeats, and the segregation of this chromosome from the Y is monitored) and by the minichromosome assay described above. This will test whether the inactive rDNA sequences can become activated by transcriptional read-through. In another line of investigation, Dr. McKee will ask whether there are sequences outside of the "spacer promoter" that are needed for pairing, such as the binding site for topisomerase I known to be in the intergenic spacer. Finally, he will investigate the effect of base-pair mismatches on the pairing competence of rDNA intragenic spacers. The effect of mismatches at different locations throughout the spacers should reflect the actual mechanism underlying the pairing. For example, if mismatches throughout the spacers disrupt pairing, this suggests that homologous pairing is part of the process, whereas more localized effects would indicate the involvement of site-specific recombination, mediation by interactions between DNA-bound proteins or by RNA or DNA bridges, depending on the nature and location of the critical sequences.

描述：虽然同源染色体在第一次减数分裂通常是通过形成物理连接同系物的重组交叉，非交换没有交叉的染色体也可以正确分离。这是在果蝇雄性减数分裂 I 期间，同源的染色体在完全缺失的情况下相互配对形成二价体的重组。麦基博士的实验室在定义哪些序列充当配对位点以允许识别同源物果蝇初级精母细胞。他们迄今为止的结果表明常染色体异染色质在配对中起的作用很小（如果有的话）。那里弱配对位点似乎在整个系统中广泛分布常染色体常染色质，但除此之外，还有一些配对位点是高度优选的。其中之一位于染色体臂 2L 的基部，并且可能对应于组蛋白基因重复。在 X 染色体上，大多数常色序列和大多数异色序列在雄性减数分裂配对（即使在其他地方存在X染色体DNA的情况下）它可能与之配对的基因组）。相反，之间配对 X 和 Y 染色体仅限于 rRNA 基因阵列中心异染色质。麦基实验室的最新结果表明负责的序列位于基因间间隔区内分离相邻的 rDNA 转录单位。这些垫片包括包含转录活性的 240 bp 序列的多次重复发起人。这些“间隔启动子”中的突变会废除两种转录以及调解配对的能力。该数据表明了工作假设配对位点由活跃转录的序列组成或到活跃的启动子，特别强的配对位点是那些含有高密度转录基因，例如 rDNA 和组蛋白基因簇。本提案中描述的研究的第一个主要目标是更详细地确定哪些类型的序列可以充当配对位点。这一特定目标下的大部分工作将利用迷你染色体测定，其中待测试配对活性的序列将被“跳跃” 到自由 X 重复 Dp(1;f)1187 上。两份未改变的 Dp 副本彼此随机分离，或与相同的 X-Y 随机分离精母细胞，因此该区域没有功能性雄性减数分裂配对位点微型染色体。添加到 Dp 的序列将测试其能力促进两条微型染色体或常规染色体的正确分离微型染色体与基因组其他地方的相关 DNA 的分离。该测定法要测试的序列包括：（1）rDNA，重点关注基因间间隔重复； (2)组蛋白基因重复单元； (3)随机常染色体 DNA 片段； (4) 星序列； (5)启动子阵列序列，特别是那些活性可以控制的序列；和（6）蛋白质结合位点阵列（例如 Polycomb 复合体中的结合位点）与启动子相关的。在第二种方法中，麦基博士将利用 X-4 易位和末端 4 号染色体缺失来识别配对位点位于较小的第四染色体上。第二个具体目标是了解一些管理规则配对网站的使用。首先，缺乏配对能力的rDNA序列将被放置在强启动子的下游，这些启动子本身也是无法促进配对。这些建筑将被改造进入果蝇基因组并使用两种方法检查它们的配对活性目前的检测（其中转基因被放置在缺乏 rDNA 重复，该染色体与 Y 的分离是监测）并通过上述微型染色体测定。这将测试失活的 rDNA 序列是否可以通过转录激活通读。在另一项调查中，麦基博士会问是否在“间隔启动子”之外还有一些序列是必需的配对，例如已知的拓扑异构酶 I 的结合位点基因间间隔区。最后，他将研究碱基对的影响 rDNA 基因内间隔区配对能力的不匹配。效果整个垫片不同位置的不匹配应反映配对背后的实际机制。例如，如果不匹配整个间隔破坏了配对，这表明同源配对是这个过程的一部分，而更多的局部效应会表明位点特异性重组的参与，介导 DNA 结合蛋白之间或 RNA 或 DNA 桥之间的相互作用，具体取决于关键序列的性质和位置。