MECHANISM OF MEIOTIC PAIRING IN DROSOPHILA MALES

雄性果蝇减数分裂配对机制

• 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
• 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/2396055
• 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

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：尽管同源染色体的分离在 通常，通常通过形成第一的减数分裂划分 重组的chiasmata物理加入同源物，不换取 没有Chiasmata的染色体也可以正确隔离。 这是 在果蝇男性的减数分裂I期间，我很清楚 染色体彼此配对以在完全不存在的情况下形成二价 重组。 麦基博士的实验室有助于定义 哪个序列充当配对位点，可以识别同源物 果蝇原发性精子细胞。 他们迄今为止的结果表明 常染色体异染色质在配对中几乎没有任何作用。 那里 整个整个弱配对位点的分布似乎是广泛的分布 常染色体白染色质，但此外，还有一些配对站点 非常喜欢。 其中之一是在染色体臂2L的底部，而 可能对应于组蛋白基因重复。 在X染色体上，大多数 圣体序列和大多数异光序列在 男性减数分裂配对（即使在其他地方存在X染色体DNA的情况下 它可能配对的基因组）。 相反，在之间配对 X和Y染色体仅限于RRNA基因的阵列 中心异染色质。 麦基实验室的最新结果表明 负责任的序列位于基因间间隔物中 单独的相邻rDNA转录单元。 这些垫片包括 240 bp序列的几个重复序列，该序列包含具有转录功能的重复 发起人。 这些“间隔启动子”中的突变废除了两个转录 以及介导配对的能力。 这些数据提出了工作 假设配对位点由主动转录序列或 到主动启动子，特别是强大的配对站点是 包含高密度转录的基因，例如 rDNA和组蛋白基因簇。 该提案中描述的研究的第一个主要目的是 更详细地确定哪些序列可以用作配对站点。 这个特定目标下的大多数工作都将使用迷你染色体 测定，在其中测试配对活动的序列将“跳跃” 在免费的X复制DP（1; F）1187上。 未更改的DP的两个副本 在相同的同一 精子细胞，因此没有功能性的男性脱生位点 微小粒子体。 将添加到DP中的序列将测试其能力 促进两个微型染色体的适当分离或常规的 与基因组其他地方相关DNA的微小色粒分离。 该分析要测试的序列包括：（1）rDNA，重点放在 基因间间隔物重复； （2）组蛋白基因重复单位； （3）随机 常染色体DNA的碎片； （4）星状序列； （5）发起人的阵列 序列，特别是那些可以控制活动的序列； （6） 蛋白质的结合位点的阵列（例如Polycomb复合物中的结合位点） 与启动子相关的。 在第二种方法中，麦基博士将 利用X-4易位和终端第四染色体删除以识别 小染色体上的配对位点。 第二个具体目的是了解一些管理的规则 配对网站的使用。 首先，缺乏配对能力的rDNA序列 将被放置在强大促进者的下游 无法促进配对。 这些结构将被转换 进入果蝇基因组及其配对活动，同时检查 当前的测定（将转基因放置在缺乏的X染色体上 rDNA重复，该染色体与Y的分离为 受监测的）和上述微小颜色小体测定法。 这将测试 非活动rDNA序列是否可以通过转录激活 读取。 在另一项调查中，麦基博士将询问是否 “间隔启动子”以外的序列需要 配对，例如我知道在 基因间间隔物。 最后，他将研究基本对的效果 RDNA内基质间隔器的配对能力不匹配。 效果 整个垫片的不同位置的不匹配应反映 配对的实际机制。 例如，如果不匹配 在整个间隔者中，这表明该同源 配对是过程的一部分，而局部效果会 指出特定地点重组的参与，通过 DNA结合蛋白或通过RNA或DNA桥之间的相互作用，取决于 关于关键序列的性质和位置。