Meiotic Kinetochores of Maize

玉米减数分裂动粒

• 批准号: 9513556
• 负责人: R Kelly Dawe
• 金额: $ 11.8万
• 依托单位: University of Georgia Research Foundation Inc
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9513556&HistoricalAwards=false
• 关键词: Meiotic; Kinetochores; Maize

9513556 Dawe The long term goal of this study is to identify the structural and molecular properties of the kinetochore that ensure normal meiotic chromosome segregation. Kinetochores have a variety of critical roles in cell division, such as ensuring that the chromatids do not separate prematurely, and acting as the biological motors that pull the chromosomes along the spindle. In meiosis, the kinetochores have more specific roles that relate to their critical function in ensuring that the genetic information is transmitted accurately from generation to generation. Maize is an excellent organism for studying kinetochores, because of the unusually large size of the chromosomes and of the kinetochores themselves. In prior studies, advanced three dimensional light microscmpy has been used to identify subtle structural features, first in the chromosomes and more recently in the kinetochores using a human antiserum that recognizes maize meiotic kinetochores. The major cell biological advantages of maize are coupled with extensive genetic resources with which to analyze kinetochore function. One such resource is an unusual variant of chromosome 10 that induces facultative centromeres ("neocentromeres") as a part of a more complex meiotic drive system. In the presence of Abnormal chromosome 10, quiescent heterochromatic regions called "knobs" are converted into meiotic kinetochores that stretch the chromosome arms towards the spindle poles. Neocentromeres and true centromeres are probably closely related, because knobs have strong sequence similarity to true centromeres. However, unlike true centromeres, mutations of neocentromere formation can be readily identified. The method for identifying neocentromeres relies on the fact that their formation is closely associated with a preferential segregation, or meiotic drive, of genetic markers that are linked to knobs. If the genetic marker affects kernel pigmentation, meiotic drive is visible as an excess of pigmented kernels. In prior stud ies, a meiotic drive mutation called Ab10-smd1 was identified, which proved to be a defect in neocentromere formation. This proposal contains experiments leading to the cloning and characterization of Ab10-smd1 by virtue of the fact that the mutation was induced by a transposable element. It should be possible to identify the genes that regulate normal meiotic kinetochore behavior by homology to Ab10-smd1 and other genes like it. Additional studies are proposed that relate to how neocentromeres promote meiotic drive, and how normal kinetochores direct meiotic chromosome segregation. It will be possible to verify a long-standing model for how neocentromeres lead to meiotic drive by directly labeling knobs with fluorescent oligonucleotides. Labeling the knobs as well as the kinetochores and spindle using specific antibodies will make it possible to study the interaction of these three components during chromosome segregation. Finally, experiments are proposed to analyze neocentromere behavior using time lapse three dimensional light microscopy. The conspicuous nature of the neocentromeres coupled with a powerful new light microscope workstation will make it possible to study meiotic kinetochores in the living state. Such time lapse microscopy will also provide an added level of resolution to the characterization of neocentromere mutations. ***

9513556 DAWE这项研究的长期目标是确定动力学的结构和分子特性，以确保正常的减数分裂染色体分离。 动力学在细胞分裂中具有多种关键作用，例如确保染色单体不会过早分离，并且充当沿纺锤体拉动染色体的生物电机。 在减数分裂中，动力学具有更具体的作用，这些作用与它们的关键功能有关，以确保遗传信息能够准确地产生。 玉米是研究动力学的出色生物，因为染色体的大小异常大和动物学本身。 在先前的研究中，已使用先进的三维光显微镜用于识别微妙的结构特征，首先是在染色体中，最近在动力学中使用人类抗血清，该抗血清识别玉米减数分裂动物学。 玉米的主要细胞生物学优势与广泛的遗传资源相结合，可以分析动力学功能。 这样的资源之一是染色体10的不寻常变体，该变体诱导了兼性的centromeres（“ Neocentromeres”）作为更复杂的减数分裂驱动系统的一部分。 在异常染色体10的情况下，称为“旋钮”的静态异质区域被转化为减数分裂动物学，将染色体臂伸向纺锤形杆。 Neopertromeres和真实的中心粒可能密切相关，因为旋钮与真实的共晶粒具有很强的序列相似性。 但是，与真实的中心粒不同，可以很容易地识别出新中心粒形成的突变。 鉴定新中心粒子的方法依赖于它们的形成与与旋钮相关的遗传标记的优先隔离或减数分裂驱动密切相关的事实。 如果遗传标记会影响核色素沉着，则可以看到减数分裂驱动，因为多余的核。 在先前的研究中，确定了一个称为AB10-SMD1的减数分裂驱动突变，事实证明是新中心粒形成的缺陷。 该建议包含实验，从而导致AB10-SMD1的克隆和表征，这是因为突变是由可转座元件诱导的。 应该可以通过与AB10-SMD1及其其他基因同源性来识别调节正常减数分裂动力学行为的基因。 提出了其他研究，该研究与新中心粒促进减数分裂驱动以及动力学如何直接减数分裂染色体分离有关。 可以通过直接用荧光寡核苷酸标记旋钮来验证新中心粒如何导致减数分裂驱动的长期模型。 使用特定抗体标记旋钮以及动力学和主轴，将使研究在染色体分离过程中研究这三个成分的相互作用。 最后，提出了实验，以使用延时三维光学显微镜分析新中心粒行为。 Necentromeres的显着性，再加上强大的新光显微镜工作站，将使在生活状态下研究减数分裂动物学。 这种时间流逝显微镜还将为新中心粒突变的表征提供增加的分辨率。 ***