Meiotic Kinetochores of Maize

9513556我们这项研究的长期目标是确定动粒的结构和分子特性，以确保正常的减数分裂染色体分离。着丝点在细胞分裂中起着各种关键作用，例如确保染色单体不会过早分离，以及充当沿着纺锤体拉染色体的生物马达。在减数分裂过程中，动点具有更特殊的作用，这与它们在确保遗传信息在世代之间准确传递的关键功能有关。玉米是研究动点的极佳生物，因为它的染色体和动点本身都非常大。在以前的研究中，先进的三维光显微技术已经被用来识别精细的结构特征，首先是在染色体上，最近是在动粒中心，使用识别玉米减数分裂动粒的人抗血清。玉米的主要细胞生物学优势与丰富的遗传资源相结合，可以用来分析动粒功能。其中一个来源是10号染色体的一种不寻常的变异，它诱导兼性着丝粒(“新着丝粒”)，作为更复杂的减数分裂驱动系统的一部分。在10号染色体异常的情况下，静止的异染色区被称为“旋钮”，转化为减数分裂动点，将染色体的手臂向纺锤体极伸展。新着丝粒和真着丝粒可能是密切相关的，因为旋钮与真着丝粒有很强的序列相似性。然而，与真正的着丝粒不同，新着丝粒形成的突变很容易识别。识别新着丝粒的方法依赖于这样一个事实，即它们的形成与与节相关的遗传标记的优先分离或减数分裂驱动密切相关。如果遗传标记影响籽粒的色素形成，则减数分裂的驱动力表现为过量的着色籽粒。在先前的研究中，发现了一种名为AB10-smd1的减数分裂驱动突变，该突变被证明是新着丝粒形成的缺陷。这项提议包含的实验导致了AB10-smd1的克隆和鉴定，这是因为AB10-smd1的突变是由转座元件诱导的。通过与AB10-smd1和其他类似基因的同源性，应该可以识别调节正常减数分裂动粒行为的基因。关于新着丝粒如何促进减数分裂驱动，以及正常着丝点如何引导减数分裂染色体分离的其他研究也被提出。通过直接用荧光寡核苷酸标记旋钮，将有可能验证一个长期存在的模型，即新着丝粒如何导致减数分裂驱动。使用特定的抗体标记旋钮以及动粒和纺锤体，将使研究这三种成分在染色体分离过程中的相互作用成为可能。最后，提出了利用时间推移三维光学显微镜分析新着丝点行为的实验。新着丝粒的显著性质加上一台功能强大的新光学显微镜工作站，将使研究活体状态下的减数分裂动粒成为可能。这种延时显微镜还将为新着丝粒突变的表征提供更高水平的分辨率。***

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. ***