Meiotic Kinetochores of Maize

小行星9513556 本研究的长期目标是确定确保正常减数分裂染色体分离的动粒的结构和分子特性。 着丝粒在细胞分裂中有着多种重要作用，例如确保染色单体不会过早分离，以及充当生物马达，沿着纺锤体沿着染色体。 在减数分裂中，动粒具有更具体的作用，这些作用与其确保遗传信息准确地代代相传的关键功能有关。 玉米是研究动粒的极好的生物，因为它的染色体和动粒本身都非常大。 在以前的研究中，先进的三维光显微镜已被用来确定微妙的结构特征，首先在染色体和最近在动粒使用人抗血清，识别玉米减数分裂动粒。 玉米的细胞生物学优势与广泛的遗传资源相结合，可以用来分析动粒功能。 其中一个这样的资源是10号染色体的一种不寻常的变体，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。 当存在异常10号染色体时，被称为“球”的静止异染色质区域被转化为减数分裂动粒，这些动粒将染色体臂向纺锤体两极伸展。 新着丝粒和真正的着丝粒可能是密切相关的，因为球有很强的序列相似性，真正的着丝粒。 然而，与真正的着丝粒不同，新着丝粒形成的突变可以容易地鉴定。 鉴定新着丝粒的方法依赖于这样一个事实，即它们的形成与与结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色素沉着，则减数分裂驱动可以作为过量的色素沉着籽粒而可见。 在先前的研究中，发现了一个称为Ab 10-smd 1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。 该提案包含导致Ab 10-smd 1的克隆和表征的实验，这是由于突变是由转座因子诱导的。 通过与Ab 10-smd 1和其他类似基因的同源性，应该有可能鉴定出调节正常减数分裂动粒行为的基因，并进一步研究新着丝粒如何促进减数分裂驱动，以及正常动粒如何指导减数分裂染色体分离。 这将是可能的，以验证一个长期存在的模型，新着丝粒如何导致减数分裂驱动直接标记旋钮与荧光寡核苷酸。 用特异性抗体标记球以及动粒和纺锤体，将使研究染色体分离过程中这三个组分的相互作用成为可能。 最后，实验提出了分析新着丝粒的行为，使用时间推移三维光学显微镜。 新着丝粒的引人注目的性质，加上一个强大的新的光学显微镜工作站将有可能在生活状态下研究减数分裂动粒。 这种延时显微镜也将为新着丝粒突变的表征提供额外水平的分辨率。 ***

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. ***