Meiotic Kinetochores of Maize

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着丝点的结构和分子特性。着丝点在细胞分裂过程中具有多种关键作用，例如确保染色单体不过早分离，以及充当染色体沿纺锤体移动的生物马达。在减数分裂中，着丝点有更具体的作用，这与其确保遗传信息准确地代代相传的关键功能有关。玉米是研究着丝点的极好生物，因为它的染色体和着丝点本身都非常大。在先前的研究中，先进的三维光学显微镜已被用于识别细微的结构特征，首先是在染色体中，最近使用人类抗血清识别玉米减数分裂着丝点。玉米的主要细胞生物学优势与广泛的遗传资源相结合，可用于分析着丝点功能。其中一种资源是10号染色体的不寻常变异，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。在异常的10号染色体存在时，称为“旋钮”的静止异色区域被转化为减数分裂着丝点，使染色体臂向纺锤极伸展。新着丝粒和真着丝粒可能有密切的关系，因为旋钮与真着丝粒具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可以很容易地识别。鉴定新着丝粒的方法依赖于这样一个事实，即它们的形成与与旋钮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，减数分裂驱动表现为籽粒色素沉着过剩。在先前的研究中，发现了一种称为Ab10-smd1的减数分裂驱动突变，这被证明是新着丝粒形成中的缺陷。由于Ab10-smd1突变是由转座因子诱导的，因此本研究包含了克隆和鉴定Ab10-smd1的实验。通过与Ab10-smd1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着丝粒行为的基因。新的着丝粒如何促进减数分裂驱动，以及正常着丝点如何直接减数分裂染色体分离的研究被提出。这将有可能验证一个长期存在的模型，即新中心粒如何通过荧光寡核苷酸直接标记旋钮来导致减数分裂驱动。利用特异性抗体标记这些旋钮以及着丝点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。新着丝粒的显著性与强大的新型光学显微镜工作站相结合，将使研究活态减数分裂着丝点成为可能。这种延时显微镜也将为新着丝粒突变的表征提供更高的分辨率。＊＊＊

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. ***