Analysis of Chromosome Behavior of Arabidopsis Mutants Defective in Reproductive Processes

From Arabidopsis mutant collections, more than 30 meiotic mutants have been isolated. However, the molecular mechanism of plant meiosis is still largely obscure. For the purpose of further understanding, we searched for new Arabidopsis meiotic mutants. As a results of our collaboration, we found several new mutants, which were defective in reproductive processes. Since our main interest was in meiosis, we selected one meiotic mutant among them, and analyzed its chromosome behavior during meiosis

Analysis of Chromosome Behavior of Arabidopsis Mutants Defective in Reproductive Processes II

In a previous work, we showed that a mutation in AtSPO11-2 resulted in the production of abnormal sterile pollens and that AtSPO11-2 protein was required for meiotic homologous recombination. Atspoll-2 mutant was used with other meiotic mutants to examine the regulatory system of centromere roles during meiosis in this research. Yeast and Arabidopsis centromeres have been shown to couple with each other at early prophase I to promote homolog pairing, and to direct the polarity of sister chromatids at metaphase I. The present research revealed that centromere coupling at early prophase I was independent of meiotic homologous recombination, but that the decision of sister centromere polarity was dependent on recombination

<所内学術研究成果報告>T. シロイヌナズナ花粉母細胞の減数分裂時の染色体に対する FISH 法の確立

染色体を個別に追跡しその挙動を調べることのできる方法,FISH法をシロイヌナズナの花粉母細胞の減数分裂時の染色体に適用することを目的に条件検討を行った。その結果,以下の条件でFISHを行うのが最良と結論された。花序の固定はカルノイ溶液(75%エタノール,25%酢酸)を用い,室温で16時間程度行う。細胞壁とカロース層の消化は0.4%Cellulase,0.4%Pectolyase,0.4%Cytohelicase,1370U/ml β-Glucronidaseを用いて37℃で3時間処理する。スライドガラス上に消化した細胞を展開した後,再度カルノイ溶液で固定する。0.1mg/ml RNaseAで37℃30分間,0.0025%ペプシンで37℃3分間,4%パラフォルムアルデヒドで室温10分間処理した後,エタノールシリーズを通し風乾させる。ビオチン標識したプローブDNAとスライドガラス上の染色体DNAと同時に72℃3分間熱変性させた後,37℃16時間反応させハイブリッドを形成させる。洗浄後,アビジン-FITCを反応させ,ビオチン化抗アビジンDを用いて,シグナルの増幅を行う。なお,本研究においてはBACクローンF10B6よりプローブを作成し用いた

Virus-Infection or 5′ppp-RNA Activates Antiviral Signal through Redistribution of IPS-1 Mediated by MFN1

In virus-infected cells, RIG-I-like receptor (RLR) recognizes cytoplasmic viral RNA and triggers innate immune responses including production of type I and III interferon (IFN) and the subsequent expression of IFN-inducible genes. Interferon-β promoter stimulator 1 (IPS-1, also known as MAVS, VISA and Cardif) is a downstream molecule of RLR and is expressed on the outer membrane of mitochondria. While it is known that the location of IPS-1 is essential to its function, its underlying mechanism is unknown. Our aim in this study was to delineate the function of mitochondria so as to identify more precisely its role in innate immunity. In doing so we discovered that viral infection as well as transfection with 5′ppp-RNA resulted in the redistribution of IPS-1 to form speckle-like aggregates in cells. We further found that Mitofusin 1 (MFN1), a key regulator of mitochondrial fusion and a protein associated with IPS-1 on the outer membrane of mitochondria, positively regulates RLR-mediated innate antiviral responses. Conversely, specific knockdown of MFN1 abrogates both the virus-induced redistribution of IPS-1 and IFN production. Our study suggests that mitochondria participate in the segregation of IPS-1 through their fusion processes

Analysis of Chromosome Dynamics during Meiosis I of Arabidopsis thaliana Pollen Mother Cells by Fluorescent In Situ Hybridization(FISH)

Since insertion mutagenesis methods, which enabled us to identify the mutagenized genes routinely, were developed for plants, Arabidopsis thaliana has been playing a central role in plant meiosis research. Though several techniques to analyze meiotic chromosome behavior have been introduced into Arabidopsis research since Ross et al. reported the method to observe male meiotic chromosomes of this plant through light microscope in 1996 (Chromosome Res. 4-507-516), intimate analysis of the chromosome behavior has not been accomplished. Taking advantage of the recent development of new nucleotides labeled with fluorescent dyes, we investigated chromosome behavior during male meiosis by multicolor FISH. Telomeres found around nucleoli in premeiotic interphase cells dispersed after entering meiosis, then clustered in a bouquet-like configuration. Statistically, telomeres of homologous chromosomes paired earlier than centromeres, but when respective chromosomes were examined, the telomeres were not always quick to pair. At early prophase I, possibly at around the zygotene stage, the signals from telomeres reduced to less than ten. This reduction suggests that the paired telomeres of homologous chromosomes temporally associate with other telomeres to look for their real partners. When homologous chromosomes separated at anaphase I, telomeres were always last to segregate. This suggested that there was unknown interaction between the telomeres of homologs, connecting them until anaphase I started

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization