The final specimens were obtained after washing and centrifuging. The isolated interphase nuclei and metaphase chromosomes were suspended in Tris buffer (2m M Tris-HCl, 50 mM NaCl, 10 mM MgCl 2, 1 mM PMSF, pH 7.4), digested with 200 μg/ml DNase I at 37 ☌ for 30 min and disposed with 2 M NaCl at room temperature for 20 min. The procedure of isolating chromosomes was as follows: the synchronized plasmodia were monitored with light microscope at regular intervals, and the plasmodia at metaphase was put into the isolation solution precooled in ice-water metaphase nuclei were isolated according to the method of Mohberg and Rusch 22, the isolated metaphase nuclei were resuspended in the isolation solution, disposed twice with ultrasonic waves (each for 30 sec), centrifuged at 3000 g for 20 min, and chromosomes were obtained by purifying the sediments. Nuclei were isolated from the plasmodia of suspension culture referred to the method of Mohberg and Rusch 22. Preparations of nuclear matrix and chromosome scaffold Philipe Albert, Cytobiology Laboratory of Reims University, France, and the culture method was referred to Daniel and Baldwin 21. In order to gain a clear idea whether tropomyosin exists in the nuclear matrix and chromosome scaffold, we analysed the protein composition of the nuclear matrix and chromosome scaffold of P.polycephalum with SDS-PAGE and localized tropomyosin in the nuclear matrix and chromosome scaffold with immunodotting, immunofluorescence and immunoelectron microscopy. Some previous biochemical analyses indicated that a polypeptide of about 37 kD, which is equivalent to tropomyosin in molecular weight, existed in the nuclear matrix and chromosome scaffold 15, 17, 18, 19, 20 and this polypeptide might represent tropomyosin 19.
Although many biochemical and immunocytochemical analyses have been conducted concerning protein composition of the nuclear matrix and chromosome scaffold, only a few proteins (such as topoisomerase II) have been identified 11, 12, 13, 14, 15, 16. The main component of nuclear matrix and chromosome scaffold is nonhistone proteins 1, 2, 3, 4, 10. These two structures are dynamic during the cell cycle and play important roles in various activities essential for the cell life 7, 8, 9. All rights reserved.The nuclear matrix and chromosome scaffold are the residual fibrillar network structures of the nucleus and chromosome which are depleted of DNA, histone and most of nonhistone proteins 1, 2, 3, 4, 5, 6. Using the locations of gold nanoparticles to visualize the underlying structure, the tomograms we obtained reveal the patterns of chromosome scaffold organization, which appears to consist of a helical structure that serves to organize chromatin loops into the metaphase chromosome.Ĭhromosome scaffold Condensin Electron tomography Immuno-electroscopy.Ĭrown Copyright © 2019. The chromosome was stained with immunogold-labeled condensin complex, one of the major chromosome scaffold proteins and then observed in three dimensions using ET. Here we present a new technique that enables the observation of the chromosome scaffold structure in metaphase chromosomes from any direction, by transferring an isolated chromosome to a 360° rotational holder for electron tomography (ET). However, it remains to be elucidated how the chromosome scaffold organizes the mitotic chromosome and how it supports shaping the structure of the chromosome during metaphase. It plays a vital role in chromosome condensation, shaping the X-shaped structure of the mitotic chromosome, and also provides flexibility for chromosome movement during cell division. The chromosome scaffold is considered to be a key structure of the mitotic chromosome.
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