Otobe Kazunori, Shichiri Motoharu, Fukushi Daisuke, Yoshino Tomoyuki, Nakao Hidenobu, Sugiyama Shigeru, Ohtani Toshio
Department of Farm Mechanization and Engineering, National Agricultural Research Center, Tsukuba, Ibaraki, Japan.
Arch Histol Cytol. 2002 Dec;65(5):457-63. doi: 10.1679/aohc.65.457.
The present study investigated the mechanical elongation of the centromere in the barley chromosomes by a microneedle manipulation method for the structural analysis of the chromosomes. Chromosomes were extracted from barley root cells, affixed on a cover slip by a standard preparation method, and elongated in either distilled water, phosphate buffered saline (PBS), or 2 x sodium saline citrate (SSC). The mechanical property of the chromosome elongation was assessed by the measurement of the force required for the elongation of chromosomes. This assessment has shown that the chromosomes in distilled water were much firmer than those in the PBS or 2 x SSC. To confirm the elongation of the centromere, the elongated chromosomes were investigated by fluorescence in situ hybridization with a centromere probe. The fluorescence information indicated that the extent of the loosening of the centromere during elongation differed depending on the buffers used; the centromere elongated in 2 x SSC was more loosened than that in the PBS. Atomic force microscopy also revealed the structure of the unpacked centromere after the mechanical elongation, when rows of fibrous structures about 30 to 50 nm thick were clearly observed in the centromere elongated in 2 x SSC. The investigation of elongated chromosomes should prove useful for an understanding of the structural analysis of chromosomes.
本研究采用微针操作方法对大麦染色体着丝粒的机械伸长进行了研究，以用于染色体的结构分析。从大麦根细胞中提取染色体，通过标准制备方法固定在盖玻片上，并在蒸馏水、磷酸盐缓冲盐水（PBS）或2×柠檬酸钠盐水（SSC）中进行伸长。通过测量染色体伸长所需的力来评估染色体伸长的力学性质。该评估表明，蒸馏水中的染色体比PBS或2×SSC中的染色体更硬。为了确认着丝粒的伸长，用着丝粒探针通过荧光原位杂交对伸长后的染色体进行研究。荧光信息表明，伸长过程中着丝粒的松弛程度因所用缓冲液而异；在2×SSC中伸长的着丝粒比在PBS中伸长的着丝粒更松弛。原子力显微镜还揭示了机械伸长后解压缩着丝粒的结构，在2×SSC中伸长的着丝粒中清晰地观察到了约30至50纳米厚的纤维结构行。对伸长染色体的研究应有助于理解染色体的结构分析。
