Popp S, Scholl H P, Loos P, Jauch A, Stelzer E, Cremer C, Cremer T
Institute of Human Genetics and Anthropology, University of Heidelberg, Federal Republic of Germany.
Exp Cell Res. 1990 Jul;189(1):1-12. doi: 10.1016/0014-4827(90)90249-a.
In situ hybridization of human chromosome 18 and X-specific alphoid DNA-probes was performed in combination with three dimensional (3D) and two dimensional (2D) image analysis to study the interphase distribution of the centric heterochromatin (18c and Xc) of these chromosomes in cultured human cells. 3D analyses of 18c targets using confocal laser scanning microscopy indicated a nonrandom disposition in 73 amniotic fluid cell nuclei. The shape of these nuclei resembled rather flat cylinders or ellipsoids and targets were preferentially arranged in a domain around the nuclear center, but close to or associated with the nuclear envelope. Within this domain, however, positionings of the two targets occurred independently from each other, i.e., the two targets were observed with similar frequencies at the same (upper or lower) side of the nuclear envelope as those on opposite sides. This result strongly argues against any permanent homologous association of 18c. A 2D analytical approach was used for the rapid evaluation of 18c positions in over 4000 interphase nuclei from normal male and female individuals, as well as individuals with trisomy 18 and Bloom's syndrome. In addition to epithelially derived amniotic fluid cells, investigated cell types included in vitro cultivated fibroblastoid cells established from fetal lung tissue and skin-derived fibroblasts. In agreement with the above 3D observations 18c targets were found significantly closer (P less than 0.01) to the center of the 2D nuclear image (CNI) and to each other in all these cultures compared to a random distribution derived from corresponding ellipsoid or cylinder model nuclei. For comparison, a chromosome X-specific alphoid DNA probe was used to investigate the 2D distribution of chromosome X centric heterochromatin in the same cell types. Two dimensional Xc-Xc and Xc-CNI distances fit a random distribution in diploid normal and Bloom's syndrome nuclei, as well as in nuclei with trisomy X. The different distributions of 18c and Xc targets were confirmed by the simultaneous staining of these targets in different colors within individual nuclei using a double in situ hybridization approach.
采用人18号染色体和X染色体特异性α卫星DNA探针原位杂交技术，并结合三维（3D）和二维（2D）图像分析，研究这些染色体的着丝粒异染色质（18c和Xc）在培养的人细胞间期的分布。使用共聚焦激光扫描显微镜对18c靶点进行的3D分析表明，在73个羊水细胞核中存在非随机分布。这些细胞核的形状类似于相当扁平的圆柱体或椭球体，靶点优先排列在围绕核中心的区域，但靠近或与核膜相关联。然而，在这个区域内，两个靶点的定位相互独立，即观察到两个靶点在核膜同一侧（上侧或下侧）的频率与在相对侧的频率相似。这一结果有力地反驳了18c存在任何永久性同源关联的观点。采用二维分析方法快速评估了正常男性和女性个体以及18三体和布卢姆综合征个体的4000多个间期核中18c的位置。除了上皮来源的羊水细胞外，研究的细胞类型还包括从胎儿肺组织建立的体外培养的成纤维样细胞和皮肤来源的成纤维细胞。与上述3D观察结果一致，在所有这些培养物中，与从相应的椭球体或圆柱体模型核得出的随机分布相比，发现18c靶点明显更靠近二维核图像（CNI）的中心且彼此更靠近（P小于0.01）。为作比较，使用X染色体特异性α卫星DNA探针研究了相同细胞类型中X染色体着丝粒异染色质的二维分布。在二倍体正常核、布卢姆综合征核以及X三体核中，二维Xc - Xc和Xc - CNI距离符合随机分布。通过使用双重原位杂交方法在单个核内以不同颜色同时对这些靶点进行染色，证实了18c和Xc靶点的不同分布。
