Bischoff A, Albers J, Kharboush I, Stelzer E, Cremer T, Cremer C
Institute of Applied Physics, University of Heidelberg, Federal Republic of Germany.
Microsc Res Tech. 1993 May 1;25(1):68-77. doi: 10.1002/jemt.1070250110.
It is a widely held belief that the inactive X-chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X-chromosome (Xa). We have reconsidered this problem and painted X-chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46,XX and 46,XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X-chromosome specific library DNA. FITC-conjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two-dimensional (2D) and three-dimensional (3D) analyses. 2D analyses of Xi- and Xa-domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI-stained nuclei prior to CISS-hybridization was confirmed by its colocalization with one of the two painted X-domains. In 31 of the 34 nuclei the area AXi for the inactive X-domain was smaller than the area AXa for the active domain (mean ratio AXa/AXi = 1.9 +/- 0.8 SD, range 1.0-4.3). The signed rank test showed a highly significant (P < .0001) difference both between AXa and AXi and between the ratios r(Xa) and r(Xi), calculated by dividing the maximum length L of each X-domain by its maximum width W. In most nuclei (26/34) we found r(Xa) > r(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI-image, FITC-image) were registered with equal spacings (approx. 0.4 microns). A thresholding procedure was applied to determine the PI-labeled nuclear and FITC-labeled X-domain areas in each section. Estimated slice volumes were used to compute total nuclear and X-domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X-chromosome domains comprised (3.7 +/- 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 +/- 0.2 SD (range 1.1-2.3) was found for the volumes of the larger and the smaller X-domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X-chromosome domain volume comprised (4.0 +/- 2.6 S.D.)%.(ABSTRACT TRUNCATED AT 400 WORDS)
人们普遍认为，与大多处于解聚状态的活性X染色体（Xa）相比，雌性细胞核中的失活X染色体（Xi）高度浓缩。我们重新审视了这个问题，并通过与生物素化的人类X染色体特异性文库DNA进行染色体原位抑制（CISS）杂交，对亚汇合状态的雌性和雄性人类羊水细胞培养物（46,XX和46,XY）细胞核中的X染色体区域进行了描绘。使用异硫氰酸荧光素（FITC）偶联的抗生物素蛋白进行探针检测，并用碘化丙啶（PI）对细胞核进行复染。这些形状类似扁平椭球体或椭圆圆柱体的细胞核适用于二维（2D）和三维（3D）分析。通过使用明视描绘器勾勒出描绘区域，对34个雌性细胞核中的Xi和Xa区域进行了二维分析。在CISS杂交之前，通过其与两个描绘的X区域之一的共定位，证实了在DAPI染色的细胞核中性染色质体的识别。在34个细胞核中的31个中，失活X区域的面积AXi小于活性区域的面积AXa（平均比率AXa/AXi = 1.9 +/- 0.8标准差，范围1.0 - 4.3）。符号秩检验显示AXa与AXi之间以及通过将每个X区域的最大长度L除以其最大宽度W计算得到的比率r(Xa)和r(Xi)之间存在高度显著（P <.0001）差异。在大多数细胞核（26/34）中，我们发现r(Xa) > r(Xi)，表明Xa通常具有更细长的结构。对于三维分析，使用了共聚焦扫描激光荧光显微镜（CSLFM）。以相等间距（约0.4微米）记录10至20个光学切片（PI图像、FITC图像）。应用阈值化程序确定每个切片中PI标记的细胞核和FITC标记的X区域面积。估计的切片体积用于计算细胞核和X区域的总体积。在一系列35个雌性细胞核中，大多数区域从细胞核的顶部延伸到底部切片。两个X染色体区域中较大的那个占细胞核体积（%）（3.7 +/- 1.7标准差）。在这些雌性细胞核中，较大和较小X区域的体积平均比率为1.2 +/- 0.2标准差（范围1.1 - 2.3）。在一系列27个雄性羊水细胞核中，相对X染色体区域体积占（%）（4.0 +/- 2.6标准差）。（摘要截断于400字）
