Heterochromatic features of an 11-megabase transgene in brain cells.
Heterochromatic features of an 11-megabase transgene in brain cells.
复制标题
DOI：
10.1073/pnas.88.3.1049
复制
发表时间：
1991-02
影响因子：
11.1
通讯作者：
L. Manuelidis
中科院分区：
文献类型：
--
作者：
L. Manuelidis
转基因小鼠为核结构的研究提供了非凡的实验环境。通过使用高分辨率原位杂交，可以方便地跟踪小鼠基因组内外源 DNA 的三维定位和精细结构。具有特定特征（例如碱基偏倚、序列基序和大小）设计的外源 DNA 可以稳定地整合到宿主染色体上的有限位置。因此，可以在不同的细胞类型中评估这些特征中的每一个在确定转基因的三维核位置和详细形态方面的相对重要性。本研究的目的是评估具有可能有助于间期细胞核中异染色质从头形成的序列特征的转基因。在成年脑细胞中确定了表型沉默的 11 兆碱基转基因的结构，该转基因包含整合到小鼠 3 号染色体同源物的周粒区域中的 β-珠蛋白-pBR 序列的串联重复序列。大部分为常染色质的神经元在转基因位置的三维研究中尤其具有丰富的信息。这两个转基因位点的行为很像来自单个染色体的、长度相当的富含着丝粒或旁着丝粒A+T的卫星DNA。一个或两个转基因结构域与着丝粒卫星 DNA 一起定位在核仁上。对于不包含大量组成型异染色质卫星 DNA 的端粒或染色体臂区域来说，这是一个不寻常的核位置。 G + C 丰富度并没有阻止这些区域组装成体积约为 1 微米的致密异色体。从超微结构上看，转基因结构域通常与组成型异染色质紧密相连，并且高度浓缩。标记的超螺旋由大约 250 nm 宽的离散纤维形成，在穿过域中心的倾斜薄片中观察到。结构数据与该基因座检测到的可忽略不计的转录活性以及组成型异染色质的预测构象一致。有趣的是，在转基因小鼠而非对照小鼠中，大量大神经元，包括大约 30% 的小脑浦肯野细胞，显示出核膜过度内陷。
Transgenic mice provide a remarkable experimental setting for the study of nuclear architecture. The three-dimensional localization and fine structure of a foreign DNA within the mouse genome can be conveniently followed by using high-resolution in situ hybridization. Foreign DNAs designed with specific characteristics, such as base bias, sequence motif(s), and size can stably integrate into finite positions on host chromosomes. Thus the relative importance of each of these characteristics in determining the three-dimensional nuclear position and the detailed morphology of the transgene can be evaluated in different cell types. The aim of this study was to evaluate a transgene with sequence characteristics that might contribute to the de novo formation of heterochromatin in interphase nuclei. The structure of a phenotypically silent 11-megabase transgene, containing tandem repeats of beta-globin-pBR sequences integrated into the peritelomeric region of both mouse chromosome 3 homologs, was determined in adult brain cells. Neurons that are largely euchromatic were especially informative in three-dimensional studies of transgene position. The two transgenic loci behaved much like centromeric or paracentromeric A + T-rich satellite DNAs of comparable length from a single chromosome; one or both transgene domains localized together with centromeric satellite DNA on the nucleolus. This is an unusual nuclear position for a telomeric or chromosome arm region that does not contain a substantial amount of constitutively heterochromatic satellite DNA. G + C richness did not prevent these regions from assembling as dense heterochromatic bodies of approximately 1 micron3 in volume. Ultrastructurally, transgenic domains were often intimately connected with constitutive heterochromatin and were highly condensed. Labeled supercoils, formed by a discrete approximately 250-nm-wide fiber, were observed in oblique thin sections through the center of the domain. The structural data were consistent with negligible transcriptional activity detected for this locus, as well as the predicted conformation of constitutive heterochromatin. Interestingly, in transgenic but not control mice, a substantial number of large neurons, including approximately 30% of cerebellar Purkinje cells, showed excessive invaginations of the nuclear membrane.