在整个果蝇胚胎中进行FISH。RNA:转录基因座的激活,DNA:基因结构和表达

Mark J Gemkow, Peter Buchenau, Donna J Arndt-Jovin
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引用次数: 8

摘要

通过与RNA和DNA序列杂交的例子,我们证明了整个果蝇胚胎是通过FISH和共聚焦激光扫描显微镜相结合来研究核结构和功能问题的优秀对象。荧光标记的29个碱基的寡核苷酸用于探测已知在热休克时强烈诱导的基因座的转录。该基因座的转录物显然起到稳定和保护核蛋白的作用,而核蛋白可能是热或化学应激后核过程所需的。我们发现,在正常生长条件下,只有不到5%的蛋白质与转录物共定位,但在热休克期间,超过50%的蛋白质被转录物螯合。双胸复合体中基因的DNA探针用于检测同源配对与晚期原肠胚中基因表达之间的关系。通过分析P1载体中克隆的探针在整个原肠胚形成过程中在胚胎中的分布,我们可以得出结论,极化的核组织在胚细胞阶段后分解。双胸复合体基因的同源配对在原肠胚形成过程中进行,因此在胚带收缩时,两个等位基因总是非常接近,而与基因的表达或沿着身体前后轴的区域无关。最后,我们证明,通过酪酰胺荧光团沉积增强FISH信号,可以在整个胚胎中观察到较小的DNA靶标。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
FISH in whole-mount Drosophila embryos. RNA: activation of a transcriptional locus, DNA: gene architecture and expression

Using examples of hybridization both to RNA and to DNA sequences we demonstrate that whole-mount Drosophila embryos are excellent objects with which to study questions about nuclear structure and function by combining FISH and confocal laser scanning microscopy. A fluorescently labelled 29-base oligonucleotide was used to probe transcription at a locus known to be strongly induced upon heat shock. The transcript from this locus apparently serves to stabilize and protect nuclear proteins which may be needed for nuclear processes after heat or chemical stress. We found that less than 5% of the protein is colocalized with the transcript under normal growth conditions, but more than 50% is sequestered by the transcript during heat shock. DNA probes to genes in the bithorax complex were used to examine the relationship between homologous pairing and gene expression in late stage gastrulating embryos. Analysis of the disposition of probes cloned in P1 vectors in embryos from mid-blastoderm throughout gastrulation allowed us to conclude that polarized nuclear organization breaks down after the blastoderm stage. Homologous pairing of the bithorax complex genes proceeds during gastrulation so that at the time of germ band retraction the two alleles are always in close proximity independent of expression of the gene or the region along the anterior–posterior axis of the body. Finally, we demonstrate that smaller DNA targets can be visualized in whole mount embryos by enhancement of the FISH signal by tyramide-fluorophore deposition.

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