Szuperák Milán, Zvara Agnes, Erdélyi Miklós
Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged.
Gene Expr Patterns. 2005 Jun;5(5):717-23. doi: 10.1016/j.modgep.2005.02.004. Epub 2005 Apr 9.
The development of embryonic germ cells in Drosophila depends on the germ plasm, the most posterior part of the ooplasm. The germ plasm is devoted to the formation of future germ cells and is known to contain all the factors that are necessary to induce germ cell fate. Besides having a characteristic organelle and protein distribution, the germ plasm also contains a large number of localized RNA species that have been shown to play crucial roles in germ cell determination. To identify germ plasm-enriched, localized transcripts, we used a two-step method composed of cDNA microarray (containing 3200 annotated Drosophila cDNAs) and in situ RNA hybridization techniques. We compared germ plasm deficient, normal and ectopic germ plasm conditions in the cDNA microarray experiments. RNA species whose concentration increased when ectopic germ plasm was present and decreased when the germ plasm was missing were selected. These candidates were then subjected to a second screen which compared the distribution of the given RNA in wild type embryos and in eggs with ectopic germ plasm. Finally, 17 RNA species were found to be enriched in the germ plasm. Based on these data, we estimate that around 1% of the Drosophila genes encode for germ plasm-enriched, localized transcripts. We conclude that this combination of microarray and in situ hybridization techniques is a simple but powerful experimental design for the genome-wide identification of genes coding for germ plasm localized transcripts.
果蝇胚胎生殖细胞的发育依赖于生殖质，即卵质最靠后的部分。生殖质致力于未来生殖细胞的形成，并且已知其包含诱导生殖细胞命运所需的所有因子。除了具有独特的细胞器和蛋白质分布外，生殖质还含有大量已被证明在生殖细胞决定中起关键作用的定位RNA种类。为了鉴定富集于生殖质的定位转录本，我们采用了一种两步法，该方法由cDNA微阵列（包含3200个注释的果蝇cDNA）和原位RNA杂交技术组成。在cDNA微阵列实验中，我们比较了生殖质缺陷、正常和异位生殖质的情况。选择那些在存在异位生殖质时浓度增加而在生殖质缺失时浓度降低的RNA种类。然后对这些候选物进行第二次筛选，比较给定RNA在野生型胚胎和具有异位生殖质的卵中的分布。最后，发现有17种RNA种类在生殖质中富集。基于这些数据，我们估计果蝇约1%的基因编码富集于生殖质的定位转录本。我们得出结论，这种微阵列和原位杂交技术的组合是一种简单但强大的实验设计，可用于全基因组鉴定编码生殖质定位转录本的基因。
