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使用生物素标记的探针通过原位杂交将核糖体基因定位到秀丽隐杆线虫染色体上。

Localization of the ribosomal genes in Caenorhabditis elegans chromosomes by in situ hybridization using biotin-labeled probes.

作者信息

Albertson D G

出版信息

EMBO J. 1984 Jun;3(6):1227-34. doi: 10.1002/j.1460-2075.1984.tb01957.x.

Abstract

The site of the ribosomal gene cluster on embryonic metaphase chromosomes of Caenorhabditis elegans has been mapped by in situ hybridization using probe DNAs that have been nick-translated to incorporate biotin-labeled UTP. The hybridized probe DNA was detected by a double-layer fluorescent antibody technique. Since chromosomes from wild-type C. elegans embryos are indistinguishable, in situ hybridization was carried out with chromosomes from C. elegans strains carrying cytologically distinct translocation or duplication chromosomes in order to identify the right end of linkage group I as the site of the ribosomal genes. Chromosomes carrying a lethal mutation, let-209 I displayed smaller hybridization signals than wild-type, suggesting that these chromosomes carried a partial deficiency of the ribosomal gene cluster. A duplication of the ribosomal genes, eDp20(I;II) rescued let-209 homozygotes. Chromosomes carrying the alterations in the ribosomal genes were combined with mnT12(IV;X) to facilitate the mapping of genes in C. elegans by in situ hybridization. Linkage groups I and II are then labeled by the distinctive hybridization signals from the ribosomal probes, linkage groups IV and X are together distinguishable morphologically and linkage group V is labeled by hybridization to a 5S gene probe.

摘要

利用已进行缺口平移以掺入生物素标记的尿苷三磷酸(UTP)的探针DNA,通过原位杂交技术对秀丽隐杆线虫胚胎中期染色体上核糖体基因簇的位点进行了定位。杂交的探针DNA通过双层荧光抗体技术进行检测。由于野生型秀丽隐杆线虫胚胎的染色体无法区分,因此对携带细胞学上不同的易位或重复染色体的秀丽隐杆线虫菌株的染色体进行原位杂交,以确定连锁群I的右端为核糖体基因的位点。携带致死突变let-209的染色体I显示出比野生型更小的杂交信号,表明这些染色体携带核糖体基因簇的部分缺失。核糖体基因的重复eDp20(I;II)挽救了let-209纯合子。将携带核糖体基因改变的染色体与mnT12(IV;X)组合,以通过原位杂交促进秀丽隐杆线虫中基因的定位。然后,连锁群I和II通过核糖体探针独特的杂交信号进行标记,连锁群IV和X在形态上一起可区分,连锁群V通过与5S基因探针杂交进行标记。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e4bd/557503/f4099c0079f3/emboj00310-0023-a.jpg

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