HO核酸内切酶增强拟南芥体细胞染色体内同源重组
Enhancement of somatic intrachromosomal homologous recombination in Arabidopsis by the HO endonuclease.
作者信息
Chiurazzi M, Ray A, Viret J F, Perera R, Wang X H, Lloyd A M, Signer E R
机构信息
Department of Biology, Massachusetts Institute of Technology, Cambridge 02138, USA.
出版信息
Plant Cell. 1996 Nov;8(11):2057-66. doi: 10.1105/tpc.8.11.2057.
The HO endonuclease promotes gene conversion between mating-type alleles in yeast by a DNA double-strand break at the site of conversion (the MAT-Y/Z site). As a first step toward understanding the molecular basis of homologous recombination in higher plants, we demonstrate that expression of HO in Arabidopsis enhances intrachromosomal recombination between inverted repeats of two defective beta-glucuronidase (gus) genes (GUS- test construct). One of these genes has the Y/Z site. The two genes share 2.5 kb of DNA sequence homology around the HO cut site. Somatic recombination between the two repeats was determined by using a histochemical assay of GUS activity. The frequency of Gus+ sectors in leaves of F1 plants from a cross between parents homozygous for the GUS- test construct and HO, respectively, was 10-fold higher than in F1 plants from a cross between the same plant containing the GUS- test construct and a wild-type parent. Polymerase chain reaction analysis showed restoration of the 5' end of the GUS gene in recombinant sectors. The induction of intrachromosomal gene conversion in Arabidopsis by HO reveals the general utility of site-specific DNA endonucleases in producing targeted homologous recombination in plant genomes.
HO核酸内切酶通过在转换位点（MAT-Y/Z位点）产生DNA双链断裂，促进酵母中交配型等位基因之间的基因转换。作为理解高等植物同源重组分子基础的第一步，我们证明在拟南芥中HO的表达增强了两个缺陷型β-葡萄糖醛酸酶（gus）基因（GUS测试构建体）的反向重复序列之间的染色体内重组。其中一个基因具有Y/Z位点。这两个基因在HO切割位点周围共享2.5 kb的DNA序列同源性。通过对GUS活性进行组织化学分析来确定两个重复序列之间的体细胞重组。分别对GUS测试构建体和HO纯合的亲本之间杂交产生的F1植株叶片中Gus+扇形区域的频率进行测定，结果显示，该频率比含有GUS测试构建体的同一植株与野生型亲本杂交产生的F1植株高10倍。聚合酶链反应分析表明，重组扇形区域中GUS基因的5'端得以恢复。HO在拟南芥中诱导染色体内基因转换，揭示了位点特异性DNA内切酶在植物基因组中产生靶向同源重组方面的普遍效用。
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