Mutations in the mariner transposase: The D,D(35)E consensus sequence is nonfunctional
Mutations in the mariner transposase: The D,D(35)E consensus sequence is nonfunctional
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DOI：
10.1073/pnas.94.4.1293
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发表时间：
1997-02-18
影响因子：
11.1
通讯作者：
Hartl, DL
中科院分区：
文献类型：
--
作者：
Lohe, AR;DeAguiar, D;Hartl, DL
真核生物转座酶的遗传分析以及与原核生物转座酶的比较由于分离突变的困难而受到很大阻碍。我们描述了一个简单的眼睛的颜色屏幕，有利于分离和分析果蝇的水手转座酶的突变。使用甲磺酸乙酯和定点诱变已经确定了18个残基，这些残基对于靶mariner元件的体内切除是关键的。当在杂合突变/非突变基因型中检查突变时，发现超过一半的突变转座酶蛋白质降低野生型转座酶的活性，如通过靶元件的生殖系切除的频率所测定的。值得注意的是，当D，D（34）D酸性离子结合结构域被线虫Tc 1转座酶和超家族中许多其他转座酶中发现的共有序列D，D（34）E取代时，转座酶功能消失。许多突变以显性负性方式强烈互补野生型转座酶，提示切除反应中的亚基相互作用;这些突变位于包括D，D（34）D基序的一部分的小区域中。转座酶的功能也会被推断的起始密码子的突变和推定的核定位信号的突变所消除。
Genetic analysis of eukaryote transposases and comparison with their prokaryote counterparts have been greatly hindered by difficulty in isolating mutations. We describe a simple eye-color screen that facilitates isolation and analysis of mutations in the mariner transposase in Drosophila melanogaster. Use of ethyl methanesulfonate and site-directed mutagenesis has identified 18 residues that are critical for in vivo excision of a target mariner element. When the mutations were examined in heterozygous mutant/nonmutant genotypes, more than half of the mutant transposase proteins were found to reduce the activity of the wild-type transposase, as assayed by the frequency of germline excision of a target element. Remarkably, transposase function is obliterated when the D,D(34)D acidic, ion-binding domain is replaced with the consensus sequence D,D(34)E found in the nematode Tc1 transposase and in many other transposases in the superfamily. A number of mutations strongly complement wild-type transposase in a dominant-negative manner, suggestive of subunit interactions in the excision reaction; these mutations are located in a small region that includes part of the D,D(34)D motif. Transposase function also is eliminated by a mutation in the inferred initiation codon and by a mutation in a putative nuclear localization signal.