Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum.
Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum.
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DOI：
10.1016/j.funbio.2012.11.004
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发表时间：
2013-01
期刊：
影响因子：
2.5
通讯作者：
Rappleye, Chad A.
中科院分区：
文献类型：
--
作者：
Kemski, Megan M.;Stevens, Bryan;Rappleye, Chad A.
农杆菌介导的转化正越来越多地用于真菌的插入突变。为了更好地评估其作为真菌病原体荚膜组织胞浆菌诱变剂的有效性，我们分析了随机选择的T-DNA插入突变体的集合。对用于真菌转化的不同T-DNA元件载体的测试表明，pBHt 2提供了最高的转化效率和最低的载体骨架携带率。68个单独的T-DNA整合的特征在于T-DNA末端和侧翼基因组序列的恢复。T-DNA的右边界末端大部分被保留，而左边界末端经常被截短。T-DNA插入位点的分析证实了组织胞浆菌基因组中缺乏任何整合热点。相对于基因，T-DNA整合以编码序列为代价显示出对启动子区域的显著偏好。考虑到潜在的启动子中断和内含子插入的有效性，61%的定位T-DNA插入应该会损害基因表达或功能。T-DNA侧翼序列的定位表明，67%的T-DNA整合是在单个染色体位点的整合，31%的T-DNA整合与大规模的染色体重排有关。在不考虑表型的情况下选择的突变体中的T-DNA插入的这种表征支持将农杆菌介导的转化作为插入诱变剂用于组织胞浆菌中基因的基于基因组的筛选和功能发现。
Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border end of the T-DNA is largely preserved whereas the left border end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61% of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67% of T-DNA integrations are integrations at a single chromosomal site and 31% of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.
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影响因子：
4.3
作者：
Curtis MJ;Belcram K;Bollmann SR;Tominey CM;Hoffman PD;Mercier R;Hays JB
通讯作者：
Hays JB
影响因子：
7.2
作者：
Kononov, ME;Bassuner, B;Gelvin, SB
通讯作者：
Gelvin, SB
影响因子：
56.9
作者：
Alonso, JM;Stepanova, AN;Ecker, JR
通讯作者：
Ecker, JR
影响因子：
56.9
作者：
BEIJERSBERGEN, A;DENDULKRAS, A;HOOYKAAS, PJJ
通讯作者：
HOOYKAAS, PJJ
影响因子：
56.9
作者：
HENSEL, M;SHEA, JE;HOLDEN, DW
通讯作者：
HOLDEN, DW