Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays
Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays
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DOI：
10.1073/pnas.0408203102
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发表时间：
2005-03-22
影响因子：
11.1
通讯作者：
Sussman, MR
中科院分区：
文献类型：
--
作者：
Stolc, V;Samanta, MP;Sussman, MR
使用无掩模的光刻方法，我们生产了跨植物拟南芥基因组的探针序列的DNA寡核苷酸微阵列。通过平铺500万36-Mer探针来确定完整的核，线粒体和叶绿体基因组的RNA表达。将这些探针杂交以从未分化的拟南芥细胞培养线中从液体生长的T87细胞中分离出的mRNA。从近26,330个注释的基因中，至少60％从60％的人中检测到了转录本，其中包括151个预测基因，这些基因先前尚未通过类似的全基因组杂交研究在四种不同的细胞系上鉴定出来。与先前发表的结果相比，基于铬掩蔽的光刻技术生产的25-Mer平铺阵列，发现36-Mer寡核苷酸探针在识别内含子外观边界方面更有用。使用二维HPLC串联质谱法，对相同的细胞进行了小规模的蛋白质组学分析。在已知基因的“反义”区域发现了大量强杂交RNA。 25-MER和36-MER数据集之间反义活动的相似性表明它是实验的可再现和固有特性。还检测到许多基因间区域和小的RNA（包括tRNA，小核RNA，小核仁RNA和microRNA）的转录活性。 TRNA的表达与全基因组氨基酸的使用相关。
Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.