State Key Laboratory for Molecular Virology and Genetic Engineering, Institute of Pathogen Biology, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China.
PLoS One. 2011 Apr 5;6(4):e18509. doi: 10.1371/journal.pone.0018509.
The completion of numerous genome sequences introduced an era of whole-genome study. However, many genes are missed during genome annotation, including small RNAs (sRNAs) and small open reading frames (sORFs). In order to improve genome annotation, we aimed to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery.
METHODOLOGY/PRINCIPAL FINDINGS: We identified 64 sRNAs in Shigella, which were experimentally validated in other bacteria based on sequence conservation. We employed computer-based and tiling array-based methods to search for sRNAs, followed by RT-PCR and northern blots, to identify nine sRNAs in Shigella flexneri strain 301 (Sf301) and 256 regions containing possible sRNA genes. We found 29 candidate sORFs using bioinformatic prediction, array hybridization and RT-PCR verification. We experimentally validated 557 (57.9%) DOOR operon predictions in the chromosomes of Sf301 and 46 (76.7%) in virulence plasmid.We found 40 additional co-expressed gene pairs that were not predicted by DOOR.
CONCLUSIONS/SIGNIFICANCE: We provide an updated and comprehensive annotation of the Shigella genome. Our study increased the expected numbers of sORFs and sRNAs, which will impact on future functional genomics and proteomics studies. Our method can be used for large scale reannotation of sRNAs and sORFs in any microbe with a known genome sequence.
大量基因组序列的完成开创了全基因组研究的时代。然而，在基因组注释过程中会遗漏许多基因，包括小 RNA（sRNA）和小开放阅读框（sORF）。为了改进基因组注释，我们旨在鉴定志贺氏菌中的新 sRNA 和 sORF，志贺氏菌是细菌性痢疾的主要病原体。
方法/主要发现：我们在志贺氏菌中鉴定了 64 个 sRNA，这些 sRNA 基于序列保守性在其他细菌中进行了实验验证。我们采用基于计算机和基于芯片的方法来搜索 sRNA，然后通过 RT-PCR 和 northern blot，在福氏志贺菌 301 株（Sf301）中鉴定了 9 个 sRNA 和 256 个可能包含 sRNA 基因的区域。我们使用生物信息学预测、芯片杂交和 RT-PCR 验证，发现了 29 个候选 sORF。我们在 Sf301 染色体中实验验证了 557 个（57.9%）DOOR 操纵子预测，在毒力质粒中验证了 46 个（76.7%）。我们发现了 40 对额外的共表达基因对，这些基因对不是由 DOOR 预测的。
结论/意义：我们提供了志贺氏菌基因组的更新和全面注释。我们的研究增加了预期的 sORF 和 sRNA 的数量，这将对未来的功能基因组学和蛋白质组学研究产生影响。我们的方法可用于任何具有已知基因组序列的微生物的大规模重新注释 sRNA 和 sORF。
