Dataset Information

Bug mapping and fitness testing of chemically synthesized chromosome X.

ABSTRACT: Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1, and a loxPsym site affecting promoter function of ATP2 PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.

SUBMITTER: Wu Y

PROVIDER: S-EPMC5679077 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Bug mapping and fitness testing of chemically synthesized chromosome X.

Wu Yi Y Li Bing-Zhi BZ Zhao Meng M Mitchell Leslie A LA Xie Ze-Xiong ZX Lin Qiu-Hui QH Wang Xia X Xiao Wen-Hai WH Wang Ying Y Zhou Xiao X Liu Hong H Li Xia X Ding Ming-Zhu MZ Liu Duo D Zhang Lu L Liu Bao-Li BL Wu Xiao-Le XL Li Fei-Fei FF Dong Xiu-Tao XT Jia Bin B Zhang Wen-Zheng WZ Jiang Guo-Zhen GZ Liu Yue Y Bai Xue X Song Tian-Qing TQ Chen Yan Y Zhou Si-Jie SJ Zhu Rui-Ying RY Gao Feng F Kuang Zheng Z Wang Xuya X Shen Michael M Yang Kun K Stracquadanio Giovanni G Richardson Sarah M SM Lin Yicong Y Wang Lihui L Walker Roy R Luo Yisha Y Ma Ping-Sheng PS Yang Huanming H Cai Yizhi Y Dai Junbiao J Bader Joel S JS Boeke Jef D JD Yuan Ying-Jin YJ

Science (New York, N.Y.) 20170301 6329

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that ...[more]

PMID: 28280152

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Project description:The methylglyoxal-derived hydroimidazolones (MG-Hs) comprise the most prevalent class of non-enzymatic, post-translational modifications of protein arginine residues found in nature. These adducts form spontaneously in the human body, and are also present at high levels in the human diet. Despite numerous lines of evidence suggesting that MG-H-arginine adducts play critical roles in both healthy and disease physiology in humans, detailed studies of these molecules have been hindered by a lack of general synthetic strategies for their preparation in chemically homogeneous form, and on scales sufficient to enable detailed biochemical and cellular investigations. To address this limitation, we have developed efficient, multigram-scale syntheses of all MG-H-amino acid building blocks, suitably protected for solid-phase peptide synthesis, in 2-3 steps starting from inexpensive, readily available starting materials. Thus, MG-H derivatives were readily incorporated into oligopeptides site-specifically using standard solid-phase peptide synthesis. Access to synthetic MG-H-peptide adducts has enabled detailed investigations, which have revealed a series of novel and unexpected findings. First, one of the three MG-H isomers, MG-H3, was found to possess potent, pH-dependent antioxidant properties in biochemical and cellular assays intended to replicate redox processes that occur in vivo. Computational and mechanistic studies suggest that MG-H3-containing constructs are capable of participating in mechanistically distinct H-atom-transfer and single-electron-transfer oxidation processes. Notably, the product of MG-H3 oxidation was unexpectedly observed to disassemble into the fully unmodified arginine residue and pyruvate in aqueous solution. We believe these observations provide insight into the role(s) of MG-H-protein adducts in human physiology, and expect the synthetic reagents reported herein to enable investigations into non-enzymatic protein regulation at an unprecedented level of detail.

Dataset Information

Bug mapping and fitness testing of chemically synthesized chromosome X.

Publications

Bug mapping and fitness testing of chemically synthesized chromosome X.

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