Volatility of Mutator Phenotypes at Single Cell Resolution

Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.     

DRSP：蛋白质单残基替换结构数据库

蛋白质残基替换是基因突变的产物之一,它可能改变蛋白质三维结构,对其生物学功能产生重大影响,因此研究蛋白质残基替换与结构改变的关系具有重要意义．随着实验解析蛋白质结构的数量迅猛增长,越来越多的野生型-突变体被应用于结构生物学的比较研究中．本研究从蛋白质三维结构数据库(PDB)出发,收集和计算了大量结构特征数据,构建了一个目前已知最大的野生型-突变体(单残基差异)的结构对数据库DRSP,展示出氨基酸类型和主链偏好性对结构保守性的相关性．DRSP的开放使用可为高精度的蛋白质结构分析预测提供有用信息,它的数据库网址是http://www.labshare.cn/drsp/index.php．     

Structural Characterization of Tau in Fuzzy Tau:Tubulin Complexes

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Structural Polymorphism of Actin

Information on the structural polymorphism of a protein is essential to understand the mechanisms of how it functions at an atomic level. Numerous studies on actin have accumulated substantial amounts of information about its polymorphism, and there are over 200 published atomic structures of different forms of actin using crystallography, fiber diffraction, and electron microscopy. To characterize all the reported structures, we proposed simple parameters based on the discrete rigid bodies within the actin molecule and identified four conformation groups by cluster analysis: the F-form in naked F-actin, the C-form in cofilactin, the O-form in profilin–actin, and the G-form in the majority of actin-containing crystal structures. The G-form group included the most variations, but each conformational variation was convertible via a thermal fluctuation, whereas the F- and C-forms were not accessible from the G-form. The convertibility and accessibility of the structures were evaluated using molecular dynamics simulations. Information about conformational conversion among each group is useful for understanding the mechanisms of actin function.     

DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele Resolution

Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation.     

Structural Determinants of Yeast Protein-Protein Interaction Interface Evolution at the Residue Level

Interfaces of contact between proteins play important roles in determining the proper structure and function of protein–protein interactions (PPIs). Therefore, to fully understand PPIs, we need to better understand the evolutionary design principles of PPI interfaces. Previous studies have uncovered that interfacial sites are more evolutionarily conserved than other surface protein sites. Yet, little is known about the nature and relative importance of evolutionary constraints in PPI interfaces. Here, we explore constraints imposed by the structure of the microenvironment surrounding interfacial residues on residue evolutionary rate using a large dataset of over 700 structural models of baker’s yeast PPIs. We find that interfacial residues are, on average, systematically more conserved than all other residues with a similar degree of total burial as measured by relative solvent accessibility (RSA). Besides, we find that RSA of the residue when the PPI is formed is a better predictor of interfacial residue evolutionary rate than RSA in the monomer state. Furthermore, we investigate four structure-based measures of residue interfacial involvement, including change in RSA upon binding (ΔRSA), number of residue-residue contacts across the interface, and distance from the center or the periphery of the interface. Integrated modeling for evolutionary rate prediction in interfaces shows that ΔRSA plays a dominant role among the four measures of interfacial involvement, with minor, but independent contributions from other measures. These results yield insight into the evolutionary design of interfaces, improving our understanding of the role that structure plays in the molecular evolution of PPIs at the residue level.     

Crystallographic Studies of Prion Protein (PrP) Segments Suggest How Structural Changes Encoded by Polymorphism at Residue 129 Modulate Susceptibility to Human Prion Disease

A single nucleotide polymorphism (SNP) in codon 129 of the human prion gene, leading to a change from methionine to valine at residue 129 of prion protein (PrP), has been shown to be a determinant in the susceptibility to prion disease. However, the molecular basis of this effect remains unexplained. In the current study, we determined crystal structures of prion segments having either Met or Val at residue 129. These 6-residue segments of PrP centered on residue 129 are “steric zippers,” pairs of interacting β-sheets. Both structures of these “homozygous steric zippers” reveal direct intermolecular interactions between Met or Val in one sheet and the identical residue in the mating sheet. These two structures, plus a structure-based model of the heterozygous Met-Val steric zipper, suggest an explanation for the previously observed effects of this locus on prion disease susceptibility and progression.     

Energetic Frustrations in Protein Folding at Residue Resolution: A Homologous Simulation Study of Im9 Proteins

Energetic frustration is becoming an important topic for understanding the mechanisms of protein folding, which is a long-standing big biological problem usually investigated by the free energy landscape theory. Despite the significant advances in probing the effects of folding frustrations on the overall features of protein folding pathways and folding intermediates, detailed characterizations of folding frustrations at an atomic or residue level are still lacking. In addition, how and to what extent folding frustrations interact with protein topology in determining folding mechanisms remains unclear. In this paper, we tried to understand energetic frustrations in the context of protein topology structures or native-contact networks by comparing the energetic frustrations of five homologous Im9 alpha-helix proteins that share very similar topology structures but have a single hydrophilic-to-hydrophobic mutual mutation. The folding simulations were performed using a coarse-grained Gō-like model, while non-native hydrophobic interactions were introduced as energetic frustrations using a Lennard-Jones potential function. Energetic frustrations were then examined at residue level based on φ-value analyses of the transition state ensemble structures and mapped back to native-contact networks. Our calculations show that energetic frustrations have highly heterogeneous influences on the folding of the four helices of the examined structures depending on the local environment of the frustration centers. Also, the closer the introduced frustration is to the center of the native-contact network, the larger the changes in the protein folding. Our findings add a new dimension to the understanding of protein folding the topology determination in that energetic frustrations works closely with native-contact networks to affect the protein folding.     

单核苷酸多态性在植物研究中的应用

本文概述了SNP的研究现状及其优越性,SNP在植物遗传育种、进化、种质资源遗传多样性研究和保存等方面的应用潜力.     

单核苷酸多态性检测技术的研究进展

单核苷酸多态性是指在基因组水平上单个核苷酸变异引起的一种DNA序列多态性。因其具有密度高,遗传稳定,易于进行自动化、规模化分析等优势它已成为第三代分子标记,因此其检测技术也在近几年得到了快速的发展。将对未知单核苷酸突变位点的检测方法和已知单核苷酸突变位点的检测方法这两部分的研究进展作一综述。     

Genetic Analysis of the Cardiac Methylome at Single Nucleotide Resolution in a Model of Human Cardiovascular Disease

Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.     

针对tau蛋白PET 示踪剂研究进展

阿尔茨海默病(Alzheimer's disease,AD)是一种常见的中枢神经系统退行性疾病,脑内主要病理变化为神经元外由β-淀粉样蛋白(Amyloidβ,Aβ)沉积形成的老年斑(Senile Plaques,SP)和神经元内由异常过度磷酸化的微管相关tau蛋白构成的神经原纤维缠结(Neurofibrillary Tangles,NFTs),对这两种病理改变的特异性识别有助于AD早期发现与诊断。既往针对AD的PET示踪剂多数作用于Aβ,近年tau蛋白类PET示踪剂发展迅速,以[18F]THK-523、[18F]THK-5105和[18F]THK-5117为代表的tau蛋白靶向PET示踪剂在AD的诊断与治疗方面显示出巨大的发展潜力与应用前景。     

Direct Observation of Single DNA Structural Alterations at Low Forces with Surface-Enhanced Raman Scattering

DNA experiences numerous mechanical events, necessitating single-molecule force spectroscopy techniques to provide insight into DNA mechanics as a whole system. Inherent Brownian motion limits current force spectroscopy methods from observing possible bond level structural changes. We combine optical trapping and surface-enhanced Raman scattering to establish a direct relationship between DNA’s extension and structure in the low force, entropic regime. A DNA molecule is trapped close to a surface-enhanced Raman scattering substrate to facilitate a detectable Raman signal. DNA Raman modes shift in response to applied force, indicating phosphodiester mechanical alterations. Molecular dynamic simulations confirm the local structural alterations and the Raman sensitive band identified experimentally. The combined Raman and force spectroscopy technique, to our knowledge, is a novel methodology that can be generalized to all single-molecule studies.