• 【全基因组关联研究确定了与人类阿片类药物敏感性相关的有效基因座。】 复制标题 收藏 收藏
    DOI:10.1038/mp.2012.164 复制DOI
    作者列表:
    BACKGROUND & AIMS: :Opioids, such as morphine and fentanyl, are widely used as effective analgesics for the treatment of acute and chronic pain. In addition, the opioid system has a key role in the rewarding effects of morphine, ethanol, cocaine and various other drugs. Although opioid sensitivity is well known to vary widely among individual subjects, several candidate genetic polymorphisms reported so far are not sufficient for fully understanding the wide range of interindividual differences in human opioid sensitivity. By conducting a multistage genome-wide association study (GWAS) in healthy subjects, we found that genetic polymorphisms within a linkage disequilibrium block that spans 2q33.3-2q34 were strongly associated with the requirements for postoperative opioid analgesics after painful cosmetic surgery. The C allele of the best candidate single-nucleotide polymorphism (SNP), rs2952768, was associated with more analgesic requirements, and consistent results were obtained in patients who underwent abdominal surgery. In addition, carriers of the C allele in this SNP exhibited less vulnerability to severe drug dependence in patients with methamphetamine dependence, alcohol dependence, and eating disorders and a lower 'Reward Dependence' score on a personality questionnaire in healthy subjects. Furthermore, the C/C genotype of this SNP was significantly associated with the elevated expression of a neighboring gene, CREB1. These results show that SNPs in this locus are the most potent genetic factors associated with human opioid sensitivity known to date, affecting both the efficacy of opioid analgesics and liability to severe substance dependence. Our findings provide valuable information for the personalized treatment of pain and drug dependence.
    背景与目标: 阿片类药物,例如吗啡和芬太尼,被广泛用作治疗急性和慢性疼痛的有效镇痛药。此外,阿片样物质系统在吗啡,乙醇,可卡因和其他各种药物的奖励作用中也起着关键作用。尽管众所周知,阿片类药物敏感性在各个受试者之间差异很大,但迄今为止报道的几种候选遗传多态性不足以全面了解人类阿片类药物敏感性之间的广泛个体差异。通过在健康受试者中进行多阶段全基因组关联研究(GWAS),我们发现跨越2q33.3-2q34的连锁不平衡区内的遗传多态性与痛苦的整容手术后对阿片类镇痛药的需求密切相关。最佳候选单核苷酸多态性(SNP)的C等位基因rs2952768与更多的止痛要求相关,并且在接受腹部手术的患者中获得了一致的结果。此外,在健康受试者中,具有甲基苯丙胺依赖,酒精依赖和进食障碍的患者中,该SNP中的C等位基因携带者对严重药物依赖的耐受性较低,并且在个性问卷上的“奖励依赖”评分较低。此外,此SNP的C / C基因型与邻近基因CREB1的表达升高显着相关。这些结果表明,该基因座中的SNP是迄今为止已知的与人类阿片类药物敏感性相关的最有效的遗传因素,既会影响阿片类镇痛药的功效,又会严重依赖药物。我们的发现为个性化治疗疼痛和药物依赖性提供了有价值的信息。
  • 【通过随机扩增多态性DNA分析鉴定巴西副球菌基因组中编码适应蛋白样蛋白的基因。】 复制标题 收藏 收藏
    DOI:10.1099/jmm.0.47127-0 复制DOI
    作者列表:Andreotti PF,Monteiro da Silva JL,Teixeira EC,Bertolini MC,Soares CP,Benard G,Mendes-Giannini MJS
    BACKGROUND & AIMS: :Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pb18) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pb18a by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.
    背景与目标: :巴西对虾的分离株在动物中的致病性和对上皮细胞的粘附方式不均一。在这项研究过程中,观察到两次培养过多次的巴西假单胞菌菌株18酵母相(Pb18)的基因型差异,一个样品是在接种动物之前(Pb18a),另一个是在接种之后(Pb18b)。使用引物OPJ4进行的随机扩增多态性DNA分析通过一个308 bp DNA片段将Pb18b与Pb18a区别开来,该片段在克隆和测序后显示编码与蛋白质β-adaptin同源的多肽序列。与其他微生物相比,该蛋白质可能在巴西假单胞菌的毒力中起重要作用。该结果证明了体外传代培养对该生物的基因型的影响。
  • 【全基因组表达分析伯克霍尔德菌在急性类鼻疮的仓鼠模型中的感染。】 复制标题 收藏 收藏
    DOI:10.1128/IAI.00737-06 复制DOI
    作者列表:Tuanyok A,Tom M,Dunbar J,Woods DE
    BACKGROUND & AIMS: :Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In the current studies we have examined gene expression in B. pseudomallei in an animal model of acute melioidosis using whole-genome microarrays. Gene expression profiles were generated by comparing transcriptional levels of B. pseudomallei-expressed genes in infected hamster organs including liver, lung, and spleen following intraperitoneal and intranasal routes of infection to those from bacteria grown in vitro. Differentially expressed genes were similar in infected livers irrespective of the route of infection. Reduced expression of a number of housekeeping genes suggested a lower bacterial growth rate during infection. Energy production during growth in vivo involved specific biochemical pathways such as isomerization of 3-phosphoglycerate, catabolism of d-glucosamine and inositol, and biosynthesis of particular amino acids. In addition, the induction of genes known to be involved in oxidative phosphorylation including ubiquinol oxidase, ferredoxin oxidoreductase, and formate dehydrogenase enzymes suggested the use of alternative pathways for energy production, while the expression of genes coding for ATP-synthase and NADH-dehydrogenase enzymes was reduced. Our studies have identified differentially expressed genes which include potential virulence genes such as those for a putative phospholipase C and a putative two-component regulatory system, and they have also provided a better understanding of bacterial metabolism in response to the host environment during acute melioidosis.
    背景与目标: 假性伯克霍尔德菌(Burkholderia pseudomallei)是类鼻疮的病原体,代表潜在的生物恐怖主义威胁。在当前的研究中,我们已经使用全基因组微阵列检查了在急性类鼻oid病的动物模型中假性疟原虫中的基因表达。通过比较经腹膜内和鼻内感染途径感染的仓鼠器官(包括肝,肺和脾脏)中假苹果芽孢杆菌表达的基因的转录水平与体外培养的细菌的转录水平,从而产生基因表达谱。无论感染途径如何,差异表达的基因在受感染的肝脏中都是相似的。许多管家基因的表达降低表明感染期间细菌的生长速率较低。体内生长过程中的能量产生涉及特定的生化途径,例如3-磷酸甘油酸酯的异构化,d-葡萄糖胺和肌醇的分解代谢以及特定氨基酸的生物合成。此外,诱导诱导参与氧化磷酸化的基因包括泛醇氧化酶,铁氧还蛋白氧化还原酶和甲酸脱氢酶建议使用替代途径产生能量,而编码ATP合酶和NADH脱氢酶的基因的表达减少了。我们的研究已鉴定出差异表达的基因,其中包括潜在的毒力基因,例如推定的磷脂酶C和推定的两组分调节系统,并且它们还提供了对急性类li虫病响应宿主环境的细菌代谢的更好理解。
  • 【在人类上皮细胞内化后,金黄色葡萄球菌整个基因组表达的全局视图。】 复制标题 收藏 收藏
    DOI:10.1186/1471-2164-8-171 复制DOI
    作者列表:Garzoni C,Francois P,Huyghe A,Couzinet S,Tapparel C,Charbonnier Y,Renzoni A,Lucchini S,Lew DP,Vaudaux P,Kelley WL,Schrenzel J
    BACKGROUND & AIMS: BACKGROUND:Staphylococcus aureus, a leading cause of chronic or acute infections, is traditionally considered an extracellular pathogen despite repeated reports of S. aureus internalization by a variety of non-myeloid cells in vitro. This property potentially contributes to bacterial persistence, protection from antibiotics and evasion of immune defenses. Mechanisms contributing to internalization have been partly elucidated, but bacterial processes triggered intracellularly are largely unknown. RESULTS:We have developed an in vitro model using human lung epithelial cells that shows intracellular bacterial persistence for up to 2 weeks. Using an original approach we successfully collected and amplified low amounts of bacterial RNA recovered from infected eukaryotic cells. Transcriptomic analysis using an oligoarray covering the whole S. aureus genome was performed at two post-internalization times and compared to gene expression of non-internalized bacteria. No signs of cellular death were observed after prolonged internalization of Staphylococcus aureus 6850 in epithelial cells. Following internalization, extensive alterations of bacterial gene expression were observed. Whereas major metabolic pathways including cell division, nutrient transport and regulatory processes were drastically down-regulated, numerous genes involved in iron scavenging and virulence were up-regulated. This initial adaptation was followed by a transcriptional increase in several metabolic functions. However, expression of several toxin genes known to affect host cell integrity appeared strictly limited. CONCLUSION:These molecular insights correlated with phenotypic observations and demonstrated that S. aureus modulates gene expression at early times post infection to promote survival. Staphylococcus aureus appears adapted to intracellular survival in non-phagocytic cells.
    背景与目标: 背景:尽管反复报道金黄色葡萄球菌在体外被多种非髓样细胞内在化,但金黄色葡萄球菌是慢性或急性感染的主要原因,传统上被认为是一种细胞外病原体。该特性可能有助于细菌持久性,保护免受抗生素侵害和逃避免疫防御。促成内在化的机制已被部分阐明,但在细胞内触发的细菌过程在很大程度上是未知的。
    结果:我们开发了一种使用人肺上皮细胞的体外模型,该模型显示了长达2周的细胞内细菌持久性。使用原始方法,我们成功地收集并扩增了从感染的真核细胞中回收的少量细菌RNA。在两个内在化后的时间使用覆盖整个金黄色葡萄球菌基因组的寡核苷酸阵列进行转录组学分析,并将其与非内在化细菌的基因表达进行比较。在上皮细胞中金黄色葡萄球菌6850长时间内在化后,未观察到细胞死亡的迹象。内化后,观察到细菌基因表达的广泛改变。尽管主要的代谢途径(包括细胞分裂,营养物质运输和调节过程)被大幅下调,但许多与铁清除和毒力有关的基因却被上调。在最初的适应之后,是几种代谢功能的转录增加。但是,已知影响宿主细胞完整性的几种毒素基因的表达似乎受到严格限制。
    结论:这些分子洞察力与表型观察结果相关,并证明金黄色葡萄球菌在感染后早期调节基因表达以促进存活。金黄色葡萄球菌似乎适合非吞噬细胞中的细胞内存活。
  • 【BRIE:单细胞中转录组范围的剪接定量。】 复制标题 收藏 收藏
    DOI:10.1186/s13059-017-1248-5 复制DOI
    作者列表:Huang Y,Sanguinetti G
    BACKGROUND & AIMS: :Single-cell RNA-seq (scRNA-seq) provides a comprehensive measurement of stochasticity in transcription, but the limitations of the technology have prevented its application to dissect variability in RNA processing events such as splicing. Here, we present BRIE (Bayesian regression for isoform estimation), a Bayesian hierarchical model that resolves these problems by learning an informative prior distribution from sequence features. We show that BRIE yields reproducible estimates of exon inclusion ratios in single cells and provides an effective tool for differential isoform quantification between scRNA-seq data sets. BRIE, therefore, expands the scope of scRNA-seq experiments to probe the stochasticity of RNA processing.
    背景与目标: :单细胞RNA-seq(scRNA-seq)提供了转录随机性的全面测量方法,但该技术的局限性使其无法用于分析RNA加工事件(例如剪接)中的变异性。在这里,我们介绍BRIE(用于同等型估计的贝叶斯回归),它是一种贝叶斯层次模型,可以通过从序列特征中学习有用的先验分布来解决这些问题。我们表明,BRIE产生单细胞中外显子包含率的可再现估计,并为scRNA-seq数据集之间的差异同工型定量提供了有效的工具。因此,BRIE扩大了scRNA-seq实验的范围,以探测RNA加工的随机性。
  • 【在两个形成蘑菇的茶农物种线粒体基因组的特定区域中,多态性微卫星基因座的异常积累。】 复制标题 收藏 收藏
    DOI:10.1111/j.1574-6968.2007.00771.x 复制DOI
    作者列表:Mouhamadou B,Férandon C,Chazoule S,Barroso G
    BACKGROUND & AIMS: :The cob/tRNA(Tyr) mitochondrial regions of Agrocybe aegerita and of the related species Agrocybe chaxingu display an unusual clustering of four microsatellite loci constituted by motifs of one to six nucleotides whose number of repeats varied from three to 18. In A. chaxingu, these microsatellite loci are followed by a small region bearing one additional microsatelite and one minisatellite locus constituted by an octanucleotide motif repeated 13-18 times. In A. aegerita, this latter region is deleted. This is the first evidence of such an accumulation of microsatellites in mitochondrial genomes. The analyses of the microsatellite loci in 11 A. aegerita and in four A. chaxingu wild strains have shown extensive intraspecific and interspecific variations in the number of tandem repeats (VNTRs), suggesting that these loci could represent powerful molecular markers for strain fingerprinting. Up to 23 different alleles were present in the 15 Agrocybe studied strains, allowing the definition of 12 different haplotypes.
    背景与目标: :Agrocybe aegerita和相关物种Agrocybe chaxingu的cob / tRNA(Tyr)线粒体区域显示出一个由四个到六个微卫星基因座的异常簇,这些基因座由一个到六个核苷酸的基序组成,其重复数从三个到18个不等。在这些微卫星基因座之后是一个小区域,该区域带有一个额外的微卫星和一个由八核苷酸基序构成的小卫星基因座,重复了18-18次。在Aegerita中,后一个区域被删除。这是线粒体基因组中微卫星积累的第一个证据。在11个Aegerita和四个A.chaxingu野生菌株中的微卫星基因座分析表明,串联重复序列(VNTR)的数量在种内和种间差异很大,表明这些基因座可以代表菌株指纹的强大分子标记。在15个土壤杆菌研究菌株中,存在多达23个不同的等位基因,从而可以定义12种不同的单倍型。
  • 【微生物文化学对全球健康的应用:非连续完成的基因组序列和马氏假单胞菌菌株CB-1T sp的描述。十一月在巴西。】 复制标题 收藏 收藏
    DOI:10.1089/omi.2017.0027 复制DOI
    作者列表:Bardet L,Cimmino T,Buffet C,Michelle C,Rathored J,Tandina F,Lagier JC,Khelaifia S,Abrahão J,Raoult D,Rolain JM
    BACKGROUND & AIMS: :Culturomics is a new postgenomics field that explores the microbial diversity of the human gut coupled with taxono-genomic strategy. Culturomics, and the microbiome science more generally, are anticipated to transform global health diagnostics and inform the ways in which gut microbial diversity contributes to human health and disease, and by extension, to personalized medicine. Using culturomics, we report in this study the description of strain CB1T ( = CSUR P1334 = DSM 29075), a new species isolated from a stool specimen from a 37-year-old Brazilian woman. This description includes phenotypic characteristics and complete genome sequence and annotation. Strain CB1T is a gram-negative aerobic and motile bacillus, exhibits neither catalase nor oxidase activities, and presents a 98.3% 16S rRNA sequence similarity with Pseudomonas putida. The 4,723,534 bp long genome contains 4239 protein-coding genes and 74 RNA genes, including 15 rRNA genes (5 16S rRNA, 4 23S rRNA, and 6 5S rRNA) and 59 tRNA genes. Strain CB1T was named Pseudomonas massiliensis sp. nov. and classified into the family Pseudomonadaceae. This study demonstrates the usefulness of microbial culturomics in exploration of human microbiota in diverse geographies and offers new promise for incorporating new omics technologies for innovation in diagnostic medicine and global health.
    背景与目标: :Culturomics是一个新的后基因组学领域,它探索人类肠道微生物的多样性以及分类基因组策略。预计文化学以及更广泛的微生物学科学将改变全球健康诊断方法,并为肠道微生物多样性促进人类健康和疾病以及扩展至个性化医学的方式提供信息。使用文化组学,我们在这项研究中报告了菌株CB1T(= CSUR P1334 = DSM 29075)的描述,该菌株是从一名37岁的巴西妇女的粪便样本中分离得到的新物种。该描述包括表型特征以及完整的基因组序列和注释。菌株CB1T是革兰氏阴性好氧和运动型芽孢杆菌,既不具有过氧化氢酶活性,也不具有氧化酶活性,并且与恶臭假单胞菌具有98.3%的16S rRNA序列相似性。该基因组长4,723,534 bp,包含4239个蛋白质编码基因和74个RNA基因,其中包括15个rRNA基因(5个16S rRNA,4个23S rRNA和6个5S rRNA)和59个tRNA基因。菌株CB1T被命名为马氏假单胞菌。十一月并归类为假单胞菌科(Pseudomonadaceae)。这项研究证明了微生物菌群学在探索人类在不同地区的微生物群中的有用性,并为结合新的组学技术在诊断医学和全球卫生领域的创新提供了新的希望。
  • 【假结核杆菌Biovar equi 258菌株的基因组序列和抗原靶标的预测,以提高生物技术疫苗的生产。】 复制标题 收藏 收藏
    DOI:10.1016/j.jbiotec.2012.11.003 复制DOI
    作者列表:
    BACKGROUND & AIMS: :Corynebacterium pseudotuberculosis is the causative agent of several veterinary diseases in a broad range of economically important hosts, which can vary from caseous lymphadenitis in sheep and goats (biovar ovis) to ulcerative lymphangitis in cattle and horses (biovar equi). Existing vaccines against C. pseudotuberculosis are mainly intended for small ruminants and, even in these hosts, they still present remarkable limitations. In this study, we present the complete genome sequence of C. pseudotuberculosis biovar equi strain 258, isolated from a horse with ulcerative lymphangitis. The genome has a total size of 2,314,404 bp and contains 2088 predicted protein-coding regions. Using in silico analysis, eleven pathogenicity islands were detected in the genome sequence of C. pseudotuberculosis 258. The application of a reverse vaccinology strategy identified 49 putative antigenic proteins, which can be used as candidate vaccine targets in future works.
    背景与目标: :假结核杆菌是多种重要经济宿主中几种兽医疾病的病原体,从绵羊和山羊的干酪性淋巴结炎(biovar ovis)到牛和马的溃疡性淋巴管炎(biovar equi)不等。现有的针对假结核杆菌的疫苗主要用于小型反刍动物,即使在这些宿主中,它们仍然存在明显的局限性。在这项研究中,我们介绍了从患有溃疡性淋巴管炎的一匹马中分离出来的假结核梭菌biovar equi菌株258的完整基因组序列。基因组的总大小为2,314,404 bp,包含2088个预测的蛋白质编码区。通过计算机分析,在假结核梭菌258的基因组序列中检测到11个致病岛。反向疫苗学策略的应用确定了49种推定的抗原蛋白,这些蛋白可以用作未来工作中的候选疫苗靶标。
  • 【凝血因子(F)IX水平正常变异的遗传决定因素:全基因组扫描和FIX结构基因检查。】 复制标题 收藏 收藏
    DOI:10.1111/j.1538-7836.2006.02024.x 复制DOI
    作者列表:Khachidze M,Buil A,Viel KR,Porter S,Warren D,Machiah DK,Soria JM,Souto JC,Ameri A,Lathrop M,Blangero J,Fontcuberta J,Warren ST,Almasy L,Howard TE
    BACKGROUND & AIMS: BACKGROUND:High-normal and elevated plasma FIX activity (FIX:C) levels are associated with increased risk for venous- and possibly arterial-thrombosis. OBJECTIVE:Because the broad normal range for FIX:C involves a substantial unknown genetic component, we sought to identify quantitative-trait loci (QTLs) for this medically important hemostasis trait. METHODS:We performed a genome-wide screen and a resequencing-based variation scan of the known functional regions of every distinct FIX gene (F9) in the genetic analysis of idiopathic thrombophilia project (GAIT), a collection of 398 Spanish-Caucasians from 21 pedigrees. RESULTS:We found no evidence for linkage (LOD scores <1.5) despite genotyping more than 540 uniformly-spaced microsatellites. We identified 27 candidate F9 polymorphisms, including three in cis-elements responsible for the increase in FIX:C that occurs with aging, but found no significant genotype-specific differences in mean FIX:C levels (P-values > or = 0.11) despite evaluating every polymorphism in GAIT by marginal multicovariate measured-genotype association analysis. CONCLUSIONS:The heritable component of interindividual FIX:C variability likely involves a collection of QTLs with modest effects that may reside in genes other than F9. Nevertheless, because the alleles of these 27 polymorphisms exhibited a low overall degree of linkage disequilibrium, we are currently defining their haplotypes to interrogate several highly-conserved non-exonic sequences and other F9 segments not examined here.
    背景与目标: 背景:血浆FIX活性(FIX:C)水平较高和较高与静脉血栓形成和可能的动脉血栓形成的风险增加有关。
    目的:由于FIX:C的广泛正常范围涉及大量未知的遗传成分,因此我们试图确定这种医学上重要的止血性状的定量性状基因座(QTL)。
    方法:在特发性血友病计划(GAIT)的遗传分析中,我们对每个不同的FIX基因(F9)的已知功能区进行了全基因组筛选和基于测序的变异扫描,该研究收集了来自21个国家的398名西班牙高加索人家谱。
    结果:尽管我们对540多个均匀分布的微卫星进行了基因分型,但我们没有发现连锁的证据(LOD得分<1.5)。我们确定了27个候选F9多态性,包括三个与衰老有关的FIX:C增加的顺式元素,但尽管平均FIX:C水平(P值>或= 0.11)没有发现明显的基因型特异性差异(尽管通过边缘多协变量测量基因型关联分析评估GAIT中的每个多态性。
    结论:个体FIX:C变异性的可遗传成分可能涉及具有适度作用的QTL集合,这些QTL可能存在于F9以外的基因中。但是,由于这27个多态性的等位基因表现出较低的整体连锁不平衡程度,因此我们目前正在定义其单倍型,以讯问几个高度保守的非外显子序列和此处未检查的其他F9片段。
  • 【马铃薯的完整叶绿体基因组序列以及与茄科物种的比较分析确定了栽培马铃薯叶绿体DNA序列中存在241 bp的缺失。】 复制标题 收藏 收藏
    DOI:10.1007/s00299-006-0196-4 复制DOI
    作者列表:Chung HJ,Jung JD,Park HW,Kim JH,Cha HW,Min SR,Jeong WJ,Liu JR
    BACKGROUND & AIMS: :The complete nucleotide sequence of the chloroplast genome of potato Solanum tuberosum L. cv. Desiree was determined. The circular double-stranded DNA, which consists of 155,312 bp, contains a pair of inverted repeat regions (IRa, IRb) of 25,595 bp each. The inverted repeat regions are separated by small and large single copy regions of 18,373 and 85,749 bp, respectively. The genome contains 79 proteins, 30 tRNAs, 4 rRNAs, and unidentified genes. A comparison of chloroplast genomes of seven Solanaceae species revealed that the gene content and their relative positions of S. tuberosum are similar to the other six Solanaceae species. However, undefined open reading frames (ORFs) in LSC region were highly diverged in Solanaceae species except N. sylvestris. Detailed comparison was identified by numerous indels in the intergenic regions that were mostly located in the LSC region. Among them, a single large 241-bp deletion, was not associated with direct repeats and found in only S. tuberosum, clearly discriminates a cultivated potato from wild potato species Solanum bulbocastanum. The extent of sequence divergence may provide the basis for evaluating genetic diversity within the Solanaceae species, and will be useful to examine the evolutionary processes in potato landraces.
    背景与目标: :马铃薯马铃薯L.cv.的叶绿体基因组的完整核苷酸序列。西瑞确定了。环状双链DNA长度为155,312 bp,包含一对分别为25,595 bp的反向重复区域(IRa,IRb)。反向重复区分别被大小分别为18,373和85,749 bp的大单拷贝区域隔开。基因组包含79种蛋白质,30个tRNA,4个rRNA和未鉴定的基因。比较了7种茄科植物的叶绿体基因组,发现马铃薯的基因含量及其相对位置与其他6种茄科植物相似。然而,除樟脑猪笼草外,茄科物种在LSC区域的未定义开放阅读框(ORF)差异很大。详细的比较通过基因间区域中的大量插入缺失进行鉴定,这些插入片段大多位于LSC区域中。其中,单个241 bp的大缺失与直接重复无关联,仅在马铃薯中发现,可从野生马铃薯物种Solanum bulbocastanum中清楚地辨别出栽培马铃薯。序列差异的程度可为评估茄科物种内的遗传多样性提供基础,并将有助于研究马铃薯地方品种的进化过程。
  • 【用于靶标特异性筛选的CAS9转录激活因子和用于协作基因组工程的配对切口酶。】 复制标题 收藏 收藏
    DOI:10.1038/nbt.2675 复制DOI
    作者列表:Mali P,Aach J,Stranges PB,Esvelt KM,Moosburner M,Kosuri S,Yang L,Church GM
    BACKGROUND & AIMS: :Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.
    背景与目标: :原核II型CRISPR-Cas系统可进行改造,以实现跨一系列真核生物的靶向基因组修饰。在这里,我们设计了这个系统,通过将转录激活结构域直接束缚至无核酸酶的Cas9蛋白或适配体修饰的单向导RNA(sgRNA),从而实现人细胞中RNA指导的基因组调控。使用此功能,我们开发了一种基于转录激活的测定法,以确定sgRNA:Cas9复合物脱靶结合的情况,并将其与转录激活剂样(TALs)效应子的脱靶活性进行了比较。我们的结果表明,特异性谱是sgRNA依赖性的,并且sgRNA:Cas9复合物和18-mer TAL效应子可以分别耐受1-3和1-2个靶错配。通过设计基因组编辑的偏移切口或通过多个协同sgRNA进行鲁棒的转录激活来设计对协同性的要求,我们提出了减轻脱靶现象的方法。我们的结果扩展了sgRNA:Cas9工具的多功能性,并强调了工程改造更高特异性的关键需求。
  • 【绿藻小菜蛾的叶绿体基因组序列:倒节菌科中倒位重复序列的多次丢失和广泛的基因组重排。】 复制标题 收藏 收藏
    DOI:10.1186/1471-2164-8-213 复制DOI
    作者列表:de Cambiaire JC,Otis C,Turmel M,Lemieux C
    BACKGROUND & AIMS: BACKGROUND:In the Chlorophyta--the green algal phylum comprising the classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae--the chloroplast genome displays a highly variable architecture. While chlorophycean chloroplast DNAs (cpDNAs) deviate considerably from the ancestral pattern described for the prasinophyte Nephroselmis olivacea, the degree of remodelling sustained by the two ulvophyte cpDNAs completely sequenced to date is intermediate relative to those observed for chlorophycean and trebouxiophyte cpDNAs. Chlorella vulgaris (Chlorellales) is currently the only photosynthetic trebouxiophyte whose complete cpDNA sequence has been reported. To gain insights into the evolutionary trends of the chloroplast genome in the Trebouxiophyceae, we sequenced cpDNA from the filamentous alga Leptosira terrestris (Ctenocladales). RESULTS:The 195,081-bp Leptosira chloroplast genome resembles the 150,613-bp Chlorella genome in lacking a large inverted repeat (IR) but differs greatly in gene order. Six of the conserved genes present in Chlorella cpDNA are missing from the Leptosira gene repertoire. The 106 conserved genes, four introns and 11 free standing open reading frames (ORFs) account for 48.3% of the genome sequence. This is the lowest gene density yet observed among chlorophyte cpDNAs. Contrary to the situation in Chlorella but similar to that in the chlorophycean Scenedesmus obliquus, the gene distribution is highly biased over the two DNA strands in Leptosira. Nine genes, compared to only three in Chlorella, have significantly expanded coding regions relative to their homologues in ancestral-type green algal cpDNAs. As observed in chlorophycean genomes, the rpoB gene is fragmented into two ORFs. Short repeats account for 5.1% of the Leptosira genome sequence and are present mainly in intergenic regions. CONCLUSION:Our results highlight the great plasticity of the chloroplast genome in the Trebouxiophyceae and indicate that the IR was lost on at least two separate occasions. The intriguing similarities of the derived features exhibited by Leptosira cpDNA and its chlorophycean counterparts suggest that the same evolutionary forces shaped the IR-lacking chloroplast genomes in these two algal lineages.
    背景与目标: 背景:在绿藻(绿藻门,包括草藻科,绿藻科,绿藻科和绿藻科)中,叶绿体基因组显示出高度可变的结构。尽管叶绿藻叶绿体DNA(cpDNA)与描述的古藻类Nephroselmis olivacea的祖先模式有很大差异,但迄今为止,两个完全被测序的ulvophyte cpDNA所维持的重塑程度相对于叶绿藻和trebouxiophyte cpDNA所观察到的中等。小球藻(Chlorellales)是目前唯一已报道完整cpDNA序列的光合藻类植物。为了深入了解细叶藻科中叶绿体基因组的进化趋势,我们对丝状藻类海藻Leptosira terrestris(Ctenocladales)的cpDNA进行了测序。
    结果:195081bp的叶绿体叶绿体基因组类似于150613bp的小球藻基因组,但缺少大的反向重复序列(IR),但基因顺序差异很大。小球藻cpDNA中存在的六个保守基因在Leptosira基因库中缺失。 106个保守基因,四个内含子和11个自由开放阅读框(ORF)占基因组序列的48.3%。这是迄今为止在绿藻cpDNA中观察到的最低基因密度。与小球藻中的情况相反,但与斜叶绿藻场景中的情况相似,该基因的分布在Leptosira的两条DNA链上高度偏向。与小球藻中的三个基因相比,九个基因相对于其祖先型绿色藻类cpDNA中的同源物具有显着扩展的编码区。如在叶绿素基因组中所观察到的,rpoB基因被片段化为两个ORF。短重复序列占Leptosira基因组序列的5.1%,主要存在于基因间区域。
    结论:我们的结果突显了藻藻科中叶绿体基因组的可塑性,并表明至少在两次不同的情况下IR丢失了。 Leptosira cpDNA及其对应的叶绿素对应物表现出的令人着迷的相似之处表明,相同的进化力塑造了这两个藻类谱系中缺少IR的叶绿体基因组。
  • 【李斯特菌属物种的基因组组织和毒力基因基因座的进化。】 复制标题 收藏 收藏
    DOI:10.1016/S1438-4221(00)80086-7 复制DOI
    作者列表:Chakraborty T,Hain T,Domann E
    BACKGROUND & AIMS: :The chromosomal region of Listeria monocytogenes harboring the gene cluster prfA-plcA-hly-mpl-actA-plcB (virulence gene cluster; vgc) harbors virulence genes critical for the survival of the bacteria following infection. Previous studies have implicated it as an ancestral pathogenicity island, derivatives of which are present in the species L. ivanovii and L. seeligeri, but absent in non-pathogenic species such as L. innocua. We cloned the corresponding region from L. innocua and L. welshimeri and compared its sequences to those from L. monocytogenes, L. ivanovii and L. seeligeri. The analysis allowed exact determination of delineation and size of the vgc and suggests that these genes may have been acquired by bacteriophage transduction. Thus, here we present an alternative view of the evolution of Listeria spp. and suggest that L. monocytogenes may be the primordial species of this genus.
    背景与目标: :单核细胞增生性李斯特菌的染色体区域带有prfA-plcA-hly-mpl-actA-plcB(毒性基因簇; vgc)基因簇,其中含有对细菌感染后的存活至关重要的毒力基因。先前的研究表明它是祖先的致病岛,其衍生物存在于伊凡诺氏杆菌和Seeligeri物种中,但在非致病性物种(如无害L. innocua)中却不存在。我们从无毒李斯特菌和welshimeri克隆了相应的区域,并将其序列与单核细胞增生李斯特菌,伊万诺维杆菌和seeligeri的序列进行了比较。分析允许精确确定vgc的轮廓和大小,并暗示这些基因可能已经通过噬菌体转导获得。因此,在这里,我们提出了李斯特菌属物种进化的另一种观点。并提示单核细胞增生李斯特氏菌可能是该属的原始种。
  • 【恶性疟原虫中染色体相互作用的全基因组分布图表征了与抗原变异相关的核结构和重构。】 复制标题 收藏 收藏
    DOI:10.1111/mmi.12381 复制DOI
    作者列表:Lemieux JE,Kyes SA,Otto TD,Feller AI,Eastman RT,Pinches RA,Berriman M,Su XZ,Newbold CI
    BACKGROUND & AIMS: :Spatial relationships within the eukaryotic nucleus are essential for proper nuclear function. In Plasmodium falciparum, the repositioning of chromosomes has been implicated in the regulation of the expression of genes responsible for antigenic variation, and the formation of a single, peri-nuclear nucleolus results in the clustering of rDNA. Nevertheless, the precise spatial relationships between chromosomes remain poorly understood, because, until recently, techniques with sufficient resolution have been lacking. Here we have used chromosome conformation capture and second-generation sequencing to study changes in chromosome folding and spatial positioning that occur during switches in var gene expression. We have generated maps of chromosomal spatial affinities within the P. falciparum nucleus at 25 Kb resolution, revealing a structured nucleolus, an absence of chromosome territories, and confirming previously identified clustering of heterochromatin foci. We show that switches in var gene expression do not appear to involve interaction with a distant enhancer, but do result in local changes at the active locus. These maps reveal the folding properties of malaria chromosomes, validate known physical associations, and characterize the global landscape of spatial interactions. Collectively, our data provide critical information for a better understanding of gene expression regulation and antigenic variation in malaria parasites.
    背景与目标: :真核内的空间关系对于适当的核功能至关重要。在恶性疟原虫中,染色体的重新定位与调节负责抗原变异的基因的表达有关,单个核周核仁的形成导致rDNA聚集。尽管如此,染色体之间的精确空间关系仍然知之甚少,因为直到最近,仍缺乏具有足够分辨率的技术。在这里,我们已经使用染色体构象捕获和第二代测序来研究在var基因表达转换期间发生的染色体折叠和空间定位的变化。我们已经以25 Kb的分辨率生成了恶性疟原虫核内染色体空间亲和力的图谱,揭示了结构核仁,染色体区域的缺失,并确认了先前确定的异染色质病灶聚类。我们表明,在var基因表达中的开关似乎并不涉及与远距离增强子的相互作用,但确实会导致活性基因座的局部改变。这些图谱揭示了疟疾染色体的折叠特性,验证了已知的物理关联,并刻画了空间相互作用的全球格局。总体而言,我们的数据为更好地理解疟原虫的基因表达调控和抗原变异提供了关键信息。
  • 【嗜热嗜酸清除剂嗜酸嗜热菌的基因组序列。】 复制标题 收藏 收藏
    DOI:10.1038/35035069 复制DOI
    作者列表:Ruepp A,Graml W,Santos-Martinez ML,Koretke KK,Volker C,Mewes HW,Frishman D,Stocker S,Lupas AN,Baumeister W
    BACKGROUND & AIMS: :Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives at 59 degrees C and pH 2, which was isolated from self-heating coal refuse piles and solfatara fields. Species of the genus Thermoplasma do not possess a rigid cell wall, but are only delimited by a plasma membrane. Many macromolecular assemblies from Thermoplasma, primarily proteases and chaperones, have been pivotal in elucidating the structure and function of their more complex eukaryotic homologues. Our interest in protein folding and degradation led us to seek a more complete representation of the proteins involved in these pathways by determining the genome sequence of the organism. Here we have sequenced the 1,564,905-base-pair genome in just 7,855 sequencing reactions by using a new strategy. The 1,509 open reading frames identify Thermoplasma as a typical euryarchaeon with a substantial complement of bacteria-related genes; however, evidence indicates that there has been much lateral gene transfer between Thermoplasma and Sulfolobus solfataricus, a phylogenetically distant crenarchaeon inhabiting the same environment. At least 252 open reading frames, including a complete protein degradation pathway and various transport proteins, resemble Sulfolobus proteins most closely.
    背景与目标: :Thermoplasma acidophilum是一种嗜热嗜酸古菌,在59摄氏度和pH 2下2,壮成长,它是从自热的煤refuse石堆和solfatara田野中分离出来的。嗜热单胞菌属的物种不具有刚性的细胞壁,而仅由质膜界定。来自热质体的许多大分子装配,主要是蛋白酶和分子伴侣,在阐明其更复杂的真核同源物的结构和功能方面起着关键作用。我们对蛋白质折叠和降解的兴趣促使我们通过确定生物体的基因组序列来寻求更完整地表示这些途径中涉及的蛋白质。在这里,我们使用一种新策略,仅在7,855个测序反应中就对1,564,905个碱基对的基因组进行了测序。 1,509个开放阅读框确定Thermoplasma为典型的euryarchaeon,具有大量与细菌相关的基因。然而,有证据表明,嗜热单胞菌和Sulfolobus solfataricus(一种在同一环境中发生的系统发育距离很近的火棘鱼)之间存在大量的侧向基因转移。至少252个开放阅读框,包括完整的蛋白质降解途径和各种转运蛋白,与Sulfolobus蛋白最为相似。

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