Overview of DNA chip technology

DNA chip technology utilizes microscopic arrays (microarrays) of molecules immobilized on solid surfaces for biochemical analysis. Microarrays can be used for expression analysis, polymorphism detection, DNA resequencing, and genotyping on a genomic scale. Advanced arraying technologies such as photolithograpy, micro-spotting and ink jetting, coupled with sophisticated fluorescence detection systems and bioinformatics, permit molecular data gathering at an unprecedented rate. Microarray-based characterization of plant genomes has the potential to revolutionize plant breeding and agricultural biotechnology. This review provides an overview of DNA chip technology, focusing on manufacturing approaches and biological applications.     

DNA微阵列技术及其在肿瘤生物学中的应用

人类基因组测序工作的完成使人们可以方便地调用任何基因序列,但仅有基因序列并不能解释众多的生物学问题,这要求发展一种高通量的技术用于研究基因的生物学功能以及基因的相互作用。DNA微阵列技术以其高通量的特点,已经在肿瘤生物学的研究中逐渐被采用。由于癌症是源于基因表达谱改变的基因疾病,通过DNA微阵列技术研究癌症细胞和对应的正常细胞的基因表达差异,将会使人们更好地了解肿瘤的形成和发展过程。     

Microarray technology GEM microarrays and drug discovery

Incyte Genomics' GEM™ Gene Expression Microarray is a proven genomics tool used by a large number of pharmaceutical companies to speed up the drug discovery and development process. The development and integration of this technology, together with Incyte's sequence databases and clone resources, have resulted in GEM microarrays that span approximately 60,000 human genes as well as approximately 60,000 plant, rat, mouse, yeast, and bacterial genes. The technology underlying the use of these arrays and their application to the drug discovery process is highlighted. Journal of Industrial Microbiology & Biotechnology (2002) 28, 180–185 DOI: 10.1038/sj/jim/7000136 Received 16 November 2000/ Accepted in revised form 01 March 2001     

Efficient use of DNA molecular markers to construct industrial yeast strains

Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phase duration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50-170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure.     

酿酒酵母染色体设计与合成研究进展

随着合成生物学的蓬勃发展,基因组学的研究正在由读取基因组信息拓展到以编写基因组信息为主的合成基因组学时代。2009年,由Jef D. Boeke教授提出的人工合成酵母基因组计划(Sc2.0)旨在合成世界上首个真核生物基因组。在美、中、英、法、澳大利亚、新加坡等多国科学家的努力下,目前已经完成1/3的酵母染色体的人工合成。本文从合成基因组学领域的发展历程出发,介绍了Sc2.0计划中酿酒酵母(Saccharomyces cerevisiae)染色体设计与合成的最新进展,包括酿酒酵母9号染色体右臂、3号染色体、2号染色体、5号染色体、6号染色体、10号染色体和12号染色体的设计与合成过程,阐述了其各自的合成策略以及生物学意义,以期为合成基因组学的深入开展提供借鉴与参考。     

副溶血性弧菌全基因组DNA芯片的研制和质量评价

【目的】研制副溶血性弧菌全基因组芯片,建立芯片杂交方法,并对芯片质量进行评价。【方法】利用副溶血性弧菌全基因组序列,挑选出4770条基因,PCR扩增各基因并将PCR产物纯化,点样制备芯片;设计了两个质控杂交组合,采用双色荧光杂交策略,对芯片质量进行评价;PCR方法验证部分芯片结果。【结果】芯片杂交与理论预期结果以及PCR验证结果完全一致。【结论】成功的研制了一批质量良好的副溶血性弧菌全基因组DNA芯片,并建立了基于DNA芯片的副溶血性弧菌比较基因组学技术平台,建立了一套系统的芯片数据分析的标准方法。     

A negative relationship between mutation pleiotropy and fitness effect in yeast

It is generally thought that random mutations will, on average, reduce an organism's fitness because resulting phenotypic changes are likely to be maladaptive. This relationship leads to the prediction that mutations that alter more phenotypic traits, that is, are more pleiotropic, will impose larger fitness costs than mutations that affect fewer traits. Here we present a systems approach to test this expectation. Previous studies have independently estimated fitness and morphological effects of deleting all nonessential genes in Saccharomyces cerevisiae. Using datasets generated by these studies, we examined the relationship between the pleiotropic effect of each deletion mutation, measured as the number of morphological traits differing from the parental strain, and its effect on fitness. Pleiotropy explained approximately 18% of variation in fitness among the mutants even once we controlled for correlations between morphological traits. This relationship was robust to consideration of other explanatory factors, including the number of protein-protein interactions and the network position of the deleted genes. These results are consistent with pleiotropy having a direct role in affecting fitness.     

重组蛋白质在酵母表达体系中的高效表达策略

酵母由于其本身的一些优良特性和容易操作性,可以高水平表达重组蛋白,近年来已经有多个酵母表达的蛋白质多肽类药物上市。作为宿主的酵母最常用的是毕赤氏酵母和酿酒酵母。本文对酵母的一般特性、酵母表达操作、密码子、载体和表达策略、两种不同酵母表达系统的特点等进行了论述,供研究者进行酵母高效表达体系的选择与操作参考。     

新型酵母表达系统的研究

酵母表达系统是在酿酒酵母质粒的发现和酵母转化技术的成熟基础上建立起来的真核生物表达系统。许多有应用价值的外源基因成功地在其中表达,但也存在一些局限性。为了开发新的酵母表达系统,人们在酿酒酵母以外的酵母中寻找合适的宿主以建立新的载体－宿主系统。本文综述了建立新的酵母表达系统涉及的有关问题和近几年来的研究进展。     

The effect of genetic and environmental variation on metabolic gene expression

What is the relationship between genetic or environmental variation and the variation in messenger RNA (mRNA) expression? To address this, microarrays were used to examine the effect of genetic and environmental variation on cardiac mRNA expression for metabolic genes in three groups of Fundulus heteroclitus: (i) individuals sampled in the field (field), (ii) field individuals acclimated for 6 months to laboratory conditions (acclimated), or (iii) individuals bred for 10 successive generations in a laboratory environment (G10). The G10 individuals have significantly less genetic variation than individuals obtained in the field and had a significantly lower variation in mRNA expression across all genes in comparison to the other two groups (P = 0.001). When examining the gene specific variation, 22 genes had variation in expression that was significantly different among groups with lower variation in G10 individuals than in acclimated individuals. Additionally, there were fewer genes with significant differences in expression among G10 individuals vs. either acclimated or field individuals: 66 genes have statistically different levels of expression vs. 107 or 97 for acclimated or field groups. Based on the permutation of the data, these differences in the number of genes with significant differences among individuals within a group are unlikely to occur by chance (P < 0.01). Surprisingly, variation in mRNA expression in field individuals is lower than in acclimated individuals. Relative to the variation among individual within a group, few genes have significant differences in expression among groups (seven, 2.3%) and none of these are different between acclimated and field individuals. The results support the concept that genetic variation affects variation in mRNA expression and also suggests that temporal environmental variation associated with estuarine environments does not increase the variation among individuals or add to the differences among groups.     

A New System for Comparative Functional Genomics of Saccharomyces Yeasts

Whole-genome sequencing, particularly in fungi, has progressed at a tremendous rate. More difficult, however, is experimental testing of the inferences about gene function that can be drawn from comparative sequence analysis alone. We present a genome-wide functional characterization of a sequenced but experimentally understudied budding yeast, Saccharomyces bayanus var. uvarum (henceforth referred to as S. bayanus), allowing us to map changes over the 20 million years that separate this organism from S. cerevisiae. We first created a suite of genetic tools to facilitate work in S. bayanus. Next, we measured the gene-expression response of S. bayanus to a diverse set of perturbations optimized using a computational approach to cover a diverse array of functionally relevant biological responses. The resulting data set reveals that gene-expression patterns are largely conserved, but significant changes may exist in regulatory networks such as carbohydrate utilization and meiosis. In addition to regulatory changes, our approach identified gene functions that have diverged. The functions of genes in core pathways are highly conserved, but we observed many changes in which genes are involved in osmotic stress, peroxisome biogenesis, and autophagy. A surprising number of genes specific to S. bayanus respond to oxidative stress, suggesting the organism may have evolved under different selection pressures than S. cerevisiae. This work expands the scope of genome-scale evolutionary studies from sequence-based analysis to rapid experimental characterization and could be adopted for functional mapping in any lineage of interest. Furthermore, our detailed characterization of S. bayanus provides a valuable resource for comparative functional genomics studies in yeast.     

副溶血性弧菌全基因组DNA芯片的研制和芯片质量的评价

摘要 目的 研制副溶血性弧菌全基因组芯片,建立芯片杂交方法,并对芯片质量进行评价。方法 利用副溶血性弧菌全基因组序列,挑选出4770条基因,PCR扩增各基因并将PCR产物纯化,点样制备芯片;设计了两个质控杂交组合,采用双色荧光杂交策略,对芯片质量进行评价;PCR方法验证部分芯片结果。结果 芯片杂交与理论预期结果以及PCR验证结果完全一致。结论 成功的研制了一批质量良好的副溶血性弧菌全基因组DNA芯片,并建立了基于DNA芯片的副溶血性弧菌比较基因组学技术平台,建立了一套系统的芯片数据分析的标准方法。     

The quest for a message: budding yeast, a model organism to study the control of pre-mRNA splicing

Removal of introns during pre-mRNA splicing is a critical processin gene expression, and understanding its control at both single-geneand genomic levels is one of the great challenges in Biology.Splicing takes place in a dynamic, large ribonucleoprotein complexknown as the spliceosome. Combining Genetics and Biochemistry,Saccharomyces cerevisiae provides insights into its mechanisms,including its regulation by RNA–protein interactions.Recent genome-wide analyses indicate that regulated splicingis broad and biologically relevant even in organisms with arelatively simple intronic structure, such as yeast. Furthermore,the possibility of coordination in splicing regulation at genomiclevel is becoming clear in this model organism. This shouldprovide a valuable system to approach the complex problem ofthe role of regulated splicing in genomic expression.      

机械点样DNA微点阵技术及其在基因表达分析上的应用(I)

从芯片制作、芯片杂交、芯片扫读与图像分析、基因表达数据分析等方面,详细介绍了机械点样DNA微点阵技术及其应用于多基因表达分析的基本步骤与原理。     

机械点样DNA微点阵技术及其在基因表达分析上的应用(II)

从芯片制作、芯片杂交、芯片扫读与图像分析、基因表达数据分析等方面,详细介绍了机械点样DNA微点阵技术及其应用于多基因表达分析的基本步骤与原理。     

A population genomics insight into the Mediterranean origins of wine yeast domestication

The domestication of the wine yeast Saccharomyces cerevisiae is thought to be contemporary with the development and expansion of viticulture along the Mediterranean basin. Until now, the unavailability of wild lineages prevented the identification of the closest wild relatives of wine yeasts. Here, we enlarge the collection of natural lineages and employ whole‐genome data of oak‐associated wild isolates to study a balanced number of anthropic and natural S. cerevisiae strains. We identified industrial variants and new geographically delimited populations, including a novel Mediterranean oak population. This population is the closest relative of the wine lineage as shown by a weak population structure and further supported by genomewide population analyses. A coalescent model considering partial isolation with asymmetrical migration, mostly from the wild group into the Wine group, and population growth, was found to be best supported by the data. Importantly, divergence time estimates between the two populations agree with historical evidence for winemaking. We show that three horizontally transmitted regions, previously described to contain genes relevant to wine fermentation, are present in the Wine group but not in the Mediterranean oak group. This represents a major discontinuity between the two populations and is likely to denote a domestication fingerprint in wine yeasts. Taken together, these results indicate that Mediterranean oaks harbour the wild genetic stock of domesticated wine yeasts.