Genome-wide survey of protein kinases required for cell cycle progression

Cycles of protein phosphorylation are fundamental in regulating the progression of the eukaryotic cell through its division cycle. Here we test the complement of Drosophila protein kinases (kinome) for cell cycle functions after gene silencing by RNA-mediated interference. We observed cell cycle dysfunction upon downregulation of 80 out of 228 protein kinases, including most kinases that are known to regulate the division cycle. We find new enzymes with cell cycle functions; some of these have family members already known to phosphorylate microtubules, actin or their associated proteins. Additionally, depletion of several signalling kinases leads to specific mitotic aberrations, suggesting novel roles for familiar enzymes. The survey reveals the inter-digitation of systems that monitor cellular physiology, cell size, cellular stress and signalling processes with the basic cell cycle regulatory machinery.     

Genome-wide genetic analysis of polyploidy in yeast

Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution. Despite its prevalence, little is known about the physiological alterations that accompany polyploidy. We previously described 'ploidy-specific lethality', where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids. Here we report a genome-wide screen to identify ploidy-specific lethal functions. Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality. Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function. We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability. We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole. We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores. Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.     

Genome-wide detection and characterization of positive selection in human populations

With the advent of dense maps of human genetic variation, it is now possible to detect positive natural selection across the human genome. Here we report an analysis of over 3 million polymorphisms from the International HapMap Project Phase 2 (HapMap2). We used 'long-range haplotype' methods, which were developed to identify alleles segregating in a population that have undergone recent selection, and we also developed new methods that are based on cross-population comparisons to discover alleles that have swept to near-fixation within a population. The analysis reveals more than 300 strong candidate regions. Focusing on the strongest 22 regions, we develop a heuristic for scrutinizing these regions to identify candidate targets of selection. In a complementary analysis, we identify 26 non-synonymous, coding, single nucleotide polymorphisms showing regional evidence of positive selection. Examination of these candidates highlights three cases in which two genes in a common biological process have apparently undergone positive selection in the same population:LARGE and DMD, both related to infection by the Lassa virus, in West Africa;SLC24A5 and SLC45A2, both involved in skin pigmentation, in Europe; and EDAR and EDA2R, both involved in development of hair follicles, in Asia.     

Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia

Chromosomal aberrations are a hallmark of acute lymphoblastic leukaemia (ALL) but alone fail to induce leukaemia. To identify cooperating oncogenic lesions, we performed a genome-wide analysis of leukaemic cells from 242 paediatric ALL patients using high-resolution, single-nucleotide polymorphism arrays and genomic DNA sequencing. Our analyses revealed deletion, amplification, point mutation and structural rearrangement in genes encoding principal regulators of B lymphocyte development and differentiation in 40% of B-progenitor ALL cases. The PAX5 gene was the most frequent target of somatic mutation, being altered in 31.7% of cases. The identified PAX5 mutations resulted in reduced levels of PAX5 protein or the generation of hypomorphic alleles. Deletions were also detected in TCF3 (also known as E2A), EBF1, LEF1, IKZF1 (IKAROS) and IKZF3 (AIOLOS). These findings suggest that direct disruption of pathways controlling B-cell development and differentiation contributes to B-progenitor ALL pathogenesis. Moreover, these data demonstrate the power of high-resolution, genome-wide approaches to identify new molecular lesions in cancer.     

Genome-wide analysis of nucleotide-binding site disease resistance genes in Medicago truncatula

The class of nucleotide-binding site （NBS）- Leucine-rich repeat （LRR） disease resistance genes play an important role in defending plants from a variety of pathogens and insect pests. Consequently, many NBS-LRR genes have been identified in various plant species. In this study, we identified 617 NBS-encoding genes in the Medicago truncatula genome （Mt3.5v5） and divided them into two groups, regular （490） and non-regular （127） NBS- LRR genes. The regular NBS-LRR genes were character- ized on the bases of structural diversity, chromosomal location, gene duplication, conserved protein motifs, and EST expression profiling. According to N-terminal motifs and LRR motifs, the 490 regular NBS-LRR genes were then classified into 10 types： CC-NBS （4）, CC-NBS-LRR （212）, TIR-NBS （20）, TIR-NBS-LRR （160）, TIR-NBS-TIR （1）, TIR-NBS-TIR-LRR （2）, NBS-TIR （7）, NBS-TIR-LRR （1）, NBS （10）, and NBS-LRR （73）. Analysis of the phys- ical location and duplications of the regular NBS-LRR genes revealed that the M. truncatula genome is similar to rice. Interestingly, we found that TIR-type genes are more frequently expressed than non-TIR-type genes in M. trun- catula, whereas the number of non-TIR-type regular NBS- LRR genes was greater than TIR-type genes, suggesting the gene expression was not associated with the total number of NBS-LRR genes. Moreover, we found that the phylogenetic tree supported our division of the regular NBS-LRR genes into two distinct clades （TIR-type and non-TIR-type）, but some of the non-TIR-type lineages contain TIR-type genes. These analyses provide a robust database of NBS-LRR genes in M. truncatula that will facilitate the isolation of new resistance genes and breeding strategies to engineer disease resistance in leguminous crop     

Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes

Regulation of body fat storage involves signalling between centres that regulate feeding in the brain and sites of fat storage and use in the body. Here we describe an assay for analysing fat storage and mobilization in living Caenorhabditis elegans. By using RNA-mediated interference (RNAi) to disrupt the expression of each of the 16,757 worm genes, we have systematically screened the C. elegans genome for genes necessary for normal fat storage. We identify 305 gene inactivations that cause reduced body fat and 112 gene inactivations that cause increased fat storage. Analysis of the fat-reducing gene inactivations in insulin, serotonin and tubby signalling mutants of C. elegans, which have increased body fat, identifies a core set of fat regulatory genes as well as pathway-specific fat regulators. Many of the newly identified worm fat regulatory genes have mammalian homologues, some of which are known to function in fat regulation. Other C. elegans fat regulatory genes that are conserved across animal phylogeny, but have not previously been implicated in fat storage, may point to ancient and universal features of fat storage regulation, and identify targets for treating obesity and its associated diseases.     

Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae

A functional genomics approach has revealed that caveolae/raft-mediated endocytosis is subject to regulation by a large number of kinases. Here we explore the role of some of these kinases in caveolae dynamics. We discover that caveolae operate using principles different from classical membrane trafficking. First, each caveolar coat contains a set number (one 'quantum') of caveolin-1 molecules. Second, caveolae are either stored as in stationary multi-caveolar structures at the plasma membrane, or undergo continuous cycles of fission and fusion with the plasma membrane in a small volume beneath the surface, without disassembling the caveolar coat. Third, a switch mechanism shifts caveolae from this localized cycle to long-range cytoplasmic transport. We have identified six kinases that regulate different steps of the caveolar cycle. Our observations reveal new principles in caveolae trafficking and suggest that the dynamic properties of caveolae and their transport competence are regulated by different kinases operating at several levels.     

Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene

The chief limitation of phenotype-based genetic screening in mammalian systems is the diploid nature of the genome. Cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of loss of heterozygosity. Here we have used a tetracycline-regulated Blm allele (Blm(tet)) to introduce bi-allelic mutations across the genome in mouse embryonic stem (ES) cells. Transient loss of Blm expression induces homologous recombination not only between sister chromatids but also between homologous chromosomes. We considered that the phenotype of ES cells bearing bi-allelic mutations would be maintained after withdrawal of the tetracycline analogue doxycycline. Indeed, a combination of N-ethyl-N-nitrosourea mutagenesis and transient loss of Blm expression enabled us to generate an ES cell library with genome-wide bi-allelic mutations. The library was evaluated by screening for mutants of glycosylphosphatidylinositol-anchor biosynthesis, which involves at least 23 genes distributed throughout the genome. Mutants derived from 12 different genes were obtained and two unknown mutants were simultaneously isolated. Our results indicate that phenotype-based genetic screening with Blm(tet) is very efficient and raises possibilities for identifying gene functions in ES cells.     

高等动植物网格蛋白介导的内吞

主要阐述了网格蛋白、接头蛋白AP2复合体的组成及其功能、动物网格蛋白介导内吞的分子机制和植物网格蛋白介导内吞的一些最新进展;阐述了植物网格蛋白介导的内吞在生长素极性运输、胚胎发育及逆境响应等中的作用,总结了植物网格蛋白介导内吞的生物学意义及展望．     

应用nanoLC/MS/MS对人羊水的蛋白质组学分析

建立应用nano LC/MS/MS技术寻找羊水特异性表达蛋白质的蛋白质组学方法.羊膜穿刺获取两份正常妊娠晚期的羊水,通过Cibacron blue和Protein A吸附去除血清白蛋白和IgGs,样品经变性、还原和烷基化后酶解,nano LC/MS/MS分析,鉴定了62种蛋白质.通过与已有的羊水和血浆蛋白质表达数据库比对,从中新发现22种只在羊水中表达的蛋白质,其中4种已知是子宫或妊娠特异的.本研究建立了nano LC/MS/MS对羊水进行蛋白组学分析的方法并发现了一些可能的羊水特异表达蛋白.     

全基因组关联分析在大豆遗传学上的研究进展

全基因组关联分析(GWAS)是挖掘作物重要性状遗传信息的主要手段。随着测序技术的快速发展,开发单核苷酸多态性(SNP)标记的成本大幅降低,以连锁不平衡(LD)为遗传基础的GWAS技术已被广泛用于研究作物的农艺、品质和抗性等复杂性状。近年来GWAS技术已成功运用到对大豆重要性状的遗传解析上,并取得了一系列的研究进展。在简要介绍GWAS技术的原理和实施方法的基础上,总结分析了近年来其在大豆重要农艺性状、品质性状和逆境抗性性状上的遗传学研究进展及应用前景。     

Genome-wide non-mendelian inheritance of extra-genomic information in Arabidopsis

A fundamental tenet of classical mendelian genetics is that allelic information is stably inherited from one generation to the next, resulting in predictable segregation patterns of differing alleles. Although several exceptions to this principle are known, all represent specialized cases that are mechanistically restricted to either a limited set of specific genes (for example mating type conversion in yeast) or specific types of alleles (for example alleles containing transposons or repeated sequences). Here we show that Arabidopsis plants homozygous for recessive mutant alleles of the organ fusion gene HOTHEAD (HTH) can inherit allele-specific DNA sequence information that was not present in the chromosomal genome of their parents but was present in previous generations. This previously undescribed process is shown to occur at all DNA sequence polymorphisms examined and therefore seems to be a general mechanism for extra-genomic inheritance of DNA sequence information. We postulate that these genetic restoration events are the result of a template-directed process that makes use of an ancestral RNA-sequence cache.     

大豆BGLU基因家族全基因组鉴定与表达分析

在植物中,β-葡萄糖苷酶(BGLU)参与调控许多重要的生理过程.利用大豆基因组数据库,共鉴定了42个大豆BGLU家族基因,对该家族成员的基因特征、蛋白结构、系统进化、染色体定位、启动子调控元件和表达模式等进行了全面分析.为今后研究大豆BGLU家族基因的功能提供了基因信息.     

DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage

Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome. It is also enriched in segmental duplications, ranking third in density among the autosomes. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.