Cohesin establishes sister-chromatid cohesion from S phase until mitosis or meiosis. To allow chromosome segregation, cohesion has to be dissolved. In vertebrate cells, this process is mediated in part by the protease separase, which destroys a small amount of cohesin, but most cohesin is removed from chromosomes without proteolysis. How this is achieved is poorly understood. Here, we show that the interaction between cohesin and chromatin is controlled by Wapl, a protein implicated in heterochromatin formation and tumorigenesis. Wapl is associated with cohesin throughout the cell cycle, and its depletion blocks cohesin dissociation from chromosomes during the early stages of mitosis and prevents the resolution of sister chromatids until anaphase, which occurs after a delay. Wapl depletion also increases the residence time of cohesin on chromatin in interphase. Our data indicate that Wapl is required to unlock cohesin from a particular state in which it is stably bound to chromatin.

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem URL.The BACFinder clone search and oligo design tool is available online at http://www.mitocheck.org/cgi-bin/BACfinder.Database accession codes. The ChIP/chip data has been submitted to the Gene Expression Omnibus database with accession number GSE10845. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http:// www.nature.com/naturemethods/. Europe PMC Funders GroupAuthor Manuscript Nat Methods. Author manuscript; available in PMC 2010 May 17. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.At a time when the 'thousand-dollar genome' seems a realistic goal for the near future, methods for dissecting the functions of the encoded genetic information lag far behind the genome sequence, both in throughput and in quality of the produced data. Genome sequencing and subsequent bioinformatics analysis have made it possible to study the function of genes in mammalian tissue culture cells using systematic reverse-genetic approaches1-3 and have radically improved researchers' ability to identify human disease genes. Such studies typically identify single genes, whose biological function has often not yet been described. In order to place the proteins these genes encode in pathways, these studies must be followed by detailed molecular-level analysis, of which the most powerful types are protein localization and protein-protein interaction. The power of protein localization and protein-protein interaction studies can be seen from the genome-wide application of GFP localization and tandem affinity tag-based complex purification in the yeast Saccharomyces cerevisiae, which has produced a comprehensive picture of the core proteome of a simple, well-studied model system4-8. The key advantage of yeast for these studies was their efficient intrinsic homologous recombination, which allowed the same tagcoding sequence to be introduced at the endogenous locus of nearly every gene of the genome. The tagged proteins were then systematically analyzed through standardized, generic, tag-based assays.To transfer this approach to mammali...

Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference (RNAi) screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization and tandem affinity purificationmass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex (APC/C) and the γ-tubulin ring complex (γ-TuRC), large complexes which are essential †

The human Augmin complex (HAUS) is a critical and evolutionary conserved multisubunit protein complex that regulates centrosome and spindle integrity.

We have conducted a comprehensive metabolic profiling on tomato (Lycopersicon esculentum) leaf and developing fruit tissue using a recently established gas chromatography-mass spectrometry profiling protocol alongside conventional spectrophotometric and liquid chromatographic methodologies. Applying a combination of these techniques, we were able to identify in excess of 70 small-M r metabolites and to catalogue the metabolite composition of developing tomato fruit. In addition to comparing differences in metabolite content between source and sink tissues of the tomato plant and after the change in metabolite pool sizes through fruit development, we have assessed the influence of hexose phosphorylation through fruit development by analyzing transgenic plants constitutively overexpressing Arabidopsis hexokinase AtHXK1. Analysis of the total hexokinase activity in developing fruits revealed that both wild-type and transgenic fruits exhibit decreasing hexokinase activity with development but that the relative activity of the transgenic lines with respect to wild type increases with development. Conversely, both point-by-point and principal component analyses suggest that the metabolic phenotype of these lines becomes less distinct from wild type during development. In summary, the data presented in this paper demonstrate that the influence of hexose phosphorylation diminishes during fruit development and highlights the importance of greater temporal resolution of metabolism.Hexokinase (E.C. 2.7.1.1) catalyzes the phosphorylation of hexoses to form hexose monophosphates. This reaction is especially important in plants because the use of free phosphates is particularly complex in higher plants ( Kruger, 1997). There have been many reports on the presence of glucokinase and hexokinase enzymes in a wide variety of plant species including tomato (Lycopersicon esculentum; Martinez-Barajaz and Randall, 1998), maize (Zea mays; Doehlert, 1989;Schnarrenberger, 1990; Galina et al., 1995), potato (Solanum tuberosum; Renz and Stitt, 1993;Veramendi et al., 1999), pea (Pisum sativum; Turner et al., 1977;Turner and Copeland, 1981) Recently, transgenic manipulations of the activity of hexokinase have been carried out in tomato, potato, and Arabidopsis (Jang et al., 1997; Dai et al., 1999;Veramendi et al., 1999Veramendi et al., , 2002. The results of these manipulations varied greatly between species. Transgenic Arabidopsis seeds that exhibited decreased or increased activities of hexokinase 1 displayed hyposensitive or hypersensitive responses to growth on high (6% [w/v]) Glc containing agar (Jang et al., 1997). The authors concluded that the hexokinase protein acts as a sensor for Glc in an analogous manner to those operating in yeast (Saccharomyces cerevisiae) and that the modulation in the abundance of this sensor led to changes in gene expression that were responsible for the phenotype observed. The overexpression of this Arabidopsis hexokinase isoform in tomato plants led to growth inhibition, reduced photosynthesis, and a...