The rockefeller university

We describe a highly efficient method for exact gene replacement in budding yeast. Induction of rapid and efficient recombination in an entire cell population results in at least 50% of the recombinants undergoing a switch of the endogenous copy to a specific mutated allele, with no remaining markers or remnant of foreign DNA, without selection. To accomplish this, a partial copy of the replacement allele, followed by an HO cut site, is installed adjacent to the wild-type locus, in a GAL-HO MATa-inc background. HO induction results in near-quantitative site cleavage and recombination/gene conversion, resulting in either regeneration of wild-type or switch of the endogenous allele to the mutant, with accompanying deletion of intervening marker sequences, yielding an exact replacement. Eliminating the need for selection (over days) of rare recombinants removes concerns about second-site suppressor mutations and also allows direct phenotypic analysis, even of lethal gene replacements, without the need of a method to make the lethality conditional or to employ regulated promoters of unknown strength compared to the endogenous promoter. To test this method, we tried two known lethal gene replacements, substituting the non-essential CDH1 gene with a dominantly lethal version mutated for its Cdk phosphorylation sites and substituting the essential CDC28 gene with two recessively lethal versions, one containing an early stop codon and another inactivating Cdc28 kinase activity. We also tested a gene replacement of unknown phenotypic consequences: replacing the non-essential CLB3 B-type cyclin with a version lacking its destruction box. Copyright. (c) 2010 John Wiley & Sons, Ltd.

Background: Epidemiologic data have shown that obesity independently increases colorectal cancer (CRC) risk, but the mechanisms are poorly understood. Obesity is an inflammatory state, and chronic colonic inflammation induces CRC. Objective: We conducted this proof-of-principle study to seek evidence of obesity-associated colorectal inflammation and to evaluate effects of diet-induced weight loss. Design: We measured inflammatory cytokines, gene arrays, and macrophage infiltration in rectosigmoid mucosal biopsies of 10 obese premenopausal women [mean +/- SD body mass index (in kg/m(2)): 35 +/- 3.5] before and after weight loss induced by a very-low-calorie diet. Results: Subjects lost a mean (+/- SD) of 10.1 +/- 1% of their initial weight. Weight loss significantly reduced fasting blood glucose, total cholesterol, triglycerides, LDL, tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-8 concentrations (P < 0.05). After weight loss, rectosigmoid biopsies showed a 25-57% reduction in TNF-alpha, IL-1 beta, IL-8, and monocyte chemotactic protein 1 concentrations (P < 0.05). T cell and macrophage counts decreased by 28% and 42%, respectively (P < 0.05). Gene arrays showed dramatic down-regulation of proinflammatory cytokine and chemokine pathways, prostaglandin metabolism, and the transcription factors STAT3 (signal transducer and activator of transcription 3) and nuclear transcription factor kappa B. Weight loss reduced expression of FOS and JUN genes and down-regulated oxidative stress pathways and the transcription factors ATF (activating transcription factor) and CREB (cyclic AMP response element-binding). Conclusions: Our data show that diet-induced weight loss in obese individuals reduces colorectal inflammation and greatly modulates inflammatory and cancer-related gene pathways. These data imply that obesity is accompanied by inflammation in the colorectal mucosa and that diet-induced weight loss reduces this inflammatory state and may thereby lower CRC risk. Am J Clin Nutr 2011;93:234-42.

It has often been suggested that the high curvature of transport intermediates in cells may be a sufficient means to segregate different lipid populations based on the relative energy costs of forming bent membranes. In this review, we present in vitro experiments that highlight the essential physics of lipid sorting at thermal equilibrium: It is driven by a trade-off between bending energy, mixing entropy, and interactions between species. We collect evidence that lipid sorting depends strongly on lipid-lipid and protein-lipid interactions, and hence on the underlying composition of the membrane and on the presence of bound proteins.

NK cells are multicompetent lymphocytes of the innate immune system with a central role in host defense and immune regulation. Studies in experimental animal models of multiple sclerosis (MS) provided evidence for both pathologic and protective effects of NK cells. Humans harbor two functionally distinct NK-cell subsets exerting either predominantly cytotoxic (CD56(dim)CD16(+)) or immunoregulatory (CD56(bright)CD16(-)) functions. We analyzed these two subsets and their functions in the peripheral blood of untreated patients with relapsing-remitting MS compared with healthy blood donors. While ex vivo frequencies of CD56(bright)CD16(-) and CD56(dim)CD16(+) NK cells were similar in patients and controls, we found that cytokine-driven in vitro accumulation and IFN-gamma production of CD56(bright)CD16(-) NK cells but not of their CD56(dim)CD16(+) counterparts were substantially diminished in MS. Impaired expansion of CD56(bright)CD16(-) NK cells was cell intrinsic because the observed effects could be reproduced with purified NK cells in an independent cohort of patients and controls. In contrast, cytolytic NK-cell activity toward the human erythromyeloblastoid leukemia cell line K562, the allogeneic CD4(+) T cell line CEM and allogeneic primary CD4(+) T-cell blasts was unchanged. Thus, characteristic functions of CD56(bright)CD16(-) NK cells, namely cytokine-induced NK cell expansion and IFN-gamma production, are compromised in the NK cell compartment of MS patients.

Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) heavy-chain (IgH) class switch recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating cytidines on template and nontemplate strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA-processing/degradation complex, in targeting AID to both DNA strands. In B lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID-dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID-and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for noncoding RNA surveillance machinery in generating antibody diversity.

Regulation of chromatin structure involves histone posttranslational modifications that can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Notably, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence-based method, we found that uH2B acts through a mechanism distinct from H4 tail acetylation, a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acetylated H4 resulted in synergistic inhibition of higher-order chromatin structure formation, possibly a result of their distinct modes of action.

Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors(1). At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia(2). Mutations in thirteen distinct Fanconi anemia genes(3) have been shown to interfere with the DNA-replication-dependent repair of lesions involving crosslinked DNA at stalled replication forks(4). Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase(5-7), results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.

Histone H3K4 methylation is associated with active genes and, along with H3K27 methylation, is part of a bivalent chromatin mark that typifies poised developmental genes in embryonic stem cells (ESCs). However, its functional roles in ESC maintenance and differentiation are not established. Here we show that mammalian Dpy-30, a core subunit of the SET1/MLL histone methyltransferase complexes, modulates H3K4 methylation in vitro, and directly regulates chromosomal H3K4 trimethylation (H3K4me3) throughout the mammalian genome. Depletion of Dpy-30 does not affect ESC self-renewal, but significantly alters the differentiation potential of ESCs, particularly along the neural lineage. The differentiation defect is accompanied by defects in gene induction and in H3K4 methylation at key developmental loci. Our results strongly indicate an essential functional role for Dpy-30 and SET1/MLL complex-mediated H3K4 methylation, as a component of the bivalent mark, at developmental genes during the ESC fate transitions.

Stem and progenitor cells use asymmetric cell divisions to balance proliferation and differentiation. Evidence from invertebrates shows that this process is regulated by proteins asymmetrically distributed at the cell cortex during mitosis: Par3-Par6-aPKC, which confer polarity, and G alpha(i)-LGN/AGS3-NuMA-dynein/dynactin, which govern spindle positioning. Here we focus on developing mouse skin, where progenitor cells execute a switch from symmetric to predominantly asymmetric divisions concomitant with stratification. Using in vivo skin-specific lentiviral RNA interference, we investigate spindle orientation regulation and provide direct evidence that LGN (also called Gpsm2), NuMA and dynactin (Dctn1) are involved. In compromising asymmetric cell divisions, we uncover profound defects in stratification, differentiation and barrier formation, and implicate Notch signalling as an important effector. Our study demonstrates the efficacy of applying RNA interference in vivo to mammalian systems, and the ease of uncovering complex genetic interactions, here to gain insights into how changes in spindle orientation are coupled to establishing proper tissue architecture during skin development.

We study the behavior of thin elastic sheets that are bent and strained under a weak, smooth confinement. We show that the emerging shapes exhibit the coexistence of two types of domains. A focused-stress patch is subject to a geometric, piecewise-inextensibility constraint, whereas a diffuse-stress region is characterized by a mechanical constraint-the dominance of a single component of the stress tensor. We discuss the implications of our findings for the analysis of elastic sheets under various types of forcing.