Gene mapping in the physical map of the Triticum aestivum chromosome arm 1BL
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ABSTRACT: To improve our understanding of the organization and evolution of the wheat gene space, we established the first map of genes of the wheat chromosome 1BL by hybridizing the newly developed INRA GDEC Triticum aestivum NimbleGen 12x40k unigenes microarray (A-MEXP-2314) with BAC pools from the 1BL physical map as well as with genomic DNA of the sorted chromosome 1BL. By hybridizing the BAC pools with the wheat NimbleGen 40K unigenes chip we managed to map almost 1615 unigenes on the wheat chromosome 1BL BACs. By hybridizing the genomic DNA of the 1BL sorted chromosome and by comparison with 454 sequences from the sorted chromosome 1BL, we confirmed the assignation of 1223 unigenes in individual BACs from the chromosome 1BL. This data allowed us to study the organization of the wheat gene space along chromosome 1BL. The sequences of the unigenes helped to perform synteny and evolutionary analyses of these unigenes.
Project description:To improve our understanding of the organization and evolution of the wheat gene space, we established the first map of genes of the wheat chromosome 1BS by hybridizing the newly developed INRA GDEC Triticum aestivum NimbleGen 12x40k unigenes microarray (A-MEXP-2314) with 3D-pools of MTP BACs of from the 1BS physical map. By hybridizing the BAC pools with the wheat NimbleGen 40K unigenes chip we managed to map almost 1063 unigenes on the wheat chromosome 1BS BACs. By comparison with 454 sequences and Illumina survey sequence contigs from the sorted chromosome 1BS, we confirmed the assignation of 849 unigenes in individual BACs from the chromosome 1BS. This data allowed us to study the organization of the wheat gene space along chromosome 1BS. The sequences of the unigenes helped to perform synteny and evolutionary analyses of these unigenes. DNA from MTP clones were pooled into 3D manner: library of MTP clones was stored in 17 plates of 384 wells (24 columns x 16 rows); plate1 pool consist of mixture of DNA from all MTP clones situated in plate 1, Row A pool consist of mixture of DNA from all MTP clones situated in Rows A (from all 17 plates, etc). The set of positive plate, column and row pools for the unigene (represented in microarray) allow to detect the list of putative positive clones (clones from the intersection of positive pools, cleaned using information on physical intersection clones based on clone fingerprints). Hence, all 57 experiments (17 for plate pools, 24 for column pools, and 16 for row pools) have the same experimental factor.
Project description:The development of leptomeningeal melanoma metastases (LMM) is a rare and devastating complication of the late-stage disease, for which no effective treatments exist. Here, we performed a multi-omics analysis of the CSF from LMM patients to determine how the leptomeningeal microenvironment shapes the biology and therapeutic responses of melanoma cells. A total of 45 serial CSF samples were collected from 16 patients, 8 of these with confirmed LMM. Of those with LMM, 7 had poor survival (<4 months) and one was an extraordinary responder (still alive with survival >32 months). CSF samples were analyzed by mass spectrometry and incubated with melanoma cells, that were subjected to RNA-Seq analysis. Functional assays were performed to validate the pathways identified. Mass spectrometry analyses showed the CSF of most LMM patients to be enriched for pathways involved in innate immunity, protease-mediated damage, and IGF-related signaling. All of these were anti-correlated in the extraordinary responder. RNA-Seq analysis showed CSF to induce PI3K/AKT, integrin, B-cell activation, S-phase entry, TNFR2, TGF- and oxidative stress responses in the melanoma cells. ELISA assays confirmed that TGF- expression increased in the CSF of patients progressing with LMM. CSF from poorly responding patients conferred tolerance to BRAF inhibitor therapy in apoptosis assays. These analyses identified proteomic/transcriptional signatures in the CSF of patients who succumbed to LMM. We further showed that the CSF from LMM patients has the potential to modulate BRAF inhibitor responses and may contribute to drug resistance.
Project description:Deciphering the role of alternative splicing in developmental processes relies on the identification of key genes whose expression is controlled by splicing regulators throughout growth of a whole organism. Targeting expression of five SR proteins in the developing eye of Drosophila allowed us to show that these splicing factors induce various phenotypic alterations concerning eye organogenesis and viability. Although both dASF/SF2 and B52 caused defects in ommatidia structure, only B52 impairs normal photoreceptor axons projection and neurogenesis in visual ganglia. Consistently, microarray analyses revealed that many of the B52 targets are involved in brain organogenesis and we show that their splicing profile is altered both in B52 loss and gain of function. Conversely, a large proportion of dASF/SF2 targets are involved in eye development. This differential effect argues that SR proteins confer accuracy to developmental gene-expression programs, thus ensuring tissue identity and supporting cell-lineage decisions. Keywords: genetic modification Experiment aimed at determining whether increased SR protein B52 expression can affect global gene expression in Drosophila. Control: GMR X GFP-NLS trangenic larvae The 2 samples correspond to dye-swap experiments.
Project description:Deciphering the role of alternative splicing in developmental processes relies on the identification of key genes whose expression is controlled by splicing regulators throughout growth of a whole organism. Targeting expression of five SR proteins in the developing eye of Drosophila allowed us to show that these splicing factors induce various phenotypic alterations concerning eye organogenesis and viability. Although both dASF/SF2 and B52 caused defects in ommatidia structure, only B52 impairs normal photoreceptor axons projection and neurogenesis in visual ganglia. Consistently, microarray analyses revealed that many of the B52 targets are involved in brain organogenesis and we show that their splicing profile is altered both in B52 loss and gain of function. Conversely, a large proportion of dASF/SF2 targets are involved in eye development. This differential effect argues that SR proteins confer accuracy to developmental gene-expression programs, thus ensuring tissue identity and supporting cell-lineage decisions. Keywords: genetic modification Experiment aimed at identifying dASF/SF2 potential mRNA targets in the Drosophila developing eye Control: GMR X GFP-NLS trangenic larvae The 2 IP- GFP cy3 vs ASF cy5 rep.1 and IP- GFP cy5 vs ASF cy3 rep.1 samples correspond to dye-swap experiments. Two replicates (rep1 and rep2) are included.
Project description:Deciphering the role of alternative splicing in developmental processes relies on the identification of key genes whose expression is controlled by splicing regulators throughout growth of a whole organism. Targeting expression of five SR proteins in the developing eye of Drosophila allowed us to show that these splicing factors induce various phenotypic alterations concerning eye organogenesis and viability. Although both dASF/SF2 and B52 caused defects in ommatidia structure, only B52 impairs normal photoreceptor axons projection and neurogenesis in visual ganglia. Consistently, microarray analyses revealed that many of the B52 targets are involved in brain organogenesis and we show that their splicing profile is altered both in B52 loss and gain of function. Conversely, a large proportion of dASF/SF2 targets are involved in eye development. This differential effect argues that SR proteins confer accuracy to developmental gene-expression programs, thus ensuring tissue identity and supporting cell-lineage decisions. Keywords: genetic modification Experiment aimed at identifying B52 potential mRNA targets in the Drosophila developing eye Control: GMR X GFP-NLS trangenic larvae The 2 IP- GFP cy3 vs B52 cy5 rep.1 and IP- GFP cy5 vs B52 cy3 rep.1 samples correspond to dye-swap experiments. Two replicates (rep1 and rep2) are included.
Project description:Deciphering the role of alternative splicing in developmental processes relies on the identification of key genes whose expression is controlled by splicing regulators throughout growth of a whole organism. Targeting expression of five SR proteins in the developing eye of Drosophila allowed us to show that these splicing factors induce various phenotypic alterations concerning eye organogenesis and viability. Although both dASF/SF2 and B52 caused defects in ommatidia structure, only B52 impairs normal photoreceptor axons projection and neurogenesis in visual ganglia. Consistently, microarray analyses revealed that many of the B52 targets are involved in brain organogenesis and we show that their splicing profile is altered both in B52 loss and gain of function. Conversely, a large proportion of dASF/SF2 targets are involved in eye development. This differential effect argues that SR proteins confer accuracy to developmental gene-expression programs, thus ensuring tissue identity and supporting cell-lineage decisions. Keywords: genetic modification Experiment aimed at determining whether increased SR protein dASF/SF2 expression can affect global gene expression in Drosophila. Control: GMR X GFP-NLS trangenic larvae The 2 samples correspond to dye-swap experiments.
Project description:We performed a genome-wide association study in pooled DNA samples from patients with severe statin myopathy and persistent symptoms post-therapy versus pooled DNAs from an age-adjusted statin-tolerant group. Affymetrix 100K SNP arrays were used according to the manufacturers instructions with two pools of 19 and 20 statin myopathy patients and two pools of 20 statin-tolerant controls.
Project description:We performed a large-scale expression analysis with inducible gain-of-function (GOF) lines which contain a C-terminal fusion protein of the TF of interest and a glucocorticoid receptor (GR) domain, driven by a constitutive 35S promoter. Such fusion proteins reside in the cytosol and can only translocate to the nucleus in the presence of dexamethasone (DEX), enabling the TF to regulate its downstream target genes (Corrado and Karali, 2009). In total 18 GOF lines were used of the following TFs: ERF-1 (AT4G17500), ERF2 (AT5G47220), ERF5 (AT5G47230), ERF6 (AT4G17490), ERF11 (AT1G28370), ERF8 (AT1G53170), ERF9 (AT5G44210), ERF59 (AT1G06160), ERF98 (AT3G23230), MYB51 (AT1G18570), STZ (AT1G27730), WRKY28 (AT4G18170), WRKY33 (AT2G38470), WRKY15 (AT2G23320), ZAT6 (AT5G04340), WRKY48 (AT5G49520), RAP2.6L (AT5G13330) and the control 35S::GFP-GR. To get an indication of which genes are direct or indirect targets of the induced TF, we performed a time-course experiment. All GOF lines were transferred at 15 DAS to DEX-containing medium and the third leaf was harvested at 1 h, 2 h, 4 h, 8 h and 24 h after transfer. The expression of 30 TFs (17 TFs listed above and WRKY6 (AT1G62300), WRKY30 (AT5G24110) and WRKY40 (AT1G80840), GA2OX6 (AT1G02400), GA20OX1 (AT4G25420), ACS6 (AT4G11280), MPK3 (AT3G45640), DEL1 (AT3G48160), UVI4 (AT2G42260), CYCA3;1 (AT5G43080), CYCB1;2 (AT5G06150), XET (AT1G10550), EXPA5 (AT3G29030)) was measured with nCounter Nanostring (Geiss et al, 2008). The time-course experiment gives an indication of whether a gene is putatively a direct, and thus induced during the early time points, or an indirect target of the induced TF.
Project description:Maternal low-protein diet increases the susceptibility of offspring to type 2 diabetes. An insult to the pancreas during development can have long-term consequences on ?-cell mass and function. Because nutrients and growth factors signaling converge on mTOR, we hypothesized that mTOR plays a central role in ?-cell programming during fetal development. In this study, we revealed that newborns of dams exposed to low-protein diet (LP0.5) throughout pregnancy exhibited decreased insulin levels, ?-cell fraction, and mTOR signaling. Adult LP0.5 offspring demonstrated enhanced insulin sensitivity but remained glucose intolerant due to an insulin secretory defect and not reduced ?-cell mass. The insulin secretory defect was distal to Ca2+ influx, at the level of proinsulin biosynthesis and insulin content. LP0.5 islets exhibited reduced mTOR protein and increased expression of specific microRNAs. The reductions in mTOR protein and insulin secretion in LP0.5 islets were restored to normal by blockade of microRNAs 199a-3p and 342. Finally, transient activation of mTORC1 signaling in ?-cells during the last week of pregnancy rescued the neonatal ?-cell fraction defect and metabolic abnormalities in LP0.5 mice. These findings identify a major role of microRNAs and mTOR in ?-cell programing by maternal low-protein diet. To investigate which microRNAs are altered in islets isolated from 2-3 months old male offspring of dams fed low-protein diet (LP0.5) or control diet throughout pregnancy Pancreatic islets were isolated from 2-3 months old male offspring of dams fed low-protein diet (LP0.5) or control diet throughout pregnancy. Islets were isolated by collagenase digestion. The pancreas was perfused via the common duct with 1 mg/ml collagenase XI (Sigma-Aldrich) in HBSS (Life Technologies). Pancreatic digestion was carried out at 37 °C for 15 min, after which cold HBSS with 2.5% FBS (Life Technologies) was added. The suspension was centrifuged at 1000 rpm for 30s, washed 3 times with HBSS with 2.5% FBS, resuspended in RPMI complete media (RPMI 1640 with 5 mM Glucose, 10% FBS,100 IU/ml penicillin, 100 g/ml streptomycin) and poured onto a 70 µm cell strainer (BD Falcon, BD Biosciences). Islets were rinsed and handpicked. Islets were allowed to recover overnight in RPMI complete media before RNA isolation using MirVana Kit (Life Technologies).
Project description:The kep1 gene encodes an RNA-binding protein containing a single maxi-KH domain. We have previously demonstrated that loss of the kep1 gene results in Drosophila females displaying a reduction in fertility and wished to further characterize the kep1 mutation in Drosophila males. Wild-type females mated to homozygous kep1- males resulted in 6% of the eggs laid hatching. This reduced reproductive success is due in part to a meiosis defect during spermatogenesis with approximately 10 % of sperm demonstrating a defect in cytokinesis. Utilizing indirect immunohistochemistry we found that the Kep1 protein is present in the nucleus of adult brain neuronal cells, suggesting a behavioural component contributing to the almost complete male sterility phenotype. We report that homozygote kep1- males display almost no courtship behaviour with a measured median courtship index of 0.005 relative to a median index of 0.77 for OreR wild-type controls. In order to better understand the behavioural role of kep1, we carried out gene profiling experiments to identify transcripts whose steady-state levels are altered in the kep1- homozygote males. Our gene profiling studies identified 61 transcripts whose steady-state levels are altered by at least 2 fold or more comparing RNA isolated from w1118 and kep1-/kep1- male heads. A large percentage of transcripts identified whose steady-state expression levels are depleted in kep1-/kep1- heads (10 out of 44) encode for proteins involved in some aspect of proteolysis. Our results show that the Kep1 protein has a pleiotropic effect in Drosophila males leading to cytokinesis defects and behavioural abnormalities. RNA was isolated from heads of 3-4 day old wild-type or homozygote mutant males. Three indepentent RNA samples were utilized to complete experiment.