1,400 research outputs found

    Physical mapping integrated with syntenic analysis to characterize the gene space of the long arm of wheat chromosome 1A

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    Background: Bread wheat (Triticum aestivum L.) is one of the most important crops worldwide and its production faces pressing challenges, the solution of which demands genome information. However, the large, highly repetitive hexaploid wheat genome has been considered intractable to standard sequencing approaches. Therefore the International Wheat Genome Sequencing Consortium (IWGSC) proposes to map and sequence the genome on a chromosome-by-chromosome basis. Methodology/Principal Findings: We have constructed a physical map of the long arm of bread wheat chromosome 1A using chromosome-specific BAC libraries by High Information Content Fingerprinting (HICF). Two alternative methods (FPC and LTC) were used to assemble the fingerprints into a high-resolution physical map of the chromosome arm. A total of 365 molecular markers were added to the map, in addition to 1122 putative unique transcripts that were identified by microarray hybridization. The final map consists of 1180 FPC based or 583 LTC based contigs. Conclusions/Significance: The physical map presented here marks an important step forward in mapping of hexaploid bread wheat. The map is orders of magnitude more detailed than previously available maps of this chromosome, and the assignment of over a thousand putative expressed gene sequences to specific map locations will greatly assist future functional studies. This map will be an essential tool for future sequencing of and positional cloning within chromosome 1A

    Enhancer hijacking determines intra- and extrachromosomal circular MYCN amplicon architecture in neuroblastoma

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    MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA. The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyzed the MYCN amplicon structure and its chromatin landscape. This revealed two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplified a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons was characterized by high structural complexity, lacked key local enhancers, and instead contained distal chromosomal fragments, which harbored CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification

    A novel genomic alteration of LSAMP associates with aggressive prostate cancer in African American men

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    Evaluation of cancer genomes in global context is of great interest in light of changing ethnic distribution of the world population. We focused our study on men of African ancestry because of their disproportionately higher rate of prostate cancer (CaP) incidence and mortality. We present a systematic whole genome analyses, revealing alterations that differentiate African American (AA) and Caucasian American (CA) CaP genomes. We discovered a recurrent deletion on chromosome 3q13.31 centering on the LSAMP locus that was prevalent in tumors from AA men (cumulative analyses of 435 patients: whole genome sequence, 14; FISH evaluations, 101; and SNP array, 320 patients). Notably, carriers of this deletion experienced more rapid disease progression. In contrast, PTEN and ERG common driver alterations in CaP were significantly lower in AA prostate tumors compared to prostate tumors from CA. Moreover, the frequency of inter-chromosomal rearrangements was significantly higher in AA than CA tumors. These findings reveal differentially distributed somaticmutations in CaP across ancestral groups, which have implications for precision medicine strategies. (C) 2015 The Authors. Published by Elsevier B.V

    A Methodological Framework for the Reconstruction of Contiguous Regions of Ancestral Genomes and Its Application to Mammalian Genomes

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    The reconstruction of ancestral genome architectures and gene orders from homologies between extant species is a long-standing problem, considered by both cytogeneticists and bioinformaticians. A comparison of the two approaches was recently investigated and discussed in a series of papers, sometimes with diverging points of view regarding the performance of these two approaches. We describe a general methodological framework for reconstructing ancestral genome segments from conserved syntenies in extant genomes. We show that this problem, from a computational point of view, is naturally related to physical mapping of chromosomes and benefits from using combinatorial tools developed in this scope. We develop this framework into a new reconstruction method considering conserved gene clusters with similar gene content, mimicking principles used in most cytogenetic studies, although on a different kind of data. We implement and apply it to datasets of mammalian genomes. We perform intensive theoretical and experimental comparisons with other bioinformatics methods for ancestral genome segments reconstruction. We show that the method that we propose is stable and reliable: it gives convergent results using several kinds of data at different levels of resolution, and all predicted ancestral regions are well supported. The results come eventually very close to cytogenetics studies. It suggests that the comparison of methods for ancestral genome reconstruction should include the algorithmic aspects of the methods as well as the disciplinary differences in data aquisition

    From Birds to Drug-Resistant Cancer, a novel In situ Methodology to Explore Divergent Genome Evolution

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    Fluorescent hybridisatio nmethodologies have not changed in principles over the past 30 years, with the increase of computational sequencing technologies causing the replacement of in situ hybridisations. Fluorescence in situ hybridisation (FISH) is in need of a refresh to be a worthwhile tool in a modern day cytogenetic laboratory to overcome short comings of these new methods. The creation of the novel multilayer FISH protocol has effectively eliminated many negative aspects of classic FISH based experiments, such as a large reduction in cost and is no longer as limited by fluorophore availability. Here presented within this thesis is the creation of this methodology and application to a wide variety of cytogenetic hypothesises. Key species from the Galliform order were investigated in order to detect previously missed intrachromosomal rearrangements within their macrochromosomes, a premise formerly overlooked. Rearrangements were found within chromosomes of the galliforme species used such as E.chinensis which displays a intrachromosomal inversion on the p-arm of chromosome 2. Furthermore, the creation of an interphase state folding prediction tool has been used to assess the arrangement of macrochromosomes during cellular growth stages within G.gallus. Here it is noted that there are particular arrangements identified which are similar across chromosomes studied. The chicken lymphoma cell line DT40 is of great importance in B-cell receptor studies along with gene disruption experiments. Presented here is an updated karyotype for the cell line. Here shows contrasting and more in-depth evidence of aberrations to further develop our understanding of the genomic arrangement of this useful cell line. The level of tumour heterogeneity in a cancer is a diagnostic tool allowing clinicians to comment on therapeutic choices and prognosis of the disease. Found to be dominant in recurrent cancers, cytotoxic resistant tumour cell populations may indeed exist within initial primary tumours at low frequency to be positively selected during chemotherapy. Within a neuroblastoma cell line,and cyto-toxic resistant derivatives lines,there has been identified a level of genomic heterogeneity which may give clues towards the generation of drug resistance mechanisms

    Fusion genes in high-grade serous ovarian cancer

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    High-grade serous ovarian cancer (HGSC) is the most common and lethal subtype of ovarian cancer. Debulking surgery is the primary treatment, supplemented by platinum and taxane combination chemotherapy. The response to treatment is generally good, but most patients eventually develop drug resistance, leading to disease progression and death. Nevertheless, the treatment strategy has remained relatively unchanged, and overall survival has hardly improved over the last 20 years. The primary events in early HGSC development are the inactivation of the tumor suppressor p53, and the homologous recombination pathway, which results in a severely damaged genome and allows the development of individual cancer cell clones. Genomic rearrangements, including gene fusions may evolve, when two separate genes join to form a new gene product. Genes can also fuse at the RNA level due to splicing. The functional changes in fusion proteins and chimeric RNAs can cause cancer initiation, tumor progression, and drug resistance. HGSC is a genetically unstable disease in which fusions are very common. Poor treatment outcomes can be associated with the emergence of treatment-resistant subclones. Deep sequencing has significantly increased the knowledge of the molecular characteristics of HGSC. Understanding the genomic and non-genomic abnormalities of HGSC tumors will help design personalized therapeutic approaches and discover novel mechanisms of drug resistance. However, at present, the role of gene fusions in HGSC is poorly studied. This Ph.D. thesis aimed to identify new potential drug targets for ovarian cancer patients. Using computational modeling, we identified 228 novel fusion genes from 107 cancer samples of 36 HGSC patients. We demonstrated by laboratory experiments the presence of the most biologically interesting fusions in cancer cells. Furthermore, we investigated the PIK3R1-CCDC178 fusion protein and its role as a tumor-promoting alteration and impact on drug response. The fusion induced HGSC cell migration and resistance to platinum and trametinib treatment through ERK1/2 activation but the fusion-expressing cells remained sensitive to the combination of the treatments. Therapy resistance was associated with rod and ring-like cellular structure formation.  Fuusiogeenit huonosti erilaistuneessa seroosissa munasarjasyövĂ€ssĂ€ Huonosti erilaistunut seroosi munasarjasyöpĂ€ (HGSC) on yleisin ja tappavin munasarjasyövĂ€n muoto. Perushoitomuoto on leikkaus, jota tĂ€ydennetÀÀn platinan ja taksaanin yhdistelmĂ€hoidolla. Hoitovaste on usein hyvĂ€, mutta suurimmalle osalle potilaista kehittyy lÀÀkeresistenssi, mikĂ€ johtaa syövĂ€n etenemiseen ja kuolemaan. Hoitostrategia on pysynyt melko muuttumattomana eikĂ€ potilaiden kokonaiselossaoloajassa ole tapahtunut juurikaan edistystĂ€ viimeisten 20 vuoden aikana. HGSC:n alkuvaiheessa p53-kasvunrajoiteproteiinin sekĂ€ homologisen rekombinaatiokorjausreitin toimimattomuus johtavat vaikeasti vioittuneeseen genomiin ja mahdollistavat yksilöllisten syöpĂ€solukloonien kehittymisen. Geenifuusiot voivat syntyĂ€ genomin uudelleenjĂ€rjestĂ€ytymisen seurauksena, jolloin kaksi erillistĂ€ geeniĂ€ muodostavat uuden geenituotteen. Geenit voivat fuusioitua myös RNA-tasolla silmukoitumisen seurauksena. Fuusioproteiinien ja -RNA:iden toiminnalliset muutokset voivat edesauttaa syövĂ€n syntymistĂ€, etenemistĂ€ sekĂ€ lÀÀkeresistenssin kehittymistĂ€. HGSC on geneettisesti hyvin epĂ€vakaa tauti, jossa fuusiot ovat erittĂ€in yleisiĂ€. Huono hoitotulos voidaan yhdistÀÀ hoitoresistenttien alakloonien syntymiseen. SyvĂ€sekvensointi on lisĂ€nnyt merkittĂ€vĂ€sti tietoa HGSC:n molekulaarisista ominaisuuksista. Kasvainten genomisten ja ei-genomisten poikkeavuuksien ymmĂ€rtĂ€minen auttaa yksilöllisten terapeuttisten ratkaisujen suunnittelussa ja uusien lÀÀkeresistenssimekanismien löytĂ€misessĂ€. Toistaiseksi geenifuusioiden merkitystĂ€ HGSC:ssĂ€ on tutkittu melko vĂ€hĂ€n. VĂ€itöskirjatutkimukseni tarkoituksena oli löytÀÀ uusia potentiaalisia syöpĂ€lÀÀkekohteita munasarjasyöpĂ€potilaille. Tunnistimme 228 uutta fuusiogeeniĂ€ 36:n HGSCpotilaan 107:sta syöpĂ€nĂ€ytteestĂ€ tietokonemallinnusta hyödyntĂ€en. Osoitimme laboratoriokokein biologisesti mielenkiintoisimpien fuusioiden olemassaolon syöpĂ€soluissa. Tutkimme tarkemmin PIK3R1-CCDC178 fuusioproteiinin biologiaa ylituottosoluissa. Fuusio aikaansai ERK1/2 viestireitin aktivaation ja indusoi HGSCsolujen migraatiota ja resistenssin platina- ja trametinibihoidolle. Solut olivat kuitenkin herkkiĂ€ yhdistelmĂ€hoidolle. LÀÀkeresistenssi liittyi sauvamaisten ja rengasmaisten solunsisĂ€isten filamenttirakenteiden muodostumiseen

    Using DNA microarrays to study host-microbe interactions.

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    Complete genomic sequences of microbial pathogens and hosts offer sophisticated new strategies for studying host-pathogen interactions. DNA microarrays exploit primary sequence data to measure transcript levels and detect sequence polymorphisms, for every gene, simultaneously. The design and construction of a DNA microarray for any given microbial genome are straightforward. By monitoring microbial gene expression, one can predict the functions of uncharacterized genes, probe the physiologic adaptations made under various environmental conditions, identify virulence-associated genes, and test the effects of drugs. Similarly, by using host gene microarrays, one can explore host response at the level of gene expression and provide a molecular description of the events that follow infection. Host profiling might also identify gene expression signatures unique for each pathogen, thus providing a novel tool for diagnosis, prognosis, and clinical management of infectious disease

    The little skate genome and the evolutionary emergence of wing-like fins

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    Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins—including gene expression, chromatin occupancy and three-dimensional conformation—we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait
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