| 1. |
- García, Patricia, et al.
(författare)
-
Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor
- 2020
-
Ingår i: Biological Research. - : Springer Science and Business Media LLC. - 0716-9760 .- 0717-6287. ; 53
-
Tidskriftsartikel (refereegranskat)abstract
- © 2020 The Author(s). Background: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. Results: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. Conclusions: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.
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| 2. |
- Abrahamsson, Sanna, et al.
(författare)
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Comparison of online learning designs during the COVID-19 pandemic within bioinformatics courses in higher education
- 2021
-
Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1460-2059. ; 37:Suppl 1
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Tidskriftsartikel (refereegranskat)abstract
- Motivation: Due to the worldwide COVID-19 pandemic, new strategies had to be adopted to move from classroom-based education to online education, in a very short time. The lack of time to set up these strategies, hindered a proper design of online instructions and delivery of knowledge. Bioinformatics-related training and other onsite practical education, tend to rely on extensive practice, where students and instructors have a face-to-face interaction to improve the learning outcome. For these courses to maintain their high quality when adapted as online courses, different designs need to be tested and the students' perceptions need to be heard. Results: This study focuses on short bioinformatics-related courses for graduate students at the University of Gothenburg, Sweden, which were originally developed for onsite training. Once adapted as online courses, several modifications in their design were tested to obtain the best fitting learning strategy for the students. To improve the online learning experience, we propose a combination of: (i) short synchronized sessions, (ii) extended time for own and group practical work, (iii) recorded live lectures and (iv) increased opportunities for feedback in several formats.
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| 3. |
- Abrahamsson, Sanna, et al.
(författare)
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PΨFinder: a practical tool for the identification and visualization of novel pseudogenes in DNA sequencing data
- 2022
-
Ingår i: BMC Bioinformatics. - : Springer Science and Business Media LLC. - 1471-2105. ; 23
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Processed pseudogenes (PΨgs) are disabled gene copies that are transcribed and may affect expression of paralogous genes. Moreover, their insertion in the genome can disrupt the structure or the regulatory region of a gene, affecting its expression level. These events have been identified as occurring mutations during cancer development, thus being able to identify PΨgs and their location will improve their impact on diagnostic testing, not only in cancer but also in inherited disorders. Results: We have implemented PΨFinder (P-psy-finder), a tool that identifies PΨgs, annotates known ones and predicts their insertion site(s) in the genome. The tool screens alignment files and provides user-friendly summary reports and visualizations. To demonstrate its applicability, we scanned 218 DNA samples from patients screened for hereditary colorectal cancer. We detected 423 PΨgs distributed in 96% of the samples, comprising 7 different parent genes. Among these, we confirmed the well-known insertion site of the SMAD4-PΨg within the last intron of the SCAI gene in one sample. While for the ubiquitous CBX3-PΨg, present in 82.6% of the samples, we found it reversed inserted in the second intron of the C15ORF57 gene. Conclusions: PΨFinder is a tool that can automatically identify novel PΨgs from DNA sequencing data and determine their location in the genome with high sensitivity (95.92%). It generates high quality figures and tables that facilitate the interpretation of the results and can guide the experimental validation. PΨFinder is a complementary analysis to any mutational screening in the identification of disease-causing mutations within cancer and other diseases.
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| 4. |
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| 5. |
- Alm Rosenblad, Magnus, 1957, et al.
(författare)
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Inventory and analysis of the protein subunits of the ribonucleases P and MRP provides further evidence of homology between the yeast and human enzymes.
- 2006
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Ingår i: Nucleic acids research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 34:18, s. 5145-56
-
Tidskriftsartikel (refereegranskat)abstract
- The RNases P and MRP are involved in tRNA and rRNA processing, respectively. Both enzymes in eukaryotes are composed of an RNA molecule and 9-12 protein subunits. Most of the protein subunits are shared between RNases P and MRP. We have here performed a computational analysis of the protein subunits in a broad range of eukaryotic organisms using profile-based searches and phylogenetic methods. A number of novel homologues were identified, giving rise to a more complete inventory of RNase P/MRP proteins. We present evidence of a relationship between fungal Pop8 and the protein subunit families Rpp14/Pop5 as well as between fungal Pop6 and metazoan Rpp25. These relationships further emphasize a structural and functional similarity between the yeast and human P/MRP complexes. We have also identified novel P and MRP RNAs and analysis of all available sequences revealed a K-turn motif in a large number of these RNAs. We suggest that this motif is a binding site for the Pop3/Rpp38 proteins and we discuss other structural features of the RNA subunit and possible relationships to the protein subunit repertoire.
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| 6. |
- Alm Rosenblad, Magnus, 1957, et al.
(författare)
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The enigmatic RNase MRP of kinetoplastids
- 2021
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Ingår i: Rna Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 18:Supplement 1, s. 139-147
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Tidskriftsartikel (refereegranskat)abstract
- The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.
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| 7. |
- Andersson, Björn, 1977, et al.
(författare)
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Development of a machine learning framework for radiation biomarker discovery and absorbed dose prediction.
- 2023
-
Ingår i: Frontiers in oncology. - 2234-943X. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Molecular radiation biomarkers are an emerging tool in radiation research with applications for cancer radiotherapy, radiation risk assessment, and even human space travel. However, biomarker screening in genome-wide expression datasets using conventional tools is time-consuming and underlies analyst (human) bias. Machine Learning (ML) methods can improve the sensitivity and specificity of biomarker identification, increase analytical speed, and avoid multicollinearity and human bias.To develop a resource-efficient ML framework for radiation biomarker discovery using gene expression data from irradiated normal tissues. Further, to identify biomarker panels predicting radiation dose with tissue specificity.A strategic search in the Gene Expression Omnibus database identified a transcriptomic dataset (GSE44762) for normal tissues radiation responses (murine kidney cortex and medulla) suited for biomarker discovery using an ML approach. The dataset was pre-processed in R and separated into train and test data subsets. High computational cost of Genetic Algorithm/k-Nearest Neighbor (GA/KNN) mandated optimization and 13 ML models were tested using the caret package in R. Biomarker performance was evaluated and visualized via Principal Component Analysis (PCA) and dose regression. The novelty of ML-identified biomarker panels was evaluated by literature search.Caret-based feature selection and ML methods vastly improved processing time over the GA approach. The KNN method yielded overall best performance values on train and test data and was implemented into the framework. The top-ranking genes were Cdkn1a, Gria3, Mdm2 and Plk2 in cortex, and Brf2, Ccng1, Cdkn1a, Ddit4l, and Gria3 in medulla. These candidates successfully categorized dose groups and tissues in PCA. Regression analysis showed that correlation between predicted and true dose was high with R2 of 0.97 and 0.99 for cortex and medulla, respectively.The caret framework is a powerful tool for radiation biomarker discovery optimizing performance with resource-efficiency for broad implementation in the field. The KNN-based approach identified Brf2, Ddit4l, and Gria3 mRNA as novel candidates that have been uncharacterized as radiation biomarkers to date. The biomarker panel showed good performance in dose and tissue separation and dose regression. Further training with larger cohorts is warranted to improve accuracy, especially for lower doses.
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| 8. |
- Banyai, Gabor, et al.
(författare)
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Mediator Can Regulate Mitotic Entry and Direct Periodic Transcription in Fission Yeast
- 2014
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 34:21, s. 4008-4018
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Tidskriftsartikel (refereegranskat)abstract
- Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events.
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| 9. |
- Bhadury, Joydeep, et al.
(författare)
-
Hypoxia-regulated gene expression explains differences between melanoma cell line-derived xenografts and patient-derived xenografts.
- 2016
-
Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 7:17
-
Tidskriftsartikel (refereegranskat)abstract
- Cell line-derived xenografts (CDXs) are an integral part of drug efficacy testing during development of new pharmaceuticals against cancer but their accuracy in predicting clinical responses in patients have been debated. Patient-derived xenografts (PDXs) are thought to be more useful for predictive biomarker identification for targeted therapies, including in metastatic melanoma, due to their similarities to human disease. Here, tumor biopsies from fifteen patients and ten widely-used melanoma cell lines were transplanted into immunocompromised mice to generate PDXs and CDXs, respectively. Gene expression profiles generated from the tumors of these PDXs and CDXs clustered into distinct groups, despite similar mutational signatures. Hypoxia-induced gene signatures and overexpression of the hypoxia-regulated miRNA hsa-miR-210 characterized CDXs. Inhibition of hsa-miR-210 with decoys had little phenotypic effect in vitro but reduced sensitivity to MEK1/2 inhibition in vivo, suggesting down-regulation of this miRNA could result in development of resistance to MEK inhibitors.
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| 10. |
- Bhadury, Joydeep, et al.
(författare)
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Identification of tumorigenic and therapeutically actionable mutations in transplantable mouse tumor cells by exome sequencing.
- 2013
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Ingår i: Oncogenesis. - : Springer Science and Business Media LLC. - 2157-9024. ; 2
-
Tidskriftsartikel (refereegranskat)abstract
- Cancer development occurs in response to the successive accumulation of mutations that eventually targets key regulators of cell proliferation. As most mutations likely occur randomly, cancer driver mutations can only be found if they are recurrent. Here we use exome sequencing of the mouse cell lines Panc02, L1210 and Colon 26 to identify genetic alterations (single-nucleotide polymorphisms and small insertion and deletions) that occurred in three different strains of mice and that resulted in tumorigenesis. We identify known mutations in genes like Kras, Cdkn2a/b, Smad4 and Trp53 and a large list of genes whose causal link to cancer is unknown. Interestingly, by screening a compound library we find that the identified oncogenic Kras mutation in Colon 26 cells correlates with its sensitivity to MEK inhibitors in vitro and in vivo. Our analysis of these mouse tumor exomes show that their manageable number of mutations could facilitate the identification of novel mutations or pathways driving tumor development. Furthermore, their use as tools is now enhanced as they can be used to create syngenic transplant models for utilization in drug discovery and validation. Finally, by showing that Kras mutant Colon 26 cells are sensitive to MEK inhibitors, we provide one proof-of-principle experiment that a platform containing targeted resequencing and drug screens could be a valuable addition in the clinic to devise anti-cancer drug schemes.
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| 11. |
- Börjesson, Vanja, et al.
(författare)
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TC-hunter: identification of the insertion site of a transgenic gene within the host genome
- 2022
-
Ingår i: Bmc Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 23:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Transgenic animal models are crucial for the study of gene function and disease, and are widely utilized in basic biological research, agriculture and pharma industries. Since the current methods for generating transgenic animals result in the random integration of the transgene under study, the phenotype may be compromised due to disruption of known genes or regulatory regions. Unfortunately, most of the tools that predict transgene insertion sites from high-throughput data are not publicly available or not properly maintained. Results: We implemented TC-hunter, Transgene-Construct hunter, an open tool that identifies transgene insertion sites and provides simple reports and visualization aids. It relies on common tools used in the analysis of high-throughput data and makes use of chimeric reads and discordant read pairs to identify and support the transgenic insertion site. To demonstrate its applicability, we applied TC-hunter to four transgenic mice samples harboring the human PPM1D gene, a model used in the study of malignant tumor development. We identified the transgenic insertion site in each sample and experimentally validated them with Touchdown-polymerase chain reaction followed by Sanger sequencing. Conclusions: TC-hunter is an accessible bioinformatics tool that can automatically identify transgene insertion sites from DNA sequencing data with high sensitivity (98%) and precision (92.45%). TC-hunter is a valuable tool that can aid in evaluating any potential phenotypic complications due to the random integration of the transgene and can be accessed at https://github.com/bcfgothenburg/SSF.
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| 12. |
- Carlsten, Jonas O P, et al.
(författare)
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Loss of the Mediator subunit Med20 affects transcription of tRNA and other non-coding RNA genes in fission yeast
- 2016
-
Ingår i: Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms. - : Elsevier BV. - 1874-9399. ; 1859:2, s. 339-347
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Tidskriftsartikel (refereegranskat)abstract
- Mediator is a co-regulator of RNA polymerase II (Pol II), transducing signals from regulatory elements and transcription factors to the general transcription machinery at the promoter. We here demonstrate that Med20 influences ribosomal protein expression in fission yeast. In addition, loss of Med20 leads to an accumulation of aberrant, readthrough tRNA transcripts. These transcripts are polyadenylated and targeted for degradation by the exosome. Similarly, other non-coding RNA molecules, such as snRNA, snoRNA and rRNA, are also enriched in the polyadenylate preparations in the absence of Med20. We suggest that fission yeast Mediator takes part in a regulatory pathway that affects Pol III-dependent transcripts.
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| 13. |
- Carlsten, Jonas O P, et al.
(författare)
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Mediator Promotes CENP-A Incorporation at Fission Yeast Centromeres
- 2012
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Ingår i: Molecular and Cellular Biology. - : Informa UK Limited. - 0270-7306 .- 1098-5549. ; 32:19, s. 4035-4043
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Tidskriftsartikel (refereegranskat)abstract
- At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A(Cnp1), which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-A(Cnp1) from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20 Delta cells appears to directly block CENP-A(Cnp1) incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.
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| 14. |
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| 15. |
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| 16. |
- Davila Lopez, Marcela, et al.
(författare)
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Computational screen for spliceosomal RNA genes aids in defining the phylogenetic distribution of the major and minor spliceosomal components
- 2008
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Ingår i: RNA Society Meeting 2008.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- An essential step of gene expression is the removal of non-coding sequences (introns) from the pre-mRNA and the ligation of coding sequences (exons) to form the mature RNA (mRNA). It is known that the pre-mRNA splicing occurs by two sequential trans-esterification reactions and is catalyzed by a multicomponent complex, the spliceosome. Its assembly involves five small nuclear ribonucleoprotein particles (snRNPs) as well as an array of protein factors, which are determined by the class of intron to be spliced1. To date, two intron classes are known, U2- and U12-type introns. The U2-dependent spliceosome (panel a) is formed by the interaction of the U1, U2, U4/U6 and U5 snRNPs and numerous non-snRNP proteins with the pre-mRNA. The U12-dependent spliceosome (also referred to as the minor spliceosome, panel b), in contrast, consists of the U11, U12, U4atac/U6atac and U5 snRNPs with an unknown number of non-snRNP proteins. Thus, of the main spliceosomal subunits, only the U5 snRNA is common to both spliceosomes2.
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| 17. |
- Davila Lopez, Marcela, et al.
(författare)
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Conserved and variable domains of RNase MRP RNA
- 2009
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Ingår i: RNA Biology. - 1547-6286. ; 6:3, s. 208-221
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Tidskriftsartikel (refereegranskat)abstract
- Ribonuclease MRP is a eukaryotic ribonucleoprotein complex consisting of one RNA molecule and 7-10 protein subunits. One important function of MRP is to catalyze an endonucleolytic cleavage during processing of rRNA precursors. RNase MRP is evolutionary related to RNase P which is critical for tRNA processing. A large number of MRP RNA sequences that now are available have been used to identify conserved primary and secondary structure features of the molecule. MRP RNA has structural features in common with P RNA such as a conserved catalytic core, but it also has unique features and is characterized by a domain highly variable between species. Information regarding primary and secondary structure features is of interest not only in basic studies of the function of MRP RNA, but also because mutations in the RNA give rise to human genetic diseases such as cartilage-hair hypoplasia.
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| 18. |
- Davila Lopez, Marcela, et al.
(författare)
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Early evolution of histone mRNA 3' end processing.
- 2008
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 14:1, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.
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| 19. |
- Davila Lopez, Marcela, et al.
(författare)
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eGOB: eukaryotic Gene Order Browser.
- 2011
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Ingår i: Bioinformatics (Oxford, England). - : Oxford University Press (OUP). - 1367-4811 .- 1460-2059 .- 1367-4803. ; 27:8, s. 1150-1
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Tidskriftsartikel (refereegranskat)abstract
- A large number of genomes have been sequenced, allowing a range of comparative studies. Here, we present the eukaryotic Gene Order Browser with information on the order of protein and non-coding RNA (ncRNA) genes of 74 different eukaryotic species. The browser is able to display a gene of interest together with its genomic context in all species where that gene is present. Thereby, questions related to the evolution of gene organization and non-random gene order may be examined. The browser also provides access to data collected on pairs of adjacent genes that are evolutionarily conserved. AVAILABILITY: eGOB as well as underlying data are freely available at http://egob.biomedicine.gu.se SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: tore.samuelsson@medkem.gu.se.
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| 20. |
- Davila Lopez, Marcela
(författare)
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Evolution of protein and non-coding RNA genes studied with comparative genomics
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The identification of protein and non-coding RNA (ncRNA) genes is one important step in the analysis of a genome. This thesis focuses on the identification and analysis of proteins and ncRNAs homologues by exploiting a variety of computational methods in order to reach conclusions as to their structure, function, evolution and regulation. This work is composed of two different parts. One deals with computational prediction of protein and ncRNA homologues from different ribonucleoprotein (RNP) complexes and the other addresses problems related to non-random gene order in eukaryotes. In the first part RNPs that were previously not well explored with respect to their phylogenetic distribution were examined. Thus, homology-based methods were employed to analyze the RNP complexes of RNase P, RNase MRP and the spliceosome as well as RNPs and RNA structures involved in the 3' end processing of histone mRNAs. We identified a large number of previously unrecognized homologues that improved our understanding of the evolution of the different RNPs. For example, homology relationships of the RNases P and MRP proteins were identified providing further evidence of homology between the human and the yeast RNPs. We presented evidence that the histone 3’ end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We presented a detailed map of the distribution of the spliceosomal U12-type RNA genes, supporting an early origin of the minor spliceosome and pointing to a number of occasions where it was lost during evolution. In the second part we generated gene order maps to show the localization of both protein and ncRNA genes in a wide range of eukaryotic organisms. Non-random gene order was then examined to identify the most important determinants of gene order conservation. One important conclusion was that gene pairs that are evolutionarily conserved and that are divergently transcribed are much more likely to be related by function as compared to poorly conserved gene pairs. The genes of such pairs are likely to be related also in terms of transcriptional control. Moreover, we presented the eukaryotic Gene Order Browser (eGOB), where data related to this project is available and where researchers can visualize and compare the evolution of gene organization in different organisms. In addition, the browser may be used to identify pairs of adjacent genes that are evolutionarily conserved and likely to be transcriptionally linked. eGOB is available at http://egob.bioimedicine.gu.se.
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| 21. |
- Doimo, Mara, et al.
(författare)
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Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells.
- 2023
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Ingår i: Nucleic acids research. - : Oxford University Press. - 1362-4962 .- 0305-1048. ; 51:14, s. 7392-7408
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.
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| 22. |
- Einarsdottir, Berglind Osk, 1979, et al.
(författare)
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Melanoma patient-derived xenografts accurately model the disease and develop fast enough to guide treatment decisions.
- 2014
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 5:20, s. 9609-18
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Tidskriftsartikel (refereegranskat)abstract
- The development of novel therapies against melanoma would benefit from individualized tumor models to ensure the rapid and accurate identification of biomarkers of therapy response. Previous studies have suggested that patient-derived xenografts (PDXes) could be useful. However, the utility of PDXes in guiding real-time treatment decisions has only been reported in anecdotal forms. Here tumor biopsies from patients with stage III and IV metastatic malignant melanoma were transplanted into immunocompromised mice to generate PDXes. 23/26 melanoma biopsies generated serially transplantable PDX models, and their histology, mutation status and expression profile resembled their corresponding patient biopsy. The potential treatment for one patient was revealed by an in vitro drug screen and treating PDXes with the MEK inhibitor trametinib. In another patient, the BRAF mutation predicted the response of both the patient and its corresponding PDXes to MAPK-targeted therapy. Importantly, in this unselected group of patients, the time from biopsy for generation of PDXes until death was significantly longer than the time required to reach the treatment phase of the PDXes. Thus, it could be clinically meaningful to use this type of platform for melanoma patients as a pre-selection tool in clinical trials.
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| 23. |
- Heckmann Barbalho de Figueroa, Laiz, et al.
(författare)
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A Modeling Approach for Bioinformatics Workflows
- 2019
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Ingår i: The Practice of Enterprise Modeling - 12th {IFIP} Working Conference, PoEM 2019, Luxembourg, Luxembourg, November 27-29, 2019, Proceedings. - Cham : Springer.
-
Konferensbidrag (refereegranskat)
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| 24. |
- Jemt, Elisabeth, et al.
(författare)
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Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element
- 2015
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 43:19, s. 9262-9275
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Tidskriftsartikel (refereegranskat)abstract
- The majority of mitochondrial DNA replication events are terminated prematurely. The nascent DNA remains stably associated with the template, forming a triple-stranded displacement loop (D-loop) structure. However, the function of the D-loop region of the mitochondrial genome remains poorly understood. Using a comparative genomics approach we here identify two closely related 15 nt sequence motifs of the D-loop, strongly conserved among vertebrates. One motif is at the D-loop 5'-end and is part of the conserved sequence block 1 (CSB1). The other motif, here denoted coreTAS, is at the D-loop 3'-end. Both these sequences may prevent transcription across the D-loop region, since light and heavy strand transcription is terminated at CSB1 and coreTAS, respectively. Interestingly, the replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication. Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication.
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| 25. |
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| 26. |
- Nilsson, Johanna, et al.
(författare)
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Polyglucosan body myopathy caused by defective ubiquitin ligase RBCK1.
- 2013
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Ingår i: Annals of neurology. - : Wiley. - 1531-8249 .- 0364-5134. ; 74:6, s. 914-919
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Tidskriftsartikel (refereegranskat)abstract
- Glycogen storage diseases are important causes of myopathy and cardiomyopathy. We describe ten patients from eight families with childhood or juvenile onset of myopathy, eight of whom also had rapidly progressive cardiomyopathy requiring heart transplant in four. The patients were homozygous or compound heterozygous for missense or truncating mutations in the ubiquitin ligase RBCK1 and had extensive polyglucosan accumulation in skeletal muscle and in the heart in cases of cardiomyopathy. We conclude that RBCK1 deficiency is a frequent cause of polyglucosan storage myopathy associated with progressive muscle weakness and cardiomyopathy. ANN NEUROL 2013. © 2013 American Neurological Association.
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| 27. |
- Oldfors Hedberg, Carola, 1969, et al.
(författare)
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Cardiomyopathy with lethal arrhythmias associated with inactivation of KLHL24
- 2019
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Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 28:11, s. 1919-1929
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Tidskriftsartikel (refereegranskat)abstract
- Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder, yet the genetic cause of up to 50% of cases remains unknown. Here, we show that mutations in KLHL24 cause HCM in humans. Using genome-wide linkage analysis and exome sequencing, we identified homozygous mutations in KLHL24 in two consanguineous families with HCM. Of the 11 young affected adults identified, 3 died suddenly and 1 had a cardiac transplant due to heart failure. KLHL24 is a member of the Kelch-like protein family, which acts as substrate-specific adaptors to Cullin E3 ubiquitin ligases. Endomyocardial and skeletal muscle biopsies from affected individuals of both families demonstrated characteristic alterations, including accumulation of desmin intermediate filaments. Knock-down of the zebrafish homologue klhl24a results in heart defects similar to that described for other HCM-linked genes providing additional support for KLHL24 as a HCM-associated gene. Our findings reveal a crucial role for KLHL24 in cardiac development and function.
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| 28. |
- Olsson Lindvall, Martina, et al.
(författare)
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A Comprehensive Sequencing-Based Analysis of Allelic Methylation Patterns in Hemostatic Genes in Human Liver
- 2020
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Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 120:2, s. 229-242
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Tidskriftsartikel (refereegranskat)abstract
- Characterizing the relationship between genetic, epigenetic (e.g., deoxyribonucleic acid [DNA] methylation), and transcript variation could provide insights into mechanisms regulating hemostasis and potentially identify new drug targets. Several hemostatic factors are synthesized in the liver, yet high-resolution DNA methylation data from human liver tissue is currently lacking for these genes. Single-nucleotide polymorphisms (SNPs) can influence DNA methylation in cis which can affect gene expression. This can be analyzed through allele-specific methylation (ASM) experiments. We performed targeted genomic DNA- and bisulfite-sequencing of 35 hemostatic genes in human liver samples for SNP and DNA methylation analysis, respectively, and integrated the data for ASM determination. ASM-associated SNPs (ASM-SNPs) were tested for association to gene expression in liver using in-house generated ribonucleic acid-sequencing data. We then assessed whether ASM-SNPs associated with gene expression, plasma proteins, or other traits relevant for hemostasis using publicly available data. We identified 112 candidate ASM-SNPs. Of these, 68% were associated with expression of their respective genes in human liver or in other human tissues and 54% were associated with the respective plasma protein levels, activity, or other relevant hemostatic genome-wide association study traits such as venous thromboembolism, coronary artery disease, stroke, and warfarin dose maintenance. Our study provides the first detailed map of the DNA methylation landscape and ASM analysis of hemostatic genes in human liver tissue, and suggests that methylation regulated by genetic variants in cis may provide a mechanistic link between noncoding SNPs and variation observed in circulating hemostatic proteins, prothrombotic diseases, and drug response.
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| 29. |
- Olsson Lindvall, Martina, et al.
(författare)
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Comparison of DNA Methylation Profiles of Hemostatic Genes between Liver Tissue and Peripheral Blood within Individuals
- 2021
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Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 121:5, s. 573-583
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Tidskriftsartikel (refereegranskat)abstract
- DNA methylation has become increasingly recognized in the etiology of complex diseases, including thrombotic disorders. Blood is often collected in epidemiological studies for genotyping and has recently also been used to examine DNA methylation in epigenome-wide association studies. DNA methylation patterns are often tissue-specific, thus, peripheral blood may not accurately reflect the methylation pattern in the tissue of relevance. Here, we collected paired liver and blood samples concurrently from 27 individuals undergoing liver surgery. We performed targeted bisulfite sequencing for a set of 35 hemostatic genes primarily expressed in liver to analyze DNA methylation levels of >10,000 cytosine-phosphate-guanine (CpG) dinucleotides. We evaluated whether DNA methylation in blood could serve as a proxy for DNA methylation in liver at individual CpGs. Approximately 30% of CpGs were nonvariable and were predominantly hypo- (<25%) or hypermethylated (>70%) in both tissues. While blood can serve as a proxy for liver at these CpGs, the low variability renders these unlikely to explain phenotypic differences. We therefore focused on CpG sites with variable methylation levels in liver. The level of blood-liver tissue correlation varied widely across these variable CpGs; moderate correlations (0.5 <= r <0.75) were detected for 6% and strong correlations ( r 0.75) for a further 4%. Our findings indicate that it is essential to study the concordance of DNA methylation between blood and liver at individual CpGs. This paired blood-liver dataset is intended as a resource to aid interpretation of blood-based DNA methylation results.
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| 30. |
- Olsson Lindvall, Martina, et al.
(författare)
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Hemostatic Genes Exhibit a High Degree of Allele-Specific Regulation in Liver
- 2019
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Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 119:7, s. 1072-1083
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Tidskriftsartikel (refereegranskat)abstract
- Objective Elucidating the genetic basis underlying hepatic hemostatic gene expression variability may contribute to unraveling genetic factors contributing to thrombotic or bleeding disorders. We aimed to identify novel cis-regulatory variants involved in regulating hemostatic genes by analyzing allele-specific expression (ASE) in human liver samples. Study Design Biopsies of human liver tissue and blood were collected from adults undergoing liver surgery at the Sahlgrenska University Hospital (n =20). Genomic deoxyribonucleic acid (gDNA) and total ribonucleic acid (RNA) were isolated. A targeted approach was used to enrich and sequence 35 hemostatic genes for single nucleotide polymorphism (SNP) analysis (gDNAseq) and construct individualized genomes for transcript alignment. The allelic ratio of transcripts from targeted RNAseq was determined via ASE analysis. Public expression quantitative trait loci (eQTL) and genome-wide association study (GWAS) data were used to assess novelty and importance of the ASE SNPs (and proxies, r(2) >= 0.8) for relevant traits/diseases. Results Sixty percent of the genes studied showed allelic imbalance across 53 SNPs. Of these, 7 SNPs were previously validated in liver eQTL studies. For 32 with eQTLs in other cell/tissue types, this is the first time genotype-specific expression is demonstrated in liver, and for 14 ASE SNPs, this is the first ever reported genotype-expression association. A total of 29 ASE SNPs were previously associated with the respective plasma protein levels and 17 ASE SNPs to other relevant GWAS traits including venous thromboembolism, coronary artery disease, and stroke. Conclusion Our study provides a comprehensive ASE analysis of hemostatic genes and insights into the regulation of hemostatic genes in human liver.
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| 31. |
- Oltean, Mihai, 1976, et al.
(författare)
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The proteomic signature of intestinal acute rejection in the mouse
- 2022
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Ingår i: Metabolites. - : MDPI AG. - 2218-1989. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Intestinal acute rejection (AR) lacks a reliable non-invasive biomarker and AR surveillance is conducted through frequent endoscopic biopsies. Although citrulline and calprotectin have been suggested as AR biomarkers, these have limited clinical value. Using a mouse model of intestinal transplantation (ITx), we performed a proteome-wide analysis and investigated rejection-related proteome changes that may eventually be used as biomarkers. ITx was performed in allogenic (Balb/C to C57Bl) and syngeneic (C57Bl) combinations. Graft samples were obtained three and six days after transplantation (n = 4/time point) and quantitative proteomic analysis with iTRAQ-labeling and mass spectrometry of whole tissue homogenates was performed. Histology showed moderate AR in all allografts post-transplantation at day six. Nine hundred and thirty-eight proteins with at least three unique peptides were identified in the intestinal grafts. Eighty-six proteins varying by >20% between time points and/or groups had an alteration pattern unique to the rejecting allografts: thirty-seven proteins and enzymes (including S100-A8 and IDO-1) were significantly upregulated whereas forty-nine (among other chromogranin, ornithine aminotransferase, and arginase) were downregulated. Numerous proteins showed altered expression during intestinal AR, several of which were previously identified to be involved in acute rejection, although our results also identified previously unreported proteome changes. The metabolites and downstream metabolic pathways of some of these proteins and enzymes may become potential biomarkers for intestinal AR.
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| 32. |
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| 33. |
- Samuelsson, Tore, 1951, et al.
(författare)
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A histone 3' end processing machinery in protozoa
- 2007
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Ingår i: 5th Annual International Conference on Intelligent Systems for Molecular Biology (ISMB) & 6th European Conference on Computational Biology (ECCB).
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- In metazoa replication-dependent histone mRNAs are not polyadenylated. At the 3' end of these mRNAs there is a conserved sequence that contains a 16 nucleotide stem loop (SL) besides a purine rich sequences, called the histone downstream element (HDE) located several nucleotides downstream. This is necessary for endonucleolytic cleavage of pre-mRNA to release the mature mRNA. For this task also trans-acting factors are needed, such as a U7 snRNP, a stem loop binding protein (SLBP) and a subunit of the cleavage/polyadenylation specificity factor (CPSF-73)1,2. In order to understand the evolutionary origin of this molecular machinery we have computationally searched for all the transacting factors as well as the histone mRNA stem-loop structure in histone mRNAs in a range of eukaryotic species. We have used profile HMMs and covariance models to identify SL motifs as well as U7 RNA homologues in lower metazoa. Profile-based searches were used to identify proteins homologous to the mammalian SLBP. Surprisingly, a number of protozoan species seem to have the histone 3' end processing machinery characteristic of metazoa. These results show that this machinery is more ancient than previously anticipated.
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| 34. |
- Scherer, D., et al.
(författare)
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RNA Sequencing of Hepatobiliary Cancer Cell Lines: Data and Applications to Mutational and Transcriptomic Profiling.
- 2020
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 12:9
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Tidskriftsartikel (refereegranskat)abstract
- Cancer cell lines allow the identification of clinically relevant alterations and the prediction of drug response. However, sequencing data for hepatobiliary cancer cell lines in general, and particularly gallbladder cancer (GBC), are sparse. Here, we apply RNA sequencing to characterize 10 GBC, eight hepatocellular carcinoma, and five cholangiocarcinoma (CCA) cell lines. RNA extraction, quality control, library preparation, sequencing, and pre-processing of sequencing data were implemented using state-of-the-art techniques. Public data from the MSK-IMPACT database and a large cohort of Japanese biliary tract cancer patients were used to illustrate the usage of the released data. The total number of exonic mutations varied from 7207 for the cell line NOZ to 9760 for HuCCT1. Researchers planning experiments that require TP53 mutations could use the cell lines NOZ, OCUG-1, SNU308, or YoMi. Mz-Cha-1 showed mutations in ATM, SNU308 presented SMAD4 mutations, and the only investigated cell line that showed ARID1A mutations was GB-d1. SNU478 was the cell line with the global gene expression pattern most similar to GBC, intrahepatic CCA, and extrahepatic CCA. EGFR, KMT2D, and KMT2C generally presented a higher expression in the investigated cell lines than in Japanese primary GBC tumors. We provide the scientific community with detailed mutation and gene expression data, together with three showcase applications, with the aim of facilitating the design of future in vitro cell culture assays for research on hepatobiliary cancer.
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| 35. |
- Sofou, Kalliopi, et al.
(författare)
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Whole exome sequencing reveals mutations in NARS2 and PARS2, encoding the mitochondrial asparaginyl-tRNA synthetase and prolyl-tRNA synthetase, in patients with Alpers syndrome.
- 2015
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Ingår i: Molecular genetics & genomic medicine. - : Wiley. - 2324-9269. ; 3:1, s. 59-68
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Tidskriftsartikel (refereegranskat)abstract
- Alpers syndrome is a progressive neurodegenerative disorder that presents in infancy or early childhood and is characterized by diffuse degeneration of cerebral gray matter. While mutations in POLG1, the gene encoding the gamma subunit of the mitochondrial DNA polymerase, have been associated with Alpers syndrome with liver failure (Alpers-Huttenlocher syndrome), the genetic cause of Alpers syndrome in most patients remains unidentified. With whole exome sequencing we have identified mutations in NARS2 and PARS2, the genes encoding the mitochondrial asparaginyl-and prolyl-tRNA synthetases, in two patients with Alpers syndrome. One of the patients was homozygous for a missense mutation (c.641C>T, p.P214L) in NARS2. The affected residue is predicted to be located in the stem of a loop that participates in dimer interaction. The other patient was compound heterozygous for a one base insertion (c.1130dupC, p.K378 fs*1) that creates a premature stop codon and a missense mutation (c.836C>T, p.S279L) located in a conserved motif of unknown function in PARS2. This report links for the first time mutations in these genes to human disease in general and to Alpers syndrome in particular.
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| 36. |
- Szilagyi, Zsolt, et al.
(författare)
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Cyclin dependent kinase 8 regulates mitotic commitment in fission yeast
- 2012
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Ingår i: Molecular and Cellular Biology. - : Informa UK Limited. - 0270-7306 .- 1098-5549. ; 32:11, s. 2099-2109
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Tidskriftsartikel (refereegranskat)abstract
- Temporal changes in transcription programs are coupled to control of cell growth and division. We here report that Mediator, a conserved coregulator of eukaryotic transcription, is part of a regulatory pathway that controls mitotic entry in fission yeast. The Mediator subunit cyclin-dependent kinase 8 (Cdk8) phosphorylates the forkhead 2 (Fkh2) protein in a periodic manner that coincides with gene activation during mitosis. Phosphorylation prevents degradation of the Fkh2 transcription factor by the proteasome, thus ensuring cell cycle-dependent variations in Fkh2 levels. Interestingly, Cdk8-dependent phosphorylation of Fkh2 controls mitotic entry, and mitotic entry is delayed by inactivation of the Cdk8 kinase activity or mutations replacing the phosphorylated serine residues of Fkh2. In addition, mutations in Fkh2, which mimic protein phosphorylation, lead to premature mitotic entry. Therefore, Fkh2 regulates not only the onset of mitotic transcription but also the correct timing of mitotic entry via effects on the Weel kinase. Our findings thus establish a new pathway linking the Mediator complex to control of mitotic transcription and regulation of mitotic entry in fission yeast.
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| 37. |
- Terzioglu, M., et al.
(författare)
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MTERF1 Binds mtDNA to Prevent Transcriptional Interference at the Light-Strand Promoter but Is Dispensable for rRNA Gene Transcription Regulation
- 2013
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Ingår i: Cell Metabolism. - : Elsevier BV. - 1550-4131 .- 1932-7420. ; 17:4, s. 618-626
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial transcription termination factor 1, MTERF1, has been reported to couple rRNA gene transcription initiation with termination and is therefore thought to be a key regulator of mammalian mitochondrial ribosome biogenesis. The prevailing model is based on a series of observations published over the last two decades, but no in vivo evidence exists to show that MTERF1 regulates transcription of the heavy-strand region of mtDNA containing the rRNA genes. Here, we demonstrate that knockout of Mterf1 in mice has no effect on mitochondrial rRNA levels or mitochondrial translation. Instead, loss of Mterf1 influences transcription initiation at the light-strand promoter, resulting in a decrease of de novo transcription manifested as reduced 7S RNA levels. Based on these observations, we suggest that MTERF1 does not regulate heavy-strand transcription, but rather acts to block transcription on the opposite strand of mtDNA to prevent transcription interference at the light-strand promoter.
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| 38. |
- Uribe, Pamela, 1988, et al.
(författare)
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Soluble silica stimulates osteogenic differentiation and gap junction communication in human dental follicle cells
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Several studies have indicated that dietary silicon (Si) is beneficial for bone homeostasis and skeletal health. Furthermore, Si-containing bioactive glass biomaterials have positive effects on bone regeneration when used for repair of bone defects. Si has been demonstrated to stimulate osteoblast differentiation and bone mineralisation in vitro. However, the mechanisms underlying these effects of Si are not well understood. The aim of the present study was to investigate the effects of soluble Si on osteogenic differentiation and connexin 43 (CX43) gap junction communication in cultured pluripotent cells from human dental follicles (hDFC). Neutral Red uptake assay demonstrated that 25 mu g/ml of Si significantly stimulated hDFC cell proliferation. Dosages of Si above 100 mu g/ml decreased cell proliferation. Alizarin Red staining showed that osteogenic induction medium (OIM) by itself and in combination with Si (25 mu g/ml) significantly increased mineralisation in hDFC cultures, although Si alone had no such effect. The expression of osteoblast-related markers in hDFC was analysed with RT-qPCR. OSX, RUNX2, BMP2, ALP, OCN, BSP and CX43 genes were expressed in hDFC cultured for 1, 7, 14 and 21 days. Expression levels of BMP-2 and BSP were significantly upregulated by OIM and Si (25 mu g/ml) and were also induced by Si alone. Notably, the expression levels of OCN and CX43 on Day 21 were significantly increased only in the Si group. Flow cytometric measurements revealed that Si (50 mu g/ml) significantly increased CX43 protein expression and gap junction communication in hDFC. Next-generation sequencing (NGS) and bioinformatics processing were used for the identification of differentially regulated genes and pathways. The influence of OIM over the cell differentiation profile was more prominent than the influence of Si alone. However, Si in combination with OIM increased the magnitude of expression (up or down) of the differentially regulated genes. The gene for cartilage oligomeric matrix protein (COMP) was the most significantly upregulated. Genes for the regulator of G protein signalling 4 (RGS4), regulator of G protein signalling 2 (RGS2), and matrix metalloproteinases (MMPs) 1, 8, and 10 were also strongly upregulated. Our findings reveal that soluble Si stimulates Cx43 gap junction communication in hDFC and induces gene expression patterns associated with osteogenic differentiation. Taken together, the results support the conclusion that Si is beneficial for bone health.
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| 39. |
- Zhu, Xuefeng, et al.
(författare)
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Histone modifications influence mediator interactions with chromatin.
- 2011
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Ingår i: Nucleic acids research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 39:19, s. 8342-54
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Tidskriftsartikel (refereegranskat)abstract
- The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild-type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator-histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization.
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