| 1. |
- Fasterius, Erik, et al.
(författare)
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A novel RNA sequencing data analysis method for cell line authentication
- 2017
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 12:2
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a novel analysis method that can interrogate the authenticity of biological samples used for generation of transcriptome profiles in public data repositories. The method uses RNA sequencing information to reveal mutations in expressed transcripts and subsequently confirms the identity of analysed cells by comparison with publicly available cell-specific mutational profiles. Cell lines constitute key model systems widely used within cancer research, but their identity needs to be confirmed in order to minimise the influence of cell contaminations and genetic drift on the analysis. Using both public and novel data, we demonstrate the use of RNA-sequencing data analysis for cell line authentication by examining the validity of COLO205, DLD1, HCT15, HCT116, HKE3, HT29 and RKO colorectal cancer cell lines. We successfully authenticate the studied cell lines and validate previous reports indicating that DLD1 and HCT15 are synonymous. We also show that the analysed HKE3 cells harbour an unexpected KRAS-G13D mutation and confirm that this cell line is a genuine KRAS dosage mutant, rather than a true isogenic derivative of HCT116 expressing only the wild type KRAS. This authentication method could be used to revisit the numerous cell line based RNA sequencing experiments available in public data repositories, analyse new experiments where whole genome sequencing is not available, as well as facilitate comparisons of data from different experiments, platforms and laboratories.
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| 2. |
- Forslund, Kristoffer, 1980-
(författare)
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The relationship between orthology, protein domain architecture and protein function
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lacking experimental data, protein function is often predicted from evolutionary and protein structure theory. Under the 'domain grammar' hypothesis the function of a protein follows from the domains it encodes. Under the 'orthology conjecture', orthologs, related through species formation, are expected to be more functionally similar than paralogs, which are homologs in the same or different species descended from a gene duplication event. However, these assumptions have not thus far been systematically evaluated. To test the 'domain grammar' hypothesis, we built models for predicting function from the domain combinations present in a protein, and demonstrated that multi-domain combinations imply functions that the individual domains do not. We also developed a novel gene-tree based method for reconstructing the evolutionary histories of domain architectures, to search for cases of architectures that have arisen multiple times in parallel, and found this to be more common than previously reported. To test the 'orthology conjecture', we first benchmarked methods for homology inference under the obfuscating influence of low-complexity regions, in order to improve the InParanoid orthology inference algorithm. InParanoid was then used to test the relative conservation of functionally relevant properties between orthologs and paralogs at various evolutionary distances, including intron positions, domain architectures, and Gene Ontology functional annotations. We found an increased conservation of domain architectures in orthologs relative to paralogs, in support of the 'orthology conjecture' and the 'domain grammar' hypotheses acting in tandem. However, equivalent analysis of Gene Ontology functional conservation yielded spurious results, which may be an artifact of species-specific annotation biases in functional annotation databases. I discuss possible ways of circumventing this bias so the 'orthology conjecture' can be tested more conclusively.
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| 3. |
- Ström, Cecilia E., et al.
(författare)
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Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate
- 2011
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:8, s. 3166-3175
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Tidskriftsartikel (refereegranskat)abstract
- Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.
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| 4. |
- Uhlén, Mathias, et al.
(författare)
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Tissue-based map of the human proteome
- 2015
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 347:6220, s. 1260419-
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Tidskriftsartikel (refereegranskat)abstract
- Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.
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