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Megaprimer-based methodology for deletion of a large fragment within a repetitive polypyrimidine-rich DNA
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2006 (English)In: Molecular Biotechnology, ISSN 1073-6085, E-ISSN 1559-0305, Vol. 32, no 1, 65-71 p.Article in journal (Refereed) Published
Abstract [en]

Site-directed mutagenesis is often a prerequisite for elucidation of the functional significance of cis- and trans-factors involved in gene regulation. The aim of this study was to delete the primary binding site for heterogeneous nuclear ribonucleoprotein I (hnRNPI) within the inducible nitric oxide synthase (iNOS) 3' untranslated region mRNA. The binding site consists of a 53-nucleotide CU-rich region within a long stretch of polypyrimidines. As a result of primer pair annealing, the repetitive sequence limited the use of several deletion methods based on polymerase chain reaction. Therefore, a megaprimer approach was chosen. The megaprimer was produced by a forward primer outside the polypyrimidine-rich region, and a mutagenic reverse primer annealing to flanking regions of the desired deletion, thereby looping out the target sequence. Subsequently, this megaprimer was used to create the final deletion recombinant. The deletion was verified by sequencing and by ultraviolet cross-linking mouse liver protein extracts with radiolabeled mutant and wild-type RNAs. In conclusion, the megaprimer method offers a solution for generating large internal deletions in repetitive sequences, which facilitates investigations on large repetitive DNA or RNA regions interacting with trans-factors.

Place, publisher, year, edition, pages
2006. Vol. 32, no 1, 65-71 p.
Keyword [en]
3' Untranslated Regions/genetics, Animals, Binding Sites/genetics, Cytoplasm/metabolism, DNA/genetics/metabolism, Gene Deletion, Heterogeneous-Nuclear Ribonucleoproteins/metabolism, Humans, Liver/metabolism, Male, Mice, Mice; Inbred DBA, Mutagenesis; Site-Directed/*methods, Nitric Oxide Synthase Type II/genetics, Polymerase Chain Reaction/methods, Protein Binding/genetics, Pyrimidine Nucleotides/*genetics, RNA; Messenger/genetics/metabolism, Repetitive Sequences; Nucleic Acid/*genetics, Reproducibility of Results
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-93759DOI: 10.1385/MB:32:1:065PubMedID: 16382183OAI: oai:DiVA.org:uu-93759DiVA: diva2:167338
Available from: 2005-11-25 Created: 2005-11-25 Last updated: 2013-03-22Bibliographically approved
In thesis
1. Post-Transcriptional Regulation of the Murine Inducible Nitric Oxide Synthase Gene
Open this publication in new window or tab >>Post-Transcriptional Regulation of the Murine Inducible Nitric Oxide Synthase Gene
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented.

Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA.

Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. Trans-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs.

In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 101 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 22
Pharmaceutical biochemistry, iNOS, inducible nitric oxide synthase, gene regulation, post-transcriptional, mRNA stability, RNA-protein interactions, dexamethasone, murine, heterogeneous nuclear ribonucleoprotein, hnRNPI, hnRNPL, Farmaceutisk biokemi
National Category
Pharmaceutical Sciences
urn:nbn:se:uu:diva-6137 (URN)91-554-6402-5 (ISBN)
Public defence
2005-12-16, Room C4:305, BMC, Husargatan 3, UPPSALA, 09:00
Available from: 2005-11-25 Created: 2005-11-25Bibliographically approved

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