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dc.contributor.authorHampson, Lynne
dc.contributor.authorHampson, Ian N
dc.contributor.authorBabichuk, Charolyn K
dc.contributor.authorCotter, Laura A
dc.contributor.authorBleackley, R Chris
dc.contributor.authorDexter, T Michael
dc.contributor.authorCross, Michael A
dc.date.accessioned2009-11-06T16:16:43Z
dc.date.available2009-11-06T16:16:43Z
dc.date.issued2001
dc.identifier.citationA minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells. 2001, 2 (3):150-60 Hematol. J.en
dc.identifier.issn1466-4860
dc.identifier.pmid11920240
dc.identifier.doi10.1038/sj/thj/6200102
dc.identifier.urihttp://hdl.handle.net/10541/85585
dc.description.abstractINTRODUCTION: The serine protease inhibitor Serpin 2A is highly expressed in ex vivo bipotent granulocyte/macrophage progenitor cells and in cultured myeloid stem cells. The gene undergoes rapid down-regulation as these cells are induced to differentiate, and constitutive expression in cultured myeloid stem cells retards maturation. Serpin 2A is also expressed in T cells as a consequence of activation. We now report analysis of the upstream regulatory elements that control Serpin 2A transcription. MATERIALS AND METHODS: Using primer extension and rapid amplification of cDNA ends the transcription start site of the Serpin 2A gene was mapped, and a 1.2 Kb genomic upstream fragment cloned and sequenced. Promoter activity and protein binding of deletion and site-directed mutant constructs were analysed by transient transfection and by electrophoretic mobility shift assays. RESULTS: A minimal promoter fragment was identified with high activity dependent on NF-kappa and Moloney murine leukaemia enhancer factor LVa binding sites in both myeloid stem cells and activated T cells. NF-kappa was shown to be the main DNA binding protein in T cells, whereas that in haematopoietic stem cells appears to be novel. CONCLUSION: Serpin 2A promoter activity in T cells is due predominantly to NF-kappa binding to its consensus site. Activity in haematopoietic stem cells appears to be mediated by a novel protein, which recognises the NF-kappa consensus only in the context of flanking sequences. This concise regulatory element may be of potential value in gene therapeutic applications.
dc.language.isoenen
dc.subjectHaematopoietic Stem Cellsen
dc.subject.meshAnimals
dc.subject.meshBase Sequence
dc.subject.meshBinding Sites
dc.subject.meshCells, Cultured
dc.subject.meshChromosome Mapping
dc.subject.meshConsensus Sequence
dc.subject.meshCosmids
dc.subject.meshDNA-Binding Proteins
dc.subject.meshElectrophoretic Mobility Shift Assay
dc.subject.meshExons
dc.subject.meshGene Expression Regulation
dc.subject.meshGenes, Reporter
dc.subject.meshHematopoietic Stem Cells
dc.subject.meshLymphocyte Activation
dc.subject.meshMice
dc.subject.meshMice, Inbred BALB C
dc.subject.meshMice, Inbred CBA
dc.subject.meshMolecular Sequence Data
dc.subject.meshMutagenesis, Site-Directed
dc.subject.meshNF-kappa B
dc.subject.meshOrgan Specificity
dc.subject.meshPromoter Regions, Genetic
dc.subject.meshRNA, Messenger
dc.subject.meshReverse Transcriptase Polymerase Chain Reaction
dc.subject.meshSequence Deletion
dc.subject.meshSerpins
dc.subject.meshT-Lymphocytes
dc.subject.meshTranscription, Genetic
dc.subject.meshTransfection
dc.titleA minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells.en
dc.typeArticleen
dc.contributor.departmentCRC Department of Hematopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, UK. lynne.hampson@man.ac.uken
dc.identifier.journalThe Hematology Journalen
html.description.abstractINTRODUCTION: The serine protease inhibitor Serpin 2A is highly expressed in ex vivo bipotent granulocyte/macrophage progenitor cells and in cultured myeloid stem cells. The gene undergoes rapid down-regulation as these cells are induced to differentiate, and constitutive expression in cultured myeloid stem cells retards maturation. Serpin 2A is also expressed in T cells as a consequence of activation. We now report analysis of the upstream regulatory elements that control Serpin 2A transcription. MATERIALS AND METHODS: Using primer extension and rapid amplification of cDNA ends the transcription start site of the Serpin 2A gene was mapped, and a 1.2 Kb genomic upstream fragment cloned and sequenced. Promoter activity and protein binding of deletion and site-directed mutant constructs were analysed by transient transfection and by electrophoretic mobility shift assays. RESULTS: A minimal promoter fragment was identified with high activity dependent on NF-kappa and Moloney murine leukaemia enhancer factor LVa binding sites in both myeloid stem cells and activated T cells. NF-kappa was shown to be the main DNA binding protein in T cells, whereas that in haematopoietic stem cells appears to be novel. CONCLUSION: Serpin 2A promoter activity in T cells is due predominantly to NF-kappa binding to its consensus site. Activity in haematopoietic stem cells appears to be mediated by a novel protein, which recognises the NF-kappa consensus only in the context of flanking sequences. This concise regulatory element may be of potential value in gene therapeutic applications.


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