| 1. |
- Liberg, David, et al.
(författare)
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Differentiation-specific, octamer-dependent costimulation of κ transcription
- 1998
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 160:8, s. 3899-3907
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Tidskriftsartikel (refereegranskat)abstract
- By mutational analysis of the octamer-TATA box intervening region in the mouse SP6 κ promoter, we have mapped two octamer-dependent, costimulatory regions, A and B. The A region was active in late B cells only, while the B region was active throughout B cell differentiation. The B region was TATA proximal and contained a heptamer and an E box of the E2A type that is common in Vκ promoters. Mutation of the heptamer element did not decrease transcriptional stimulation from this region, but mutations in, or immediately 5' of, the E box core sequence did. A protein binding to this region could be detected in nuclear extracts. The complex could only partially be competed with a μE5 binding site and could not be supershifted with Abs raised to E2A gene products, indicating that it may represent a novel E-box binding complex. The A region was located proximal to the octamer and contained a CCCT element that is conserved both with regard to position and sequence in human VκII promoters. By mutational analysis, the transcriptional stimulatory activity was mapped to the CCCT element that also is part of an early B cell factor (EBF) binding site. In late B cells, a novel protein (FA), which did not bind to the EBF binding site in the mb1 promoter, interacted with the A region. This protein was found to be expressed at lower levels in early B cells as well as in HeLa cells. Thus, the octamer-flanking sequence contains positive control elements that may act independently but that differ in the stage of B cell differentiation at which they are active. One of these factors is an example of an ubiquitously expressed transcription factor that participate in differentiation-specific transcriptional activation.
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| 2. |
- Sigvardsson, Mikael, et al.
(författare)
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Pentadecamer-binding proteins : Definition of two independent protein-binding sites needed for functional activity
- 1995
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Ingår i: Molecular and Cellular Biology. - 0270-7306. ; 15:3, s. 1343-1352
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Tidskriftsartikel (refereegranskat)abstract
- The SP6 κ-promoter pentadecamer (pd) element was found to be unable to stimulate transcription when present in one copy as the only promoter element in a minimal promoter but showed weak stimulatory activity when present as a multimer (four copies). One copy of the pd element acted synergistically with an octamer element, but not with a SP1 site, to stimulate transcription. The effect was orientation dependent with regard to the pd element. Gel shift analysis showed that pd-binding proteins were expressed in transformed as well as nontransformed B lymphocytes, irregardless of their differentiation stage, and in HeLa cells. Two major complexes, binding to different sites within the pd element, were observed in gel shifts. A low-molecular-weight form dominated in resting cells, while a higher-molecular-weight form appeared after mitogenic stimulation. Southwestern analysis showed that the low-molecular-weight pd-binding protein had a molecular mass of 35 kDa, which was confirmed by fractionation by denaturating polyacrylamide gel electrophoresis and molecular sieving. The higher-molecular-weight complex was sensitive to detergent treatment, while the low-molecular-weight complex was not. Mutation analysis showed that the two pd-binding complexes interacted with distinct sites within the element and that dual occupancy was required for functional activity. The functional synergy between the pd element and the octamer was more pronounced in plasmacytomas than in B-cell lymphomas.
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| 3. |
- Sigvardsson, Mikael, et al.
(författare)
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Stimulation of χ transcription by a decamer‐dependent, synergistic mechanism
- 1995
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Ingår i: European Journal of Immunology. - : Wiley. - 0014-2980 .- 1521-4141. ; 25:1, s. 298-301
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Tidskriftsartikel (refereegranskat)abstract
- The intact SP6 χ promoter stimulated transcription 30 times more efficiently than did a control promoter consisting of a TATA motif as the only promoter element. Mutation of the SP6 χ promoter decamer in two positions reduced the transcriptional stimulation activity by over 90%. Promoters containing the SP6 χ promoter octamer or a consensus octamer in front of a TATA box were ineffective immunoglobulin promoters and stimulated at the most 15% of maximal transcription. Identical results were obtained after transfection of untransformed mouse splenic B cells stimulated by lipopolysaccharide, that express high levels of Oct2A, or of S194 cells that express negligible levels of Oct2A. Selective mutations in the penta‐decamer (pd), χY or early B cell factor (EBF) elements of the promoter reduced transcriptional stimulation by 20–30% in untransformed B cells. In S194 plasmacytoma cells the EBF mutation was functionally silent while the χY and pd mutations reduced transcriptional activation by 60‐70% in this cell line. A mutation in a TATA‐proximal E‐box motif did not alter the functional activity of the promoter in either cell population. It can be concluded that χ promoter function is highly dependent on complex interactions between individual promoter elements and that the decamer motif is pivotal for these interactions. The relative functional activity of a given promoter varied according to the target cell population used for the functional assay.
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