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The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.  相似文献   

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Regulation of the immunoglobulin gene transcription   总被引:2,自引:0,他引:2  
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Multiple nuclear factors interact with the immunoglobulin enhancer sequences   总被引:536,自引:0,他引:536  
R Sen  D Baltimore 《Cell》1986,46(5):705-716
To characterize proteins that bind to the immunoglobulin (Ig) heavy chain and the kappa light chain enhancers, an electrophoretic mobility shift assay with end-labeled DNA fragments was used. Three binding proteins have been found. One is NF-A, a factor found in all tested cell types that binds to the octamer sequence found upstream of all Ig variable region gene segments and to the same octamer in the heavy chain enhancer. The second, also ubiquitous, protein binds to a sequence in both the heavy chain and the kappa enhancers that was previously shown to be protected from methylation in vivo. Other closely related sites do not compete for this binding, implying a restriction enzyme-like binding specificity. The third protein binds to a sequence in the kappa enhancer (and to an identical sequence in the SV40 enhancer) and is restricted in its occurrence to B cells.  相似文献   

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We previously described a domain in the 5'' half of the human immunoglobulin kappa enhancer which could bind nuclear proteins in vitro, as detected by a lambda exonuclease protection assay. A second more 3'' binding domain in the enhancer has now been detected by a similar assay employing a different exonuclease, the T7 gene 6 exonuclease. Using this assay and starting with a pig spleen nuclear extract, we have purified 5000-fold a protein that binds to the 3'' domain. In a DNase I footprint experiment the partially purified protein protects a 27 bp segment in the enhancer centered around the sequence CAGGTGGC, which corresponds to the kappa E2 sequence motif described in the mouse kappa enhancer. The protein, designated NF-kappa E2, also appears to bind at a position downstream of kappa E2, at or near the kappa E3 site. Proteins capable of binding at kappa E2 are found in several mammalian species and are expressed in both lymphoid and non-lymphoid tissues.  相似文献   

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We have systematically investigated the functional role of protein binding sites within the mouse immunoglobulin heavy chain enhancer which we previously identified by in vitro binding studies (1,2). Each binding site was deleted, mutant enhancers were cloned 3' of the chloramphenicol acetyl transferase gene in the vector pA10CAT2 and transfected into plasmacytoma cells. We demonstrate that the newly identified site E, located at 324-338 bp, is important for enhancer function; previously identified sites B(uE1), Cl(uE2), C2(uE3) and C3 were also shown to be important for enhancer activity. Sites A and D are not required for IgH enhancer function, as assayed by our methods. Thus, including the octamer site, six protein binding sites which bind at least six different proteins are important for enhancer function in vivo.  相似文献   

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