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In contrast to the mouse immunoglobulin heavy chain and kappa light chain genes, very little is known about the regulation of expression of the immunoglobulin lambda light chain locus. To identify elements responsible for lambda gene regulation we mapped DNaseI hypersensitive sites associated with a functionally rearranged lambda 1 gene in nuclei from the myeloma cell line J558L. Tissue-specific hypersensitive sites were identified 2.3 to 2.5 kb upstream of the CAP site of both the lambda 1 gene and the unrearranged variable (V) lambda 2 gene segments. DNA sequences flanking the lambda 1 gene were isolated and tested for their influence on expression of the lambda 1 gene after transfection into myeloma cells and after injection into fertilized mouse eggs. Two enhancer elements were identified downstream of the lambda 1 gene. A proximal element (located 4 to 10 kb 3' of the gene) enhanced expression of a lambda 1 gene in stable myeloma cell transfectants but had no effect on the expression of a heterologous reporter gene in transient assays. A second, distal element, located approximately 30 kb 3' of the gene, enhanced heterologous expression in J558L cells expressing a lambda gene but not in a non-lambda myeloma cell line (SP2/0-Ag14). Co-injection of cosmids containing the lambda 1 gene and both the proximal and distal downstream elements into fertilized mouse eggs resulted in high-level expression of the lambda 1 transgene in B cells of transgenic mice. The identification of these lambda regulatory elements, in addition to contributing to an understanding of lambda gene regulation per se, will facilitate the study of the regulation of differential expression of kappa and lambda light chain genes in the immune system. 相似文献
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We reported previously that composite DNA constructed from a mammalian plasmid (L factor) and foreign gene can be reestablished as a plasmid in mouse embryonal carcinoma (F9) cells after transfection and the plasmid-bearing F9 cells undergo normal in vitro differentiation in response to retinoic acid, an inducer for F9 cell differentiation. We constructed F9 cells bearing plasmidal L factor DNA in which a reporter (chloramphenicol acetyltransferase; CAT) gene was placed under the control of a differentiation-responsive viral (Moloney murine leukemia virus or simian virus 40) enhancer-promoter. When such plasmid-bearing cells were treated with retinoic acid, the CAT gene was inducibly expressed. These results indicate that mammalian gene expression can be studied with the plasmidal expression vector which is structurally dissociated from complex chromosomes. 相似文献
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Determinant differences between the rabbit and mouse immunoglobulin kappa enhancers impair the activity of the rabbit enhancer in mouse myeloma cells.
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Enhancer activity of the rabbit immunoglobulin kappa light chain gene intron conserved region (KICR) was examined in mouse myeloma cells using transient expression experiments. Compared to the homologous region of the mouse kappa light chain gene, the rabbit KICR shows nearly no stimulatory effect on expression of the indicator gene, cat. Experiments with mouse-rabbit chimeric KICRs indicated that differences in the region around the NF-kappa B binding site are responsible for the impaired activity of the rabbit KICR whereas mouse sequences covering the kappa E2 and kappa E3 motifs can be replaced by the equivalent rabbit fragment without affecting enhancer function. Creation of a perfect mouse NF-kappa B target sequence in the rabbit gene only partially restores enhancer activity. Furthermore, mouse and rabbit DNA fragments encompassing the NF-kappa B target sequence behave in an identical manner in an electrophoretic mobility shift assay. The results indicate species-related functional differences in the immunoglobulin kappa light chain gene enhancer and suggest that although the NF-kappa B binding site plays a crucial role in enhancer activity surrounding gene elements are also necessary for full enhancer effect. 相似文献
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Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells 总被引:119,自引:44,他引:75
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Neuberger MS 《The EMBO journal》1983,2(8):1373-1378
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Plasmidal maintenance of composite DNA derived from polyoma related plasmid, L factor. 总被引:2,自引:1,他引:1
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Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed. 相似文献
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Two rearranged immunoglobulin kappa light chain genes in one mouse myeloma. 总被引:18,自引:12,他引:6
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The organization of immunoglobulin gene segments coding for kappa light chains has been studied in uncloned and cloned DNA from mouse liver and a mouse myeloma. It is known that the C (constant, ref. 2) gene segment is present in the tumor DNA on two EcoRI fragments of 14 and 20 kb and in liver DNA on a 15 kb fragment. The 14 kb myeloma and the 15 kb liver fragment have been cloned previously. Here we report on the cloning of the 20 kb myeloma fragment and present detailed restriction maps covering about 22 kb of DNA surrounding the C gene segment in liver and tumor DNA. The region on the 20 kb fragment has been localized where a DNA rearrangement had occurred. The presence of two rearranged kappa light chain genes in one tumor is discussed in regard to the molecular basis of allelic exclusion. 相似文献
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H Yoshimura Y Ikeda J Fujiwara K Nishimori K Kotoji S Tamaki K Hanada M Oishi 《Plasmid》1992,28(2):115-122
It has previously been reported that composite DNAs derived from L factor, a polyoma-related mammalian plasmid, can be established in several mouse cell lines after transfection. Here, we report that the copy number of a plasmid composite DNA consisting of L factor, pBR DNA, dihydrofolate reductase (dhfr) gene, and gamma-interferon (gamma-IFN) gene was increased more than 10-fold after two successive adaptations of the plasmid-bearing mouse L cells to increasing concentrations of methotrexate (MTX), an inhibitor of dhfr. The structure of the amplified L factor plasmid remained intact during prolonged cell culture, but the copy number remained to be amplified only when the selective pressure (presence of MTX in the medium) has been exerted during the culture. Cells bearing the amplified plasmid produced a higher level of gamma-IFN compared with the original clone, which was likely to be derived from the plasmid gamma-IFN gene amplified along with L factor and the dhfr gene. 相似文献
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Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell cultures. 总被引:5,自引:2,他引:3
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T V Gopal 《Molecular and cellular biology》1985,5(5):1188-1190
A new method was developed to study transient gene expression, stable transformation, and cotransformation in suspension cells, such as mouse myeloma and erythroleukemia cells. This method involves attachment of cells to a concanavalin A-coated tissue culture dish, treatment of cells with DEAE-dextran to adsorb plasmid DNA to the attached cells, and finally treatment with a 40% solution of polyethylene glycol to facilitate the uptake of DNA by the cells. Plasmids pSV2cat and pSV2neo were used as markers to optimize the conditions for transient gene expression and stable transformation, respectively, of mouse myeloma and erythroleukemia cells. This method was successfully used to obtain cotransformants of mouse myeloma cells. 相似文献
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Functionally rearranged immunoglobulin heavy-chain (gamma 2b) and light-chain (lambda 1 and kappa) genes were introduced into mouse L tk- cells by co-transformation with the Herpes virus tk gene. Cloned cell lines were selected in HAT medium and tested for the presence of transfected immunoglobulin gene sequences by Southern blotting analysis. It was found that the gamma 2b gene was accurately transcribed at a low level in transfected mouse L cells and cytoplasmic gamma 2b, heavy-chain protein was detected by immunoprecipitation of cell extracts. Light-chain genes, on the other hand, were not accurately transcribed. Instead, lambda 1 or kappa RNA species were detected which were approximately 200 to 300 bases longer than the authentic mRNAs. These results suggest that the expression of rearranged heavy-chain and light-chain genes are controlled differently and that these differences can be seen in transfected, non-lymphoid cells. 相似文献
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Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse 总被引:5,自引:0,他引:5
The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes. 相似文献
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F G Falkner E Neumann H G Zachau 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1984,365(11):1331-1343
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DNA repair in cells sensitive and resistant to cis-diamminedichloroplatinum(II): host cell reactivation of damaged plasmid DNA 总被引:6,自引:0,他引:6
cis-Diamminedichloroplatinum(II) (cis-DDP) has a broad clinical application as an effective anticancer drug. However, development of resistance to the cytotoxic effects is a limiting factor. In an attempt to understand the mechanism of resistance, we have employed a host cell reactivation assay of DNA repair using a cis-DDP-damaged plasmid vector. The efficiency of DNA repair was assayed by measuring the activity of an enzyme coded for by the plasmid vector. The plasmid expression vector pRSVcat contains the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in a configuration which permits expression in mammalian cells. The plasmid was transfected into repair-proficient and -deficient Chinese hamster ovary cells, and CAT activity was subsequently measured in cell lysates. In the repair-deficient cells, one cis-DDP adduct per cat gene was sufficient to eliminate expression. An equivalent inhibition of CAT expression in the repair-proficient cells did not occur until about 8 times the amount of damage was introduced into the plasmid. These results implicate DNA intrastrand cross-links as the lesions responsible for the inhibition of CAT expression. This assay was used to investigate the potential role of DNA repair in mediating cis-DDP resistance in murine leukemia L1210 cells. The parent cell line L1210/0 resembled repair-deficient cells in that about one adduct per cat gene eliminated expression. In three resistant L1210 cell lines, 3-6-fold higher levels of damage were required to produce an equivalent inhibition. This did not correlate with the degree of resistance as these cells varied from 10- to 100-fold resistant.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Rearranged and germline immunoglobulin kappa genes: different states of DNase I sensitivity of constant kappa genes in immunocompetent and nonimmune cells 总被引:28,自引:0,他引:28
The rearrangement of a variable (V) and a constant (C) gene appears to be a necessary prerequisite for immunoglobulin gene expression. Multiple different rearranged kappa genes were found in several mouse myelomas, although these cells produce only one type of kappa chain [Wilson, R., Miller, J., & Storb, U. (1979) Biochemistry 18, 5013--5021]. It is therefore of interest to understand how only one allele within a lymphoid cell becomes expressed, while the other allele remains nonfunctional ("allelic exclusion"). We have studied the chromatin conformation of kappa genes by making use of the preferential digestion of potentially active genes by DNase I described, for example, for globin genes [Weintraub, H., & Groudine, M. (1976) Science (Washington, D.C.) 193, 848--856]. The DNase I sensitivity of kappa genes in myeloma tumors, in a B cell lymphoma, and in liver was determined by hybridization with DNA on Southern blots. It was found that rearranged C kappa genes are DNase I sensitive in myelomas in which several kappa genes are rearranged, regardless of whether the rearranged genes code for the kappa chains synthesized by the cell. Furthermore, the C kappa gene in germline configuration is also DNase I sensitive in a B cell lymphoma; i.e., it is in the same chromatin state as the rearranged C kappa gene which probably codes for the kappa chains produced by the cell. The altered chromatin state appears to be localized: V kappa genes in germline context are not DNase I sensitive in myeloma or B lymphoma cells while C kappa genes present in a kappa gene cluster on the same chromosomes are sensitive. When rearranged, however, the V kappa genes are as sensitive to DNase I as are rearranged C kappa genes. V lambda and C lambda genes are not DNase I sensitive in kappa myelomas. Thus, commitment to kappa gene expression is apparently correlated with a chromatin conformation which confers increased DNase I sensitivity to the DNA in the vicinity of all C kappa genes in the cell. "Allelic exclusion" does not operate on the level of chromatin conformation which can be detected by altered DNase I sensitivity. 相似文献
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