共查询到20条相似文献,搜索用时 15 毫秒
1.
M Kawaichi C Oka R Reeves M Kinoshita T Honjo 《The Journal of biological chemistry》1991,266(27):18387-18394
We have constructed a plasmid, pLTR100, which contains human interleukin 2 receptor light (IL-2R L) chain cDNA in the inverted orientation relative to the upstream SV40 promoter. The cDNA segment is flanked by the immunoglobulin gene recombination signal sequences so that the cDNA segment can invert and the human IL-2R L chain is subsequently expressed under the control of the SV40 promoter. A murine pre-B cell line, 38B9, transfected with pLTR100 began to express the human IL-2R L chain on the cell surface. The frequency of human IL-2R L chain positive cells increased almost linearly up to 50% for 60 days of culture after transfection. Southern blot analysis and sequencing of the DNA fragments at the recombination junction confirmed that the cDNA segment was inverted in a signal sequence-dependent manner by the variable-diversity-joining recombination process. Transgenic mice bearing the recombination substrate DNA similar to pLTR100 expressed the human IL-2 L chain in the spleen, thymus, and bone marrow, but not in the other tissues examined at the detectable level. Both IgM- and CD3-positive cells expressed the human IL-2R L chain, indicating that this artificial DNA can serve as a substrate for recombination both in B- and T-cells and that another DNA segment may be necessary to confer the cell-type specificity on the substrate DNA. 相似文献
2.
H Sugiyama S Akira N Yoshida S Kishimoto Y Yamamura P Kincade T Honjo T Kishimoto 《Journal of immunology (Baltimore, Md. : 1950)》1982,128(6):2793-2797
Eighteen Abelson virus-transformed immature B cell lines were established and immunoglobulin biosynthesis, expression of a B lymphocyte antigen detected by a monoclonal antibody, and rearrangement of immunoglobulin genes in these cell lines were studied. Only one cell line (A1) synthesized micro-chains but no light chains, and the other cell lines synthesized no detectable immunoglobulins. None of the cell lines established had detectable membrane-associated IgM. Fifteen cell lines expressed a B lymphocyte antigen on their cell surfaces. In three cell lines, however, the majority (greater than 99%) of cells did not express this antigen. Heavy chain genes were rearranged on both chromosomes in all the cell lines, although one heavy chain gene was deleted in three cell lines. In 12 of 18 cell lines, one or both kappa-chain genes were rearranged. In six cell lines, however, both kappa-chain genes remained in embryonic form; lambda-chain genes were in embryonic form in all the cell lines. These results suggested the hierarchy of Ig gene rearrangements, beginning with mu and proceeding to kappa and then to lambda. JH rearrangement was also shown to precede the appearance of a B lymphocyte antigen. In three cell lines (A1-A3), which were considered subclones derived from a single common precursor, it was suggested that one rearranged JH gene was functional, and the other was nonfunctional, indicating that allelic exclusion already operated in pre-B cells. 相似文献
3.
Abnormal recombination of Igh D and J gene segments in transformed pre-B cells of scid mice 总被引:14,自引:0,他引:14
M G Kim W Schuler M J Bosma K B Marcu 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(4):1341-1347
Studies of Ig and TCR genes in transformed lymphocytes of scid mice have revealed aberrant DNA rearrangements. Here we present a more detailed analysis of the Igh gene recombination in nine scid pre-B cell lines transformed by Abelson murine leukemia virus. We found 85% of the rearranged Igh alleles to contain abnormal Dh-Jh deletions of varying size. All of these deletions encompassed Jh elements and extended into the Igh enhancer region, occasionally involving the switch (S) region of the C mu gene. Some of these rearrangements removed most of the Dh elements, but none appeared to extend to the Vh genes. DNA sequence analysis of the two abnormally rearranged Igh alleles in one pre-B cell line showed that no Dh or Jh coding sequences were retained at the recombination sites though heptamer-like (CACTGTG) recognition signal sequences were present in the absence of nonamer (GGTTTTTGT) recognition signal sequences. These results imply that a deregulated recombinase activity may be responsible for the abnormal Dh-Jh deletions and the absence of Vh-Dh joining in established lines of Abelson murine leukemia virus-transformed scid pre-B cells. 相似文献
4.
Distinct intracellular fates of membrane and secretory immunoglobulin heavy chains in a pre-B cell line 总被引:2,自引:0,他引:2
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The intracellular fates of membrane and secretory immunoglobulin heavy chains were examined in a pre-B cell line that has switched to the gamma isotype. The membrane form of the heavy chain (gamma m) was rapidly degraded while the secretory form (gamma s) was retained intracellularly in association with BiP. The degradation of gamma m could not be inhibited by ammonium chloride, chloroquine, or monensin suggesting that it occurred in a nonlysosomal compartment. The inability to detect any Endo H-resistant form of gamma m before its degradation suggested that degradation occurs before entry into the Golgi compartment. Degradation of gamma m could be inhibited by incubation at 24 degrees C. In a derivative of this cell line expressing a transfected kappa gene, gamma s formed disulfide linked tetramers with kappa and was secreted, while gamma m, although associated with kappa, continued to be rapidly degraded. These observations suggest that membrane and secretory heavy chain proteins are retained by distinct intracellular mechanisms. Although masking of the CH1 domain abrogates gamma s retention, this domain does not influence the intracellular fate of gamma m. 相似文献
5.
E E Moore 《In vitro》1984,20(6):463-472
Cell lines corresponding to various cell lineages of the mouse embryo have been isolated from murine teratocarcinomas. Embryonal carcinoma cell lines are developmentally equivalent to the embryonic ectoderm or inner cell mass. Most of these cell lines have a modal chromosome number equal or close to 40, the normal mouse complement. However, cell lines corresponding to more advanced cell lineages (e.g., endoderm) are tetraploid or hypotetraploid and display multiple chromosomal rearrangements. This paper describes the isolation of a near-diploid differentiated cell line (LT-D) from an LT teratocarcinoma. The modal chromosome number of LT-D is 40, and this number is stable during at least 12 mo of continuous culture. LT-D cells are morphologically distinct from embryonal carcinoma cells and no longer express the SSEA-1 cell surface antigen or high alkaline phosphatase activity characteristic of embryonal carcinoma cells. LT-D cells can be induced to fuse into structures resembling myotubes. The formation of these structures is accompanied by expression of the muscle-specific isozyme of creatine phosphokinase and desmin, a muscle-specific component of intermediate filaments. Lastly, LT-D cells do not form tumors in syngenetic mice. 相似文献
6.
Emma E. Moore 《In vitro cellular & developmental biology. Plant》1984,20(6):463-472
Summary Cell lines corresponding to various cell lineages of the mouse embryo have been isolated from murine teratocarcinomas. Embryonal carcinoma cell lines are developmentally equivalent to the embryonic ectoderm or inner cell mass. Most of these cell lines have a modal chromosome number equal or close to 40, the normal mouse complement. However, cell lines corresponding to more advanced cell lineages (e. g., endoderm) are tetraploid or hypotetraploid and display multiple chromosomal rearrangements. This paper describes the isolation of a near-diploid differentiated cell line (LT-D) from an LT teratocarcinoma. The modal chromosome number of LT-D is 40, and this number is stable during at least 12 mo of continuous culture. LT-D cells are morphologically distinct from embryonal carcinoma cells and no longer express the SSEA-1 cell surface antigen or high alkaline phosphatase activity characteristic of embryonal carcinoma cells. LT-D cells can be induced to fuse into structures resembling myotubes. The formation of these structures is accompanied by expression of the muscle-specific isozyme of creatine phosphokinase and desmin, a muscle-specific component of intermediate filaments. Lastly, LT-D cells do not form tumors in syngenetic mice. This work was supported in part by National Institutes of Health, Bethesda, MD, Grant CA-15823 and by a departmental gift from R. J. Reynolds Industries, Inc. 相似文献
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Control of IgM synthesis in the murine pre-B cell line, 70Z/3' 总被引:5,自引:0,他引:5
The murine 70Z/3 tumor resembles a pre-B cell in synthesizing only intracellular mu-chains and no detectable light chain. However, one kappa gene is already rearranged, and after overnight incubation with lipopolysaccharide (LPS), most of the cells are induced to synthesize light chain. The induced cells display IgM on their surface, but do not secrete IgM. Thus, 70Z/3 cells resemble cells poised at the pre-B cell/B lymphocyte border. We have examined synthesis and post-translational modification of mu-chains in uninduced and induced 70Z/3 cells. Isolation of mu-chains and peptide maps demonstrated that both populations synthesize intracellular forms that correspond to membrane-specific mum and secretion-specific mus chains. These intracellular forms have completed only the first of the two glycosylation steps characteristic of eukaryotic cells. After induction by LPS, L chain synthesis commences, mum and mus synthesis are both increased twofold to threefold (due to an increased rate of synthesis rather than decreased degradation), and both complex with L chain to form mu2L2 tetramers. Furthermore, the glycosylation of a subset of the mum chains is completed, and these are placed on the membrane. However, unglycosylated mu2L2 tetramers can be placed on the membrane, so glycosylation is not a requirement. These data suggest that L chain may not be sufficient for externalization of mum and mus chains. These data support the idea that the controls of membrane placement and secretion of mu chains are post-translational and that different mechanisms operate for mum and mus chains. 相似文献
9.
Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products. 总被引:23,自引:22,他引:23
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We describe experiments designed to measure the efficiency of intermolecular recombination between mutant herpesvirus thymidine kinase (tk) genes introduced into mouse L cells. Recombinants were scored as stable transformants containing a functional tk gene. The two recombination substrates used were ptkB8, a pBR322-based plasmid containing a mutant tk gene, with a BamHI linker in an SphI restriction site that is centrally located within the gene, and mp10tk delta 3' delta 5', an mp10 vector with a tk gene deleted at both the 3' and 5' ends. The only homology shared by the two DNAs is 885 base pairs within the tk gene. To determine whether the double-strand break repair model that has been used to explain recombination in yeast cells (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) can account for recombination during the introduction of these DNAs into mammalian cells, we transformed cells with BamHI-linearized ptkB8 and supercoiled mp10tk delta 3' delta 5' replicative-form DNA. These two DNAs should recombine efficiently according to that model and should generate gene conversion products. In this reaction, the supercoiled DNA acts as the donor of information to repair the cleaved tk gene. Our results indicated that the efficiency of this reaction was very low (less than 10 transformants were obtained per 0.1 microgram of each DNA used in the reaction per 10(6) cells). In contrast, if BamHI-cleaved ptkB8 DNA was cotransformed into cells along with a circular DNA molecule containing a tk gene deleted only at its 3' end or only at its 5' end (mp10tk delta 3' or mp10tk delta 5'), then the efficiency of recombination could be more than 4 orders of magnitude higher than it was with circular mp10tk delta 3' delta 5' DNA. Recombination frequencies were highest when the tk delta 3' or tk delta 5' DNA used was cleaved at the tk deletion junction. Southern analyses of DNA from TK+ transformants generated with BamHI-cleaved ptkB8 and BamHI-cleaved mp10tk delta 3' DNAs indicated that recombination was almost always associated with the reassortment of markers flanking the reconstructed tk DNA. Together, these results are more consistent with the nonconservative single-strand annealing model for recombination that we proposed several years ago (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984) than they are with the double-strand break repair model. 相似文献
10.
Glucocorticoids induce focus formation and increase sarcoma viral expression in a mink cell line that contains a murine sarcoma viral genome.
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Dexamethasone (3 X 10(-10) to 3 X 10(-6) M) induced foci of morphologically transformed cells in a small proportion of a mink cell line that contains the Moloney murine sarcoma viral genome (S+L-). The induction was glucocorticoid specific, since other steroids with glucocorticoid activity (prednisolone, cortisol, and aldosterone) induced foci with an efficiency that paralleled their glucocorticoid activity, and steroids lacking glucocorticoid activity (17B-estradiol, testosterone, and progesterone) failed to induce foci. Viral antigen, as measured by specific immunofluorescence, was localized to the foci. The induction of foci by dexamethasone (3 X 10(-7)) was accompanied by an approximately 10-fold increase in intracellular Moloney murine sarcoma virus-specific RNA and viral p30 antigen. Removal of dexamethasone was followed by the disappearance of foci and a decrease in viral RNA and p30. In this cell system, therefore, glucocorticoids can affect the intracellular levels of type C viral RNA and protein. 相似文献
11.
Maës J Caspi Y Rougeon F Haimovich J Goodhardt M 《Journal of immunology (Baltimore, Md. : 1950)》2000,165(2):703-709
It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD- murine B cell line, 38C-13, which has previously been found to undergo kappa light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vkappa-Jkappa and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD- B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination. 相似文献
12.
A Mackel-Vandersteenhoven G R Vasta M J Waxdal J J Marchalonis 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1985,178(3):476-485
To determine precisely the nature of serological determinants shared between T-cell surface molecules and immunoglobulin variable regions, the capacity of antisera directed against a synthetic peptide corresponding to the entire JH 1 region of classical immunoglobulin plus five residues of the D region were tested for their capacity to bind to T-cell membranes and isolated T-cell products. The anti-JH 1 antisera reacted with normal and monoclonal in vitro grown T-cell lines as judged by microhemagglutination and binding in enzyme-linked immunosorbent assays. Immunologically cross-reactive membrane components disclosed by immunoblot transfer analysis ("Western blots") consisted of major components in the molecular weight range 30-35,000 and minor components in the range 65-70,000. The major product of the human T-cell leukemia line MOLT-3 had an approximate mass of 34,000 Da, a value consistent with the predicted size of the molecule specified by the recently described putative T-cell receptor gene YT35. The 65 to 70,000-Da components are most probably tightly associated dimers of the 30 to 35,000-Da forms. It was possible to align the JH sequences of molecules reactive with the anti-JH 1 antisera and other characterized VH sequences of molecules known to be cross-reactive with T-cell products. This facilitated a comparison disclosing clear segmental homology between the protein sequence derived from the YT35 gene and immunoglobulin VH framework regions sharing approximately 50% of sequence identity. The identification of VH-related T-cell products (termed VT-bearing molecules) with products of putative T-cell receptor genes gained further support by N-terminal sequence of the 68,000-Da product of the 70-N2 T-cell line which showed homology to the predicted N-terminal region of the YT35 product. These serological and protein chemical data, coupled with the comparison to gene sequence, show that T-cell components that bear serological determinants cross-reactive with VH show segmental homology with products of putative T-cell receptor genes and immunoglobulin VH. 相似文献
13.
The sheep immunoglobulin heavy chain Igh-J locus has been characterized in order to determine the genomic organization of JH segments and their contribution to heavy chain diversity. The locus contains six segments, of which two are functional and
four are apparently pseudogenes. These segments span a 1.8 kilobase (kb) region. The distance between JH-ps4 (the 3′-most segment) and the first domain of the μ-chain encoding constant gene is about 5 kb. The two functional JH segments have a standard upstream recombination signal sequence, including heptamer and nonamer sequences separated by a
22–23 nucleotide spacer, and end with a RNA donor splice site. These two segments possess all the characteristic JH invariant residues and are found in expressed μ heavy chain variable regions. The 5′ functional JH1 segment is used in more than 90% of the cDNAs sequenced to date. The contribution of JH segment germline multiplicity to variable regions diversity appears therefore to be minimal. Comparison with other mammalian
JH segments shows that all loci are very closely related and probably have evolved from a common ancestral locus.
Received: 19 November 1996 / Revised: 17 March 1997 相似文献
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15.
Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome.
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A culture of mouse cells containing a 1,000-nucleotide deletion mutant of Moloney murine leukemia virus has been isolated. The deletion did not affect the size or function of the 21S mRNA that encodes the env gene products. Both the deleted RNA and the 21S mRNA were recovered in polyribosomes. Cells containing the deleted virus made no detectable Pr180gag-pol. Pr65gag synthesis with also absent, but a 45,000-molecular-weight gag gene product was found that might be encoded by the deleted genome. Biosynthesis of Pr80env proceeded normally in these cells; the intracellular precursor was cleaved and migrated to the cell surface as gp70. The cells could not be superinfected by homologous Moloney murine leukemia virus presumably because of surface restriction due to the gp70. Although the cells express the Moloney murine leukemia virus gp70 on their surface, they will not make pseudotypes after infection with vesicular stomatitis virus implying that Pr65gag may play a critical role in pseudotype formation. Induction of endogenous virus expression in the cells carrying the deletion mutant generated an N-tropic murine leukemia virus that can fuse XC cells. This may represent a recombinant between the deletion mutant and an endogenous virus. 相似文献
16.
S Tsukada H Sugiyama Y Oka S Kishimoto 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(10):4053-4059
We established SPL2-1-2, a murine immature B cell line transformed by tsOS-59, a temperature-sensitive mutant of Abelson murine leukemia virus. SPL2-1-2 has a VHDJH-/G (germ-line) configuration, and can continuously generate D to JH joinings from the germ-line allele during culture. The D to JH joinings are strongly promoted by the shift of the culture temperature from permissive (35 degrees C) to non-permissive temperature (39 degrees C). Using this H chain gene rearrangement system, we succeeded in the estimation of the frequencies of D segment usage in initial D to JH joinings by deletion-mapping analysis. The results demonstrated the preferential utilization of DFL16.1, the most 5' D segment and DQ52, the most JH-proximal D segment in initial D to JH joinings. 相似文献
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Akiyoshi Hiragun Mayumi Sato Hiromi Mitsui 《In vitro cellular & developmental biology. Plant》1980,16(8):685-693
Summary From the fibroblastic cells of murine mammary tumor tissue we isolated a clonal cell line that could be induced to differentiate into fat-accumulating cells in vitro. Differentiation began after the cultures had reached confluence and was accompanied by (a) an increase in the incorporation of sodium acetate into the triglyceride (TG) fraction of cellular lipids, (b) a more than 50-fold increase in cellular TG content per milligram cell-layer protein basis and (c) the cessation of cellular DNA synthesis. The addition of insulin to the culture medium enhanced lipid accumulation and increased the fraction of cells that differentiated into adipocytes. When insulin (5 to 10 μg/ml) was added exogenously, 80% or more of the cells were induced to differentiate into mature adipocytes within 2 weeks. This cell line can be used as a model for mammalian cell differentiation and also as a convenient material for the study of lipid metabolism in adipocytes. This work was supported by Grants in Aid from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare of Japan. 相似文献