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1.
Chinese hamster cells with a minichromosome containing the centromere region of human chromosome 1 总被引:2,自引:0,他引:2
Kathleen Carine Joseph Solus Elysa Waltzer Jean Manch-Citron Barbara A. Hamkalo Immo E. Scheffler 《Somatic Cell and Molecular Genetics》1986,12(5):479-491
We describe a series of primary and secondary hamster-human hybrids which have selectively retained a small amount of human DNA. The hybrid XJM12.1.3 contains an estimated 4000–8000 kb of human DNA, and for a secondary hybrid derived from it, XEW8.2.3, our estimate is 1000–2000 kb. The hybridization of Southern blots of DNA from these hybrids with a variety of human satellite DNA probes reveals that these lines include centromere sequences of human chromosome 1. The identifiable human DNA is in the form of a minichromosome, as detected by in situ hybridization in the light microscope and in the electron microscope. At mitosis, the minichromosome can be observed to have kinetochores and to be associated with microtubules. Therefore, it can segregate in a stable fashion. It may be significant that in the selection of the hybrids we had selected for a human gene which has been mapped on human chromosome 1. 相似文献
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3.
Molecular characterization of human minichromosomes with centromere from chromosome 1 in human-hamster hybrid cells 总被引:4,自引:0,他引:4
Kathleen Carine Alain Jacquemin-Sablon Elysa Waltzer Jim Mascarello Immo E. Scheffler 《Somatic Cell and Molecular Genetics》1989,15(5):445-460
In this study we examine the amounts of four different human satellite DNA sequences in a series of human-hamster hybrid cells, which contain a human minichromosome including the centromere of human chromosome 1. Comparisons with the corresponding amounts in an intact human chromosome 1 suggest that the minichromosomes have lost satellite DNA sequences, and in one case a substantial fraction of several satellite DNAs is lost, without affecting the stability and normal mitotic segregation of the minichromosome. The smallest minichromosome appears to have lost all of the long arm and a significant portion of centromeric heterochromatin, while retaining 1000–2000 kb of the short arm of human chromosome 1. The satellite sequences examined include: a chromosome 1-specific satellite III probe, a chromosome 1-specific alpha satellite DNA, another alpha satellite DNA originally derived from the X chromosome, and an alphoid EcoRI dimer whose isolation from one of the minichromosomes and characterization is also described in this paper. One interpretation of these data indicates that an interspersion of blocks of satellite sequences occurs in the centromere region of chromosome 1. If these satellite sequences have functional significance, then there may be redundancy in the system that allows for a variation in the size of the kinetochore and the number of attachment sites for microtubules. 相似文献
4.
T23 are hybrids derived from the fusion between an IL-2-dependent mouse cell line, C10 and the rat lymphoma C58NTD. Supernatants from exponentially growing T23 cells induce the growth of CTLL2, and IL-2-dependent cell line, suggesting that these hybrids secrete interleukin 2. Addition of recombinant IL-2 to slowly growing T23 cells increases the rate of growth. Using an 125I IL-2 binding assay, a low number of cell surface IL-2 receptors were detected. T23 hybrids contain mouse but not rat IL-2 receptor genes as revealed by Southern blot analysis. These receptors are functional because the growth of exponentially growing hybrids is inhibited by an anti-mouse IL-2 receptor antibody. These data suggest an autocrine-like mechanism as responsible for the growth of these T cell hybridomas. 相似文献
5.
A linear mammalian artificial chromosome (MAC) will require at least three types of functional element: a centromere, two telomeres and origins of replication. As yet, our understanding of these elements, as well as many other aspects of structure and organization which may be critical for a fully functional mammalian chromosome, remains poor. As a way of defining these various requirements, minichromosome reagents are being developed and analysed. Approaches for minichromosome generation fall into two broad categories: de novo assembly from candidate DNA sequences, or the fragmentation of an existing chromosome to reduce it to a minimal size. Here we describe the generation of a human minichromosome using the latter, top-down, approach. A human X chromosome, present in a DT40-human microcell hybrid, has been manipulated using homologous recombination and the targeted seeding of a de novo telomere. This strategy has generated a linear approximately 2.4 Mb human X centromere-based minichromosome capped by two artificially seeded telomeres: one immediately flanking the centromeric alpha-satellite DNA and the other targeted to the zinc finger gene ZXDA in Xp11.21. The chromosome retains an alpha-satellite domain of approximately 1. 8 Mb, a small array of gamma-satellite repeat ( approximately 40 kb) and approximately 400 kb of Xp proximal DNA sequence. The mitotic stability of this minichromosome has been examined, both in DT40 and following transfer into hamster and human cell lines. In all three backgrounds, the minichromosome is retained efficiently, but in the human and hamster microcell hybrids its copy number is poorly regulated. This approach of engineering well-defined chromosome reagents will allow key questions in MAC development (such as whether a lower size limit exists) to be addressed. In addition, the 2.4 Mb minichromosome described here has potential to be developed as a vector for gene delivery. 相似文献
6.
A monoclonal antibody inhibiting leucocyte adhesion blocks induction of IL-2 production but not IL-2 receptor expression. 总被引:1,自引:0,他引:1
Monoclonal antibody 60.3 defines the leucocyte antigen CD 18 and recognizes a cell surface glycoprotein with an apparent molecular weight (MW) of 90,000 expressed by most human peripheral blood and bone marrow cells. This antibody can, among other things, block phorbol ester-induced adhesion among human mononuclear leucocytes. We show in this study that phorbol esters alone can induce peripheral blood mononuclear cells (PBL) to secrete interleukin-2 (IL-2) and that the IL-2-dependent cell line CTLL can be used for measuring this lymphokine without influence of the phorbol esters themselves. These findings make it possible to analyse the capacity of antibody 60.3 to interfere with IL-2 production and receptor expression by phorbol ester or phytohaemagglutinin (PHA)-treated human PBL. A significant positive correlation between blockage of induced cell aggregation by antibody 60.3 and reduction in IL-2 release was observed. The addition of interleukin-1 (IL-1) restored IL-2 secretion in PHA-treated, but not in 4-beta-phorbol 12, 13-dibutyrate [P(Bu)2]-treated, cells in the presence of this antibody. In parallel, IL-2 receptor expression was determined by immunofluorescence using biotinylated anti-IL-2 receptor (Tac) antibodies. FACS analysis showed that IL-2 receptor expression was unaffected by antibody 60.3, whereas DNA synthesis of the same P(Bu)2-treated PBL was inhibited. However, addition of external recombinant IL-2 overcame this proliferation blockade. These results indicate that a cell-to-cell adhesion step is necessary for the production of IL-2, but not for the expression of its receptor on both PHA- and P(Bu)2-treated human PBL. 相似文献
7.
Philip A. Emrie Carol Jones Talia Hofmann James H. Fisher 《Somatic Cell and Molecular Genetics》1988,14(1):105-110
The 18-kd Hydrophobic pulmonary surfactant protein (PSP-B) is a developmentally regulated protein which is important for normal lung function. A complementary DNA probe for 221 NH2 terminal amino acids of PSP-B was used to determine the chromosomal location of this gene and identify a restriction fragment length polymorphism (RFLP). Southern blot hybridization to genomic DNA isolated from a panel of human-CHO somatic cell hybrids unambiguously maps this gene to chromosome 2. Human DNA cut with BamHI yields a RFLP with variable bands at 2.8 and 2.6 kb. Since there is a relative lack of polymorphic markers for chromosome 2, this sequence may be useful in linkage analysis. 相似文献
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9.
IL-2 release from mouse splenocytes was measured by assaying the IL-2 on an IL-2-dependent cytotoxic T-lymphocyte line in culture (CTLL). Proliferation of the CTLL cells was monitored indirectly with the dye thiazolyl blue. The slow-acting anti-rheumatic drug auranofin at concentrations below 0.1 microM potentiated concanavalin A (Con A)-induced IL-2 release. Similar potentiation of Con A-induced IL-2 release was obtained with D-penicillamine, 1 microM-1 mM, and with the angiotensin-converting enzyme-inhibitor captopril, 10 nM-1 microM. Potentiation of Con A-induced IL-2 release was obtained with concentrations of the drugs likely to be achieved in vivo during therapy. Auranofin but not D-penicillamine and captopril inhibited Con A-induced IL-2 release at high concentrations (greater than 0.3 microM). 相似文献
10.
A macrophage-derived factor that inhibits the production and action of interleukin 2 总被引:4,自引:0,他引:4
T Krakauer 《Journal of leukocyte biology》1985,38(3):429-439
The human monocytic leukemia cell line THP-1 produces an immunosuppressive factor that inhibits interleukin 1 (IL-1)-dependent proliferation of mouse thymocytes as well as the mitogenic effects of concanavalin A (Con A) and phytohemagglutinin (PHA) on human peripheral blood mononuclear cells. The mechanism of action of this factor includes interference with both the production of interleukin 2 (IL-2) and its effects on target cells. Thus, the suppressor abrogates the proliferation of an IL-2-dependent cytotoxic T cell line (CTLL), but not of IL-2 independent cells like the L929 fibroblasts or the EL4 T lymphoma and U937 histiocytic lymphoma lines. It also suppresses IL-2 production by human peripheral blood enriched T cells and mouse splenocytes. The mediator has a molecular weight of 60,000-70,000 dalton, as determined by gel filtration chromatography, is heat labile, and is sensitive to trypsin, chymotrypsin, and protease. 相似文献
11.
人源化IL—2免疫毒素的构建和鉴定 总被引:2,自引:0,他引:2
利用基因工程技术在大肠杆菌表达成功人白细胞介素2(IL-2)和人血管生成素(Angiogenin)的融合蛋白(IL-2-Ang)。活性检测证明,它具有IL-2免疫毒素的作用,在体外可以杀伤IL-2依赖性CTLL细胞株和经ConA活化的小鼠脾脏IL-2R阳性细胞。在双相混合淋巴细胞培养试验中,IL-2-Ang可抑制淋巴细胞的增殖。在体内实验中,IL-2-Ang注射小鼠可使其抗原活化的脾细胞数量减少。由于IL-2和Ang均为正常人体成分,IL-2-Ang融合分子作为人源化免疫毒素可望具有更好的应用前景。 相似文献
12.
Expression of human CD antigens, including CD1 and CD25, by human x mouse interlineage leukaemia hybrids
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The expression of human cell-membrane antigens by hybrid cell lines derived by fusing a human B-ALL and mouse BW 5147 T-lymphoma cells has been studied. Using monoclonal antibodies (mAb), the phenotypes of 19 of the 24 hybrids which grew 11-44 days post-fusion have been analysed by indirect membrane immunofluorescence (IF). These uncloned hybrid cells were assayed early after outgrowth, prior to extensive human chromosome and antigen loss. Nonetheless, cytogenetic analysis showed that all hybrids contained variable numbers of human chromosomes. Phenotypic analysis showed that the hybrids could be grouped as follows: a high frequency expressing CD25 (IL-2 receptor), human T200, HLA class I alpha and beta 2microglobulin, and reacting with the mAb H207 and 12E7; an intermediate frequency expressing CD1 and CD2; and a low frequency expressing CD3, CD4, CD5, CD7, CD8 and CD9. This pattern of antigen expression resembled the frequency of these cells in the human B-ALL parent line. Cell sorting was used to immunoselect hybrids expressing CD1 and CD2, but CD1 expression was unstable during subsequent culture. 相似文献
13.
The lymphokine interleukin-2 (IL-2), which is necessary for the generation of an optimal cell-mediated immune response, has recently been shown to have lectinlike properties, with specificity for high-mannose groups. Therefore, the ability of IL-2 to bind to the mannose-rich fungus Candida albicans was examined. Heat-killed fungi preincubated with IL-2 stimulated, in a dose-dependent manner, proliferation of the IL-2-dependent cell line CTLL20. Soluble mannan, which is rich in exposed mannose groups, inhibited binding of IL-2 to C. albicans by approximately 60%, suggesting that the lectinlike properties of IL-2 are partially responsible for its fungal binding capacity. Binding of IL-2 to fungi appeared to be reversible, as C. albicans preincubated with IL-2 stimulated CTLL20 proliferation even when the fungi and cells were separated by an 0.4-microns-pore-size membrane. The lymphoproliferative response of normal human peripheral blood mononuclear cells to C. albicans was augmented when the fungus was preincubated with IL-2. Binding of 125I-IL-2 could not be inhibited by unlabeled IL-2, suggesting the absence of high-affinity receptors on C. albicans for IL-2. While the in vivo relevance remains to be determined, these data demonstrate that IL-2 can bind to C. albicans in vitro and thereby influence the host response to this medically important fungus. 相似文献
14.
We studied the effects of the gold compound sodium aurothionalate (SATM) on the responses of murine CTLL2 cells, and human T cells to Interleukin-2 (IL-2). SATM inhibited tritiated thymidine (3HTdR) incorporation by CTLL2 cells stimulated with human recombinant IL-2. Human T cells were cultured with phytohemagglutinin (PHA) in separate experiments and IL-2 receptor expression measured by using immunofluorescent anti-Tac serum; SATM inhibited IL-2 receptor expression. Furthermore, SATM when added concurrently with PHA, and IL-2 inhibited 3HTdR incorporation by human T cells in 5 day cultures. The kinetics of inhibition were further studied by adding PHA to T cells for 48 hours fllowed by the addition of SATM and IL-2; SATM inhibited 3HTdR incorporation even though receptor expression had occurred. These results suggest that SATM inhibits the stimulatory effects of IL-2 on T cells partly by interfering with IL-2 receptor expression, and partly by other mechanisms of action. These effects of SATM may explain some of the conflicting data in the literature on T cell responses to IL-2 in rheumatoid arthritis (RA), and suggest a possible mechanism of action for the drug in the treatment of RA. 相似文献
15.
OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor. 总被引:1,自引:0,他引:1
The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation. 相似文献
16.
Bryan R. G. Williams Mary E. Saunders Huntington F. Willard 《Somatic Cell and Molecular Genetics》1986,12(4):403-408
The low-molecular-weight human 2–5A synthetase gene has been assigned to chromosome 12 using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. A cDNA probe representing almost all the coding sequences of the 2–5A synthetase gene hybridizes to four fragments of human DNA digested with the restriction enzyme EcoR1. By correlating the presence of these fragments in somatic cell hybrid DNA with the human chromosome content of the hybrids, the 2–5A synthetase gene can be mapped to chromosome 12. This contrasts with a previous assignment of this gene to chromosome 11 using an enzyme activity assay. The reason for this discrepancy remains unclear. 相似文献
17.
David Owerbach William J. Rutter James L. Roberts Peter Whitfeld John Shine Peter H. Seeburg Thomas B. Shows 《Somatic Cell and Molecular Genetics》1981,7(3):359-369
The proopiocortin gene is located on chromosome 2 in humans. A 13-kb DNA fragment containing proopiocortin gene sequences was identified in human cells while proopiocortin-related gene sequences of 9.8 and 6.2 kb were present in mouse cells. In human-mouse cell hybrids which contained reduced numbers of human chromosomes and a complete set of mouse chromosomes, the 9.8- and 6.2-kb fragments were always present while the 13-kb fragment segregated with human chromosome 2 and the chromosome 2 enzyme markers acid phosphatase-1 (ACP1), malate dehydrogenase-1 (MDH1), and isocitrate dehydrogenase-1 (IDH1). Analysis of a single cell hybrid with a broken chromosome 2 indicates that the proopiocortin andACP1 genes are closely linked and in the distal region of the short arm of chromosome 2. 相似文献
18.
IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce T-Bet and IL-12Rbeta2 in naive T cells. 总被引:8,自引:0,他引:8
Linda Hibbert Stefan Pflanz Rene De Waal Malefyt Robert A Kastelein 《Journal of interferon & cytokine research》2003,23(9):513-522
Interleukin-27 (IL-27) supports proliferation of naive CD4(+) T cells and enhances interferon-gamma (IFN-gamma) production by activated T cells and natural killer (NK) cells. We report here that IL-27 induces Stat1 and Stat3 phosphorylation and activation in human and murine cell lines and primary human T cells. IL-27 also induces T-Bet, a Stat1-dependent gene crucial to Th1 cell commitment. Similarly, IFN-alpha activates Stat1 and Stat3 and T-Bet expression in naive T cells. Induction of T-Bet results in upregulation of IL-12Rbeta2 on naive T cells, which is essential for responsiveness to IL-12 and differentiation to a Th1 phenotype. Both IL-27 and IFN-alpha induce expression of IL-12Rbeta2 in T cells. In contrast, IFN-gamma, which activates Stat1 but not Stat3, induces expression of T-Bet but not IL-12Rbeta2 in naive T cells. We propose that IL-27 and IFN-alpha are important for early Th1 commitment and act upstream of IL-12 and IFN-gamma in this pathway. 相似文献
19.
《Immunopharmacology and immunotoxicology》2013,35(2):141-156
AbstractWe studied the effects of the gold compound sodium aurothionalate (SATM) on the responses of murine CTLL2 cells, and human T cells to Interleukin-2 (IL-2). SATM inhibited tritiated thymidine (3HTdR) incorporation by CTLL2 cells stimulated with human recombinant IL-2. Human T cells were cultured with phytohemagglutinin (PHA) in separate experiments and IL-2 receptor expression measured by using immunofluorescent anti-Tac serum; SATM inhibited IL-2 receptor expression. Furthermore, SATM when added concurrently with PHA, and IL-2 inhibited 3HTdR incorporation by human T cells in 5 day cultures. The kinetics of inhibition were further studied by adding PHA to T cells for 48 hours fllowed by the addition of SATM and IL-2; SATM inhibited 3HTdR incorporation even though receptor expression had occurred. These results suggest that SATM inhibits the stimulatory effects of IL-2 on T cells partly by interfering with IL-2 receptor expression, and partly by other mechanisms of action. These effects of SATM may explain some of the conflicting data in the literature on T cell responses to IL-2 in rheumatoid arthritis (RA), and suggest a possible mechanism of action for the drug in the treatment of RA. 相似文献
20.
M. Ala-Kapee H. Nevanlinna M. Mali M. Jalkanen J. Schröder 《Somatic Cell and Molecular Genetics》1990,16(5):501-505
Syndecan, a cell surface proteoglycan, is an integral membrane protein acting as a receptor for the extracellular matrix. For chromosomal localization of the human syndecan gene, a panel of mouse-human somatic cell hybrids was analyzed by Southern blotting using the cDNA probe for human syndecan. The hybrids were karyotyped at the time of DNA extraction. A band corresponding to the human syndecan gene in Southern blots was found only in a hybrid cell line containing human chromosome 2. This hybrid was subcloned and its subclones were analyzed by Southern blotting and karyotyped. Subclones carrying human chromosome 2 contained the syndecan gene, while subclones not carrying this chromosome did not. The human syndecan gene is thus assigned to chromosome 2. 相似文献