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Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression. 总被引:5,自引:3,他引:5
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B D Crawford M D Enger B B Griffith J K Griffith J L Hanners J L Longmire A C Munk R L Stallings J G Tesmer R A Walters et al. 《Molecular and cellular biology》1985,5(2):320-329
We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins. 相似文献
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Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification. 总被引:3,自引:2,他引:1
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R L Stallings A C Munk J L Longmire C E Hildebrand B D Crawford 《Molecular and cellular biology》1984,4(12):2932-2936
Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines. 相似文献
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Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cdr2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd2+ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd2+-resistant Cdr2C10 subline to induce synthesis of the Cd2+- and Zn2+-binding protein(s), metallothionein(s) (MT). Evidence that Cd2+ behaves as an analog of the essential trace metal, Zn2+, especially as an inducer of MT synthesis, suggested that the Cdr and CHO cell types could be employed to investigate cellular Zn2+ metabolism. In the present study, measurements were made to compare CHO and Cdr cell types for (a) growth as a function of the level of ZnCl2 added to the culture medium, (b) uptake and subcellular distribution of Zn2+, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cdr cell types grew normally (T
d≊16–18 h) during exposures to Zn2+ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn2+ levels, (b) Cdr cells incorporate fourfold more Zn2+ during a 24-h exposure to the maximal subtoxic level of Zn2+ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cdr cell is proficient in this response during exposure to the maximal subtoxic Zn2+ level. These findings suggest that (a) the CHO and Cdr cell systems will be useful in further studies of cellular Zn2+ metabolism, especially in comparisons of Zn2+ metabolism in the presence and absence of induction of the Zn2+-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular
Zn2+ uptake. 相似文献
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Masa-aki Okuyama Yoshihiro Kobayashi Masahiro Inouhe Hiroshi Tohoyama Masanori Joho 《Biometals》1999,12(4):307-314
Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu2+
in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd2+ and Mn2+. By adding Cd2+ and Mn2+ at 10 M concentration, the -galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO4, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu2+ and 1 M-Cd2+. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd2+ environment. In contrast, the presence of Mn2+ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu2+. The recovery from growth inhibition by Mn2+ was due to decreased Cu2+ accumulation. Inhibitory concentrations of Co2+, Ni2+ and Zn2+ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd2+ and Mn2+. These results are discussed in relation to Cu2+ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae. 相似文献
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Stefania Cigliano Paolo Remondelli Liliana Minichiello Maria Cristina Mellone Gianluca Martire Stefano Bonatti Arturo Leone 《Experimental cell research》1996,228(2):173
The expression of metallothionein (MT) and heat shock protein gene families was investigated in normal and in HeLa-derived cadmium-resistant cells, named H454. In the absence of amplification of MT genes H454 cells accumulated elevated concentrations of cadmium ions and synthesized higher levels of MT proteins than unselected HeLa cells. Northern blot analyses revealed higher levels of MT mRNAs in the resistant cells than in wild-type cells after Cd2+and Zn2+exposure. Evaluation of the cytotoxic potential of the different metals confirmed the high resistance to cadmium of the H454 cells. Two proteins of the heat shock family, hsp70 and GRP78, were synthesized in Cd2+-exposed H454 cells at levels comparable to the ones present in Cd2+-treated normal cells. Northern blot analyses of the mRNA levels corresponding to these proteins revealed elevated expression of both hsp70 and GRP78 mRNAs in H454 cells upon exposure to cadmium ions and no response to zinc induction. These data suggest the existence in the H454 cells of a cadmium-specific pathway of regulation of MT and heat shock genes. 相似文献
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A stably transformed BHK cell line, engineered to produce a human transferrin half-molecule under the control of a mouse metallothionein (MT) promoter, was used as a model system to develop strategies to increase inducible recombinant protein production. Gene expression regulated by the MT promoter is induced by heavy metals (e.g. Zn+2 or Cd+2) in a dose dependent fashion. However, at high concentrations these metals are toxic to cells. Culture protocols which balance these counteractive effects are needed to maximize transferrin production. Fully induced cells produced up to 0.7 pg transferrin/cell·h, a 3-fold increase in production over uninduced levels. Cell growth was inhibited at Cd+2 dosages above 1 fmol/cell; prolinged exposure at this dosage was cytotoxic. Cell specific transferrin productivities decreased within 48 h following induction with Cd+2 although cell-associated Cd+2 levels remain high. Further addition of Cd+2 to cultures restored cell specific transferrin production rates. This suggests that cell associated Cd+2 is sequestered into a form which does not stimulate the MT promoter. Cd+2 dosing regimes which maintained cell associated Cd+2 concentrations between 0.2 and 0.35 fmol/cell ensured cell growth and high cell specific productivities which maximized final product titers. For routine batch culture, initial Cd+2 loadings of 0.8 fmol/cell gave near-maximum transferrin production levels. For extended culture, repeated small doses of 0.5 fmol/cell every 24 to 48 h maximized transferrin synthesis with this cell line. 相似文献
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A cadmium-resistant variant of the Chinese hamster (CHO) cell with increased metallothionein induction capacity 总被引:12,自引:0,他引:12
C. E. Hildebrand R. A. Tobey E. W. Campbell M. D. Enger 《Experimental cell research》1979,124(2):237-246
The toxic trace metal Cd2+ has been used to select a variant (designated Cdr) of the Chinese hamster cell (line CHO) resistant to the growth-inhibitory and cytotoxic effects of Cd2+. Resistance of the Cdr cell to Cd2+-mediated cytotoxicity is not due to a decreased capability of the Cdr cell to accumulate Cd2+ since Cd2+ uptake in the Cdr cell is indistinguishable from that in the CHO cell at both toxic and subtoxic Cd2+ exposures. Comparison of the relative capacities of these two cell types to induce specific low molecular weight Cd2+-binding proteins (metallothioneins) reveals that the Cdr cell has an increased capacity to induce metallothionein and to sequester intracellular Cd2+ in metallothioneins. These results suggest that the greater competence of the Cdr cell to induce metallothionein is a major factor in the Cd2+-resistant phenotype of the variant. 相似文献
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Metallothioneins (MTs) are ubiquitous, low molecular mass and cysteine-rich proteins that play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting the cells against oxidative damages. MTs are able to bind metal ions through the thiol groups of their cysteine residues. Plants have several MT isoforms which are classified into four types based on the arrangement of cysteine residues. In the present study, a rice (Oryza sativa) gene encoding type 1 MT isoform, OsMTI-1b, was inserted in vector pET41a and overexpressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). The recombinant protein GST-OsMTI-1b was purified using affinity chromatography and its ability to bind with Ni2+, Cd2+, Zn2+ and Cu2+ ions was analyzed. The results demonstrated that this isoform has ability to bind Ni2+, Cd2+ and Zn2+ ions in vitro, whereas it has no substantial ability to bind Cu2+ ions. From competitive reaction with 5,5′-dithiobis(2-nitrobenzoic acid), DTNB, the affinity of metal ions for recombinant form of GST-OsMTI-1b was as follows: Ni2+/Cd2+ > Zn2+ > Cu2+ 相似文献
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Masahiro Inouhe Madoka Mitsumune Hiroshi Tohoyama Masanori Joho Tetsuo Murayama 《Journal of plant research》1991,104(3):217-229
Possible roles of cell wall and cytoplasmic peptides in the tolerance of cells to Cu2+ and Cd2+ ions were studied in suspension-cultured cells of tomato (Lycopersicon esculentum L. cv. Palace). Cu2+ and Cd2+ ions inhibited growth of wild type cells at concentrations more than 100 and 200 μM, respectively. Tomato cells readily developed
tolerance to Cd2+ ions up to 1 mM but not to Cu2+ ions, after repeated subculturings in the presence of the respective ions. Such a metal-specific adaptation of cells was
not due to the difference in the total uptakes between Cd2+ and Cu2+ ions by cells. Wild-type cells accumulated Cd2+ preferentially into the cytoplasmic peptide fraction and Cu2+ into the cell-wall fraction, when grown under the subtoxic metal conditions. Under excess metal conditions, Cd-tolerant cells
produced greater amounts of Cd-binding peptides in the cytoplasm and retained lesser amounts of Cd2+ ions in the cell wall than did wild-type cells. In contrast, tomato cells grown in the presence of Cu2+ ions synthesized no detectable amounts of Cu-binding peptides in the cytoplasm and retained most of the Cu2+ in the cell-wall fraction, irrespective of cell lines.
These results suggested that the cytoplasmic peptides rather than cell wall properties have a primary role in the response
of tomato cells to excess metal environments. 相似文献
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We describe a region of human DNA containing four metallothionein (hMT) genes. One of these genes, hMT-IA, was found to encode a functional protein that confers heavy metal resistance to NIH 3T3 cells after transfer on a bovine papilloma virus-derived vector. This gene is expressed in cultured human cell lines, but at a lower basal level than the hMT-IIA gene; it shows a different induction response to heavy metals and glucocorticoids than the hMT-IIA gene. Induction of the human MT family therefore does not represent an equivalent elevation in the level of expression of individual genes, but is the sum of the differential responses of active members. The differential response is due to functional differences of the respective promoter/regulatory regions of the genes as shown by gene-fusion experiments. While the hMT-IIA promoter is responsive to Cd++, Zn++, and glucocorticoids, the hMT-IA promoter mediates response only to Cd++. 相似文献
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Metalloadsorption by Escherichia coli Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB 总被引:8,自引:0,他引:8
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Carolina Sousa Pavel Kotrba Tomas Ruml Angel Cebolla Víctor De Lorenzo 《Journal of bacteriology》1998,180(9):2280-2284
Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB153-YMT and LamB153-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. coli cells to bind Cd2+ 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions. 相似文献
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Summary
Kluyveromyces spp. have been found to be more efficient than a CUP1R strain of S. cerevisiae in heavy metal resistance and accumulation. The present study describes the subcellular distribution of the accumulated metals (Ag, Cd, Cu) in S. cerevisiae and K. marxianus. Absorption by insoluble cellular material of the metals appears as the main mechanism of metal accumulation in both organisms. 相似文献