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L Gedamu

Publications and source records attributed to L Gedamu.

At least 55 records · Page 3Linked to original sources
PubMed1989

Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

Animals↗
PubMed1989

Metal-specific posttranscriptional control of human metallothionein genes.

During the initial 4 h of treatment, copper and zinc similarly activated the rates of transcription and mRNA accumulation from the two human metallothionein (MT) genes, viz., MTI-G and MTII-A, in the hepatoblastoma cell line HepG2. The levels of copper-induced MT mRNAs remained at a plateau for up to 15 h. In contrast, the levels of zinc-induced MT mRNAs gradually declined after about 4 h, despite substantial transcription. The decrease in the zinc-induced MT mRNA half-life is probably due to a posttranscriptional event(s).

Actins↗
PubMed1989

Calcium phosphate-mediated transfection alters metallothionein gene expression in response to Cd2+ and Zn2+.

The level of expression of a transfected metallothionein (MT)-IGcat fusion gene in response to cadmium differed from that of the endogenous MT-IG gene. Atomic absorption analysis indicated that the total cellular content of cadmium and zinc increased upon calcium phosphate-mediated transfection. Thus, changes in the influx/efflux of metals may regulate the level of MT gene expression.

Cadmium↗
PubMed1988

Structure and expression of the human metallothionein-IG gene. Differential promoter activity of two linked metallothionein-I genes in response to heavy metals.

The human metallothionein (MT)-IG gene (hMT-IG) is tandemly linked in a head-to-head fashion with the hMT-IF gene. The hMT-IG gene encodes a MT-I polypeptide and has a tripartite structure. The 5'-flanking region of the hMT-IG gene has a TATAA box, four GC motifs, and at least four metal responsive elements. The 3'-untranslated region has a variation of the polyadenylation signal, AATTAA, and the 3'-flanking region a YGTGTTYY RNA processing signal. This gene is expressed in hepatoma-derived cell lines (Hep G2 and Hep3B2) in response to the heavy metals (cadmium, copper, and zinc) but not to the glucocorticoid analogue dexamethasone. In contrast, the lymphoblastoid cell line (Wi-L2) does not express the hMT-IG gene. These results suggest that the hMT-IG gene is regulated differentially and in a cell type-specific manner. Transient expression studies of the chloramphenicol acetyltransferase (CAT) gene under the transcriptional control of either the hMT-IG or hMT-IF promoter in Hep G2 cells has demonstrated that both promoters contain all the necessary cis-acting elements to elicit a similar pattern of heavy metal inducibility. However, the hMT-IG promoter in all instances is five times more active than the hMT-IF promoter. The differences in promoter activity of these genes could possibly be due to inherent differences in their basal level regulatory sequences. The expression of MT-IGcat in transfected Wi-L2 cells demonstrates that the hMT-IG promoter is not cell type-specific.

Base Sequence↗
PubMed1988

Regulation of human metallothionein (MT) genes. Differential expression of MTI-F, MTI-G, and MTII-A genes in the hepatoblastoma cell line (HepG2).

We have analyzed the pattern of expression of three human metallothionein (MT) genes (MTI-F, MTI-G, and MTII-A) in the hepatoblastoma cell line, HepG2, in response to the metal ion inducers cadmium, copper, and zinc. The absolute number of transcripts of each of the three genes were measured, and the data clearly suggest differential regulation of these members of the MT gene family by the different inducers both in terms of the rate and the extent of transcript accumulation. The lowest levels of transcript accumulation was observed for MTI-F gene (maximum 4,000 molecules of mRNA per cell) and copper was shown to be its poorest inducer (up to 2,000 molecules per cell). Cadmium is the poorest inducer of MTI-G gene even though transcripts of this gene accumulated at comparatively higher levels than those of MTI-F. Copper- and zinc-induced MTI-G transcript accumulation was up to 12,000 molecules per cell whereas the corresponding value for cadmium was only 4,500. MTII-A was the only gene expressed in the absence of any externally added inducer. Also, in contrast to the MTI genes, the MTII-A gene was equally responsive to all the metal ions tested and the induced levels of accumulation were much higher (up to 75,000 molecules of MTII-A mRNA per cell).

Cadmium↗
PubMed1988

Structure of the rainbow trout metallothionein B gene and characterization of its metal-responsive region.

The trout metallothionein (MT) genes consist of two members. We describe the structure of the first fish MT (tMT-B) gene which shows an overall resemblance but some remarkable differences with mammalian MT genes. The similarities included (i) tripartite structure of the gene, (ii) conservation of cysteine residues, and (iii) a TATAAA signal and two copies of metal-responsive elements (MREs). The differences consisted of (i) an AT-rich tMT-B promoter compared with highly GC-rich mammalian MT promoters and (ii) a lack of SP1-binding sites in the tMT-B promoter. Functional analysis of the tMT-B 5'-flanking region following fusion with the bacterial chloramphenicol acetyltransferase gene and its transfection into the rainbow trout hepatoma cell line revealed that sequences from positions -600 to +8 are sufficient for regulation by metals. Further deletion analyses of this fragment suggested that a minimum of 100 nucleotides upstream of the transcription initiation site are required for induction by cadmium and zinc. The tMT-B promoter was also functional in the human hepatoblastoma cell line, suggesting that an MT regulatory factor(s) is conserved in phylogenetically distant species like humans and fish.

Amino Acid Sequence↗
PubMed1988

Pairwise linkage analysis of 11 loci on human chromosome 4.

New RFLPs are described for INP10 and interleukin 2. The 55 pairwise genetic linkage relationships for these two loci and nine additional loci on the long arm of chromosome 4 (4q) are reported. Fifteen new linkages are established, and new data are added to the four previously reported linkages on 4q. Tight linkage of interleukin 2 (T-cell growth factor), epidermal growth factor, and alcohol dehydrogenase is described. Significant differences were observed between male and female recombination rates. The female rate was estimated to be 1.27 times the male rate. On the basis of these pairwise results, the order for the 11 loci is D4S35-GC-(ALB/AFP)-MT2P1-D4S1-INP10-ADH3-( EGF/IL2)-(FBB/FBA/FBG)-MNS. This preliminary order can serve as a starting point for more detailed multipoint analysis.

Chromosome Mapping↗
PubMed1987

Metallothionein gene expression in fish cell lines: its activation in embryonic cells by 5-azacytidine.

We have investigated the regulation of metallothionein gene expression in two fish cell lines. Rainbow trout hepatoma (RTH) cells synthesized metallothionein in response to heavy metal exposure. The maximum level of metallothionein synthesis detected during zinc exposure was much greater than during cadmium exposure. The time-courses of metallothionein synthesis were different for the different metal inducers, suggesting that metallothionein may be differentially regulated by cadmium and zinc in these cells. The metal-induced synthesis of metallothionein was correlated with increased translational activity and accumulation of metallothionein-mRNA, suggesting that metallothionein may be regulated at the transcriptional and post-transcriptional levels in RTH cells. Chinook salmon embryo (CHSE) cells, unlike RTH cells, did not synthesize metallothionein or metallothionein-mRNA in response to heavy metal exposure. However, when these cells were treated with 5-azacytidine prior to heavy metal exposure, the synthesis of metallothionein was induced, suggesting that DNA methylation may play a role in metallothionein gene expression in fish.

Animals↗
PubMed1987

Structure and expression of the human metallothionein genes.

The human metallothioneins are represented by a multigene family consisting of about 14 members. A number of MT-like genes have been isolated from a human genomic library and in this report, four MT genes have been characterized. Our results show that two of these genes represent the MT-I and MT-II processed genes. The other two genes (MT-IF and MT-IG) are functional members of the MT-I gene family. The amino acid sequence encoded by the MT-IF and MT-IG genes differ from the amino acid sequences of the published MT-I proteins at few positions. The 5'-flanking region of these genes contain metal responsive elements. Our studies show that the MT-IF and MT-IG genes are differentially regulated in two human hepatoma cell lines, HepG2 and Hep3B2, and a human lymphoblastoid cell line, WI-L2 in response to the heavy metals cadmium, zinc and copper, and glucocorticoids. In addition, these genes also show cell-type specific expression.

Amino Acid Sequence↗
PubMed1987

Heavy metal induced protein synthesis in fish cell lines.

The regulation of heavy metal induced gene expression was investigated in two fish cell lines: the rainbow trout hepatoma (RTH) and Chinook salmon embryo (CHSE) cells. The induction of metallothionein (MT) synthesis occurred in RTH cells exposed to zinc, and to a lesser extent, following exposure to cadmium. The time courses of MT synthesis were different for the different metal inducers, suggesting that MT may be differentially regulated in these cells. CHSE cells, unlike RTH cells, did not synthesize MT in response to metal treatment. Treatment of the cells with 5-azacytidine prior to heavy metal exposure resulted in the induction of MT synthesis. Since these cells were embryonic in origin, these findings raised the possibility that MT gene expression may be developmentally regulated in fish. Analysis of the entire spectrum of cellular proteins has revealed the synthesis of an unknown, 14,000 dalton, metal-inducible protein (MIP) and various stress proteins following exposure of fish cells to heavy metals.

Animals↗
PubMed1987

The rainbow trout metallothioneins: molecular cloning and characterization of two distinct cDNA sequences.

The rainbow trout hepatoma (RTH) cell line responds to heavy metals such as zinc and cadmium by synthesizing the ubiquitous thiol-rich protein metallothionein (MT). From this cell line we have isolated two full-length cDNA clones, tMT-A and tMT-B, which encode two similar but distinct trout MTs. The clones were isolated by cross-homologies between the trout MT mRNAs and a human MT riboprobe. Clones tMT-A and tMT-B code for proteins of 61 and 60 amino acids, respectively; the one extra amino acid in tMT-A is due to an apparent insertion at position 31 of the protein. There are also two other amino acid changes between the two isoforms. Overall, the coding regions show extensive homologies to mammalian MTs, especially at the cysteine residues and at a core sequence at the boundary of the two domains. However, closer examination reveals a number of significant differences in positions usually invariant in the mammalian MTs. Northern blot analysis of RNA from metal-treated RTH cells demonstrated MT-mRNA is induced to high levels by zinc, low levels by cadmium, and minimally by copper. In contrast, intraperitoneal injections of rainbow trout demonstrated that all three metals induce MT-mRNA to comparable levels in the liver. Southern blot analysis of trout DNA cleaved with three restriction enzymes suggests that the trout family of MT genes is probably limited to these two members.

Amino Acid Sequence↗
PubMed1987

In vitro synthesis of double stranded RNA and measurement of thermal stability: effect of base composition, formamide and ionic strength.

Double stranded RNA was prepared by transcription of complementary strands followed by annealing. RNA duplexes prepared in this way showed cooperative thermal transition. Thermal denaturation of RNA duplexes of 33%-70% G+C content shows that Tm increases more sharply [0.93 degrees C/(%G+C)] with increase in G+C than that of DNA. Formamide (up to 80%) reduces Tm of RNA duplex linearly, and the rate of reduction is much less than that of DNA. The relation between salt concentration and Tm of double stranded RNA was also determined. The following relationship has been derived based on the above observations: Tm (ds RNA) = 14.88 logM + 0.93 (%G+C) + 67.62 - 0.4 (%Formamide).

Base Sequence↗
PubMed1986

Structure, organization, and regulation of human metallothionein IF gene: differential and cell-type-specific expression in response to heavy metals and glucocorticoids.

We describe a human genomic clone containing the metallothionein (MT) IF and MT IG genes. Southern blot analysis and partial DNA sequence determinations show that these genes are organized in a head-to-head fashion and are located approximately 7.0 kilobases apart from each other. Sequence analysis shows that the MT IF gene contains three exons separated by two introns. All of the intron-exon junctions are defined by the GT-AG rule. The 5' flanking region shows the presence of a duplicated metal regulatory element (TGCGC CCGGCCC) important in heavy-metal induction of this gene and a sequence for its basal level expression (GCGGGGCGGGTGCAAAG). The 5' flanking region is also highly G + C rich (approximately 75%) and contains several GC boxes (GGGCGG), probably important in the binding of transcription factors. The TATAA box and the AATAAA sequence are represented by their variants, the TATCAA box and the AATTAA sequence, respectively. This gene is functional and inducible by heavy metals but not by dexamethasone in mouse LMTK- cells after its transfer on a plasmid containing the herpes simplex virus thymidine kinase gene. Further studies on various human cell lines show that this gene is not expressed in a splenic lymphoblastoid cell line (WI-L2) but is expressed in two hepatoma cell lines (Hep 3B2 and Hep G2) in response to cadmium, zinc, and copper. Dexamethasone appears to have no significant effect on its expression. The studies suggest that the MT IF gene shows cell-type-specific expression and is differentially regulated by heavy metals and glucocorticoids.

Amino Acid Sequence↗
PubMed1986

Heat-shock gene expression in animal embryonic systems.

We have examined the expression of heat shock or stress genes in fish, echinoderm, amphibian, and mammalian embryonic systems. In a Chinook salmon embryo cell line, elevation of the incubation temperature or exposure to metal ions (e.g., cadmium and zinc) induced a set of heat-shock proteins HSPs. Transcriptional inhibitor, in vitro translation, and Northern hybridization studies suggest that fish HSP synthesis is regulated at the transcriptional level. The synthesis of HSPs during early development of Arbacia punctulata, Xenopus laevis, mouse, and rabbit is a stage-dependent phenomenon. In each of the developmental systems, HSP synthesis could not be induced until after cleavage stages. The ability of the embryo to undergo a heat-shock response (i.e., HSP synthesis) was correlated with the ability to detect HSP mRNA accumulation by either in vitro translation or Northern hybridization assays. Thus, the stage-dependent synthesis of HSPs appears to be controlled at the transcriptional level. Finally, in all of the organisms studied, the capacity to synthesize HSPs and accumulate HSP mRNA also coincides with acquisition of thermotolerance.

Animals↗