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L Gedamu

Publications and source records attributed to L Gedamu.

At least 73 records · Page 4Linked to original sources
PubMed1986

Heavy metal-induced gene expression in fish and fish cell lines.

Two isoforms of metallothionein (MT) have been isolated from rainbow trout livers following CdCl2 injections. These MTs have been identified by standard procedures and appear to be similar to mammalian MTs. Total RNA from such induced livers was shown to contain high levels of MT-mRNA activity when translated in cell free systems. This activity was demonstrated to be in the 8 to 10S region of a sucrose gradient. The RNA fractions also showed homology to a mouse MT-I cDNA probe. The exposure of rainbow trout hepatoma (RTH) cells to various concentrations of CdCl2 and ZnCl2 induced the expression of MT and MT-mRNA. Exposure of Chinook salmon embryonic (CHSE) cells to these metals, however, did not result in MT synthesis, suggesting that the MT genes have not become committed to transcription. Instead, an unknown low molecular weight (MW = 14 kDa) protein was induced. This metal-inducible protein (MIP) was capable of binding 109Cd and was stable to heating, while the binding of the metal to this protein was not. These characteristics have been reported for a protein induced in rainbow trout liver following environmental exposure to cadmium. We suggest that both MT and MIP may function in detoxification of heavy metals.

Amino Acids↗
PubMed1985

Acute treatment of mice with cadmium salts results in amplification of the metallothionein-1 gene in liver.

A variety of genes have been shown to change copy number during development, including rRNA genes in amphibians and chorion proteins in insects. Dihydrofolate reductase and metallothionein-1 (MT-1) genes are present in high copy number in cultured mammalian cells subjected to low levels of agents that will select for cells with amplified copies of specific genes. Recent studies have shown that the metallothionein-1 gene in mouse liver is regulated at the transcriptional level by treatment with heavy metals. We report here that, at cadmium concentrations 5 to 10-fold higher than that required to induce maximal transcription of the MT-1 gene, there is a 2 to 3-fold increase in MT-1 gene concentration in liver nuclear DNA by 6 hours after induction, and extra copies persist up to 3 weeks in the absence of further heavy metal treatment. The extra MT-1 gene copies that appear 6 hours after cadmium treatment are in a conformation that renders them relatively nuclease insensitive.

Animals↗
PubMed1985

Human metallothionein-II processed gene is located in region p11----q21 of chromosome 4.

Metallothionein (MT) genes comprise a multigene family encoding low-molecular-weight, heavy-metal-binding proteins. We have mapped a human MT-II processed gene to chromosome 4, using Southern blotting in combination with a human X mouse hybrid clone panel containing defined subsets of human chromosomes. We have further localized this gene to region p11----q21, using in situ hybridization.

Animals↗
PubMed1984

Induction of metallothionein and metallothionein mRNA in rainbow-trout liver following cadmium treatment.

A low-molecular-weight cadmium-binding protein was induced in the livers of rainbow trout (Salmo gairdnerii) following a series of intraperitoneal injections of cadmium chloride. The subsequent purification and amino acid analysis of this protein showed it to be a true metallothionein. As in higher organisms, two major forms of metallothionein appeared to be present in the liver following cadmium treatment. Following a similar induction procedure total RNA was also isolated and shown to contain high levels of metallothionein-mRNA activity when assayed in a wheat-germ cell-free translation system. This activity was present in the Poly-A+-containing fraction of the total RNA. The bulk of this mRNA activity was shown to be in the 8-10S region of a sucrose gradient.

Animals↗
PubMed1984

The ontogeny of expression of murine metallothionein: comparison with the alpha-fetoprotein gene.

The ontogeny of expression of mouse metallothionein was studied by RNA dot and Northern blot hybridization using a cloned cDNA probe. In some instances the synthesis of metallothionein was analyzed by cell-free translation of RNA as well as pulse-labeling of proteins in short-term organ cultures followed by polyacrylamide gel electrophoresis. Interesting parallels between metallothionein and alpha-fetoprotein gene expression during development were noted. Like alpha-fetoprotein mRNA ( Dziadek and Andrews, 1983), metallothionein mRNA was found to be abundant in developing liver as well as in visceral yolk sac endoderm. In addition, metallothionein mRNA was abundant in parietal yolk sac. During liver development metallothionein and alpha-fetoprotein mRNAs were abundant by Day 12 of gestation, increasing to maximal levels on Day 16 and decreasing during late fetal and neonatal life to basal levels in adult. Metallothionein mRNA increased in maternal liver and was also abundant in certain hepatomas. Synthesis of metallothionein and levels of metallothionein mRNA in visceral yolk sac increased from Day 9 of gestation to maximal levels on Days 11-12 and then decreased abruptly after Day 15. RNA from differentiated teratocarcinoma cells with primitive, parietal or visceral endoderm characteristics each contained high levels of metallothionein mRNA, whereas, levels of this mRNA varied widely among embryonal carcinoma stem cell lines. alpha-Fetoprotein mRNA was not detected in embryonal carcinoma cells but was expressed in visceral endoderm-like differentiated cells. These results indicate that parietal and visceral endoderm cells actively express the metallothionein gene and further suggest that expression may be initiated at the earlier stage of primitive endoderm.

Animals↗
PubMed1984

Human metallothionein MT-I and MT-II processed genes.

Two intronless pseudogenes, corresponding to the human metallothionein MT-I and MT-II processed genes, have been isolated from a human genomic library. MT-I processed gene has accumulated a number of mutations including a nonsense mutation giving rise to a termination codon at amino acid position 21, and a single base deletion at amino acid position 47 causing a shift in the reading frame. MT-II processed gene is a full-length perfect copy of its corresponding mRNA except for a few mutations. Most of the mutations in MT-II processed gene are silent except that the amino acid glycine (GGT) at position 10 is changed to serine (AGT) due to a transition. Both MT-I and MT-II processed genes possess poly(A) sequences of 21 and 17 nucleotides, respectively, 3' to the consensus AATAAA sequence. While these genes are quite similar in their sequences at the 3'-untranslated region, they show less than 50% homology in the 5'-untranslated sequences. Two direct repeats of 16 and 18 nucleotides in length define the limits of the MT-I and MT-II processed genes, respectively, and have been confirmed by S1 nuclease mapping analysis. In both MT-I and MT-II processed genes these direct repeats towards the 5' end of the gene start with an AhaIII (TTTAAA) restriction site. Our studies suggest that these direct repeats are the results of the insertion site duplication.

Amino Acid Sequence↗
PubMed1984

A frequent restriction fragment length polymorphism in the human metallothionein-II processed gene region is evolutionarily conserved.

Genomic blot analysis of human DNA indicated that metallothioneins are represented by a multi-gene family. Clones containing metallothionein sequences have been isolated and two of these have been identified as metallothionein-I and metallothionein-II processed genes by sequence analysis. The metallothionein-II processed gene in humans shows two restriction fragment length polymorphisms of 4.5 and 4.8 kb (10(3) bases) when EcoRI-digested genomic DNA from various individuals was analysed by Southern blotting. All the three genotypes are found at a high frequency and thus the metallothionein-II processed gene represents a true polymorphic marker. Familial studies also indicate that these restriction fragment length polymorphisms follow the classical Mendelian inheritance. Detailed Southern blot analyses show that this restriction fragment length polymorphism is due to a restriction site polymorphism and is localized at the 5'-flanking region of the metallothionein-II processed gene. Sequence analysis of the suspected region in the 4.8 kb fragment shows that the sequence G*GATTC, which is found 371 nucleotides downstream from the EcoRI site on the 5' end of the 4.8 kb fragment, makes a HinfI site. A transition of *G to A in this sequence in the 4.5 kb allele has resulted in loss of the HinfI site and created an EcoRI site. Thus, this mutation has given rise to this restriction fragment length polymorphism.

Alleles↗
PubMed1983

Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2).

Cadmium-binding protein synthesis and induction by cadmium chloride were studied in the human lymphoblastoid cell line WI-L2. Lymphoblasts were adapted to growth in 5 microM cadmium chloride (Cdr) and these cells were 2.5-fold more resistant to cadmium than the parental line. There was no difference in the cellular protein profile between the parental line and lymphoblasts grown for a short period, less than 10 days, in cadmium chloride as measured by [35S]cysteine labelling and SDS-polyacrylamide gel electrophoresis. A basal level of cadmium binding protein was apparent, however, by gel filtration. The Cdr lymphoblasts were found to synthesize a substantial amount of cadmium-binding protein, approximately 25-fold more than the parental line. The cadmium-binding protein has the following properties which are consistent with its being a metallothionein: (1) [35S]Cysteine-labelled protein eluted at a Ve/Vo = 2.1 on a Sephadex G-75 column; (2) the molecular weight was estimated as 11 kDa on 7-17% SDS polyacrylamide gels; (3) the protein was heat-stable; (4) the unlabelled protein bound 109Cd2+.

Cadmium↗
PubMed1983

Mouse hepatic metallothionein-I gene cleavage by micrococcal nuclease is enhanced after induction by cadmium.

Micrococcal nuclease has been shown to preferentially cleave chromatin in the region of genes actively engaged in transcription. We have used this preferential cleavage to show that the metallothionein (MT) gene in adult mouse liver, when induced to produce mRNA by injection of cadmium, becomes more susceptible to nuclease cleavage. However, the MT gene in uninduced liver, and the alphafoetal protein (AFP) gene in both induced and uninduced liver, remain relatively resistant to nuclease cleavage. The AFP gene is not normally expressed in cadmium induced or uninduced liver. Thus, susceptibility of genes to nuclease cleavage appears to rise with increasing transcription of the gene.

Animals↗
PubMed1983

Analysis of the temperature-dependent temporal pattern of heat-shock-protein synthesis in fish cells.

Continuous exposure of Chinook salmon embryo cells to an elevated incubation temperature of 24 degrees C induces the transient expression of a set of heat-shock or stress proteins whereas maintenance of the cells at a higher incubation temperature of 28 degrees C produces a continuous synthesis of these stress proteins. In vitro translation studies suggest that the temperature-dependent temporal pattern of stress-protein synthesis is correlated with the levels of stress-protein mRNA. This was verified using a recombinant-DNA probe complementary to the 70K heat-shock-protein mRNA. A transient increase in the level of the fish heat-shock 70K mRNA was observed in RNA samples isolated from cells continuously exposed at 24 degrees C. However, a constant increase in the level of this specific mRNA was found in RNA preparations obtained from cells maintained at 28 degrees C. Therefore, the temperature-dependent pattern of fish heat-shock-protein synthesis appears to be directly related to the level of heat-shock-protein mRNA.

Animals↗
PubMed1982

Expression of a set of fish genes following heat or metal ion exposure.

Elevation of the incubation temperature of Chinook salmon embryo cells from 20 to 24 degrees C or exposure to heavy metals such as CdCl2 (5 microM) or ZnCl2 (100 to 500 microM) induces the reversible expression of a set of heat shock or stress proteins. Continuous exposure of the cells to either metal ions or heat shock results in recovery of protein synthesis to a control-like pattern. Treatment of these cells with either ZnCl2 or CdCl2 also induces the protein metallothionein. Heat shock, however, does not induce metallothionein, suggesting that it does not belong to the common group of heat shock or stress proteins. The induction of these stress proteins can be inhibited by pretreatment with actinomycin D, suggesting that their expression is regulated at the transcriptional level. The major stress proteins are detectable in the products of an in vitro translation system programmed with RNA isolated from heat shock- or metal ion-treated cells. A recombinant DNA probe complementary to Drosophila mRNA coding for the 70,000-dalton heat shock protein was found to hybridize to RNA isolated from heat shock-or metal ion-treated cells but not from control cells. The fish mRNA coding for the heat shock protein with a molecular weight of 70,000 appears to be of similar size to the corresponding Drosophila mRNA.

Animals↗
PubMed1982

Nucleotide sequence of a protamine component CII gene of Salmo gairdnerii.

We have isolated, using nick-translated cloned protamine cDNA's as probes, several genomic clones containing protamine gene sequences from a Charon 4A library of Eco R1 digested rainbow trout (Salmo gairdnerii) DNA. One clone was chosen for detailed study and the 2.5 kbp Bam HI-Eco R1 restriction fragment containing the gene was subcloned in the plasmid pBR322. A 920 bp Bg1 II - Bam HI restriction fragment contains a sequence coding for protamine component CII as well as regions 5' and 3' to the mRNA coding portion. Present in the region 5' to the mRNA coding sequence are the promoter associated signals "TATA" box and "CAAT" box. The 5' untranslated region of the mRNA whose length and sequence were not established from the cDNA clones (1) was determined by nuclease mapping and starts within a sequence similar to the "capping signal" found in other genes. The protamine gene for CII contains no introns, a situation common to most histone genes, but, unlike the histone genes does not occur close to other protamine genes in a "cluster".

Animals↗
PubMed1981

Molecular analysis of the protamine multi-gene family in rainbow trout testis.

We have synthesized a family of double-stranded cDNAs (ds cDNAs) using as a template the family of highly purified protamine mRNAs from rainbow trout testis. Individual pure protamine cDNA components were isolated by cloning this family of protamine ds cDNAs in a plasmid vector (pMB9). Clones containing protamine sequences were characterized by restriction mapping and by a positive hybrid-selected translation assay, which allowed us to correlate particular cDNAs with particular protein components. To allow more detailed comparisons, complete nucleotide sequences were determined for selected protamine clones. We have detected at least 5 distinctly different coding sequences, which nevertheless show at least 82% homology, and which have probably arisen by repeated gene duplication. These very highly conserved coding sequences do however contain a distinctly variable region near the 5'-end of the mRNA (N-terminus of the protein), corresponding to the major sites of serine phosphorylation. Since the amino acid sequences predicted by our DNA sequences were slightly different from those previously published (1), we have independently determined the amino acid sequences of protamine components CI, CII, CIII from our own source of trout testis. These new peptide sequences are completely consistent with those predicted by our nucleotide sequences. The 3'-untranslated regions of the protamine mRNAs are, surprisingly almost as highly conserved as the coding regions. Both coding and 3'-noncoding portions appear to be under a similar degree of selective pressure and evolutionary constraint to remain constant.

Animals↗
PubMed1981

Studies on the heterogeneity of the 5' ends of the protamine mRNAs from rainbow trout testis.

The structures of the 5' termini of the protamine mRNAs (PmRNAs) have been investigated by inhibiting their translation in wheat-germ extracts in the presence of 7-methyl guanosine 5'-phosphate (m7-GMP), an analogue of 'cap' structure in mRNAs. Second, the cap structures on PmRNAs were examined by labelling the RNA at the 5' end with T4 polynucleotide kinase and [gamma-32P]ATP before and after removal of these structures with tobacco acid pyrophosphatase and alkaline phosphatase. The results indicate that cap structures of the PmRNAs are heterogeneous. It appears that the mRNAs coding for protamine components CI and CIII have at least a cap 1 structure while the mRNAs coding for CII do not appear to be capped or methylated.

Animals↗
PubMed1979

Heterogeneity of biologically active deadenylated protamine mRNA components isolated from rainbow trout testes.

Poly(A)+ protamine mRNA's were isolated from rainbow trout testes and deadenylated by treatment with calf thymus RNase H. Four subcomponents of deadenylated PmRNA (PmRNA1-4) were purified by electrophoresis on a 6% polyacrylamide gel in 8 M urea. Translation of each PmRNA subcomponent in the wheat germ S-30 cell-free system showed that all subcomponents are biologically active but each codes for two or more protamine polypeptides suggesting molecular heterogeneity. However, the deadenylated mRNA's can be categorized into two groups based on the spectrum of protamines whose synthesis they stimulate.

Animals↗
PubMed1979

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.

Animals↗
PubMed1979

Protamine messenger RNA: partial purification and characterization of a heterogeneous family of polyadenylated messenger components.

Poly(A)+ protamine mRNA (pmRNA) components were isolated after separation on denaturing preparative polyacrylamide gels. The four size classes of protamine mRNA described previously were found to contain poly(A) tracts of different lengths. The pmRNA1 was found to be associated with (A)110, pmRNA2 with (A)90, pmRNA3 with (A)85, and pmRNA4 with (A)69. Following deadenylation with RNase H after duplex formation with oligo-dT, the isolated mRNAs were found to be still heterogeneous, although highly enriched in certain of the deadenylated components. DNA complementary to the isolated mRNAs (cDNA) was synthesized in vitro. Following depurination, the oligopyrimidine maps indicated that C7T4, corresponding to an Arg-Arg-Gly-Gly sequence in protamine and originally thought to be characteristic of all mRNA components, is present in only one or possibly tow of the components. Cross-hybridizations between the cDNAs and the four poly(A)+ pmRNAs indicated that a basic polynucleotide unit of substantial length is common to all four mRNAs and that the existing nucleotide sequence variations probably originate from one or both of the non-coding portions of the mRNA molecules.

Animals↗