Cloning and sequencing of cDNA for mouse liver metallothionein-I

doi:10.1016/0006-291X(81)90885-8

Biochemical and Biophysical Research Communications

Volume 103, Issue 3, 15 December 1981, Pages 825-832

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Abstract

Metallothionein mRNA was purified from liver of mice injected with cadmium. The corresponding double-stranded cDNA was prepared and inserted into the PstI site of plasmid pBR322. The resulting recombinant DNA was used to transform the RR1 strain of $Escherichia coli$ . Clones resistant to tetracycline and sensitive to ampicillin were screened for the presence of metallothionein-specific restriction fragments in their plasmids. One plasmid, called M135, contains a cDNA insert covering the entire length of the mRNA for mouse liver metallothionein-I, except for the first 18 bases at the 5′ end.

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Characterisation of DNA probes for the analysis of metallothionein gene expression in the bank vole (Clethrionomys glareolus)
2002, Environment International
Citation Excerpt :
In both sequences, the first 20 bp and the last 20 bp correspond to the PCR primers whose design was based on a comparison of the MT-I nucleotide sequences from the mouse, rat and Chinese hamster; however, these sequences are almost certainly accurate since they are totally conserved in the other rodents. The intron–exon junctions were deduced from the comparison of the genomic and the cDNA sequences for the bank vole, as well as from a comparison of these sequences with the MT-I sequences of the mouse (Durnam et al., 1980; Glanville et al., 1981; Mbikay et al., 1981), the Chinese hamster (Yamada et al., 1994) and the rat (Andersen et al., 1983, 1987); this comparison also shows that the last 6 bp of the coding sequence for the bank vole MT-I were not cloned as expected from the design of the PCR primers. It is clear from the results that the coding sequence for the MT-I gene in the bank vole is also split into three exons just like in other vertebrate MT-I genes (Palmiter, 1987).
DNA probes have been developed for subsequent use in monitoring the exposure of animals to heavy metal pollution in terrestrial environments using metallothionein (MT) gene expression in the bank vole (Clethrionomys glareolus). Three different bank vole sequences were characterised corresponding to the cDNA and the genomic DNA for MT-I and the genomic DNA for MT-II. Nucleotide sequence analysis indicates that the coding sequences of the bank vole MT-I and MT-II genes exhibit a very high degree of similarity (greater than 92%) to the corresponding genes of the Chinese hamster, the mouse and the rat. In common with other mammalian MT genes, both the MT-I and MT-II genes in the bank vole are interrupted by two introns, which are at identical positions as those in other rodent MT genes; furthermore, the sizes of these introns are similar to those in other rodents with the first intron being larger than the second and those in the MT-I gene being larger than those in the MT-II gene. The predicted amino acid sequence for the proteins shows that both proteins contain 20 cysteine residues at positions identical to those in other known mammalian MTs. The availability of these DNA sequences now provides a good opportunity to investigate MT gene expression and possible gene amplification in bank voles exposed to metal pollution.
High yield expression and single step purification of human thionein/metallothionein
2001, Protein Expression and Purification
Human metallothionein (MT), isoform 2, was expressed in Escherichia coli as an intein (protein splicing element) fusion protein in the absence of added metals and purified by $i$ ntein- $m$ ediated $p$ urification with an $a$ ffinity $c$ hitin-binding $t$ ag (IMPACT system). This procedure constitutes a novel and simple strategy to prepare thionein (T), the metal-free form, or MT when reconstituting T with metals in vitro. The yield was 8 mg of T or 6 mg of pure Cd₇– or Zn₇–MT from a 1-L culture, significantly higher than yields from any other expression system. Purified recombinant protein is indistinguishable from the native protein on the basis of its metal-binding ability, titration of its sulfhydryls, and UV and CD spectra. The MALDI-TOF mass spectrum is consistent with that of T with a free N-terminus.
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We have expressed in Escherichia coli the 36 kDa domain of the human poly(ADP-ribose) polymerase. This polypeptide comprises the C-terminal part of the DNA binding domain, as well as the automodification region of the enzyme, but lacks the zinc-finger motifs of the N-terminal region and the C-terminal catalytic domain. By probing the crude E. coli protein extracts with radioactive DNA probes (South-Western blots), we have shown that the 36 kDa domain binds a DNA probe of 222 bp but does not bind a shorter probe of 66 bp. This interaction is stronger when the polypeptide is fused to the 55 kDa catalytic domain of the enzyme.
Selection of mouse cells with amplified metallothionein genes retaining their glucocorticoid inducibility
1992, FEBS Letters
Two new mouse cell mutants, resistant to either 80 or 100 mM CdCl₂, were isolated to study the regulation of transcription by the glucocorticoid hormones. Their metallothionein mt-1⁺ and mt-2⁺ genes were amplified coordinately to a maximum of 30 copies per cell. By Southern blot analysis, no gross rearrangement was detectable near the mt⁺ loci. Contrary to other mutants previously isolated, the metallothionein-specific mRNAs of these mutants are inducible by dexamethasone.
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Three gene constructions based on a mouse metallothionein I gene (mMT-I) were introduced into tobacco using an Ri plasmid vector system to test the effectiveness of animal gene regulatory signals in plant cells. No transcription from the native mouse gene was observed. In plant cells bearing chimeric mMT-I genes in which transcription was driven by the nopaline synthase promoter, neither polyadenylation nor splicing of mMT-I pre-mRNA was observed. Detailed comparisons of mMT-I sequences with those of known plant genes were carried out; slight differences in regions of known consensus sequences may be at least partly responsible for the non-recognition of mMT-I gene regulatory signals in plant cells, though other as yet unidentified, potentially necessary sequences may also be involved.
Cloning, nucleotide sequence and molecular evolution of a rabbit processed metallothionein MT-2 pseudogene
1988, Biochemical and Biophysical Research Communications
A rabbit metallothionein-2 pseudogene (MT-2Ψ) has been isolated from a partial rabbit genomic library. Its unusual sequence shows evidence of complex rearrangements involving recombination and deletion events. There are no intervening sequences, 3′ poly A tract or 5′ regulatory DNA sequences. The pseudogene is flanked by two sets of direct repeats (CT)₃ GT(CT)₄ and CTGG(G)CTC. They are most probably the sites of insertion of MT-2Ψ in the rabbit genome. In addition, a number of repetitive DNA sequences are observed flanking the MT-2Ψ gene. These are features of a processed retrogene.

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Cloning and sequencing of cDNA for mouse liver metallothionein-I

Abstract

Toxic. Appl. Pharmacol

Biochem. Biophys. Res. Commun

Exp. Cell. Res

J. Biol. Chem

J. Biol. Chem

Methods Enzymol

J. Biol. Chem

Metallothionein

Nature (London)

Nature (London)

Biochem. J