A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.
A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.
The macro-alga Fucus vesiculosus has a broad global and estuarine distribution and exhibits exceptional resistance to toxic metals, the molecular basis of which is poorly understood. To address this issue a cDNA library was constructed from an environmental isolate of F. vesiculosus growing in an area with chronic copper pollution. Characterisation of this library led to the identification of a cDNA encoding a protein known to be synthesised in response to toxicity, a full length 14-3-3 exhibiting a 71% identity to human/mouse epsilon isoform, 70-71% identity to yeast BMH1/2 and 95 and 71% identity to the Ectocarpus siliculosus 14-3-3 isoforms 1 and 2 respectively. Preliminary characterisation of the expression profile of the 14-3-3 indicated concentration- and time-dependent inductions on acute exposure of F. vesiculosus of copper (3-30 μg/l). Higher concentrations of copper (≥150 μg/l) did not elicit significant induction of the 14-3-3 gene compared with the control even though levels of both intracellular copper and the expression of a cytosolic metal chaperone, metallothionein, continued to rise. Analysis of gene expression within environmental isolates demonstrated up-regulation of the 14-3-3 gene associated with the known copper pollution gradient. Here we report for the first time, identification of a gene encoding a putative 14-3-3 protein in a multicellular alga and provide preliminary evidence to link the induction of this 14-3-3 gene to copper exposure in this alga. Interestingly, the threshold exposure profile may be associated with a decrease in the organism's ability to control copper influx so that it perceives copper as a toxic response.
Certain 5-(arylimino)-3,4-tetramethylene-1,3,4-thiadiazolidin-2-ones (thiadiazolidines) are peroxidizing bleaching herbicides which interrupt chlorophyll biosynthesis, inhibit the activity of protoporphyrinogen oxidase, lead to accumulation of protoporphyrin IX, and induce ethane formation in the light. The same effects are caused by their isomers, the 4-aryl-1,2-tetramethylene-l,2,4-triazolidin-3-one-5-thiones (triazolidines). Couples of thiadiazolidines and corresponding triazolidine isomers were synthesized. Thiadiazolidines with a 4-bromophenylimino, 4-chlorophenylimino, 4-chloro-2-methylphenylimino, 4-chloro- 2-fluorophenylimino, 4-chloro-2-fluoro-5-propargyloxyphenylimino and 4-chloro-2-fluoro- 5-isopropoxyphenylimino moiety were converted to the corresponding triazolidines both with Echinochloa seedlings or a spinach homogenate present, depending on the 5-arylimino moiety. The 5-[4-(chlorobenzyloxy)phenylimino]-3,4-tetramethylene-1,3,4-thiadiazolidin-2- one analogue did not convert to the corresponding triazolidine under both conditions. Thiadiazolidines as well as triazolidines having a 4-chloro-2-fluoro-5-methoxycarbonylmethylthiophenyl moiety were converted to an unidentified compound whose structure is assumed to be 4-(4-chloro-2-fluoro-5-carboxymethylthiophenyl)-1,2-tetramethylene-1,2,4- triazolidin-3-one-5-thione. Apparently, the general conversion mechanism is caused by enzymatic hydrolysis of thiadiazolidines to an unstable intermediate which rapidly and spontaneously changes to the corresponding triazolidine isomer.
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