Wilson, Jonathan Robert (1995) Copper and zinc ligands of pisum sativum and expression of psMT(_A). Doctoral thesis, Durham University.
The gene, PsMT(_A) is highly expressed in the roots of the garden pea, Pisum sativum. The predicted primary structures of homologues of the PsMT(_A) gene from a range of plant species were compared. Common features are amino and carboxyl terminal domains of approximately 20 residues which are rich in cysteine residues many of which are arranged in the cysteine-Xaa-cysteine (Xaa is not a cysteine) motifs characteristic of metallothionein. The greatest degree of sequence conservation between these predicted gene products occurs within the cysteine rich domains. Two principal (most highly represented in the available sample) categories of sequence were identified on the basis of the arrangement of the cysteine residues within the amino termina domain. A secondary structure motif (β-strand) was predicted (using a computer algorithm) to occur in a conserved position in the central domain linking the two cysteine rich domains of these predicted proteins. This feature is not apparent in the structure of metallothioneins from other species. A recombinant GST-PsMT(_A) fusion protein was isolated from crude lysates of Escherichia coli. containing the plasmid pGEX3X with PsMT(_A) coding sequence. When isolated from Escherichia coli cells grown in zinc supplemented media it was demonstrated that zinc was associated with the PsMT(_A) moiety of the fusion protein. In aqueous solution the PsMT(_A) moiety of the fusion protein formed discreet degradation products. The fusion protein was insoluble at concentrations greater than 20 mg ml(^-1) which rendered it unsuitable for a structural study of the putative metal binding site(s) by (^113)Cd NMR. An antibody to the GST- PsMT(_A) fusion protein was characterised and the epitope was found to lie within the GST moiety. Comparison of the arrangement of cysteines in the amino terminal domains with the different domains of mammalian metallothionein suggested that the two principal categories of predicted plant metallothionein- like gene products may have different affinities for zinc. The predicted products of the metallothionein-like gene highly expressed in the leaves of Arabidopsis thali ana (AtMT-t2) and the PsMT(_A) gene are representative of the two principal categories identified by sequence analysis. The AtMT-t2 coding sequence was amplified from an Arabidopsis thaliana leaf cDNA library and cloned into the pGEX3X plasmid to allow expression of the protein as a fusion to GST in Escherichia coli. Zinc was associated with the AtMT-t2 moiety of the fusion protein. The pH of 50 % displacement for the GST-AtMT-t2 fusion protein with respect to zinc was 0.25 pH units lower than that for the GST-PsMT(_A) fusion protein indicating that putative metallothionein-like protein from Arabidopsis thaliana may have a higher affinity for zinc. It is feasible that this difference allows AtMT-t2 to compete with endogenous ligands of zinc more effectively than PsMT(_A). Expression of the AtMT-t2 gene in a zinc metallothionein deficient strain of Synechococcus (in collaboration with Dr. J.S. Turner) partially restored zinc tolerance to the transformed cells. The similarity of the cysteine rich domains of the predicted metallothionein-like proteins with metallothionein and the demonstration of metal binding in vitro suggested that these genes may be metal regulated. The effect of variations in the exogenous concentration of the trace metals copper zinc and iron on the expression of the PsMT(_A) gene in the roots of Pisum sativum seedlings was investigated by northern analysis. Induction of PsMT(_A) expression was seen with increasing iron concentrations up to 2.0 µM iron chelate. At this concentration of iron chelate, and above, expression of PsMT(_A), decreased. At concentrations of copper above 100 nM induction of PsMT(_A) expression was seen. Below 100 nM copper PsMT(_A) expression increased with decreasing copper concentrations. This response has not been reported for metallothioneins from other species and may be significant for the role of PsMT(_A) in root tissue. In the presence of 2.0 µM iron zinc concentrations above 5.0 µM induced expression of PsMT(_A). The response to changes in exogenous metal concentrations was rapid. Complete repression of PsMT(_A) transcription occurred within 1 h of transfer to media supplemented with 2.0 µM iron. To date no translational products of plant metallothionein-like genes (excluding the E(_c) protein from Tritticum aestivum) have been identified in plant tissue. Two copper and one zinc complex were identified following ion exchange chromatography of soluble extracts from roots of Pisum sativum. The quantity' of the zinc complex eluted from the matrix was reduced by the addition of iron chelate to the growth media. There was no consistent change in the quantity of copper complex recovered in response to iron. Two copper and one zinc component of low molecular weight were identified following gel filtration chromatography of the above complexes. Following polyacrylamide gel electrophoresis of extracts from roots of Pisum sativum, labelled in vivo with (^35)S cysteine, a band was identified with the characteristics predicted for the product of the PsMT(_A) gene. On two dimensional polyacrylamide gels a doublet of spots was identified (migrating to a low pH as predicted for PsMT(_A)) in an extract from roots of seedlings grown in media not supplemented with iron which were not observed on gels of extracts from seedlings grown in media supplemented with iron. The identity of these polypeptides was not established by sequence analysis.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||24 Oct 2012 15:08|