Urwin, Peter Edward (1992) Cd-regulated gene expression in Datura innoxia. Doctoral thesis, Durham University.
The effects of Cd on the expression of specific proteins and transcripts have been examined in Cd-resistant (Cd300) and Cd-sensitive cultures of Datura innoxia. 2D PAGE analysis of the soluble fraction of D. innoxia protein proved problematic. Proteins precipitated on the surface of lEF gels. This was overcome by loading the samples in the gel mixture prior to polymerisation. Polymerisation of gels containing protein extracted from Cd-exposed cells only occurred when PCs, Cd and other components were resolved from the proteins eluting in the void volume following fractionation by gel filtration chromatography (Sephadex G50). Two peptides, designated Cd-1 and Cd-2, were detected in the Cd300 cells only after exposure to Cd. Cd-1 and Cd-2 were also both detected following exposure of the Cd300 cells to 125 piM Cu or Zn, or HS (42 ˚C 4 h). Neither Cd-1 nor Cd-2 were observed in protein extracts from WDI cells exposed to 125 μM Cd for 8 h. Both Cd-1 and Cd-2 proved refractory to Edman degradation while the N-terminal 30 amino acids of a third, constitutively expressed protein, designated Protein-3, were determined using equivalent procedures. This protein showed sequence similarity to PR proteins. Although cleavage of Cd-1 and Cd-2 generated polypeptides which were not terminally blocked, no sequence information was obtained from these polypeptides, even following purification using standard techniques. Oligonucleotide primers designed from the amino acid sequence of Protein-3 were successfully used to amplify, from cDNA, a fragment which was cloned, sequenced and shown to encode the characterised protein. A longer fragment was also amplified from cDNA by RACE PGR. However, this product was not cloned. In order to identify cDNA sequences encoding Cd-1 and Cd-2 an expression cDNA library was prepared and antibodies raised against the two peptides. However, no antigenicity could be detected when antisera raised against Cd-1 or Cd-2, or the purified IgG fractions, were used to probe western blots. The XZAP cDNA (Cd-exposed) library was "differentially screened" in order to isolate clones corresponding to Cd-induced genes. This led to the isolation of two Cd- induced clones designated Cd-6 (949 bp) and Cd-8 (659 bp). Both clones hybridised to transcripts of approximately 900 bp. Transcripts were also detected in RNA samples extracted from D. innoxia exposed to HS (42 ˚C 4 h), however no transcripts were detected in WDI cells exposed to Cd. Southern blots revealed hybridisation to multiple bands, possibly indicating the presence of a gene family. A motif, C-C-X-C-C, found in the a-domain of metallothioneins, was identified in Cd-6. This may represent a putative metal binding site in Cd-6.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||16 Nov 2012 10:55|