Characterization of two cDNAs and identification of two proteins that accumulate in response to cadmium in cadmium-tolerant Datura innoxia (Mill.) cells

Abstract

Changes in gene expression have been investigated in two lines of cultured Datura innoxia cells, Cd-300 and WDI, which differ in tolerance to the toxic trace metal Cd2+. Following modification of standard methodology, sufficient quantities of D. innoxia cell extracts were resolved by 2-D PAGE to allow accumulated proteins to be detected. Two Cd-induced proteins, Cd-1 and Cd-2, of approximate Mr 20 000, and pi 4.35 and 4.40, respectively, were detected in extracts from tolerant Cd-300 cells exposed to Cd2+, but were not observed in extracts from non-tolerant WDI cells exposed to equivalent Cd2+ treatments. However, both Cd-1 and Cd-2 were refractory to the determination of an amino acid sequence. A cDNA library was prepared from pooled polyA+ RNA isolated from Cd-300 cells exposed to a variety of Cd2+ treatments, and screened for clones containing sequences which correspond to Cd2+ -induced transcripts. Two cDNAs, designated scaA and scaB, were isolated. Northern analyses confirm that both scaA and scaB transcripts increase in abundance in response to several Cd2+ treatments in Cd-300, but not WDI, cells. Both are also induced in Cd-300 cells in response to heat-shock. ScaA and ScaB are not similar to each other and show no significant similarity to any known proteins although several expressed sequence tags (ESTs) with similarity to scaA have been recorded in the gene banks.