Abstract
With the advent of genomics, it is increasingly evident that not all genes in a genome are expressed constitutively. Differential transcription is specific to developmental stages, tissue types and microenvironment of individual cells. Thus a plethora of genes has been shown to be expressed only under stress. Stress varies in forms, among which is the exposure to subtoxic levels of heavy metals. Cadmium is one of the heavy metals, which poses as an environmental insult to organisms through its harmful effects by damaging or changing the fidelity of metalloenzymes. Several cadmium inducible genes have recently been identified in E.coli-K12 cell. In contrast, very few cadmium inducible genes have been uncovered in higher eukaryotic cells other than the metallothioneins. It is possible that the experimental system examined and methods of detection used earlier were insensitive and inadequate for such observations. In this study, a stable cadmium resistance mutant of Chinese hamster ovary cell was used due to its differentially amplified genome, hence the likelihood of extra copies of stress genes. Also the approach of differential display and subtractive hybridization was adopted, taken advantage of their being able to capture transiently expressed and small mRNA molecules. Transient expression is one of the characteristics of stress induced genes. The results show that at least three genes, heretofore unknown to be inducible by cadmium in eukaryotes, can be identified. Two of them show homology with cyclooxygenase-2 (COX-2) and glutamate synthase (GS), which have been shown to involve in cell repair or recovery. The other contains 80 bp in its cDNA. This small gene shows no extensive identity with any known gene sequences. These genes are induced concomitant with metallothionein. Interestingly, the post-transcriptonal splicing processes of metallothionein were observed to be affected by the cadmium exposure also, resulting in the transient presence of splicing intermediates. Since only a few of the cadmium inducible sequences were analyzed in this study, the findings demonstrate that additional genes may be uncovered by the approaches used and that these genes may play a functional role in the cell in its defense to cope with stress. Metallothionein naturally binds seven gram atoms of divalent ions such as Zn and Cd through cysteines forming two metals binding clusters, each constitutes a domain. Four of the metals [M1, M5, M6, M7] are found in alpha, the C-terminal domain, and three [M2, M3, M4] in beta, the N-terminal domain. The level of avidity is site specific. By semiempirical MNDO calculations, we find the relative binding stability for Cd to be M4 > M2 > M3 in the beta-cluster and M5 > M7 > M1,M6 in the alpha-cluster. This is reflected by energy differences computed with a series of simulated structures derived from either X-ray crystallography or NMR coordinates. Thus, replacement of Zn by Cd can be expected to follow the order: M4 -> M2 -> M3 in the beta-domain and M5 -> M7 -> M1 or M6 in the alpha-domain. Bridging cysteines are stronger than terminal cysteines in their relative average binding strength. Among the terminal cysteines, the strongest binding strength is found in Cys 21 to Cd[M4] and in Cys 26 to Zn[M3], both in the beta-domain of metallothionein. Although the binding strength of cysteine in the alpha domain has not yet been determined, we are able to predict from this study differential contribution of individual cysteines to metal binding. Such a prediction can be further tested by experiments with site-directed mutagenesis.