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Characterisation of Two Metallothionein cDNAs from the Shore Crab for use as Biomarkers of Heavy Metal Pollution

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Abstract

Molecular biological procedures open up possibilities for the development of new biomarker assays for use in environmental monitoring studies. Metallothionein (MT) is a useful biomarker for monitoring pollution by heavy metals and since very little information is available on the genes for MT in marine invertebrates, studies have been initiated in order to develop probes for use in biomarker assays for MT in the shore crab (Carcinus maenas). RNA isolated from the gills of shore crabs was used to produce complementary DNA (cDNA) from which two incomplete and two complete MT cDNAs have been isolated and characterised. The first complete cDNA (cDNA-4) encodes for a protein of 58 amino acids with a predicted molecular mass of 6151 Da; the predicted amino acid sequence of this protein is identical to that determined earlier for MT-Ib isolated from cadmium-exposed crabs. The second complete cDNA (cDNA-3) encodes a protein of 41 amino acids with a predicted molecular mass of 4484 Da; only the 5 C-terminal residues of this truncated MT differ from those at the corresponding positions of MT-Ib and this may correspond to a 4100 Da MT also reported previously. The implications of these findings and the use of these cDNAs as biomarkers in ecotoxicological studies are discussed.

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References

  • Bremner, I. (1987). Nutritional and physiological significance of metallothionein. Experientia Suppl. 52, 81–107.

    Google Scholar 

  • Brouwer, M., Winge, D.R. and Gray, W.R. (1989). Structural and functional diversity of copper-metallothioneins from the American lobster Homarus americanus. J. Inorg. Biochem. 35, 289–303.

    Google Scholar 

  • Chomczynski, P. and Sacchi, N. (1987). Single step method of RNA isolation by acid guanidimium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–9.

    Google Scholar 

  • Czaja, M.J., Weiner, F.R. and Freedman, J.H. (1991). Amplification of the metallothionein-1 and metallothionein-2 genes in copper resistant hepatoma cells. J. Cell Physiol. 147, 434–8.

    Google Scholar 

  • Dallinger, R., Berger, B., Hunziker, P. and Kagi, J.H.R. (1997). Metallothionein in snail Cd and Cu metabolism. Nature 388, 237–8.

    Google Scholar 

  • Durnam, D.M. and Palmiter, R.D. (1987). Analysis of the detoxification of heavy metal ions by mouse metallothionein. Experientia Suppl. 52, 457–63.

    Google Scholar 

  • Fornwald, J.A., Kuncio, G., Peng, I. and Ordahl, C.P. (1982). The complete nucleotide-sequence of the chick β-actin gene and its evolutionary relationship to the actin gene family. Nucl. Acids Res. 10, 3861–76.

    Google Scholar 

  • Grady, D.L., Moyzis, R.K. and Hilderbrand, C.E. (1987). Molecular and cellular mechanisms of cadmium resistance in cultured cells. Experientia Suppl. 52, 447–56.

    Google Scholar 

  • Hagenbuchle, O., Krikeles, M.S. and Sprague, K.U. (1979). The nucleotide sequence adjacent to poly A in silk fibroin messenger. J. Biol. Chem. 254, 7157–62.

    Google Scholar 

  • Huang, P.C., Morris, S., Dinman, J., Pine, R. and Smith, B. (1987). Role of metallothionein in detoxification and tolerance to transition metals. Experientia Suppl. 52, 439–46.

    Google Scholar 

  • Kagi, J.H.R. (1993). Evolution, structure and chemical activity of class I metallothioneins: an overview. In K.T. Suzuki, N. Imura and M. Kimura (eds) Metallothionein III, Biological Roles and Medical Implications, pp. 29–55. Basel: Birkhauser Verlag.

    Google Scholar 

  • Kagi, J.H.R. and Kojima, Y. (1987). Chemistry and biochemistry of metallothionein. Experientia Suppl. 52, 25–62.

    Google Scholar 

  • Kagi, J.H.R. and Schaffer, A. (1988). Biochemistry of metallothionein. Biochemistry 27, 8509–8514.

    Google Scholar 

  • Kagi, J.H.R. and Vallee, B.J. (1960). Metallothionein: A cadmium-and zinc-containing protein from equine renal cortex. J. Biol. Chem. 235, 3460–5.

    Google Scholar 

  • Kojima, Y. (1991). Definitions and nomenclature of metallothioneins. Meth. Enzymol. 205, 8–10.

    Google Scholar 

  • Lerch, K., Ammer, D. and Olafson, R.W. (1982). Crab metallothionein. Primary structures of metallothioneins 1 and 2. J. Biol. Chem. 257, 2420–6.

    Google Scholar 

  • Linck, B., Klein, J.M., Mangerich, S., Keller, R. and Weidemann, M.W. (1993). Molecular cloning of crustacean red pigment concentrating hormone precursor. Biochem. Biophys. Res. Commun. 195, 807–13.

    Google Scholar 

  • Margoshes, M. and Vallee, B.L. (1957). Cadmium protein from equine kidney cortex. J. Am. Chem. Soc. 79, 4813–4.

    Google Scholar 

  • Maroni, G., Wise, J., Young, J.E. and Otto, E. (1987). Metallothionein gene duplications and metal tolerance in natural populations of Drosophila melanogaster. Genetics 117, 739–44.

    Google Scholar 

  • Mehra, R.K., Thorvaldsen, J.L., Macreadie, I.G. and Winge, D.R. (1992). Cloning system for Candida glabata using elements from the metallothionein-Iia encoding gene that confer autonomous replication. Gene 113, 119–24.

    Google Scholar 

  • Otvos, J.D., Olafson, R.W. and Armitage, I.M. (1982). Structure of an invertebrate metallothionein from Scylla serrata. J. Biol. Chem. 257, 2427–31.

    Google Scholar 

  • Palmiter, R.D. (1987). Molecular biology of metallothionein gene expression. Experientia Suppl. 52, 63–80.

    Google Scholar 

  • Peakall, D.B. (1992). Animal Biomarkers as Pollution Indicators. London: Chapman and Hall.

    Google Scholar 

  • Peakall, D.B. and Shugart, L.R. (1993). Biomarkers: Research and Application in the Assessment of Environmental Health Berlin: Springer Verlag.

    Google Scholar 

  • Pedersen, K.L., Pedersen, S.N., Hojrup, P., Andersen, J.S., Roepstorff, P., Knusden, J. and Depledge, M.H. (1994). Purification and characterization of a calcium-induced metallothionein from the shore crab Carcinus maenas. Biochem. J. 297, 609–14.

    Google Scholar 

  • Proudfoot, N.J. (1982). The end of the message. Nature 282, 516–7.

    Google Scholar 

  • Proudfoot, N.J. and Brownlee, G.G. (1976). 3′ Non-coding region sequences in eukaryotic messenger RNA. Nature 263, 211–4.

    Google Scholar 

  • Ray, S. (1986). Bioaccumulation of cadmium in marine organisms. Experientia Suppl. 50, 65–75.

    Google Scholar 

  • Roesijadi, G. (1993). Response of invertebrate metallothioneins and MT genes to metals and implications for environmental toxicology. In K.T. Suzuki, N. Imura and M. Kimura (eds) Metallothionein III, Biological Roles and Medical Implications, pp. 141–58. Basel: Birkhauser Verlag.

    Google Scholar 

  • Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988). Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–91.

    Google Scholar 

  • Sambrook, J., Fritsh, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (2nd edition). New York: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  • Searle, P.F., Davison, B.L., Stuart, G.W., Wilkie, T.M., Norstedt, G. and Palmiter, R.D. (1984). Regulation, linkage and sequence of mouse metallothionein I and II. Mol. Cell Biol. 4, 1221–30.

    Google Scholar 

  • Thibodeau, J., Desouza, C., Smorawinska, M. and Thirion, J.P. (1992). Selection of mouse cells with amplified metallothionein genes retaining their glucocorticoid inducibility. FEBS Let. 310, 75–8.

    Google Scholar 

  • Tohoyama, H., Shiraishi, E., Amano, S., Inouhe, M., Joho, M. and Murayama, T. (1996). Amplification of a gene for metallothionein by tandem repeat in a strain of cadmium resistant yeast cells. FEMS Micro Let. 136, 269–73.

    Google Scholar 

  • Wahle, E. and Keller, W. (1992). The biochemistry of 3′-end cleavage and polyadenylation of messanger RNA precursors. Ann. Rev. Biochem. 61, 419–40.

    Google Scholar 

  • Walker, C.H. (1998). Biomarker strategies to evaluate the environmental effects of chemicals. Environ. Health Persp. 106, 613–22.

    Google Scholar 

  • Wan, M., Heuchel, R., Hunziker, P.E. and Kagi, J.H.R. (1995). Regulation of metallothionein gene expression in Cd-adapted or Zn-adapted RK-13 cells. Experientia 51, 606–11.

    Google Scholar 

  • Webb, M. (1987). Toxicological significance of metallothionein. Experientia Suppl. 52, 109–34.

    Google Scholar 

  • West, A.K., Hildebrand, C.E., Karin, M. and Richards, R.I. (1990). Human metallothionein genes: Structure of the functional locus at 16q13. Genomics 8, 513–8.

    Google Scholar 

  • Wong, V.W.T. and Rainbow, P.S. (1986). Two metallothioneins in the shore crab Carcinus maenas. Comp. Biochem. Physiol. 83A, 149–56.

    Google Scholar 

  • Yamada, K., Kato, H., Kanda, N., Fujiikuriyama, Y., Utakoji, T. and Itoh, R. (1994). Sequence homology of chinese hamster metallothionein gene-I and gene-II to those of the mouse and rat and their amplification in Cd-resistant cells. Biochim. Biophys. Acta 1219, 581–91.

    Google Scholar 

  • Zakut, R., Shani, M., Givol, D., Neuman, S., Yaffe, D. and Nudel, U. (1982). Nucleotide sequence of the rat skeletal muscle actin gene. Nature 298, 857–9.

    Google Scholar 

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Correspondence to Demetris Savva.

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Savva, D., Li, B. Characterisation of Two Metallothionein cDNAs from the Shore Crab for use as Biomarkers of Heavy Metal Pollution. Ecotoxicology 8, 485–493 (1999). https://doi.org/10.1023/A:1008920206323

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