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Molecular cloning of the cDNA of molluscan metallothioneins (MT)
216
Abstracts
with metal regulation. These genes are induced by Cd, Cu, and Zn in trout tissues and cell-lines, and our cumulative results indicate that they are differentially expressed and show cell-type specific expression. The latter is correlated to the DNA-methylation status of the genes. The promoter region of the trout MT gene has been extensively studied. The cis-acting metalregulatory elements (MREs) responsible for MT regulation have been characterized by transfecting fish cell-lines. Further, these elements are shown to function in distant organisms such as humans, mouse, and frog, suggesting conservation of the trans-acting protein factor(s) during evolution. One such factor (MW -- 74 kDa) has been identified and partially characterized and is shown to require Zn in activating the trout MT promoter in an in-vitro transcription system.
Molecular Cloning of the cDNA of Molluscan Metailothioneins (MT). M I C H A E L E. UNGER, *+ THOMAS T. CHEN§ & G. ROESIJADI*§
*University of Maryland, Chesapeake Biological Laboratory, Box 38, Solomons, Maryland, USA; ~ University of Maryland Program in Toxicology, Baltimore, Maryland, USA & §University of Maryland, Center ~)f Marine Biotechnology, Baltimore, Maryland, USA. A study on the response of metallothionein (MT) to metals was initiated at the molecular level in a mollusc, the oyster Crassostrea virginica. The primary structure of MT was determined by amplifying responsible cDNA with the polymerase chain reaction (PCR), molecular cloning of the amplified cDNA, and primer extension offmRNA by using primers derived from the cloned cDNA. Two sequences that encoded MT were identified. These sequences differed from each other only in three bases at the third position of codons and the 3'-noncoding region. They encoded identical proteins that were rich in cysteine and glycine and lacked aromatic residues. Some 21 cysteinyl residues were arranged in nine Cys-X-Cys motifs, five as Cys-Lys-Cys. A single Cys-X-X-Cys motif was also observed. Comparison (FASTA algorithm) of the primary structure of the oyster MTs with that of other species indicated a higher similarity with vertebrate MTs than with that of other invertebrates. The results supported designation of the oyster MTs as Class I MT. Currently, probes derived from these cDNA sequences are being used to develop a quantitative assay for the study of the regulation of MT m R N A levels in the response of oysters to metal exposure.
Cu, Zn, Cd Content in Different Tissues of the Antarctic Scallop Adamussium colbecki(Smith 1902): Role of Metallothionein in the Homeostasis and in the
Abstracts
with metal regulation. These genes are induced by Cd, Cu, and Zn in trout tissues and cell-lines, and our cumulative results indicate that they are differentially expressed and show cell-type specific expression. The latter is correlated to the DNA-methylation status of the genes. The promoter region of the trout MT gene has been extensively studied. The cis-acting metalregulatory elements (MREs) responsible for MT regulation have been characterized by transfecting fish cell-lines. Further, these elements are shown to function in distant organisms such as humans, mouse, and frog, suggesting conservation of the trans-acting protein factor(s) during evolution. One such factor (MW -- 74 kDa) has been identified and partially characterized and is shown to require Zn in activating the trout MT promoter in an in-vitro transcription system.
Molecular Cloning of the cDNA of Molluscan Metailothioneins (MT). M I C H A E L E. UNGER, *+ THOMAS T. CHEN§ & G. ROESIJADI*§
*University of Maryland, Chesapeake Biological Laboratory, Box 38, Solomons, Maryland, USA; ~ University of Maryland Program in Toxicology, Baltimore, Maryland, USA & §University of Maryland, Center ~)f Marine Biotechnology, Baltimore, Maryland, USA. A study on the response of metallothionein (MT) to metals was initiated at the molecular level in a mollusc, the oyster Crassostrea virginica. The primary structure of MT was determined by amplifying responsible cDNA with the polymerase chain reaction (PCR), molecular cloning of the amplified cDNA, and primer extension offmRNA by using primers derived from the cloned cDNA. Two sequences that encoded MT were identified. These sequences differed from each other only in three bases at the third position of codons and the 3'-noncoding region. They encoded identical proteins that were rich in cysteine and glycine and lacked aromatic residues. Some 21 cysteinyl residues were arranged in nine Cys-X-Cys motifs, five as Cys-Lys-Cys. A single Cys-X-X-Cys motif was also observed. Comparison (FASTA algorithm) of the primary structure of the oyster MTs with that of other species indicated a higher similarity with vertebrate MTs than with that of other invertebrates. The results supported designation of the oyster MTs as Class I MT. Currently, probes derived from these cDNA sequences are being used to develop a quantitative assay for the study of the regulation of MT m R N A levels in the response of oysters to metal exposure.
Cu, Zn, Cd Content in Different Tissues of the Antarctic Scallop Adamussium colbecki(Smith 1902): Role of Metallothionein in the Homeostasis and in the