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Regulation of rainbow trout metallothionein genes by heavy metals
Abstracts
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Organic Micro-Pollutants on the Deep-Sea Floor Derived from Sewage Disposal at Dumpsite 106. HIDESHIGE TAKADA,* JOHN W. F A R R I N G T O N ~ & BRUCE W. TRIPP.~ *Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan & ~ Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA. A mass of approximately 8 x 10 6 wet tons of sewage sludge is dumped annually at Deep Water Dump Site 106, 184km off the New Jersey coast. The ultimate fates of the organic micro-pollutants derived from sludge dumped in deep water are unknown. Molecular indicators of sewage sludge (i.e. linear alkylbenzenes (LABs) and coprostanols) and toxic organic compounds (i.e. polycyclic aromatic hydrocarbons) were analyzed in samples from the dump site and a control site. LABs and coprostanols were detected in the sediments from the dump site, which clearly indicated that organic pollutants derived from dumped sludge are accumulated on the deep-sea floor. The isomeric composition of LABs suggests that no significant biodegradation of LABs occurred in water column and sediments. In the sediment core, these contaminants are detected to 6-cm depth. In artificially resuspended particles, high concentrations of these organic micro-pollutants were also detected. Sewage-derived organic pollutants on the deep-sea floor are easily dispersed in benthic ecosystems by physical and/or biological processes.
S E C T I O N 4: M E T A L S A N D M E T A L B I N D I N G P R O T E I N S
Regulation of Rainbow Trout Metallothionein Genes by Heavy Metals. L. GEDAMU. Department of Biological Sciences, The University of Calgary, 2500 University Dr NW, CaLgary, Alberta, T2N 1N4, Canada. In the past several years, our research has focused on the isolation and characterization of rainbow trout metallothionein (MT) cDNAs and the expression of salmonid genes in order to function MTs and elucidate the molecular mechanism(s) involved in the regulation of MT genes by heavymetal ions. We have thus established that the rainbow trout MTs exhibit strong overall resemblance to the mammalian MTs and that they are encoded by a simple gene family. The basal and induced level of expression of these genes is primarily regulated transcriptionally. Basal-level expression varies between different organs, and high levels of MT m R N A have been observed in ovaries and in testis. Extensive variations in the expression of genes during early embryo development and in liver and gonads during the period of sexual maturation are also observed and are closely associated
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
2l5
Organic Micro-Pollutants on the Deep-Sea Floor Derived from Sewage Disposal at Dumpsite 106. HIDESHIGE TAKADA,* JOHN W. F A R R I N G T O N ~ & BRUCE W. TRIPP.~ *Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan & ~ Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA. A mass of approximately 8 x 10 6 wet tons of sewage sludge is dumped annually at Deep Water Dump Site 106, 184km off the New Jersey coast. The ultimate fates of the organic micro-pollutants derived from sludge dumped in deep water are unknown. Molecular indicators of sewage sludge (i.e. linear alkylbenzenes (LABs) and coprostanols) and toxic organic compounds (i.e. polycyclic aromatic hydrocarbons) were analyzed in samples from the dump site and a control site. LABs and coprostanols were detected in the sediments from the dump site, which clearly indicated that organic pollutants derived from dumped sludge are accumulated on the deep-sea floor. The isomeric composition of LABs suggests that no significant biodegradation of LABs occurred in water column and sediments. In the sediment core, these contaminants are detected to 6-cm depth. In artificially resuspended particles, high concentrations of these organic micro-pollutants were also detected. Sewage-derived organic pollutants on the deep-sea floor are easily dispersed in benthic ecosystems by physical and/or biological processes.
S E C T I O N 4: M E T A L S A N D M E T A L B I N D I N G P R O T E I N S
Regulation of Rainbow Trout Metallothionein Genes by Heavy Metals. L. GEDAMU. Department of Biological Sciences, The University of Calgary, 2500 University Dr NW, CaLgary, Alberta, T2N 1N4, Canada. In the past several years, our research has focused on the isolation and characterization of rainbow trout metallothionein (MT) cDNAs and the expression of salmonid genes in order to function MTs and elucidate the molecular mechanism(s) involved in the regulation of MT genes by heavymetal ions. We have thus established that the rainbow trout MTs exhibit strong overall resemblance to the mammalian MTs and that they are encoded by a simple gene family. The basal and induced level of expression of these genes is primarily regulated transcriptionally. Basal-level expression varies between different organs, and high levels of MT m R N A have been observed in ovaries and in testis. Extensive variations in the expression of genes during early embryo development and in liver and gonads during the period of sexual maturation are also observed and are closely associated
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