Sensitive assay for molluscan metallothionein induction based on ribonuclease protection and molecular titration of metallothionein and actin mRNAs.
We developed a sensitive assay for oyster metallothionein (MT) mRNA based on ribonuclease protection and molecular titration. Deletion constructs derived from previously described MT cDNA and newly constructed actin cDNA clones were used to create templates for MT and actin riboprobe synthesis, respectively. MT induction was assessed by normalizing MT expression to that of actin. MT and actin transcripts were quantified in gills of cadmium-exposed (50 micrograms/L-1 cadmium for 2 weeks) and control oysters. Assays for actin and MT were sensitive to 150 and 35 amol mRNA, respectively. Actin was not affected by cadmium exposure and was suitable for use as a normalization factor. For detection of MT mRNA of controls, it was necessary to increase the amount of total input RNA 7.5-fold over that of cadmium-exposed samples due to lower concentrations of target. MT mRNA was induced approximately 15-fold in the cadmium-exposed sample.
Unger
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University of Maryland, Chesapeake Biological Laboratory, Solomons 20688.
Roesijadi
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D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005880:Gills / Q000737:chemistry; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010125:Ostreidae / Q000187:drug effects / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D012260:Ribonucleases / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D000199:Actins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D002104:Cadmium; D008668:Metallothionein; D012260:Ribonucleases