Molecular cloning by hybrid arrest of translation in Xenopus laevis oocytes. Identification of a cDNA encoding the type I iodothyronine 5'-deiodinase from rat liver.
The enzyme(s) catalyzing the 5'-monodeiodination of thyroxine and 3,3',5'-triiodothyronine has not yet been purified, and antibodies of demonstrated specificity are not available. Thus, molecular cloning strategies which rely on the traditional screening techniques of using cDNA probes or monospecific antibodies are problematic. We previously reported that expression of type I 5'-deiodinase can be induced in Xenopus laevis oocytes by the injection of poly (A)+ RNA prepared from rat liver (St. Germain, D.L., and Morganelli, C.M. (1989) J. Biol. Chem. 264, 3054-3056). Using this expression system, we developed a hybrid arrest assay, and with it identified a 241-base pair cDNA which encodes part of this enzyme. The cDNA inhibits translation of 5'-deiodinase activity in oocytes by greater than 99% and 5'-deiodinase mRNA from rat liver poly(A)+ RNA in hybrid selection experiments. The cDNA hybridizes to a 1.9-kilobase RNA species on Northern analysis and demonstrates no significant homology to any previously cloned protein. The application of this hybrid arrest strategy for molecular cloning may prove useful for the isolation of cDNAs for proteins that are low in abundance, difficult to purify, or are subunits of a polymeric functional unit.
St Germain
D L
DL
Department of Medicine, Dartmouth Medical School, Hanover, New Hampshire 03756.
Dittrich
W
W
Morganelli
C M
CM
Cryns
V
V
D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods; D005260:Female; D015723:Gene Library; D007453:Iodide Peroxidase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009865:Oocytes / Q000201:enzymology*; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014982:Xenopus laevis
D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A; D012313:RNA; D007453:Iodide Peroxidase