Expression of chicken hepatic type I and type III iodothyronine deiodinases during embryonic development.
In embryonic chicken liver (ECL) two types of iodothyronine deiodinases are expressed: D1 and D3. D1 catalyzes the activation as well as the inactivation of thyroid hormone by outer and inner ring deiodination, respectively. D3 only catalyzes inner ring deiodination. D1 and D3 have been cloned from mammals and amphibians and shown to contain a selenocysteine (Sec) residue. We characterized chicken D1 and D3 complementary DNAs (cDNAs) and studied the expression of hepatic D1 and D3 messenger RNAs (mRNAs) during embryonic development. Oligonucleotides based on two amino acid sequences strongly conserved in the different deiodinases (NFGSCTSecP and YIEEAH) were used for reverse transcription-PCR of poly(A+) RNA isolated from embryonic day 17 (E17) chicken liver, resulting in the amplification of two 117-bp DNA fragments. Screening of an E17 chicken liver cDNA library with these probes led to the isolation of two cDNA clones, ECL1711 and ECL1715. The ECL1711 clone was 1360 bp long and lacked a translation start site. Sequence alignment showed that it shared highest sequence identity with D1s from other vertebrates and that the coding sequence probably lacked the first five nucleotides. An ATG start codon was engineered by site-directed mutagenesis, generating a mutant (ECL1711M) with four additional codons (coding for MGTR). The open reading frame of ECL1711M coded for a 249-amino acid protein showing 58-62% identity with mammalian D1s. An in-frame TGA codon was located at position 127, which is translated as Sec in the presence ofa Sec insertion sequence (SECIS) identified in the 3'-untranslated region. Enzyme activity expressed in COS-1 cells by transfection with ECL1711M showed the same catalytic, substrate, and inhibitor specificities as native chicken D1. The ECL1715 clone was 1366 bp long and also lacked a translation start site. Sequence alignment showed that it was most homologous with D3 from other species and that the coding sequence lacked approximately the first 46 nucleotides. The deduced amino acid sequence showed 62-72% identity with the D3 sequences from other species, including a putative Sec residue at a corresponding position. The 3'-untranslated region of ECL1715 also contained a SECIS element. These results indicate that ECL1711 and ECL1715 are near-full-length cDNA clones for chicken D1 and D3 selenoproteins, respectively. The ontogeny of D1 and D3 expression in chicken liver was studied between E14 and 1 day after hatching (C1). D1 activity showed a gradual increase from E14 until C1, whereas D1 mRNA level remained relatively constant. D3 activity and mRNA level were highly significantly correlated, showing an increase from E14 to E17 and a strong decrease thereafter. These results suggest that the regulation of chicken hepatic D3 expression during embryonic development occurs predominantly at the pretranslational level.
Van der Geyten
S
S
Department of Internal Medicine III, Erasmus University Medical School, Rotterdam, The Netherlands.
Sanders
J P
JP
Kaptein
E
E
Darras
V M
VM
Kühn
E R
ER
Leonard
J L
JL
Visser
T J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002642:Chick Embryo / Q000378:metabolism / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D007453:Iodide Peroxidase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection / Q000235:genetics
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D007453:Iodide Peroxidase