EXPRESSION OF CHICKEN HEPATIC TYPE-I AND TYPE-III IODOTHYRONINE DEIODINASES DURING EMBRYONIC-DEVELOPMENT

In embryonic chicken liver (ECL) two types of iodothyronine deiodinase s 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 ha ve been cloned from mammals and amphibians and shown to contain a sele nocysteine (Sec) residue. We characterized chicken D1 and D3 complemen tary DNAs (cDNAs) and studied the expression of hepatic D1 and D3 mess enger RNAs (mRNAs) during embryonic development. Oligonucleotides base d on two amino acid sequences strongly conserved in the different deio dinases (NFGSCTSecP and YIEEAH) were used for reverse transcription-PC R of poly(A(+)) RNA isolated from embryonic day 17 (E17) chicken liver , resulting in the amplification of two 117-bp DNA fragments. Screenin g of an E17 chicken liver cDNA library with these probes led to the is olation 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 ve rtebrates and that the coding sequence probably lacked the first five nucleotides. An ATG start codon was engineered by site-directed mutage nesis, generating a mutant (ECL1711M) with four additional codons (cod ing for MGTR). The open reading frame of ECL1711M coded for a 249-amin o 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 of a Sec insertion sequence (SECIS) identified in the S'- untranslated region. Enzyme activity expressed in COS-1 cells by trans fection with ECL1711M showed the same catalytic, substrate, and inhibi tor specificities as native chicken D1. The ECL1715 clone was 1366 bp long and also lacked a translation start site. Sequence alignment show ed that it was most homologous with D3 from other species and that the coding sequence lacked approximately the first 46 nucleotides. The de duced aminoacid sequence showed 62-72% identity with the D3 sequences from other species, including a putative Sec residue at a correspondin g position. The 3'-untranslated region of ECL1715 also contained a SEC IS element. These results indicate that ECL1711 and ECL1715 are near-f ull-length cDNA clones for chicken D1 and D3 selenoproteins, respectiv ely. The ontogeny of D1 and D3 expression in chicken liver was studied between E14 and 1 day after hatching (C1). D1 activity showed a gradu al increase from E14 until C1, whereas DI mRNA level remained relative ly constant. D3 activity and mRNA level were highly significantly corr elated, showing an increase from E14 to E17 and a strong decrease ther eafter. These results suggest that the regulation of chicken hepatic D 3 expression during embryonic development occurs predominantly at the pretranslational level.