RESUMO
The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO.
Assuntos
Clonagem Molecular , DNA/análise , Iodeto Peroxidase/genética , Microssomos/imunologia , Glândula Tireoide/ultraestrutura , Sequência de Aminoácidos , Animais , Sequência de Bases , Humanos , Dados de Sequência Molecular , Peso Molecular , SuínosRESUMO
Five overlapping cDNA clones representing the entire mRNA for human thyroid peroxidase (TPO) have been isolated from a human Graves' thyroid cDNA library. The cDNA sequence has been determined. Human TPO cDNA contains 3060 bases from the start of transcription to the beginning of the poly (A) tail at the 3'-end. The derived amino acid sequence of human TPO consists of 933 amino acids with a mol wt of 102,937. The derived amino acid sequence contains five potential glycosylation sites (Asn-X-Ser/Thr), a probable transmembrane signal peptide sequence at the amino terminus, and a hydrophobic putative membrane-spanning region beginning 85 amino acid residues from the carboxyl terminal end. Comparison of the human TPO amino acid sequence to that of pig TPO shows strong homology extending from the amino terminus to within 44 amino acid residues of the carboxyl-terminus.
Assuntos
Iodeto Peroxidase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Técnicas Genéticas , Humanos , Iodeto Peroxidase/química , Dados de Sequência Molecular , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , SuínosRESUMO
We undertook the molecular cloning of porcine thyroid peroxidase (TPO). Four oligonucleotide probes were synthesized on the basis of amino acid sequences of 3 tryptic peptides from highly purified porcine TPO. These probes were used to screen a pig thyroid cDNA library. Seven of 16 selected clones (0.45-1.15 kb in size) reacted with all 4 probes. Nucleotide sequencing of the 1.15 kb at the 3'-end of the structural gene revealed the complementary sequence to all 4 probes as well as the nucleotides coding for the entire length of the 3 tryptic peptides. There is an open reading frame of 332 amino acid residues. On Northern blot analysis this gene codes for an mRNA species of 2.85 kb, corresponding to the anticipated size of the mRNA for the intact TPO molecule. We have therefore cloned and characterized a cDNA clone coding for approx. 36% of porcine thyroid peroxidase.
Assuntos
Peroxidases/genética , Glândula Tireoide/enzimologia , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Oligodesoxirribonucleotídeos/síntese química , RNA Mensageiro/genética , SuínosRESUMO
We isolated the initial 1.3 kb of the 5'-flanking region of the human thyroid peroxidase (hTPO) gene, and sub-cloned this fragment into the luciferase reporter gene expression plasmid pA3-LUC. This plasmid contruct (p1.3HTPO-LUC) was stably transfected into FRTL5 rat thyroid cells and NIH-3T3 fibroblasts. Promoter activity was detected in 3 of 8 FRTL5 stable cell lines obtained. TSH, dBcAMP and phorbol ester did not alter TPO promoter activity. TPO promoter activity was also expressed in 4 of 5 NIH-3T3 stably-transfected cell lines, and this activity was also not altered by dBcAMP and phorbol ester. These data support the emerging concept that the TPO gene is not transcriptionally regulated by TSH and cAMP.
Assuntos
Iodeto Peroxidase/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Bucladesina/farmacologia , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Ratos , Sequências Reguladoras de Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologia , Glândula Tireoide/fisiologia , Tireotropina/farmacologiaRESUMO
A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.
Assuntos
Clonagem Molecular , DNA/genética , Ferritinas/genética , RNA Mensageiro/genética , Tireoglobulina/genética , Glândula Tireoide/metabolismo , Tireotropina/farmacologia , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Genes/efeitos dos fármacos , Dados de Sequência Molecular , RNA Mensageiro/efeitos dos fármacos , Ratos , Homologia de Sequência do Ácido Nucleico , Glândula Tireoide/efeitos dos fármacosRESUMO
We have isolated and determined the nucleotide sequence of overlapping cDNA clones, representing the entire structural gene for pig thyroid peroxidase. The protein coding region extends from an ATG residue at base 252 to a termination codon at base 3030, coding for a 100.4-kDa apoprotein of 926 amino acids. The derived amino acid composition agrees well with the experimentally determined amino acid composition of purified pig thyroid peroxidase. Five potential glycosylation sites are present in the protein. Potential membrane spanning regions are present at the amino-terminal end (1-23) and near the carboxyl-terminal end (845-870) of the protein. These data indicate that pig thyroid peroxidase is synthesized as a single polypeptide that is membrane-bound.