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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Molecular cloning and expression of a full-length complementary DNA encoding the guinea pig adrenocortical estrogen sulfotransferase.

Complementary DNA for the guinea pig adrenocortical estrogen sulfotransferase (EST) has been cloned and expressed. Oligonucleotides, based on amino acid sequences of the purified 34-kilodalton protein, were synthesized and used to generate a specific probe by polymerase chain reaction for screening a guinea pig adrenal cDNA library. The polymerase chain reaction rapid amplification of cDNA ends procedure was employed to obtain the 3' and 5' cDNA ends, and a full-length cDNA was constructed. The cloned cDNA consists of 1192 base pairs and encodes a protein of 296 amino acids with a calculated molecular mass of 35,161 daltons. A computer search of the protein data banks revealed significant homology with several sulfotransferases: 71% with bovine placental estrogen sulfotransferase, 52% with rat liver phenol sulfotransferase, 35% with rat liver hydroxysteroid sulfotransferase, and 36% with rat liver senescence marker protein 2. The EST cDNA was inserted into the pcDNA I eukaryotic expression vector and transfected into COS-7 cells. The successful expression of EST cDNA in COS-7 cells was ascertained by Western blot analysis using antibody generated against the protein used to obtain the original amino acid sequence. Additionally, the expressed protein was clearly functional. Only after transfection with EST cDNA was there detectable estradiol sulfotransferase activity in COS-7 cell cytosol. The expressed EST had a single pI of 6.4, whereas native guinea pig adrenocortical EST exhibits four primary charge isoforms. The majority of adrenocortical EST activity focuses as a broad bimodal band in the pH range of 6.6-6.2; additionally, three other discrete immunocross-reactive isoforms are present with pIs of 5.5, 5.4, and 5. 2. Antibodies generated against each individual isoform cross-react with all the other isoforms and with the expressed protein. These isoforms were previously reported to be isomers of a pregnenolone-binding protein; however it is now evident that the isoforms and antibodies raised against them are EST specific. Under high stringency hybridization conditions, EST mRNA was only detected in the adrenal gland, where two mRNA species of 1.4 and 1.8 kilobases were evident; when low stringency conditions were used, a faint 1.4-kilobase band was also detected in the liver. Primer extension analysis revealed that the multiple mRNAs do not arise from differential transcription initiation sites, and genomic Southern blot analysis indicated that the multiple mRNAs arise from a single gene.[1]


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