Characterization of cDNAs for Human Estradiol 17β-Dehydrogenase and Assignment of the Gene to Chromosome 17: Evidence of two mRNA Species with Distinct 5′-Termini in Human Placenta

Abstract

Human placental estradiol 17β-dehydrogenase (E₂DH) cDNA clones were isolated from a λgt11 expression library by screening with 33 mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of E₂DH and with polyclonal antibodies raised against the enzyme purified from human placenta. Using ³²P-labeled fragments from the coding and 5′-untranslated regions, two mRNA species have been identified in poly(A)⁺ RNA from human placenta, a major species migrating at 1.3 kilobases (kb) while a minor one is found at 2.2 kb. Primer extension analysis identifies the major mRNA as starting 9–10 nucleotides upstream from the in-frame ATG initiating codon while the longer mRNA has at least 814 noncoding nucleotides at its 5′-terminus. Sequence analysis of the longest cDNA clone (2092 base pairs) shows that this clone possesses identical coding and noncoding sequences in the regions of overlap with the shorter cDNA clones. The ³²P-labeled 5′-noncoding fragment hybridized only to the 2.2 kb band, thus providing additional evidence for the existence of two distinct mRNA species which differ only in their 5′- noncoding regions. Using hpE₂DH36 cDNA as a probe for in situ hybridization, the human E₂DH gene was localized to the q11-q12 region of chromosome 17. The cloned cDNAs encode E₂DH, a 327-amino acid protein having a calculated molecular weight of 34,853. Since E₂DH is the enzyme required for the formation of 17β-estradiol, the availability of the cDNA encoding the enzyme should permit a detailed investigation of the factors regulating the expression and activity of this crucial enzyme in both normal and malignant tissues, especially breast cancer.