Abstract
A cDNA clone coding for a novel oxidoreductase was cloned from a human bone marrow-derived stromal cell line KM-102. We screened a cDNA library constructed from the mRNA of KM-102 cells stimulated with phorbol 12-myristate 13-acetate and calcium ionophore A23187 using a 32P-labeled 15-mer synthetic oligonucleotide (5'-TAAATAAATAAATAA-3') probe. This probe was designed as a complementary sequence to the three reiterated AUUUA sequences, which are contained in the 3'-untranslated regions of cytokine and some proto-oncogene mRNAs and correlate with rapid mRNA turnover. Then, we obtained one cDNA clone, and further sequence analysis revealed that it coded for a new protein exhibiting 30 to approximately 40% homology with glutathione reductase. By fusion protein analysis, this protein showed reducing activities on 2, 6-dichlorophenol-indophenol and 5,5'-dithio-bis(2-nitrobenzoic acid) but only a weak reducing activity on oxidized glutathione. Although it lacked a stretch of hydrophobic amino acids in its N terminus, it was secreted by monkey kidney-derived COS-1 cells when we introduced the expression plasmid into them and also secreted by a human lung carcinoma cell line A549. Northern blot analysis revealed that the mRNA turnover of this protein was regulated by inflammatory stimuli in KM-102 cells. These results show that this protein may have scavenging enzyme properties and has its mRNA expression regulated in a similar fashion to cytokine genes or proto-oncogenes. Thus, we named it KDRF (KM-102-derived reductase-like factor), and KDRF may play a role in scavenging reactive oxygen intermediates, which are possibly toxic to cells, in response to inflammatory stimuli.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) D88687
We describe the isolation and characterization of a cDNA clone encoding a novel oxidoreductase from a human bone marrow-derived stromal cell line KM-102 [24] using a complementary probe to the AUUUA sequences
Isolation of a cDNA Clone—An Okayama-Berg expression library constructed from poly(A)ϩ RNA of KM-102 cells, which had been stimulated with phorbol 12-myristate 13-acetate (PMA) (10 ng/ml) ϩ A23187 (0.2 M) for 3, 6, or 14 h, was screened with a synthetic oligonucleotide probe (5Ј-TAAATAAATAAATAA-3Ј)
Summary
Preparation of Poly(A)ϩ RNA from KM-102 Cells—Human stromal cell line KM-102 was a kind gift from Drs K. A cDNA library was prepared from 3.3 g of the poly(A)ϩ RNA using a gt system (Amersham Corp.). The blot was hybridized with the 32P-labeled cDNA fragment prepared from the plasmid pcD-31 (a 292-bp PstI-StuI fragment) or pUCKM31-7 (a 1006-bp SmaI-XbaI fragment; SmaI and XbaI, Takara Shuzo Co.), and the hybridization was performed overnight at 42 °C in a solution of 50% formamide, 5 ϫ SSPE (1 ϫ SSPE ϭ 150 mM NaCl, 10 mM sodium dihydrogenphosphate monohydrate, and 1 mM EDTA, pH 7.4), 5 ϫ Denhardt’s solution (1 ϫ Denhardt’s ϭ 0.2 g/liter bovine serum albumin (Sigma), 0.2 g/liter polyvinylpyrrolidone (Sigma), and 0.2 g/liter Ficoll (Sigma)), 2% SDS, and 100 g/ml of denatured salmon sperm DNA (Sigma). E. coli DH5␣ (Life Technologies) was transformed with this DNA, and the strain in which the direction of the cDNA transcription was identical to the direction of the SR␣ promoter
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