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J Biol Chem, Vol. 275, Issue 8, 5409-5415, February 25, 2000

Mammalian 5'(3')-Deoxyribonucleotidase, cDNA Cloning, and Overexpression of the Enzyme in Escherichia coli and Mammalian Cells*

Chiara RampazzoDagger §, Magnus Johansson, Lisa GallinaroDagger , Paola FerraroDagger , Ulf Hellman||, Anna Karlsson, Peter ReichardDagger §, and Vera BianchiDagger **

From the Dagger  Department of Biology, University of Padova, I-35131 Padova, Italy, the § Department of Biochemistry I, Medical Nobel Institute, Karolinska Institutet, SE-17177 Stockholm, Sweden, the  Division of Clinical Virology F68, Huddinge University Hospital, SE-14186 Huddinge, Sweden, and the || Ludwig Institute for Cancer Research, Box 595, Uppsala University, SE-75142 Uppsala, Sweden

5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.

* This work was supported in part by two short-term fellowships from the European Molecular Biology Organization (to C. R.) and by funds from the Istituto Superiore di Sanitá (AIDS Project), Associazione Italiana per la Ricerca sul Cancro, and the Italian Ministry of Research (60% Projects) (to V. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF078840 and AF154829.

** To whom correspondence should be addressed. Tel.: 39-049-8276282; Fax: 39-049-8276280; E-mail: vbianchi@civ.bio.unipd.it.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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