Skip to main content
Journal cover image

Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli.

Publication ,  Journal Article
Shiota, S; von Wronski, MA; Tano, K; Bigner, DD; Brent, TP; Mitra, S
Published in: Biochemistry
February 25, 1992

A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

February 25, 1992

Volume

31

Issue

7

Start / End Page

1897 / 1903

Location

United States

Related Subject Headings

  • Sequence Homology, Nucleic Acid
  • Restriction Mapping
  • RNA
  • Polymerase Chain Reaction
  • Open Reading Frames
  • O(6)-Methylguanine-DNA Methyltransferase
  • Nucleic Acid Hybridization
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
  • Mice, Inbred BALB C
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Shiota, S., von Wronski, M. A., Tano, K., Bigner, D. D., Brent, T. P., & Mitra, S. (1992). Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli. Biochemistry, 31(7), 1897–1903. https://doi.org/10.1021/bi00122a001
Shiota, S., M. A. von Wronski, K. Tano, D. D. Bigner, T. P. Brent, and S. Mitra. “Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli.Biochemistry 31, no. 7 (February 25, 1992): 1897–1903. https://doi.org/10.1021/bi00122a001.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

February 25, 1992

Volume

31

Issue

7

Start / End Page

1897 / 1903

Location

United States

Related Subject Headings

  • Sequence Homology, Nucleic Acid
  • Restriction Mapping
  • RNA
  • Polymerase Chain Reaction
  • Open Reading Frames
  • O(6)-Methylguanine-DNA Methyltransferase
  • Nucleic Acid Hybridization
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
  • Mice, Inbred BALB C