Biomaterial Database

Mammalian DNA (cytosine-5-)-methyltransferase expressed in Escherichia coli, purified and characterized. 1995

T O Tollefsbol, and C A Hutchison

Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599, USA.

Besides modulating specific DNA-protein interactions, methylated cytosine, frequently referred to as the fifth base of the genome, also influences DNA structure, recombination, transposition, repair, transcription, imprinting, and mutagenesis. DNA (cytosine-5-)-methyltransferase catalyzes cytosine methylation in eukaryotes. We have cloned and expressed this enzyme in Escherichia coli, purified it to apparent homogeneity, characterized its properties, and we have shown that it hemimethylates DNA. The cDNA for murine maintenance methyltransferase was reconstructed and cloned for direct expression in native form. Immunoblotting revealed a unique protein (M(r) = 190,000) not present in control cells. The mostly soluble overexpressed protein was purified by DEAE, Sephadex, and DNA cellulose chromatography. Peak methylating activity correlated with methyltransferase immunoblots. The purified enzyme preferentially transferred radioactive methyl moieties to hemimethylated DNA in assays and on autoradiograms. All of the examined properties of the purified recombinant DNA methyltransferase are consistent with the enzyme purified from mammalian cells. Further characterization revealed enhanced in vitro methylation of premethylated oligodeoxynucleotides. The cloning of hemimethyltransferase in E. coli should allow facilitated structure-function mutational analysis of this enzyme, studies of its biological effects in prokaryotes, and potential large scale methyltransferase production for crystallography, and it may have broad applications in maintaining the native methylated state of cloned DNA.

UI	MeSH Term	Description	Entries
D008322	Mammals	Warm-blooded vertebrate animals belonging to the class Mammalia, including all that possess hair and suckle their young.	Mammalia,Mammal
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009838	Oligodeoxyribonucleotides	A group of deoxyribonucleotides (up to 12) in which the phosphate residues of each deoxyribonucleotide act as bridges in forming diester linkages between the deoxyribose moieties.	Oligodeoxynucleotide,Oligodeoxyribonucleotide,Oligodeoxynucleotides
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004248	DNA (Cytosine-5-)-Methyltransferases	Enzymes that catalyzes the transfer of a methyl group from S-ADENOSYLMETHIONINE to the 5-position of CYTOSINE residues in DNA.	DNA (Cytosine-5-)-Methyltransferase,DNA Cytosine-5-Methylase,DNA (Cytosine 5) Methyltransferase,Cytosine-5-Methylase, DNA,DNA Cytosine 5 Methylase
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005822	Genetic Vectors	DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition.	Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D015151	Immunoblotting	Immunologic method used for detecting or quantifying immunoreactive substances. The substance is identified by first immobilizing it by blotting onto a membrane and then tagging it with labeled antibodies.	Dot Immunoblotting,Electroimmunoblotting,Immunoelectroblotting,Reverse Immunoblotting,Immunoblotting, Dot,Immunoblotting, Reverse,Dot Immunoblottings,Electroimmunoblottings,Immunoblottings,Immunoblottings, Dot,Immunoblottings, Reverse,Immunoelectroblottings,Reverse Immunoblottings