Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase.
Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D005796:Genes; D046911:Macromolecular Substances; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology; D051379:Mice; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D012264:Ribonucleotide Reductases; D004262:DNA Restriction Enzymes