Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.
Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. M2-related DNA sequences are present on mouse chromosomes 4, 7, 12 and 13. However, M2-overproducing mouse cells show amplification of a chromosome 12-specific, single 13 kb HindIII fragment, which probably represents the active gene. We have isolated this fragment from parental mouse cell DNA and used it to clone and characterize the functional M2 gene. The 5770 bp transcribed M2 sequence contains ten exons separated by nine 95-917 bp introns. The 501 bp of 5' flanking DNA is G + C rich and contains TTTAAA and CCAAT sequences as well as potential Sp1 binding sites. The M2-related sequence on chromosome 13, which contains only the last six exons and several internal rearrangements, is a pseudogene. Transfection of BALB/3T3 cells with the M2 gene resulted in stable transformants with a 10-fold reduction in sensitivity to hydroxyurea, compared to control cells. This confirmed that the cloned M2 genomic DNA represents the functional gene and conclusively establishes the link between hydroxyurea resistance and M2 expression in mammalian cells. M2 genomic DNA should be a valuable dominant, selectable marker for identifying and isolating stable co-transformants.
Thelander
M
M
Department of Medical Biochemistry and Biophysics, University of Umeå, Sweden.
Thelander
L
L
D000818:Animals; D001483:Base Sequence; D002232:Carbocyanines / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004351:Drug Resistance / Q000235:genetics; D005091:Exons; D005819:Genetic Markers; D006918:Hydroxyurea / Q000494:pharmacology; D007438:Introns; D008236:Lypressin / Q000378:metabolism / Q000494:pharmacology / Q000031:analogs & derivatives; D051379:Mice; D008969:Molecular Sequence Data; D011544:Pseudogenes / Q000235:genetics; D012235:Rhodamines / Q000378:metabolism / Q000494:pharmacology; D012264:Ribonucleotide Reductases / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
C061832:3,3-ditetradecyloxacarbocyanine; D002232:Carbocyanines; D005819:Genetic Markers; D012235:Rhodamines; C061831:vasopressin, 1-deamino-(8-lysine(N(6)-tetramethylrhodamylaminothiocarbonyl))-; D008236:Lypressin; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea