The gene for a novel protein, a member of the protein disulphide isomerase/form I phosphoinositide-specific phospholipase C family, is amplified in hydroxyurea-resistant cells.
Cell lines selected in multiple steps for increasing resistance to hydroxyurea have been shown to have corresponding increases in ribonucleotide reductase activity. We have isolated a number of cDNA clones from a cDNA library constructed from a highly hydroxyurea-resistant hamster cell line, 600H, in which the activity of ribonucleotide reductase is elevated more than 80-fold. These clones correspond to genomic DNA sequences amplified in the 600H cell line compared with the V79 parental line. One of these cDNA clones, termed P5, codes for a 50 kDa protein detected by in vitro translation of poly(A)+ RNA isolated by hybridization/selection. The cDNA sequence contains a single open reading frame of 1317 nucleotides which encodes a polypeptide of 439 amino acids. The amino acid sequence deduced from the cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence also occur in the active site of rat and human protein disulphide isomerase (also known as the beta-subunit of human prolyl 4-hydroxylase, tri-iodothyronine-binding protein) and in Form I phosphoinositide-specific phospholipase C, indicating that P5 falls into this newly defined superfamily of proteins. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with P5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.
Chaudhuri
M M
MM
Department of Biochemistry and Molecular Biophysics, College of Physicians, Columbia University, New York, NY 10032.
Tonin
P N
PN
Lewis
W H
WH
Srinivasan
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI; D004351:Drug Resistance; D005784:Gene Amplification; D006918:Hydroxyurea / Q000494:pharmacology*; D007535:Isomerases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics*; D014176:Protein Biosynthesis; D019704:Protein Disulfide-Isomerases; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D011506:Proteins; D012333:RNA, Messenger; C073041:P5 protein, Mesocricetus auratus; D004247:DNA; D012264:Ribonucleotide Reductases; D015246:Deoxyribonuclease EcoRI; D010727:Phosphoric Diester Hydrolases; D009955:Ornithine Decarboxylase; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D006918:Hydroxyurea