Adenylate deaminase deficiency in a mutant murine T cell lymphoma cell line.
From a population of wild type S49 cells, a clone, DTB6, was isolated in a single step from selective medium containing thymidine and dibutyryl cyclic AMP that exhibited a 60% deficiency in AMP deaminase (AMP-D) activity. The AMP-D deficiency conferred to the DTB6 cells a striking susceptibility to killing by low concentrations of either adenine or adenosine, the latter in the presence of an inhibitor of adenosine deaminase activity. This growth supersensitivity of DTB6 cells toward adenine could be ameliorated by the addition of hypoxanthine to the culture medium. Immunoprecipitation of AMP-D from wild type and mutant cells revealed that the DTB6 cell line contained markedly diminished amounts of the AMP-D isozyme which reacts with antisera to the predominant isoform expressed in adult kidney. The quantities of the AMP-D isozyme immunoprecipitated by antisera raised to the predominant isoform prepared from adult heart were equivalent in the two cell lines. Although Northern blot analyses revealed no alterations in mRNA sizes or levels encoded by either of the AMP-D genes, Southern blots of genomic DNA hybridized to a cDNA specific for the ampd2 gene revealed the presence of a new BamHI restriction fragment in the DNA of DTB6 cells. These data suggested that a point mutation has occurred in the ampd2 gene of DTB6 cells which encodes the AMP-D isozyme recognized by the kidney antisera. The DTB6 cells also possessed a virtual complete deficiency in thymidine kinase activity. The two enzyme deficiencies were distinguishable. The ability to isolate single step mutants with two seemingly independent biochemical abnormalities raises the speculation that there may be some link between cellular functions responsible for purine nucleotide and thymidine metabolism.
Hanson
S
S
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201.
McCartan
K
K
Sabina
R L
RL
Holmes
E W
EW
Ullman
B
B
D000659:AMP Deaminase / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D000225:Adenine / Q000378:metabolism; D000818:Animals; D015139:Blotting, Southern; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008223:Lymphoma; D051379:Mice; D009154:Mutation; D009709:Nucleotide Deaminases / Q000172:deficiency*; D011123:Polyploidy; D012265:Ribonucleotides / Q000378:metabolism; D013601:T-Lymphocytes; D013936:Thymidine / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*
D004273:DNA, Neoplasm; D007527:Isoenzymes; D012265:Ribonucleotides; D009709:Nucleotide Deaminases; D000659:AMP Deaminase; D000225:Adenine; D013936:Thymidine