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Analysis of mutation in the rat Pig‐a assay: I) studies with bone marrow erythroid cells
Author(s) -
Revollo Javier R.,
Pearce Mason G.,
Dad Azra,
Petibone Dayton M.,
Robison Timothy W.,
Roberts Daniel,
Dobrovolsky Vasily N.
Publication year - 2018
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.22211
Subject(s) - microbiology and biotechnology , cd59 , biology , mutant , mutagen , bone marrow , mutation , transversion , gene mutation , flow cytometry , gene , antibody , biochemistry , dna , genetics , immunology , complement system
We have established a flow cytometry‐based Pig‐a assay for rat bone marrow erythroid cells (BMEs). The BME Pig‐a assay uses a DNA‐specific stain and two antibodies: one against the transmembrane transferrin receptor (CD71 marker) and the other against the GPI‐anchored complement inhibitory protein (CD59 marker). In F344 male rats treated acutely with a total of 120 mg/kg of N ‐ethyl‐ N ‐nitrosourea (ENU) the frequency of CD59‐deficient phenotypically mutant BMEs increased approximately 24‐fold compared to the rats concurrently treated with the vehicle. Such an increase of mutant BMEs coincides with increases of CD59‐deficient reticulocytes measured in rats treated with similar doses of ENU. Sequence analysis of the endogenous X‐linked Pig‐a gene of CD59‐deficient BMEs revealed that they are Pig‐a mutants. The spectrum of ENU‐induced Pig‐a mutations in these BMEs was consistent with the in vivo mutagenic signature of ENU: 73% of mutations occurred at A:T basepairs, with the mutated T on the nontranscribed strand of the gene. T→A transversion was the most frequent mutation followed by T→C transition; no deletion or insertion mutations were present in the spectrum. Since BMEs are precursors of peripheral red blood cells, our findings suggest that CD59‐deficient erythrocytes measured in the flow cytometric erythrocyte Pig‐a assay develop from BMEs containing mutations in the Pig‐a gene. Thus, the erythrocyte Pig‐a assay detects mutation in the Pig‐a gene. Environ. Mol. Mutagen. 59:722–732, 2018. © 2018 Wiley Periodicals, Inc.