Abstract
The mRNA of the APRT gene appears to be constitutively expressed and, thus, enzyme levels will be directly affected by mutations which alter translational efficiency. Accordingly, we have generated mutants altering the ATG start codon by in vitro mutagenesis of the CHO apt gene (the 2.8 kb bam Hl-XbaI fragment) on an M13 vector. Mutagenesis was performed by the use of dut−ung− E. coli strains as described by Kunkel, et. al. (Methods in Enzymology, 1987). Mutant genes are transferred to an Epstein-Barr virus derived plasraid (p220.2). These plasmids grow episomally in human cell lines. We have also generated APRT- mutants in the HL-60 myelocytic leukemic cell line, which will be used as recipients for the plasmid. In addition to increasing our knowledge of translational efficiency of non-cannonical start codons in eucaryotes, such a system can be used as a mutagen test system for potential mutagens and carcinogens, and also to analyze functional regions of the APRT protein.
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Hershey, H., Sahota, A. & Taylor, M. 159 IN VITRO MUTAGENESIS OF THE CHO ADENINE PHOSPHORIBOSYL TRANSFERASE GENES. Pediatr Res 24, 137 (1988). https://doi.org/10.1203/00006450-198807000-00183
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DOI: https://doi.org/10.1203/00006450-198807000-00183