Open AccessExpression of Purine Overproduction in a Series of 8-Azaguanine-Resistant Diploid Human Lymphoblast Lines
Author(s) -
Julia E. Lever,
G Nuki,
J. Edwin Seegmiller
Publication year - 1974
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.71.7.2679
Subject(s) - hypoxanthine guanine phosphoribosyltransferase , hypoxanthine , purine metabolism , hypoxanthine phosphoribosyltransferase , lymphoblast , purine , biochemistry , overproduction , biology , microbiology and biotechnology , enzyme , phosphoribosyltransferase , adenine phosphoribosyltransferase , chemistry , cell culture , gene , genetics , mutant
For study of the basis of an X-linked form of gout in man, several clonal lines deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) were selected from the human lymphoblast line WI-L2 by spontaneous and mutagen-induced resistance to 10 μM 8-azaguanine. Three groups could be defined: (1 ) clones with less than 1% of normal enzyme activity, unable to incorporate [3 H]hypoxanthine detectable by radioautography, unable to tuilize exogenous hypoxanthine as a source of purines, and showing a 2- to 4-fold accelerated rate of production of early intermediates inde novo purine biosynthesis; (2 ) clones with 56-63% of normal enzyme activity, decreased incorporation per cell of [3 H]hypoxanthine measured by radioautography, able to utilize exogenous hypoxanthine, and showing 1.2- to 2.8-fold purine overproduction; (3 ) clones with 10-15% of normal enzyme activity, able to utilize hypoxanthine but not incorporating amounts detectable by radioautography, and showing a 2.3- to 2.5-fold increase in purine biosynthesis. Resistant clones generated by ICR 191 mutagenesis resembled Group 1 clones. Heat inactivation studies in crude extracts from certain clones in Group 2 suggest a structural gene mutation, but no qualitative alteration in enzyme could be detected by starch gel electrophoresis. These phenotypes have persisted over at least 300 generations of nonselective growth, with retention of a diploid karyotype.