Endogenous HPRT activity in a cryptic strain of mycoplasma and its effect on cellular resistance to selective media in infected cell lines

Abstract

A cryptic strain of mycoplasma, which contains a high level of endogenous hypoxanthine phosphoribosyltransferase (HPRT) activity, was isolated from a subclone of mouse cell mutant A9 that had been maintained for over 100 cell generations in 8-azaguanine medium. HPRT-deficient cells deliberately infected with the mycoplasma acquire an apparent wild-type level of HPRT when assayed in cell-free extracts, even when cultured in medium with 8-azaguanine or 6-thioguanine. However, both normal and HPRT-deficient cells become extremely sensitive to HAT medium after infection. As a consequence, HPRT-deficient mutants contaminated with this mycoplasma will not yield HAT-resistant colonies when hybridized with HPRT-positive cells. Biochemical analysis suggests that the resistance of the mycoplasma to purine analogues may be due to elevated substrate binding constants of the mycoplasma HPRT, which render it non-functional at the physiological concentrations of the substrates. The mycoplasma-coded HPRT has characteristic electrophoretic mobility, substrate specificity and thermolability, which together distinguish it from animal HPRT allozymes in man, mouse, rat, Chinese hamster, Syrian hamster and chicken. This mycoplasma strain can grow only in association with animal cells, fails to incorporate exogenous thymidine, bands with a density of 1.217 g/ml in sucrose gradients, and passes through 0.22 μm membrane filters. Growth medium of animal cells deliberately infected with the mycoplasma can reach a maximum titer of 10 8 infectious particles per ml within a few days. The mycoplasma was positively identified by scanning electron microscopy and typed as Mycoplasma hyorhinis with a monospecific antiserum.