Activities of in vitro-synthesized pyrazinamidase. The pyrazinamidase activities synthesized in rabbit reticulocyte lysate (A), wheat germ lysate (B), and E. coli S30 lysate (C) were examined as described in Materials and Methods. The white, hatched, and filled bars in panels A and B represented the pyrazinamidase activities obtained from the experiments using ON00-0036, ON00-0046, and ON00-0016 as forward primers, respectively. As shown in panel C, although ON00-0116 (bar 4), ON00-0016 (bar 5), and ON00-0017 (bar 6) were used as forward primers for the time course study, no pyrazinamidase activity was observed. (D) The time course study of the pyrazinamidase activities synthesized in rabbit reticulocyte lysate with ON00-0036 as the forward primer was performed. Templates used for the PCR whose results are shown in panel D were M. tuberculosis H37Rv (PZA susceptible; filled square) and M. bovis BCG (PZA resistant; filled circle). OD450, optical density at 450 nm.