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. Author manuscript; available in PMC: 2014 Jan 30.

Published in final edited form as: Biochemistry (Mosc). 2011 Jan;76(1):131–146. doi: 10.1134/s0006297911010135

Fig. 2 — Mutator effects of deaminases in spot test. a) Mutator effect of expression of lamprey CDA1 in bacteria in spot test. Rosetta or Rosetta ung1⁻ were transformed by control pET24b vector or its derivatives with cloned deaminase genes from lamprey or from Micromonas. IPTG (20 µl of 100 mM solution) was added into the center of LB + kan plate with plated 10⁸ bacteria. The bacteria were allowed to grow overnight and then were replica-plated on selective medium with rifampicin. Visible resistant colonies were scored after additional overnight incubation. b) Mutator effect of the expression of human AID in yeast in spot test. Yeast strain 1B-D770 ung1 was transformed by control pESC-LEU vector or its derivative with recoded human AID gene (“Materials and Methods”). Galactose (20 µl of 20% solution) was added into the center of the complete minimal plate selective for the plasmid marker and containing raffinose with plated ~10⁷ yeast cells. Yeast were allowed to grow for two days and were replica-plated on the three types of selective media to score forward Can^r mutations and reversion of the two auxotrophic markers, ade2-1 and trp1-289. Visible colonies of mutants or revertants were scored after four days of incubation.

Fig. 2 — Mutator effects of deaminases in spot test. a) Mutator effect of expression of lamprey CDA1 in bacteria in spot test. Rosetta or Rosetta ung1⁻ were transformed by control pET24b vector or its derivatives with cloned deaminase genes from lamprey or from Micromonas. IPTG (20 µl of 100 mM solution) was added into the center of LB + kan plate with plated 10⁸ bacteria. The bacteria were allowed to grow overnight and then were replica-plated on selective medium with rifampicin. Visible resistant colonies were scored after additional overnight incubation. b) Mutator effect of the expression of human AID in yeast in spot test. Yeast strain 1B-D770 ung1 was transformed by control pESC-LEU vector or its derivative with recoded human AID gene (“Materials and Methods”). Galactose (20 µl of 20% solution) was added into the center of the complete minimal plate selective for the plasmid marker and containing raffinose with plated ~10⁷ yeast cells. Yeast were allowed to grow for two days and were replica-plated on the three types of selective media to score forward Can^r mutations and reversion of the two auxotrophic markers, ade2-1 and trp1-289. Visible colonies of mutants or revertants were scored after four days of incubation.