Developing a genetic system in Deinococcus radiodurans for analyzing mutations.
We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the beta-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif(r) system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif(r) in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.
Kim
Mandy
M
Department of Microbiology, Immunology and Molecular Genetics and the Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Wolff
Erika
E
Huang
Tiffany
T
Garibyan
Lilit
L
Earl
Ashlee M
AM
Battista
John R
JR
Miller
Jeffrey H
JH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D034301:Deinococcus / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009153:Mutagens; D009154:Mutation; D017354:Point Mutation; D017384:Sequence Deletion
D009153:Mutagens; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit