Under natural conditions, plants are exposed to solar ultraviolet (UV) radiation, which damages chromosomal DNA. Although plant responses to UV-induced DNA damage have recently been elucidated in ...detail, revealing a set of DNA repair mechanisms and translesion synthesis (TLS), limited information is currently available on UV-induced mutations in plants. We previously reported the development of a supF-based system for the detection of a broad spectrum of mutations in the chromosomal DNA of Arabidopsis. In the present study, we used this system to investigate UV-induced mutations in plants. The irradiation of supF-transgenic plants with UV-C (500 and 1000 J/m2) significantly increased mutation frequencies (26- and 45-fold, respectively). G:C to A:T transitions (43–67% of base substitutions) dominated in the mutation spectrum and were distributed throughout single, tandem, and multiple base substitutions. Most of these mutations became undetectable with the subsequent illumination of UV-irradiated plants with white light for photoreactivation (PR). These results indicated that not only G:C to A:T single base substitutions, but also tandem and multiple base substitutions were caused by two major UV-induced photoproducts, cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). In contrast, a high proportion of A:T to T:A transversions (56% of base substitutions) was a characteristic feature of the mutation spectrum obtained from photoreactivated plants. These results define the presence of the characteristic feature of UV-induced mutations, and provide insights into DNA repair mechanisms in plants.
•The supF system was used for the analysis of UV-induced mutations in plants.•The irradiation of supF-transgenic plants with UV-C increased mutation frequencies.•G:C to A:T transitions dominated in the mutation spectrum of base substitutions.•Most of the UV-induced mutations became undetectable by photoreactivation.•High proportion of A:T to T:A is a distinctive feature in photoreactivated plants.
The complete genomic sequence of the archaeon Thermoplasma volcanium , possessing optimum growth temperature (OGT) of 60°C, is reported. By systematically comparing this genomic sequence with the ...other known genomic sequences of archaea, all possessing higher OGT, a number of strong correlations have been identified between characteristics of genomic organization and the OGT. With increasing OGT, in the genomic DNA, frequency of clustering purines and pyrimidines into separate dinucleotides rises (e.g., by often forming AA and TT, whereas avoiding TA and AT). Proteins coded in a genome are divided into two distinct subpopulations possessing isoelectric points in different ranges (i.e., acidic and basic), and with increasing OGT the size of the basic subpopulation becomes larger. At the metabolic level, genes coding for enzymes mediating pathways for synthesizing some coenzymes, such as heme, start missing. These findings provide insights into the design of individual genomic components, as well as principles for coordinating changes in these designs for the adaptation to new environments.
We previously reported the development of a Cre/lox-based gene disruption system for multiple markerless gene disruption in Thermus thermophilus; however, it was a time-consuming method because it ...functioned at 50 °C, the minimum growth temperature of T. thermophilus HB27. In the present study, we improved this system by introducing random mutations into the cre-expressing plasmid, pSH-Cre. One of the resulting mutant plasmids, pSH-CreFM allowed us to remove selection marker genes by Cre-mediated recombination at temperatures up to 70 °C. By using the thermostable Cre/lox system with pSH-CreFM, we successfully constructed two valuable pTT27 megaplasmid mutant strains, a plasmid-free strain and β-galactosidase gene deletion strain, which were produced by different methods. The thermostable Cre/lox system improved the time-consuming nature of the original Cre/lox system, but it was not suitable for multiple markerless gene disruption in T. thermophilus because of its highly efficient induction of Cre-mediated recombination even at 70 °C. However, in vivo megaplasmid manipulations performed at 65 °C were faster and easier than with the original Cre/lox system. Collectively, these results indicate that this system is a powerful tool for engineering T. thermophilus megaplasmids.
•A thermostable Cre/lox-based gene disruption system was developed for Thermus thermophilus.•The thermostable Cre/lox system improved the time-consuming nature of the original system.•Manipulation of the megaplasmid pTT27 in vivo was succeeded in Thermus thermophilus HB27.•We demonstrated a β-galactosidase reporter system using a bgl− strain produced by a large deletion of pTT27.
It remains uncertain why non-genotoxic compounds that result in liver hypertrophy cause liver tumors. In an effort to resolve this issue, we examined whether liver post-mitochondrial fraction (S9) ...prepared from rats treated with non-genotoxic compounds affected the genotoxicity of pro-mutagens. Known hepatotoxic compounds, such as piperonyl butoxide (PBO), decabromodiphenyl ether (DBDE), beta-naphthoflavone (BNF), indole-3-carbinol (I3C) and acetaminophen (AA), were orally administered to male and female F344 rats at doses sufficient to cause liver hypertrophy. Rats received diets containing each test compound for 3 days, 4 weeks or 13 weeks, and were then kept for 4 weeks without the test chemical. S9 prepared from the livers of each group was used for the Ames test with 2-amino-3,8-dimethylimidazo4,5-fquinoxaline (MeIQx), benzoapyrene (BaP) and N-nitrosodimethylamine (NDMA). In both sexes, liver hypertrophy was observed following administration of all test compounds, and was then reversed to the control state when administration ceased. The mutagenicity of MeIQx, BaP and NDMA increased with the use of S9 derived from rats treated with non-genotoxic compounds other than AA. DBDE administration had a marked effect on the mutagenicity of BaP (over a 30-fold increase in females) and NDMA (about a 20-fold increase in males). To estimate the involvement of metabolic enzymes in the alteration of mutagenicity, we measured the activity of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) (phase I enzymes), and UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) (phase II enzymes) in each S9 sample. The activity of phase I enzymes increased, even at the 3rd day following administration, and then decreased gradually, except in the case of AA, while the activity of phase II enzymes increased slightly. These results suggest that non-genotoxic hepato-hypertrophic compounds may be partly involved in carcinogenesis by modulating the metabolism of pre-carcinogens incorporated from the environment, in a manner that is dependent on sex and pre-incorporated chemicals.
Markerless gene-disruption technology is particularly useful for effective genetic analyses of
Thermus thermophilus
(
T. thermophilus
), which have a limited number of selectable markers. In an ...attempt to develop a novel system for the markerless disruption of genes in
T. thermophilus
, we applied a Cre/
lox
system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a
loxP
–
htk
–
loxP
cassette and
cre
-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination. We found that the Cre/
lox
system was compatible with the proliferation of the
T. thermophilus
HB27 strain at the lowest growth temperature (50 °C), and thus succeeded in establishing a triple gene disruptant, the (∆TTC1454::
loxP,
∆TTC1535
Kpn
I::
loxP,
∆TTC1576::
loxP
) strain, without leaving behind a selectable marker. During the process of the sequential disruption of multiple genes, we observed the undesired deletion and inversion of the chromosomal region between multiple
loxP
sites that were induced by Cre-mediated recombination. Therefore, we examined the effects of a
lox66
–
htk
–
lox71
cassette by exploiting the mutant
lox
sites,
lox66
and
lox71
, instead of native
loxP
sites. We successfully constructed a (∆TTC1535::
lox72
, ∆TTC1537::
lox72
) double gene disruptant without inducing the undesired deletion of the 0.7-kbp region between the two directly oriented
lox72
sites created by the Cre-mediated recombination of the
lox66
–
htk
–
lox71
cassette. This is the first demonstration of a Cre/
lox
system being applicable to extreme thermophiles in a genetic manipulation. Our results indicate that this system is a powerful tool for multiple markerless gene disruption in
T. thermophilus
.
Recently, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae. We further ...found that phenyl hydroquinone arrested the cell cycle at G₁ and G₂/M. In this study, we demonstrate that phenyl hydroquinone can arrest the cell cycle at the G₂/M transition as a result of stabilization of Swe1 (a Wee1 homolog), probably leading to inactivation of Cdc28 (a Cdk1/Cdc2 homolog). Furthermore, Hog1 (a p38 MAPK homolog) was robustly phosphorylated by phenyl hydroquinone, which can stabilize Swe1. On the other hand, Chk1 and Rad53 were not phosphorylated by phenyl hydroquinone, indicating that the Mec1/Tel1 DNA-damage checkpoint was not functional. Mutations of swe1 and hog1 abolished phenyl hydroquinone-induced arrest at the G₂/M transition and the cells became resistant to phenyl hydroquinone lethality and aneuploidy development. These data suggest that a phenyl hydroqionone-induced G₂/M transition checkpoint that is activated by the Hog1-Swe1 pathway plays a role in the development of aneuploidy.
We measured the generation of hydroxyl radical (OH â ) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron ...uptake (Fur) using electroparamagnetic resonance
and gas chromatography-mass spectrometry with a selected-ion monitoring method. A specific signal corresponding to OH â generation and an increase in oxidative DNA lesions such as 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine were detected
in the strain deficient in sodA sodB fur . We showed that iron metabolism deregulation in fur mutant produced a 2.5-fold iron overload. The sodA sodB fur strain was about 100-fold higher mutability than the wild-type strain. The mutation spectrum in the strain was found to induce
GC â TA and AT â CG transversions predominantly. The hypermutability of the strain was suppressed by the tonB mutation which reduces iron transport. Thus, excess iron and excess superoxide were responsible for OH â generation, oxidative DNA lesion formation, and hypermutability in E. coli.
Ortho-phenyl phenol (OPP) is broad-spectrum of fungicides and antibacterial agents. OPP tested negative in an Ames system and positive with respect to the formation of tumors in the urinary bladder ...in rats when administered in diet, showing attributes of an Ames test-negative carcinogen. It has also been demonstrated that OPP does not bind or cleave DNA in vivo or in vitro, rather dose-dependent protein binding in OPP-treated rats was observed. OPP, however, generates chromosomal aberrations including aneuploidy. Thus, the steps by which Ames test-negative carcinogens exert their effects need to be elucidated. Here, we used an assay of loss of heterozygosity (LOH) in Saccharomyces cerevisiae to determine the biological effects of OPP and its hepatic metabolite phenyl hydroquinone (PHQ). LOH was found to be induced by OPP and PHQ because of a functional chromosome loss: aneuploidy. PHQ bound to and interfered with the depolymerization of tubulin in vitro and arrested the cell-cycle at M and G1. These results indicate that OPP and PHQ damaged tubulin to cause mis-segregation of chromosome by delaying cell-cycle progression through mitosis, and as a consequence caused aneuploidy.
Thermus thermophilus
(
T
.
thermophilus
) HB27 is an extreme thermophile that grows optimally at 65–72 °C. Heat-induced DNA lesions are expected to occur at a higher frequency in the genome of
T
.
...thermophilus
than in those of mesophiles; however, the mechanisms underlying the maintenance of genome integrity at high temperatures remain poorly understood. The study of mutation spectra has become a powerful approach to understanding the molecular mechanisms responsible for DNA repair and mutagenesis in mesophilic species. Therefore, we developed a
supF
-based system to detect a broad spectrum of mutations in
T
.
thermophilus
. This system was validated by measuring spontaneous mutations in the wild type and a
udgA
,
B
double mutant deficient in uracil-DNA glycosylase (UDG) activity. We found that the mutation frequency of the
udgA
,
B
strain was 4.7-fold higher than that of the wild type and G:C→A:T transitions dominated, which was the most reasonable for the mutator phenotype associated with the loss of UDG function in
T
.
thermophilus
. These results show that this system allowed for the rapid analysis of mutations in
T
.
thermophilus
, and may be useful for studying the molecular mechanisms responsible for DNA repair and mutagenesis in this extreme thermophile.
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