Spontaneous Mutation Frequency Research Articles

Validation of a minimal panel of microsatellite markers for blueberry cultivar identification and frequency of spontaneous mutations

Validation of a minimal panel of microsatellite markers for blueberry cultivar identification and frequency of spontaneous mutations

The H2TH-like motif of the Escherichia coli multifunctional protein KsgA is required for DNA binding involved in DNA repair and the suppression of mutation frequencies

BackgroundDNA oxidatively damaged by reactive oxygen species is repaired by base excision repair (BER) pathway proteins, with DNA glycosylases removing damaged or mismatched bases in the first step of BER. KsgA is a multifunctional protein that exhibits the activities of two enzymes, DNA glycosylase and rRNA dimethyltransferase. The structure-function relationship of the KsgA protein in cellular DNA repair remains unclear because the domains required for KsgA to recognize DNA have not been identified.PurposeTo clarify the mechanisms by which KsgA recognizes damaged DNA and to identify the DNA-binding site, which exists in KsgA.MethodsA structural analysis and in vitro DNA-protein binding assay were performed. The C-terminal function of the KsgA protein was investigated in vitro and in vivo.ResultsThe 3D conformations of KsgA, MutM, and Nei were compared at UCSF Chimera. The root mean square deviation of KsgA (214-273) and MutM (148-212) and that of KsgA (214-273) and Nei (145-212) were 1.067 and 1.188 Å, both less than 2 Å, suggesting that the C terminal of KsgA is spatially similar to the H2TH domains of MutM and Nei. The full-length KsgA protein and KsgA lacking 1-8 or 214-273 amino acids were purified and used in gel mobility shift assays. KsgA exhibited DNA-binding activity, which was lost in the C-terminally deleted KsgA protein. Spontaneous mutation frequency was measured using a mutM mutY ksgA-deficient strain, and the results obtained showed that the mutation frequency was not suppressed by KsgA lacking the C-terminal region, whereas it was in KsgA. To assess dimethyltransferase activity, kasugamycin sensitivity was assessed in wild-type and ksgA-deficient strains. Plasmids carrying the full-length ksgA gene and C-terminal deletion gene were introduced into ksgA-deficient strains. KsgA lacking the C terminus restored dimethyltransferase activity in the ksgA-deficient strain as well as KsgA.ConclusionThe present results confirmed that one enzyme exhibited two activities and revealed that the C-terminal (214-273) amino acids of KsgA were highly similar to the H2TH structural domain, exhibited DNA-binding activity, and inhibited spontaneous mutations. This site is not essential for dimethyltransferase activity.

Ultra-high static magnetic field induces a change in the spectrum but not frequency of DNA spontaneous mutations in Arabidopsis thaliana.

Biological effects of magnetic fields have been extensively studied in plants, microorganisms and animals, and applications of magnetic fields in regulation of plant growth and phytoprotection is a promising field in sustainable agriculture. However, the effect of magnetic fields especially ultra-high static magnetic field (UHSMF) on genomic stability is largely unclear. Here, we investigated the mutagenicity of 24.5, 30.5 and 33.0 T UHSMFs with the gradient of 150, 95 and 0 T/m, respectively, via whole genome sequencing. Our results showed that 1h exposure of Arabidopsis dried seeds to UHSMFs has no significant effect on the average rate of DNA mutations including single nucleotide variations and InDels (insertions and deletions) in comparison with the control, but 33.0 T and 24.5 T treatments lead to a significant change in the rate of nucleotide transitions and InDels longer than 3 bp, respectively, suggesting that both strength and gradient of UHSMF impact molecular spectrum of DNA mutations. We also found that the decreased transition rate in UHSMF groups is correlated with the upstream flanking sequences of G and C mutation sites. Furthermore, the germination rate of seeds exposed to 24.5 T SMF with -150 T/m gradient showed a significant decrease at 24 hours after sowing. Overall, our data lay a basis for precisely assessing the potential risk of UHSMF on DNA stability, and for elucidating molecular mechanism underlying gradient SMF-regulated biological processes in the future.

Common wheat genotypes with null-alleles at gliadin loci

Aim. The study was aimed at identification and selection of winter common wheat genotypes with null-alleles at the gliadin loci Gli-B1 and Gli-D1. Methods. The search for spontaneous mutants with the absence of synthesis of gliadin blocks was made in winter common wheat hybrid material and cultivars. To identify mutations, APAG electrophoresis and SDS-electrophoresis of seed storage proteins were performed. Results. The frequency of spontaneous mutations resulting in the null-allele at the loci Gli-B1 and Gli-D1 in F2 was 0,1 % and 0,05 %, respectively, in the cross Odesska chervonokolosa × B-16. Via sowing the material with those mutations and marker selection, F6 lines were developed: the line OB-Bnull with the null-allele at the Gli-B1 locus and the line OB-Dnull with the null-allele at the Gli-D1 locus. Another line with the null-allele at Gli-B1 was produced from the cross B3 × D4 by marker selection, as well as a biotype with the null-allele at the Gli-D1 locus was selected from the cultivar Slaven. Conclusions. The developed lines with null-alleles at the gliadin locus Gli-B1 (without omega-5 gliadins) and Gli-D1 (without omega-1,2 gliadins) are initial material for hypoallergenic wheat breeding.

A Genetic Toxicology Study of the Rapid Detection of Nitrosamine Compounds by the rpsL Gene Mutation Assay.

In a rpsL gene mutation experiment, the mutagenicity of the nitrosamine compounds N-diethylnitrosamine (NDEA) and N-dipropylnitrosamine (NDPA) was investigated at the cellular level, as well as with PCR (polymerase chain reaction) and RCA (rolling-circle amplification) amplification systems. The experiments were set up with 10 ppm, 100 ppm, and 1000 ppm concentration gradients of NDEA and NDPA, and ethidium bromide (EB) was used as a positive control group. The results demonstrated that the mutagenic frequency of NDEA and NDPA was significantly higher than the spontaneous mutation frequency of the rpsL gene under the same conditions, but lower than the mutagenic rate of EB in the positive control, and there was a dose-effect relationship, indicating that NDEA and NDPA could induce rpsL gene mutation. The rpsL mutation system has a low spontaneous mutation background and high sensitivity, thus the system is expected to become an effective tool for the rapid detection of carcinogens in the field of food.

Functionalized Dioxonaphthoimidazoliums: A Redox Cycling Chemotype with Potent Bactericidal Activities against Mycobacterium tuberculosis.

Disruption of redox homeostasis in mycobacteria causes irreversible stress induction and cell death. Here, we report the dioxonaphthoimidazolium scaffold as a novel redox cycling antituberculosis chemotype with potent bactericidal activity against growing and nutrient-starved phenotypically drug-resistant nongrowing bacteria. Maximal potency was dependent on the activation of the redox cycling quinone by the positively charged scaffold and accessibility to the mycobacterial cell membrane as directed by the lipophilicity and conformational characteristics of the N-substituted side chains. Evidence from microbiological, biochemical, and genetic investigations implicates a redox-driven mode of action that is reliant on the reduction of the quinone by type II NADH dehydrogenase (NDH2) for the generation of bactericidal levels of the reactive oxygen species (ROS). The bactericidal profile of a potent water-soluble analogue 32 revealed good activity against nutrient-starved organisms in the Loebel model of dormancy, low spontaneous resistance mutation frequency, and synergy with isoniazid in the checkerboard assay.

Subinhibitory Levels of Oxytetracycline in Earthworm Meal Significantly Boost Resistance-Mutation Rates in Bacillus spp. within the Gut of Eisenia fetida

Aims: To estimate changes in the number of oxytetracycline -resistant strains, a dynamic population of Bacillus in the gut of Eisenia fetida cultured in processed-cow-dung (PrCD) supplemented with subinhibitory amounts of oxytetracycline, was studied.
 Study Design: Sterile water containing oxytetracycline was sprayed over PrCD at concentration of 0.012 µg per mg dehydrated PrCD per spraying. The number of oxytetracycline-resistant Bacillus spp. microbiota of E. fetida’s gut was compared to the number of oxytetracycline-resistant Bacillus spp. in its feed (PrCD). For this purpose, Luria Agar (LA) and Bacillus Agar (BA) plates amended with or without oxytetracycline were used.
 Place and Duration of Study: The study was carried out in the Omics Laboratory of Department of Biotechnology, University of North Bengal situated in Darjeeling district of West Bengal, India during 2017-18.
 Methodology: E. fetida gut content and PrCD samples were collected on different days of the experiment for bacteriological analysis. Dilution plating on LA yielded the total number of cultivable heterotrophic bacteria. LA plates amended with oxytetracycline (15 µg/ml) were used to acquire the fraction of heterotrophic bacteria resistant to oxytetracycline. BA plates were also utilized to obtain the fraction of resistant Bacillus spp. population. The frequency of mutation was determined using a conventional formula.
 Results: In the gut of E. fetida reared in PrCD supplemented with sub-inhibitory concentration of oxytetracycline, a rise in the oxytetracycline-resistant Bacillus population was observed. On day 1, the frequency of oxytetracycline-resistant Bacillus spp. (5×10-8) matched the spontaneous mutation frequency, however higher frequencies on days 2 and 7 (1.6×10-4 and 3.5 x 10-6 respectively) suggested significant dissemination of oxytetracycline resistance in the gut environment.
 Conclusion: Subinhibitory oxytetracycline concentrations in earthworm diet had a significant effect on mutation rates, showing that evolutionary forces on the gut microbiota may be determining their responses to antibiotic stress.

ATP-Dependent Ligases and AEP Primases Affect the Profile and Frequency of Mutations in Mycobacteria under Oxidative Stress

The mycobacterial nonhomologous end-joining pathway (NHEJ) involved in double-strand break (DSB) repair consists of the multifunctional ATP-dependent ligase LigD and the DNA bridging protein Ku. The other ATP-dependent ligases LigC and AEP-primase PrimC are considered as backup in this process. The engagement of LigD, LigC, and PrimC in the base excision repair (BER) process in mycobacteria has also been postulated. Here, we evaluated the sensitivity of Mycolicibacterium smegmatis mutants defective in the synthesis of Ku, Ku-LigD, and LigC1-LigC2-PrimC, as well as mutants deprived of all these proteins to oxidative and nitrosative stresses, with the most prominent effect observed in mutants defective in the synthesis of Ku protein. Mutants defective in the synthesis of LigD or PrimC/LigC presented a lower frequency of spontaneous mutations than the wild-type strain or the strain defective in the synthesis of Ku protein. As identified by whole-genome sequencing, the most frequent substitutions in all investigated strains were T→G and A→C. Double substitutions, as well as insertions of T or CG, were exclusively identified in the strains carrying functional Ku and LigD proteins. On the other hand, the inactivation of Ku/LigD increased the efficiency of the deletion of G in the mutant strain.

Genetic Variation and Unintended Risk in the Context of Old and New Breeding Techniques

For thousands of years, humans have been improving crops to better suit their needs. These enhancements are driven by changes in the genetic makeup of the plant. While this was initially unintentional, there has been a steady push to increase the pace and precision of crop breeding, something that has occurred alongside a growing understanding of genetics and an escalating capacity to thoroughly assess genomes at the molecular level. With the advent and rapid uptake of molecular breeding techniques, such as transgenics and genome editing over the past few decades, there has been much trepidation regarding the possibility of off-target effects derived from unanticipated mutations at loci other than those intended for alteration, and the unintended risks that this might confer. These concerns persist regardless of the fact that a growing number of studies indicate that the occurrence of off-target mutations derived from newer biotechnological breeding techniques are negligible compared to what is observed with many conventional breeding approaches, and even spontaneously from one generation to the next. Given the impending food security crisis that we are facing in the short-term, there is a critical need to implement a wide range of breeding tools as a means of meeting growing demand, withstanding climate change-related pressures, increasing nutrition, and providing environmental benefits. While food safety is clearly of the utmost importance, now is certainly not the time to prevent the use of particular breeding technologies based on unfounded doubts. Therefore, in this review, we attempt to shed light on these apprehensions by putting purported “risks” into the context of plant breeding as a whole by comparing frequencies of spontaneous mutations with those (both anticipated and unanticipated) that occur through various conventional and biotechnological breeding approaches, including transgenics and genome editing. We then consider how these changes may, or may not, translate into unanticipated risk, and discuss the current global regulatory asynchrony surrounding genome edited crops.

Murepavadin antimicrobial activity against and resistance development in cystic fibrosis Pseudomonas aeruginosa isolates

Murepavadin, a novel peptidomimetic antibiotic, is being developed as an inhalation therapy for treatment of Pseudomonas aeruginosa respiratory infection in people with cystic fibrosis (CF). It blocks the activity of the LptD protein in P. aeruginosa causing outer membrane alterations. To determine the in vitro activity of murepavadin against CF P. aeruginosa isolates and to investigate potential mechanisms of resistance. MIC values were determined by both broth microdilution and agar dilution and results compared. The effect of artificial sputum and lung surfactant on in vitro activity was also measured. Spontaneous mutation frequency was estimated. Bactericidal activity was investigated using time-kill assays. Resistant mutants were studied by WGS. The murepavadin MIC50 was 0.125 versus 4 mg/L and the MIC90 was 2 versus 32 mg/L by broth microdilution and agar dilution, respectively. Essential agreement was >90% when determining in vitro activity with artificial sputum or lung surfactant. It was bactericidal at a concentration of 32 mg/L against 95.4% of the strains within 1-5 h. Murepavadin MICs were 2-9 two-fold dilutions higher for the mutant derivatives (0.5 to >16 mg/L) than for the parental strains. Second-step mutants were obtained for the PAO mutS reference strain with an 8×MIC increase. WGS showed mutations in genes involved in LPS biosynthesis (lpxL1, lpxL2, bamA2, lptD, lpxT and msbA). Murepavadin characteristics, such as its specific activity against P. aeruginosa, its unique mechanism of action and its strong antimicrobial activity, encourage the further clinical evaluation of this drug.

Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3.

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the respiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency.

New Insights Into DNA Repair Revealed by NucS Endonucleases From Hyperthermophilic Archaea.

Hyperthermophilic Archaea (HA) thrive in high temperature environments and their genome is facing severe stability challenge due to the increased DNA damage levels caused by high temperature. Surprisingly, HA display spontaneous mutation frequencies similar to mesophilic microorganisms, thereby indicating that the former must possess more efficient DNA repair systems than the latter to counteract the potentially enhanced mutation rates under the harsher environment. Although a few repair proteins or enzymes from HA have been biochemically and structurally characterized, the molecular mechanisms of DNA repair of HA remain largely unknown. Genomic analyses of HA revealed that they lack MutS/MutL homologues of the mismatch repair (MMR) pathway and the recognition proteins of the nucleotide excision repair (NER) pathway. Endonucleases play an essential role in DNA repair. NucS endonuclease, a novel endonuclease recently identified in some HA and bacteria, has been shown to act on branched, mismatched, and deaminated DNA, suggesting that this endonuclease is a multifunctional enzyme involved in NER, MMR, and deaminated base repair in a non-canonical manner. However, the catalytic mechanism and the physiological function of NucS endonucleases from HA need to be further clarified to determine how they participate in the different DNA repair pathways in cells from HA. In this review, we focus on recent advances in our understanding of the function of NucS endonucleases from HA in NER, MMR, and deaminated DNA repair, and propose directions for future studies of the NucS family of endonucleases.

Genetic breakdown of a Tet-off conditional lethality system for insect population control

Genetically modified conditional lethal strains have been created to improve the control of insect pest populations damaging to human health and agriculture. However, understanding the potential for the genetic breakdown of lethality systems by rare spontaneous mutations, or selection for inherent suppressors, is critical since field release studies are in progress. This knowledge gap was addressed in a Drosophila tetracycline-suppressible embryonic lethality system by analyzing the frequency and structure of primary-site spontaneous mutations and second-site suppressors resulting in heritable survivors from 1.2 million zygotes. Here we report that F1 survivors due to primary-site deletions and indels occur at a 5.8 × 10−6 frequency, while survival due to second-site maternal-effect suppressors occur at a ~10−5 frequency. Survivors due to inherent lethal effector suppressors could result in a resistant field population, and we suggest that this risk may be mitigated by the use of dual redundant, albeit functionally unrelated, lethality systems.

Structural and Functional Characterization of a Unique AP Endonuclease From Deinococcus radiodurans.

Various endogenous and exogenous agents cause DNA damage, including apurinic/apyrimidinic (AP) sites. Due to their cytotoxic effects, AP sites are usually cleaved by AP endonuclease through the base excision repair (BER) pathway. Deinococcus radiodurans, an extraordinary radiation-resistant bacterium, is known as an ideal model organism for elucidating DNA repair processes. Here, we have investigated a unique AP endonuclease (DrXth) from D. radiodurans and found that it possesses AP endonuclease, 3′-phosphodiesterase, 3′-phosphatase, and 3′–5′ exonuclease but has no nucleotide incision repair (NIR) activity. We also found that Mg2+ and Mn2+ were the preferred divalent metals for endonuclease and exonuclease activities, respectively. In addition, DrXth were crystallized and the crystals diffracted to 1.5 Å. Structural and biochemical analyses demonstrated that residue Gly198 is the key residue involved in the substrate DNA binding and cleavage. Deletion of the drxth gene in D. radiodurans caused elevated sensitivity to DNA damage agents and increased spontaneous mutation frequency. Overall, our results indicate that DrXth is an important AP endonuclease involved in BER pathway and functions in conjunction with other DNA repair enzymes to maintain the genome stability.

Improvement of the dP-nucleoside-mediated herpes simplex virus thymidine kinase negative-selection system by manipulating dP metabolism genes

A variety of positive/negative selection systems have been exploited as genome engineering tools and screening platforms for genetic switches. While numerous positive-selection systems are available, only a handful of negative-selection systems are useful for such applications. We previously reported a powerful negative-selection system using herpes simplex virus thymidine kinase (HsvTK) and the mutagenic nucleoside analog 6-(β-d-2-deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido [4,5-c][1,2] oxazin-7-one (dP). Upon addition of 1000nMdP, cells expressing HsvTK quickly die, with unprecedented efficacy. However, this selection procedure elevates the spontaneous mutation rate of the host cells by 10-fold due to the mutagenic nature of dP. To decrease the operative concentration of dP required for negative selection, we systematically created the strains of Escherichia coli either by removing or overexpressing genes involved in DNA/RNA metabolism. We found that over-expression of NupC and NupG (nucleoside uptake-related inner membrane transporters), Tsx (outer membrane transporter), NdK (nucleotide kinase) sensitized E.coli cells to dP. Simultaneous overexpression of these three genes (ndk-nupC-tsx) significantly improved the dP-sensitivity of E.coli, lowering the necessary operative concentration of dP for negative selection by 10-fold. This enabled robust and selective elimination of strains harboring chromosomally-encoded hsvtk simply by adding as low as 100nMdP, which causes only a modest increase in the spontaneous mutation frequency as compared to the cells without hsvtk.

Pharmacological action and clinical effect of tedizolid phosphate (SIVEXTRO® Tablets 200 mg, for iv infusion 200 mg), a novel oxazolidinone-class antibacterial drug

Tedizolid, a novel oxazolidinone antibacterial agent, is a protein synthesis inhibitor that acts on bacterial ribosomes to inhibit initiation of translation. Tedizolid phosphate, a prodrug of tedizolid, is rapidly converted to the active form of tedizolid by phosphatase after administration. Tedizolid has antimicrobial activity mainly against gram-positive pathogens, and generally shows 4-8 times stronger in vitro activity than linezolid, an oxazolidinone antibacterial agent. Tedizolid has antimicrobial activity against Staphylococcus aureus (S. aureus) regardless of being methicillin-resistant or susceptible, with 90% minimum inhibitory concentrations (MIC90) ranging from 0.25-0.5 μg/mL. Although antimicrobial activity of tedizolid against linezolid-resistant S. aureus (LRSA) is generally reduced, tedizolid is still active to LRSA whose linezolid resistance is caused by cfr gene. Structure-activity relationship analysis suggests that the C-5 hydroxymethyl group, the C-ring pyridine, and the D-ring tetrazole group of tedizolid are associated with enhanced antimicrobial activity of tedizolid and its antimicrobial activity against linezolid-resistant bacteria by the cfr gene. Frequency of spontaneous resistance mutation to tedizolid is low, and about 16-fold lower than that to linezolid. Pharmacokinetic/pharmacodynamic (PK/PD) parameter most related to the efficacy of tedizolid is the area under free drug concentration-time curve/minimum inhibitory concentration (fAUC/MIC), and fAUC/MIC value required for bacteriostasis under immunocompetent conditions was calculated to be three. Phase III studies of tedizolid phosphate were conducted in Japan and overseas countries and demonstrated its efficacy and safety in patients with skin and soft tissue infections caused by gram positive organisms including methicillin-resistant S. aureus (MRSA).

Assessment of Phenotypic Variations and Correlation among Seed Composition Traits in Mutagenized Soybean Populations.

Soybean [Glycine max (L.) Merr.] seed is a valuable source of protein and oil worldwide. Traditionally, the natural variations were heavily used in conventional soybean breeding programs to select desired traits. However, traditional plant breeding is encumbered with low frequencies of spontaneous mutations. In mutation breeding, genetic variations from induced mutations provide abundant sources of alterations in important soybean traits; this facilitated the development of soybean germplasm with modified seed composition traits to meet the different needs of end users. In this study, a total of 2366 ‘Forrest’-derived M2 families were developed for both forward and reverse genetic studies. A subset of 881 M3 families was forward genetically screened to measure the contents of protein, oil, carbohydrates, and fatty acids. A total of 14 mutants were identified to have stable seed composition phenotypes observed in both M3 and M4 generations. Correlation analyses have been conducted among ten seed composition traits and compared to a collection of 103 soybean germplasms. Mainly, ethyl methanesulfonate (EMS) mutagenesis had a strong impact on the seed-composition correlation that was observed among the 103 soybean germplasms, which offers multiple benefits for the soybean farmers and industry to breed for desired multiple seed phenotypes.

Yeast Lacking the PP2A Phosphatase Regulatory Subunit Rts1 Sensitizes rad51 Mutants to Specific DNA Damaging Agents.

Rts1 is a regulatory subunit of the trimeric protein phosphatase 2A phosphatase and it participates in many biological processes by modulating the phosphorylation status of proteins. Consistent with its role, mutants lacking Rts1 display multiple phenotypes. We have previously performed a high throughput screen to search for yeast haploid mutants with altered sensitivity to the anticancer drug bleomycin, which acts by damaging the DNA to produce single and double strand breaks. RTS1 was among the genes that when singly deleted cause sensitivity to bleomycin. We investigate whether Rts1 plays a role in the repair of bleomycin-induced DNA lesions. We show that deletion of the RTS1 gene in the rad51 null background, lacking Rad51 known to be involved in the repair of bleomycin-induced DNA lesions, resulted in double mutants that were sensitized to bleomycin and not to other DNA damaging agents that creates DNA adducts. We further show that Rts1 has the ability to bind to DNA and in its absence cells displayed an increase in the frequency of both spontaneous and bleomycin-induced mutations compared to the parent. This is the first report implicating Rts1 with a role in DNA damage and repair, perhaps regulating the phosphorylation status of one or more proteins involved in the repair of DNA strand breaks.

N 4-Cytosine DNA Methylation Is Involved in the Maintenance of Genomic Stability in Deinococcus radiodurans.

DNA methylation serves as a vital component of restriction-modification (R-M) systems in bacteria, where it plays a crucial role in defense against foreign DNA. Recent studies revealed that DNA methylation has a global impact on gene expression. Deinococcus radiodurans, an ideal model organism for studying DNA repair and genomic stability, possesses unparalleled resistance to DNA-damaging agents such as irradiation and strong oxidation. However, details on the methylome of this bacterium remain unclear. Here, we demonstrate that N4-cytosine is the major methylated form (4mC) in D. radiodurans. A novel methylated motif, “C4mCGCGG” was identified that was fully attributed to M.DraR1 methyltransferase. M.DraR1 can specifically bind and methylate the second cytosine at N4 atom of “CCGCGG” motif, preventing its digestion by a cognate restriction endonuclease. Cells deficient in 4mC modification displayed higher spontaneous rifampin mutation frequency and enhanced DNA recombination and transformation efficiency. And genes involved in the maintenance of genomic stability were differentially expressed in conjunction with the loss of M.DraR1. This study provides evidence that N4-cytosine DNA methylation contributes to genomic stability of D. radiodurans and lays the foundation for further research on the mechanisms of epigenetic regulation by R-M systems in bacteria.

Spontaneous mutations in maize pollen are frequent in some lines and arise mainly from retrotranspositions and deletions

While studying spontaneous mutations at the maize bronze (bz) locus, we made the unexpected discovery that specific low-copy number retrotransposons are mobile in the pollen of some maize lines, but not of others. We conducted large-scale genetic experiments to isolate new bz mutations from several Bz stocks and recovered spontaneous stable mutations only in the pollen parent in reciprocal crosses. Most of the new stable bz mutations resulted from either insertions of low-copy number long terminal repeat (LTR) retrotransposons or deletions, the same two classes of mutations that predominated in a collection of spontaneous wx mutations [Wessler S (1997) The Mutants of Maize, pp 385-386]. Similar mutations were recovered at the closely linked sh locus. These events occurred with a frequency of 2-4 × 10-5 in two lines derived from W22 and in 4Co63, but not at all in B73 or Mo17, two inbreds widely represented in Corn Belt hybrids. Surprisingly, the mutagenic LTR retrotransposons differed in the active lines, suggesting differences in the autonomous element make-up of the lines studied. Some active retrotransposons, like Hopscotch, Magellan, and Bs2, a Bs1 variant, were described previously; others, like Foto and Focou in 4Co63, were not. By high-throughput sequencing of retrotransposon junctions, we established that retrotranposition of Hopscotch, Magellan, and Bs2 occurs genome-wide in the pollen of active lines, but not in the female germline or in somatic tissues. We discuss here the implications of these results, which shed light on the source, frequency, and nature of spontaneous mutations in maize.