Site-specific unnatural base excision via visible light.

Base excision (BE) is an important yet hard-to-control biological event. Unnatural base pairs are powerful tools to revolutionize biological studies in various areas. In this paper, we report a visible-light-induced method to construct site-specific unnatural BE and show the influence of its regulation on transcription and translation levels.

Radiotherapy is associated with a deletion signature that contributes to poor outcomes in patients with cancer.

Ionizing radiation causes DNA damage and is a mainstay for cancer treatment, but understanding of its genomic impact is limited. We analyzed mutational spectra following radiotherapy in 190 paired primary and recurrent gliomas from the Glioma Longitudinal Analysis Consortium and 3,693 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified radiotherapy-associated significant increases in the burden of small deletions (5-15 bp) and large deletions (20+ bp to chromosome-arm length). Small deletions were characterized by a larger span size, lacking breakpoint microhomology and were genomically more dispersed when compared to pre-existing deletions and deletions in non-irradiated tumors. Mutational signature analysis implicated classical non-homologous end-joining-mediated DNA damage repair and APOBEC mutagenesis following radiotherapy. A high radiation-associated deletion burden was associated with worse clinical outcomes, suggesting that effective repair of radiation-induced DNA damage is detrimental to patient survival. These results may be leveraged to predict sensitivity to radiation therapy in recurrent cancer.

Different mutational function of low- and high-linear energy transfer heavy-ion irradiation demonstrated by whole-genome resequencing of Arabidopsis mutants.

Heavy-ion irradiation is a powerful mutagen that possesses high linear energy transfer (LET). Several studies have indicated that the value of LET affects DNA lesion formation in several ways, including the efficiency and the density of double-stranded break induction along the particle path. We assumed that the mutation type can be altered by selecting an appropriate LET value. Here, we quantitatively demonstrate differences in the mutation type induced by irradiation with two representative ions, Ar ions (LET: 290 keV µm-1 ) and C ions (LET: 30.0 keV µm-1 ), by whole-genome resequencing of the Arabidopsis mutants produced by these irradiations. Ar ions caused chromosomal rearrangements or large deletions (≥100 bp) more frequently than C ions, with 10.2 and 2.3 per mutant genome under Ar- and C-ion irradiation, respectively. Conversely, C ions induced more single-base substitutions and small indels (<100 bp) than Ar ions, with 28.1 and 56.9 per mutant genome under Ar- and C-ion irradiation, respectively. Moreover, the rearrangements induced by Ar-ion irradiation were more complex than those induced by C-ion irradiation, and tended to accompany single base substitutions or small indels located close by. In conjunction with the detection of causative genes through high-throughput sequencing, selective irradiation by beams with different effects will be a powerful tool for forward genetics as well as studies on chromosomal rearrangements.

[Deletions in Mitochondrial DNA from the Peripheral Blood of Mayak PA Workers Exposed to Long-Term Ionizing Radiation].

The number of large deletions of mitochondrial DNA in whole peripheral blood of the former Mayak PA workers occupationally exposed to prolonged γ-radiation has been determined in the long term period after irradiation (mean cumulative dose 135.40 ± 22.03 cGy, age range at the time of blood sampling 67-76 years) and compared with the number of deletions in groups of "young" (19-33 years) and "adult" (66-73 years) individuals who had no contact with radiation sources. Samples of the total DNA from the peripheral blood were obtained from the Radiobiological Human Tissue Repository of the Southern Urals Biophysics Institute (Ozyorsk, Chelyabinsk region) and used for carrying out a long-distance PCR. The analysis of the data showed a statistically significant increase in the number of large deletions in the peripheral blood of "adult" donors of the control group as compared with the control group of "young" donors (51.6 and 14.3%, respec- tively). No statistically significant difference in the number of large deletions in the group of former Mayak PA workers occupationally subjected to prolonged exposure to γ-radiation as compared with the control do- nors of similar age was found (53.6 and 43.8% respectively).

Genome-Wide Deletion Screening with the Array CGH Method in Mouse Offspring Derived from Irradiated Spermatogonia Indicates that Mutagenic Responses are Highly Variable among Genes.

Until the end of the 20th century, mouse germ cell data on induced mutation rates, which were collected using classical genetic methods at preselected specific loci, provided the principal basis for estimates of genetic risks from radiation in humans. The work reported on here is an extension of earlier efforts in this area using molecular methods. It focuses on validating the use of array comparative genomic hybridization (array CGH) methods for identifying radiation-induced copy number variants (CNVs) and specifically for DNA deletions. The emphasis on deletions stems from the view that it constitutes the predominant type of radiation-induced genetic damage, which is relevant for estimating genetic risks in humans. In the current study, deletion mutations were screened in the genomes of F1 mice born to unirradiated or 4 Gy irradiated sires at the spermatogonia stage (100 offspring each). The array CGH analysis was performed using a "2M array" with over 2 million probes with a mean interprobe distance of approximately 1 kb. The results provide evidence of five molecularly-confirmed paternally-derived deletions in the irradiated group (5/100) and one in the controls (1/100). These data support a calculation, which estimates that the mutation rate is 1 × 10-2/Gy per genome for induced deletions; this is much lower than would be expected if one assumes that the specific locus rate of 1 × 10-5/locus per Gy (at 34 loci) is applicable to other genes in the genome. The low observed rate of induced deletions suggests that the effective number of genes/genomic regions at which recoverable deletions could be induced would be only approximately 1,000. This estimate is far lower than expected from the size of the mouse genome (>20,000 genes). Such a discrepancy between observation and expectation can occur if the genome contains numerous genes that are far less sensitive to radiation-induced deletions, if many deletion-bearing offspring are not viable or if the current method is substandard for detecting small deletions.

The role and synergistic effect of the light irradiation and H2O2 in photocatalytic inactivation of Escherichia coli.

Inactivation of Escherichia coli K-12 was conducted by applying a continuous supplying of commercial H2O2 to mimic the H2O2 production in a photocatalytic system, and the contribution of H2O2 in photocatalytic inactivation was investigated using a modified "partition system" and five E. coli mutants. The concentration of exogenous H2O2 required for complete inactivation of bacterial cells was much higher than that produced in-situ in common photocatalytic system, indicating that H2O2 alone plays a minor role in photocatalytic inactivation. However, the concentration of exogenously produced H2O2 required for effective inactivation of E. coli K-12 was much lower when the light irradiation was applied. To further investigate the possible physiological changes, inactivation of E. coli BW25113 (the parental strain), and its corresponding isogenic single-gene deletion mutants with light pretreatment was compared. The results indicate that light irradiation increases the bacterial intracellular Fe(2+) level and favors hydroxyl radical (OH) production via the catalytic reaction of Fe(2+), leading to increase in DNA damage. Moreover, the results indicate that the properties of light source, such as intensity and major emission wavelength, may alter the physiology of bacterial cells and affect the susceptibility to in-situ resultant H2O2 in the photocatalytic inactivation processes, leading to significant influence on the photocatalytic inactivation efficiencies of E. coli K-12.

Is radon emission in caves causing deletions in satellite DNA sequences of cave-dwelling crickets?

The most stable isotope of radon, 222Rn, represents the major source of natural radioactivity in confined environments such as mines, caves and houses. In this study, we explored the possible radon-related effects on the genome of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae) sampled in caves with different concentrations of radon. We analyzed specimens from ten populations belonging to two genetically closely related species, D. geniculata and D. laetitiae, and explored the possible association between the radioactivity dose and the level of genetic polymorphism in a specific family of satellite DNA (pDo500 satDNA). Radon concentration in the analyzed caves ranged from 221 to 26,000 Bq/m3. Specimens coming from caves with the highest radon concentration showed also the highest variability estimates in both species, and the increased sequence heterogeneity at pDo500 satDNA level can be explained as an effect of the mutation pressure induced by radon in cave. We discovered a specific category of nuclear DNA, the highly repetitive satellite DNA, where the effects of the exposure at high levels of radon-related ionizing radiation are detectable, suggesting that the satDNA sequences might be a valuable tool to disclose harmful effects also in other organisms exposed to high levels of radon concentration.

Involvement of a citrus meiotic recombination TTC-repeat motif in the formation of gross deletions generated by ionizing radiation and MULE activation.

BACKGROUND: Transposable-element mediated chromosomal rearrangements require the involvement of two transposons and two double-strand breaks (DSB) located in close proximity. In radiobiology, DSB proximity is also a major factor contributing to rearrangements. However, the whole issue of DSB proximity remains virtually unexplored. RESULTS: Based on DNA sequencing analysis we show that the genomes of 2 derived mutations, Arrufatina (sport) and Nero (irradiation), share a similar 2 Mb deletion of chromosome 3. A 7 kb Mutator-like element found in Clemenules was present in Arrufatina in inverted orientation flanking the 5' end of the deletion. The Arrufatina Mule displayed "dissimilar" 9-bp target site duplications separated by 2 Mb. Fine-scale single nucleotide variant analyses of the deleted fragments identified a TTC-repeat sequence motif located in the center of the deletion responsible of a meiotic crossover detected in the citrus reference genome. CONCLUSIONS: Taken together, this information is compatible with the proposal that in both mutants, the TTC-repeat motif formed a triplex DNA structure generating a loop that brought in close proximity the originally distinct reactive ends. In Arrufatina, the loop brought the Mule ends nearby the 2 distinct insertion target sites and the inverted insertion of the transposable element between these target sites provoked the release of the in-between fragment. This proposal requires the involvement of a unique transposon and sheds light on the unresolved question of how two distinct sites become located in close proximity. These observations confer a crucial role to the TTC-repeats in fundamental plant processes as meiotic recombination and chromosomal rearrangements.

Mitochondrial DNA copy number - but not a mitochondrial tandem CC to TT transition - is increased in sun-exposed skin.

Mitochondrial DNA (mtDNA) mutations are causatively associated with photo-ageing and are used as biomarkers of UV exposure. The most prominent mitochondrial mutation is the common deletion (CD), which is induced in many tissues by oxidative stress. More photo-specific mutations might be CC to TT tandem transitions which arise from UV-induced cyclobutane pyrimidine dimers. As nucleotide excision repair is absent in mitochondria, this DNA damage can presumably not be repaired resulting in high mitochondrial mutation levels. Here, we analysed levels of the CD, a mitochondrial and a chromosomal tandem transition in epidermis and dermis from exposed and less UV-exposed skin. We also analysed mtDNA copy number, for which changes as a result of oxidative stress have been described in different experimental settings. Whereas mitochondrial tandem transition levels were surprisingly low with no discernible correlation with UV exposure, mtDNA copy number and CD were significantly increased in UV-exposed samples.

[Induction of transpositions of hobo-elements in chronically irradiated cells of dysgenetic and non-dysgenetic individuals of Drosophila melanogaster].

This paper studies the effect of chronic γ-radiation of different intensities on the induction of hobo-elements in cells ofdysgenetic and non-dysgenetic drosophila species. The level of gonadal atrophy, DNA damage, and mutability of the mini-white locus is estimated. It is shown that the frequency of displacements of the hobo-elements increases with the increase in the chronic irradiation dose, where an essential role belongs to the maternal effect. The level of DNA damage in the cells of embryos and larvae varies and depends on the conditions of induction of hobo-transposons. Analysis of the PCR products showed that chronic irradiation in a certain range of accumulated doses is able to induce formation of new copies of the hobo-elements. At the same time, the structure of deleted hobo-sequences may vary in response to higher doses of irradiation.

Radiation hybrid QTL mapping of Tdes2 involved in the first meiotic division of wheat.

Since the dawn of wheat cytogenetics, chromosome 3B has been known to harbor a gene(s) that, when removed, causes chromosome desynapsis and gametic sterility. The lack of natural genetic diversity for this gene(s) has prevented any attempt to fine map and further characterize it. Here, gamma radiation treatment was used to create artificial diversity for this locus. A total of 696 radiation hybrid lines were genotyped with a custom mini array of 140 DArT markers, selected to evenly span the whole 3B chromosome. The resulting map spanned 2,852 centi Ray with a calculated resolution of 0.384 Mb. Phenotyping for the occurrence of meiotic desynapsis was conducted by measuring the level of gametic sterility as seeds produced per spikelet and pollen viability at booting. Composite interval mapping revealed a single QTL with LOD of 16.2 and r (2) of 25.6 % between markers wmc326 and wPt-8983 on the long arm of chromosome 3B. By independent analysis, the location of the QTL was confirmed to be within the deletion bin 3BL7-0.63-1.00 and to correspond to a single gene located ~1.4 Mb away from wPt-8983. The meiotic behavior of lines lacking this gene was characterized cytogenetically to reveal striking similarities with mutants for the dy locus, located on the syntenic chromosome 3 of maize. This represents the first example to date of employing radiation hybrids for QTL analysis. The success achieved by this approach provides an ideal starting point for the final cloning of this interesting gene involved in meiosis of cereals.

[Radiation biology of structurally different Drosophila genes. Report 2. The vestigial gene: molecular characteristics of chromosome mutations].

The results of the PCR-assay of mutation lesions at each of 16 fragments overlapping the entire vestigial (vg) gene of Drosophila melanogaster in 52 gamma-ray-, neutron- and neutron + gamma-ray-induced vg mutants having the inversion or translocation breakpoint within the vg microregion are presented. 4 from 52 mutants studied were found to have large deletions of about 200 kb covering the entire vg gene and adjacent to sca and l(2)C gene-markers as well. 23 mutants from 48 (47.9%) were found to have a wild-type gene structure showing that the exchange breakpoints are located outside of the vg gene. 25 others display the intragenic lesions of different complexity detected by PCR as the absence of(i) either one fragment or (ii) two or more (6-7) adjacent fragments and (iii) simultaneously several (i) or (i) and (ii) types separated by normal gene regions. It is important that 6 from 25 mutants have the breakpoint inside the vg gene and display the (i) or (ii) type of lesions at the gene regions containing the putative break whereas 5 others from 25 with the above lesions have the exchange breakpoint outside the vg gene. Therefore, the breakpoints underlying either inversions or translocations induced by low- and high-LET radiation are likely to be located within and outside the gene under study. Thereby, the formation of exchanges is accompanied by DNA deletions of various sizes at the exchange breakpoints. The molecular model of formation of such exchange-deletion rearrangements is elaborated and presented. Also, conception of the predominately clustered action of both low- and high-LET radiation on the germ cell genome is suggested as the summing-up of the presented results. The ability of ionizing radiation to induce the clusters of genetic alterations in the form of hidden DNA damages as well as gene/chromosome mutations is determined by the track structure and hierarchical organization of the genome. To detect the quality and frequency patterns of all components of the cluster, joint molecular, genetic and cytological techniques need to be used.

Repair of DNA double-strand breaks induced by ionizing radiation damage correlates with upregulation of homologous recombination genes in Sulfolobus solfataricus.

The mechanisms used by members of the archaeal branch of life to repair DNA damage are not well understood. DNA damage responses have been of particular interest in hyperthermophilic archaea, since these microbes live under environmental conditions that constantly elevate the potential for DNA damage. The work described here focuses on the response of four Sulfolobus solfataricus strains to ionizing radiation (IR) damage. Cellular survival of three wild-type strains and a defined deletion mutant strain was examined following exposure to various IR doses. Using pulsed-field gel electrophoresis, we determined chromosomal DNA double-strand break persistence and repair rates. Among the strains, variable responses were observed, the most surprising of which occurred with the defined deletion mutant strain. This strain displayed higher chromosomal repair rates than the parent strain and was also found to have increased resistance to IR. Using quantitative real-time PCR, we found that transcript levels of homologous recombination-related genes were strongly upregulated following damage in all the strains. The mutant strain again had an enhanced response and dramatically upregulated expression of recombination genes above levels observed for the parent strain, suggesting that increased levels of recombinational repair could account for its increased radiation resistance phenotype. Our results demonstrate a transcriptional response to IR in S. solfataricus for the first time and describe a defined deletion mutant strain that may give the first insight into a damage-based archaeal control element.

Analysis of the common deletions in the mitochondrial DNA is a sensitive biomarker detecting direct and non-targeted cellular effects of low dose ionizing radiation.

One of the key issues of current radiation research is the biological effect of low doses. Unfortunately, low dose science is hampered by the unavailability of easily performable, reliable and sensitive quantitative biomarkers suitable detecting low frequency alterations in irradiated cells. We applied a quantitative real time polymerase chain reaction (qRT-PCR) based protocol detecting common deletions (CD) in the mitochondrial genome to assess direct and non-targeted effects of radiation in human fibroblasts. In directly irradiated (IR) cells CD increased with dose and was higher in radiosensitive cells. Investigating conditioned medium-mediated bystander effects we demonstrated that low and high (0.1 and 2Gy) doses induced similar levels of bystander responses and found individual differences in human fibroblasts. The bystander response was not related to the radiosensitivity of the cells. The importance of signal sending donor and signal receiving target cells was investigated by placing conditioned medium from a bystander response positive cell line (F11-hTERT) to bystander negative cells (S1-hTERT) and vice versa. The data indicated that signal sending cells are more important in the medium-mediated bystander effect than recipients. Finally, we followed long term effects in immortalized radiation sensitive (S1-hTERT) and normal (F11-hTERT) fibroblasts up to 63 days after IR. In F11-hTERT cells CD level was increased until 35 days after IR then reduced back to control level by day 49. In S1-hTERT cells the increased CD level was also normalized by day 42, however a second wave of increased CD incidence appeared by day 49 which was maintained up to day 63 after IR. This second CD wave might be the indication of radiation-induced instability in the mitochondrial genome of S1-hTERT cells. The data demonstrated that measuring CD in mtDNA by qRT-PCR is a reliable and sensitive biomarker to estimate radiation-induced direct and non-targeted effects.

[Detection of large deletions of mitochondrial DNA in tissues of mice exposed to X-rays].

Large mtDNA deletions in mouse brain and spleen cells, induced by X-radiation at doses of 2 and 5 Gy were studied within four weeks after the exposure of animals to X-rays. Variations in the content of extracellular (deletion) mtDNA were examined in the blood plasma of mice irradiated with 5 Gy in the same postirradiation times. Ionizing radiation was shown to effectively induce large mtDNA deletions at the doses chosen. The level of deletion mtDNA was dependent on dose and postirradiation time.

Role of connexin43 and ATP in long-range bystander radiation damage and oncogenesis in vivo.

Ionizing radiation is a genotoxic agent and human carcinogen. Recent work has questioned long-held dogmas by showing that cancer-associated genetic alterations occur in cells and tissues not directly exposed to radiation, questioning the robustness of the current system of radiation risk assessment. In vitro, diverse mechanisms involving secreted soluble factors, gap junction intercellular communication (GJIC) and oxidative metabolism are proposed to mediate these indirect effects. In vivo, the mechanisms behind long-range 'bystander' responses remain largely unknown. Here, we investigate the role of GJIC in propagating radiation stress signals in vivo, and in mediating radiation-associated bystander tumorigenesis in mouse central nervous system using a mouse model in which intercellular communication is downregulated by targeted deletion of the connexin43 (Cx43) gene. We show that GJIC is critical for transmission of oncogenic radiation damage to the non-targeted cerebellum, and that a mechanism involving adenosine triphosphate release and upregulation of Cx43, the major GJIC constituent, regulates transduction of oncogenic damage to unirradiated tissues in vivo. Our data provide a novel hypothesis for transduction of distant bystander effects and suggest that the highly branched nervous system, similar to the vascular network, has an important role.

Physical mapping of a large plant genome using global high-information-content-fingerprinting: the distal region of the wheat ancestor Aegilops tauschii chromosome 3DS.

BACKGROUND: Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum), Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat. RESULTS: We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs. CONCLUSIONS: The physical map reported here is the first physical map using fingerprinting of a complete Triticeae genome. This study demonstrates that global fingerprinting of the large plant genomes is a viable strategy for generating physical maps. Physical maps allow the description of the co-linearity between wheat and grass genomes and provide a powerful tool for positional cloning of new genes.

Characterization of X-ray-generated floral mutants carrying deletions at the S-locus of distylous Turnera subulata.

To investigate the genetic architecture of distyly in Turnera subulata and test the hypothesis that a supergene determines distyly, we used X-ray mutagenesis to generate floral mutants. Based upon the crossing design, all progeny were expected to be short-styled. Of 3982 progeny screened, 10 long-styled mutants, one long homostyle and one short homostyle were recovered. Assays for molecular markers tightly linked to the S-locus showed that the mutants were missing 1-3 markers indicating they are deletion mutants. We investigated the incompatibility phenotype of the mutants and found that both their styles and pollen behaved like those of the long-styled morph. There was a variation in the absolute length of styles, stamens and pollen size of the long-styled mutants. Furthermore, long-styled mutants possessing larger deletions tended to have their anthers and stigmas in closer proximity. We explored the inheritance of the S-locus mutations and found that only one of the deletion mutations was transmitted to progeny where we recovered seven such progeny. Remarkably, our data are consistent with the supergene model (GPA/gpa) of Primula. The long homostyle mutant appears to have deletions involving both the G and P loci. The other mutants appear to have deletions of the entire S-locus. The mutants generated will serve as a valuable resource for the molecular dissection of the S-locus region, and in the identification of genes determining distyly.

Increased genomic alteration complexity and telomere shortening in B-CLL cells resistant to radiation-induced apoptosis.

B-cell chronic lymphocytic leukemia (B-CLL) results in an accumulation of mature CD5(+)/CD23(+) B cells due to an uncharacterized defect in apoptotic cell death. B-CLL is not characterized by a unique recurrent genomic alteration but rather by genomic instability giving rise frequently to several chromosomal aberrations. Besides we reported that approximately 15% of B-CLL patients present malignant B-cells resistant to irradiation-induced apoptosis, contrary to approximately 85% of patients and normal human lymphocytes. Telomere length shortening is observed in radioresistant B-CLL cells. Using fluorescence in situ hybridization (FISH) and multicolour FISH, we tested whether specific chromosomal aberrations might be associated with the radioresistance of a subset of B-CLL cells and whether they are correlated with telomere shortening. In a cohort of 30 B-CLL patients, all of the radioresistant B-CLL cell samples exhibited homozygous or heterozygous deletion of 13q14.3 in contrast to 52% of the radiosensitive samples. In addition to the 13q14.3 deletion, ten out of the 11 radioresistant B-cell samples had another clonal genomic alteration such as trisomy 12, deletion 17p13.1, mutation of the p53 gene or translocations in contrast to only three out of 19 radiosensitive samples. Telomere fusions and non-reciprocal translocations, hallmarks of telomere dysfunction, are not increased in radioresistant B-CLL cells. These findings suggest (i) that the 13q14.3 deletion accompanied by another chromosomal aberration is associated with radioresistance of B-CLL cells and (ii) that telomere shortening is not causative of increased clonal chromosomal aberrations in radioresistant B-CLL cells.

Molecular nature of intrachromosomal deletions and base substitutions induced by environmental mutagens.

Cellular DNA is exposed to a variety of exogenous and endogenous mutagens. A complete understanding of the importance of different types of DNA damage requires knowledge of the specific molecular alterations induced by different types of agents in specific target tissues in vivo. The gpt delta transgenic mouse model provides the opportunity to characterize tissue-specific DNA alterations because small and large deletions as well as base substitutions can be analyzed. Here, we summarize the characteristics of intrachromosomal deletions and base substitutions induced by ionizing radiation in liver and spleen, ultraviolet B (UVB) radiation in epidermis, mitomycin C (MMC) in bone marrow, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in colon, and aminophenylnorharman (APNH) in liver of gpt delta mice. Carbon-ion radiation, UVB, and MMC induced large deletions of more than 1 kb. About half of the large deletions occurred between short direct-repeat sequences and the remainder had flush ends, suggesting the involvement of nonhomologous end joining of double-stranded breaks (DSBs) in DNA. UV photoproducts and interstrand crosslinks by MMC may block DNA replication, thereby inducing DSBs. In contrast, PhIP and APNH mainly generated 1 bp deletions in runs of guanine bases. As for base substitutions, UVB and MMC induced G:C-->A:T transitions at dipyrimidine sites and tandem base substitutions at GG sites, respectively. PhIP and APNH induced G:C-->T:A transversions. Translesion DNA synthesis across the lesions, i.e., UV photoproducts, intrastrand crosslinks by MMC, and guanine adducts by the heterocyclic amines, may be involved in the induction of base substitutions. These results indicate the importance of sequence information to elucidate the mechanisms underlying deletions and base substitutions induced in vivo by environmental mutagens.