Stealth and opportunism: alternative lifestyles of species in the fungal genus Pneumocystis.

Pneumocystis species are ascomycetous fungi that obligatorily dwell with no apparent ill effect in the lungs of normal mammals, but they become pathogenic when host defenses are compromised. Identified more than 100 years ago, these atypical fungi manifest characteristics that are unique within the Fungi, such as the lack of ergosterol, genetic complexity of surface antigens, and antigenic variation. Thought to be confined to the severely immunocompromised host, Pneumocystis spp. are being associated with new population niches owing to the advent of immunomodulatory therapies and increased numbers of patients suffering from chronic diseases. The inability to grow Pneumocystis spp. outside the mammalian lung has thwarted progress toward understanding their basic biology, but via the use of new genetic tools and other strategies, researchers are beginning to uncover their biological and genetic characteristics including a biphasic life cycle, significant metabolic capacities, and modulation of lifestyles.

Homologous chromosomes make contact at the sites of double-strand breaks in genes in somatic G0/G1-phase human cells.

Double-strand DNA breaks (DSBs) are continuously induced in cells by endogenously generated free radicals and exogenous genotoxic agents such as ionizing radiation. DSBs activate the kinase activity in sensor proteins such as ATM and DNA-PK, initiating a complex DNA damage response that coordinates various DNA repair pathways to restore genomic integrity. In this study, we report the unexpected finding that homologous chromosomes contact each other at the sites of DSBs induced by either radiation or the endonuclease I-PpoI in human somatic cells. Contact involves short segments of homologous chromosomes and is centered on a DSB in active genes but does not occur at I-PpoI sites in intergenic DNA. I-PpoI-induced contact between homologous genes is abrogated by the transcriptional inhibitors actinomycin D and α-amanitin and requires the kinase activity of ATM but not DNA-PK. Our findings provide documentation of a common transcription-related and ATM kinase-dependent mechanism that induces contact between allelic regions of homologous chromosomes at sites of DSBs in human somatic cells.

Pneumocystis carinii sterol 14α-demethylase activity in Saccharomyces cerevisiae erg11 knockout mutant: sterol biochemistry.

Pneumocystis carinii is an unusual fungus that can cause pneumonitis in immunosuppressed laboratory rats. Reactions in sterol biosynthesis are attractive targets for development of antimycotic drugs. A key enzyme in sterol biosynthesis is sterol 14α-demethylase (14DM), which is coded by the erg11 gene. Here we describe detailed sterol analysis of wild-type Saccharomyces cerevisiae and in an erg11 knockout mutant expressing either P. carinii or S. cerevisiae 14DM from a plasmid-borne cDNA. Sterols of the three strains were qualitatively and quantitatively analyzed using thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography and mass spectrometry and nuclear magnetic resonance spectroscopy. Biochemical evidence for functional complementation was provided by detecting the same major sterols in all three strains with ergosterol being by far the most abundant. A total of 25 sterols was identified, 16 of which were identified in all three strains. The ratios of lanosterol:14-desmethyllanosterol in the three strains indicate that the mutant transformed with erg11 showed more 14DM activity than wild-type yeast. The sterol analyses also indicated that the P. carinii 14DM can utilize the sterol substrates used by the S. cerevisiae 14DM and suggested that the yeast 14DM in the yeast cell utilizes 4α-methyl sterols better than the P. carinii enzyme.

Gene position within chromosome territories correlates with their involvement in distinct rearrangement types in thyroid cancer cells.

Chromosomal rearrangements in human cancers are of two types, interchromosomal, which are rearrangements that involve exchange between loci located on different chromosomes, and intrachromosomal, which are rearrangements that involve loci located on the same chromosome. The type of rearrangement that typically activates a specific oncogene may be influenced by its nuclear location and that of its partner. In interphase nuclei, each chromosome occupies a distinct three-dimensional (3D) territory that tends to not overlap the territories of other chromosomes. It is also known that after double strand breaks in the genome, mobility of free DNA ends is limited. These considerations suggest that loci located deep within a chromosomal territory might not participate in interchromosomal rearrangements as readily as in intrachromosomal rearrangements. To test this hypothesis, we used fluorescence in situ hybridization with 3D high-resolution confocal microscopy to analyze the positions of six oncogenes known to be activated by recombination in human cancer cells. We found that loci involved in interchromosomal rearrangements were located closer to the periphery of chromosome territories as compared with the loci that were involved in intrachromosomal inversions. The results of this study provide evidence suggesting that nuclear architecture and location of specific genetic loci within chromosome territories may influence their participation in intrachromosomal or interchromosomal rearrangements in human thyroid cells.

Complexity of the MSG gene family of Pneumocystis carinii.

BACKGROUND: The relationship between the parasitic fungus Pneumocystis carinii and its host, the laboratory rat, presumably involves features that allow the fungus to circumvent attacks by the immune system. It is hypothesized that the major surface glycoprotein (MSG) gene family endows Pneumocystis with the capacity to vary its surface. This gene family is comprised of approximately 80 genes, which each are approximately 3 kb long. Expression of the MSG gene family is regulated by a cis-dependent mechanism that involves a unique telomeric site in the genome called the expression site. Only the MSG gene adjacent to the expression site is represented by messenger RNA. Several P. carinii MSG genes have been sequenced, which showed that genes in the family can encode distinct isoforms of MSG. The vast majority of family members have not been characterized at the sequence level. RESULTS: The first 300 basepairs of MSG genes were subjected to analysis herein. Analysis of 581 MSG sequence reads from P. carinii genomic DNA yielded 281 different sequences. However, many of the sequence reads differed from others at only one site, a degree of variation consistent with that expected to be caused by error. Accounting for error reduced the number of truly distinct sequences observed to 158, roughly twice the number expected if the gene family contains 80 members. The size of the gene family was verified by PCR. The excess of distinct sequences appeared to be due to allelic variation. Discounting alleles, there were 73 different MSG genes observed. The 73 genes differed by 19% on average. Variable regions were rich in nucleotide differences that changed the encoded protein. The genes shared three regions in which at least 16 consecutive basepairs were invariant. There were numerous cases where two different genes were identical within a region that was variable among family members as a whole, suggesting recombination among family members. CONCLUSION: A set of sequences that represents most if not all of the members of the P. carinii MSG gene family was obtained. The protein-changing nature of the variation among these sequences suggests that the family has been shaped by selection for protein variation, which is consistent with the hypothesis that the MSG gene family functions to enhance phenotypic variation among the members of a population of P. carinii.

Visualizing loss of heterozygosity in living mouse cells and tissues.

Loss of heterozygosity (LOH) in somatic cells can contribute to the genesis of cancer, but little is known about the frequency with which LOH occurs in normal cells of the body. To detect LOH in situ, we studied mouse shYFP embryonic stem (ES) cells and cells of the intestinal epithelia derived from these ES cells. shYFP ES cells are heterozygous at the ROSA26 locus. One copy of the locus carries a gene encoding a yellow fluorescent protein (YFP), while the other copy harbors an shRNA gene that produces a short hairpin RNA (shRNA) molecule that causes degradation of YFP mRNA. Nearly all cells in shYFP populations were faintly fluorescent, but brightly fluorescent cells arose at a rate of approximately 10(-5)bright cells/generation. Bright cells lacked the gene encoding the shRNA and contained two copies of the YFP gene. Comparison of these results to previous data on LOH in ES cells that lacked interfering shRNA showed that LOH in shYFP cells was not influenced by the presence of the shRNA. Bright cells were also seen in intestinal villi of chimeric mice made by injecting blastocysts with shYFP cells. These data demonstrate that this approach can detect LOH and suggest that it will allow detection of LOH in a broad array of tissues and cell types in transgenic mice made from shYFP cells.

Pneumocystis oryctolagi sp. nov., an uncultured fungus causing pneumonia in rabbits at weaning: review of current knowledge, and description of a new taxon on genotypic, phylogenetic and phenotypic bases.

The genus Pneumocystis comprises noncultivable, highly diversified fungal pathogens dwelling in the lungs of mammals. The genus includes numerous host-species-specific species that are able to induce severe pneumonitis, especially in severely immunocompromised hosts. Pneumocystis organisms attach specifically to type-1 epithelial alveolar cells, showing a high level of subtle and efficient adaptation to the alveolar microenvironment. Pneumocystis species show little difference at the light microscopy level but DNA sequences of Pneumocystis from humans, other primates, rodents, rabbits, insectivores and other mammals present a host-species-related marked divergence. Consistently, selective infectivity could be proven by cross-infection experiments. Furthermore, phylogeny among primate Pneumocystis species was correlated with the phylogeny of their hosts. This observation suggested that cophylogeny could explain both the current distribution of pathogens in their hosts and the speciation. Thus, molecular, ultrastructural and biological differences among organisms from different mammals strengthen the view of multiple species existing within the genus Pneumocystis. The following species were subsequently described: Pneumocystis jirovecii in humans, Pneumocystis carinii and Pneumocystis wakefieldiae in rats, and Pneumocystis murina in mice. The present work focuses on Pneumocystis oryctolagi sp. nov. from Old-World rabbits. This new species has been described on the basis of both biological and phylogenetic species concepts.

Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes.

BACKGROUND: Loss of heterozygosity (LOH) contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors. RESULTS: As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS) showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10(-4) and appeared to be produced at a rate of approximately 10(-5) variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6. CONCLUSION: Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors influencing expression from allelic genes. Similar approaches will allow these phenomena to be studied in tissues.

Gene arrays at Pneumocystis carinii telomeres.

In the fungus Pneumocystis carinii, at least three gene families (PRT1, MSR, and MSG) have the potential to generate high-frequency antigenic variation, which is likely to be a strategy by which this parasitic fungus is able to prolong its survival in the rat lung. Members of these gene families are clustered at chromosome termini, a location that fosters recombination, which has been implicated in selective expression of MSG genes. To gain insight into the architecture, evolution, and regulation of these gene clusters, six telomeric segments of the genome were sequenced. Each of the segments began with one or more unique genes, after which were members of different gene families, arranged in a head-to-tail array. The three-gene repeat PRT1-MSR-MSG was common, suggesting that duplications of these repeats have contributed to expansion of all three families. However, members of a gene family in an array were no more similar to one another than to members in other arrays, indicating rapid divergence after duplication. The intergenic spacers were more conserved than the genes and contained sequence motifs also present in subtelomeres, which in other species have been implicated in gene expression and recombination. Long mononucleotide tracts were present in some MSR genes. These unstable sequences can be expected to suffer frequent frameshift mutations, providing P. carinii with another mechanism to generate antigen variation.

Exposure of mice to arsenic and/or benzo[a]pyrene does not increase the frequency of Aprt-deficient cells recovered from explanted skin of Aprt heterozygous mice.

Exposure to inorganic arsenic in drinking water is linked to cancer in humans, but the mechanism of arsenic-induced cancer is not clear. Arsenic is not a powerful point mutagen, but can cause chromosome malsegregation and mitotic recombination, two events that can cause loss of tumor suppressor alleles and thereby contribute to the evolution of cancerous cells. To determine whether arsenic increases the frequency of allele loss due to either malsegregation or mitotic recombination in vivo, Aprt(+/-) hybrid mice were exposed to sodium arsenite (10 mg/L) in their drinking water for 10 weeks. To determine whether arsenic enhances the action of a known mutagen, half of the arsenic-treated mice were exposed to benzo[a]pyrene (BaP) for 8 weeks by skin painting (500 nmoles/week). Cells were taken from painted dorsal skin and cultured in the presence of 2,6-diaminopurine (DAP), to select colonies lacking adenosine phosphoribosyl transferase (Aprt) activity. The frequency of DAP-resistant (DAP(r)) colonies varied substantially within the treatment groups, but there was no significant difference between the groups. Analysis of DNA from DAP(r) colonies suggested that mitotic recombination contributed to the loss of wild-type Aprt allele. Whether arsenic or BaP enhanced or diminished the frequency of this process could not be deduced from these data.

Dose-dependent generation of RET/PTC in human thyroid cells after in vitro exposure to gamma-radiation: a model of carcinogenic chromosomal rearrangement induced by ionizing radiation.

Ionizing radiation is a well-known risk factor for thyroid cancer in human populations. Chromosomal rearrangements involving the RET gene, known as RET/PTC, are prevalent in thyroid papillary carcinomas from patients with radiation history. We studied the generation of RET/PTC in HTori-3 immortalized human thyroid cells exposed to a range of doses of gamma-radiation and harvested 2, 5-6, and 9 d later. RET/PTC1 and RET/PTC3 were detected by RT-PCR followed by Southern blotting and hybridization with internal oligonucleotide probes. No RET/PTC was found in cells harvested 2 and 5-6 d after irradiation, whereas 59 RET/PTC events were detected in cells collected 9 d after exposure. The average rate of RET/PTC induction was 0.1 x 10(-6) after exposure to 0.1 Gy, 1.6 x 10(-6) after 1 Gy, 3.0 x 10(-6) after 5 Gy, and 0.9 x 10(-6) after 10 Gy. When adjusted for cell survival, the rate after 10 Gy was comparable with those after 5 Gy. RET/PTC1 was more common than RET/PTC3 after each dose, comprising 80% of all rearrangements. In this study, we demonstrate a dose-dependent induction of RET/PTC rearrangements in human thyroid cells after exposure to 0.1-10 Gy gamma-radiation. This provides additional evidence for a direct link between this genetic event and radiation exposure and offers a powerful experimental system for studying radiation-induced carcinogenesis in the thyroid gland.

Characterization of novel non-clonal intrachromosomal rearrangements between the H4 and PTEN genes (H4/PTEN) in human thyroid cell lines and papillary thyroid cancer specimens.

The two main forms of RET rearrangement in papillary thyroid carcinomas (PTC) arise from intrachromosomal inversions fusing the tyrosine kinase domain of RET with either the H4 (RET/PTC1) or the ELE1/RFG genes (RET/PTC3). PTEN codes for a dual-specificity phosphatase and maps to chromosome 10q22-23. Germline mutations confer susceptibility to Cowden syndrome whereas somatic mutations or deletions are common in several sporadic human tumors. Decreased PTEN expression has been implicated in thyroid cancer development. We report the characterization of a new chromosome 10 rearrangement involving H4 and PTEN. The initial H4/PTEN rearrangement was discovered as a non-specific product of RT-PCR for RET/PTC1 in irradiated thyroid cell lines. Sequencing revealed a transcript consisting of exon 1 and 2 of H4 fused with exons 3-6 of PTEN. Nested RT-PCR with specific primers bracketing the breakpoints confirmed the H4/PTEN rearrangements in irradiated KAT-1 and KAT-50 cells. Additional H4/PTEN variants, generated by recombination of either exon 1 or exon 2 of H4 with exon 6 of PTEN, were found in non-irradiated KAK-1, KAT-50, ARO and NPA cells. Their origin through chromosomal recombination was confirmed by detection of the reciprocal PTEN/H4 product. H4/PTEN recombination was not a clonal event in any of the cell lines, as Southern blots with appropriate probes failed to demonstrate aberrant bands, and multicolor FISH of KAK1 cells with BAC probes for H4 and PTEN did not show a signal overlap in all cells. Based on PCR of serially diluted samples, the minimal frequency of spontaneous recombination between these loci was estimated to be approximately 1/10(6) cells. H4/PTEN products were found by nested RT-PCR in 4/14 normal thyroid tissues (28%) and 14/18 PTC (78%) (P<0.01). H4/PTEN is another example of recombination involving the H4 locus, and points to the high susceptibility of thyroid cells to intrachromosomal gene rearrangements. As this also represents a plausible mechanism for loss-of-function of PTEN, other thyroid neoplastic phenotypes and eventually other cancer types need to be screened for clonal H4/PTEN rearrangements.

Co-mutagenic activity of arsenic and benzo[a]pyrene in mouse skin.

Exposure to inorganic arsenic in drinking water is linked to skin, lung and bladder cancer in humans. The mechanism of arsenic-induced cancer is not clear, but exposure to arsenic and polycyclic arylhydrocarbons (PAH) is more carcinogenic than exposure to either type of carcinogen alone. Arsenic can also generate reactive oxygen species, suggesting that oxidation of DNA may play a role in carcinogenesis. Oxidization of guanosines in polyG tracts is known to cause frameshift mutations, and such events can be detected in situ using the G11 placental alkaline phosphatase (PLAP) transgenic mouse model, which reports frameshift mutations in a run of 11 G:C basepairs by generating cells containing heat-resistant alkaline phosphatase activity. PAH can also induce frameshift mutations. In the study described here, FVB/N mice carrying the G11 PLAP transgene were crossed to C57Bl/6 mice. Half of the hybrid mice were given drinking water with sodium arsenite (10 mg/L) for 10 weeks. Half of the arsenic treated mice were also exposed to benzo[a]pyrene (BaP) by skin painting (500 nmol/week) for 8 weeks. Another group of mice was exposed to BaP but not arsenic. The effect on frameshift mutation was assessed by staining sections of skin tissue to detect cells with PLAP activity. Arsenic alone had no significant effect. On average, mice given BaP alone had approximately three times more PLAP-positive (PLAP+) cells. By contrast, mice exposed to both arsenic and BaP exhibited 10-fold more PLAP+ cells in the skin, and these cells were often arranged in large clusters, suggesting derivation from stem cells. Whereas combined treatment produced more PLAP+ cells, stable BaP adduct levels and arsenic burdens were not higher in mice exposed to both agents compared to mice exposed to either one agent or the other.

Impact of mismatch repair deficiency on genomic stability in the maternal germline and during early embryonic development.

The effects of lack of the mismatch repair protein PMS2 on germline and maternal-effect mutations were studied in transgenic mice that allow mutant cells to be visualized in situ. Tg(betaA-G11PLAP) mice are transgenic for the G11 allele of a human placental alkaline phosphatase (PLAP) gene driven by a human beta-actin promoter. The G11 allele of the PLAP gene does not produce enzyme due to a frameshift induced by a mononucleotide repeat containing 11 G:C basepairs. Loss of one G:C basepair restores enzyme production. When the G11 PLAP allele was passed through the germline of female mice lacking PMS2, approximately 25% of the offspring that inherited the transgene exhibited the phenotype expected for germline mutation. The mice transmitted the germline-mutation phenotype normally and their offspring exhibited PLAP enzyme activity in at least 30% of the cells in each tissue examined. By contrast, only 1 of 32 mice that inherited the G11 PLAP transgene from a wild-type male crossed to a Pms2-/- female exhibited a high number of PLAP+ cells. Compared to germline revertants, approximately one half to one quarter as many cells were PLAP+, suggesting that a mutation occurred in one cell of an embryo containing two to four cells. These data suggest that the paternally derived Pms2 gene provided normal levels of PMS2 protein to embryos by the time they reached the eight-cell stage, but that smaller embryos formed from PMS2-deficient eggs lacked PMS2 function.

Visualization of mosaicism in tissues of normal and mismatch-repair-deficient mice carrying a microsatellite-containing transgene.

To determine the frequency of mutation in different cell types of mammals, transgenic mice that allow mutant cells to be visualized in situ were used. These mice carry a defective allele of the human placental alkaline phosphatase (PLAP) gene. The allele does not produce enzyme because the reading frame is shifted by an insertion of 7 G:C basepairs. The insertion is adjacent to four existing G:C basepairs, so the allele has a tract of 11Gs. The G11 PLAP allele was studied in wildtype mice and in mice deficient in mismatch-repair (MMR) due to lack of either Pms2 or Mlh1. PLAP(+) cells were counted in brain, heart, kidney, and liver. In wildtype mice, there was an average of between 5 and 30 PLAP(+) events per million cells. No cells with alkaline phosphatase activity were detected in tissues from mice lacking the PLAP gene. In MMR-deficient mice, the number of PLAP(+) allele was increased by at least three-order of magnitude in brain, heart and kidney, but <10-fold in liver. These data show that MMR is vital to maintaining repeat stability in brain, heart and kidney cells. The reason for the different results in the liver is not clear. Cells in the liver were shown to be capable of expressing of PLAP enzyme and PLAP mRNA was present in this organ.

Increased mutation in mice genetically predisposed to oxidative damage in the brain.

Harlequin (Hq) mice develop ataxia due to an X-linked recessive mutation in the gene encoding apoptosis-inducing factor (Aif). Brain cells in Hq mice contain the modified base 8-hydroxydeoxyguanosine (8-OHdG), suggesting that the defect in Aif causes increased DNA oxidation in these cells. Because oxidative damage is mutagenic, Hq mice might suffer increased mutation in the brain. To examine this possibility, mutation in the brain was assessed using the Tg(betaA-G11PLAP) mouse model, which allows mutant cells to be visualized in tissue sections in situ. Hq mice exhibited more and larger patches of PLAP positive tissue in the brain. PLAP+ cells were observed in all areas of the brain. No increase in the number of PLAP+ cells was seen in three other tissues, suggesting that the effect of Aif deficiency on mutation was specific to brain.

Molecular and phenotypic description of Pneumocystis wakefieldiae sp. nov., a new species in rats.

Organisms in the genus Pneumocystis are fungi that reside in the lungs of mammals that can cause a lethal pneumonia once the hosts lose immune function. The genus Pneumocystis contains many members, but only two species have been described formally to date, P. carinii, the type species found in rats, and P. jirovecii, resident in human beings. Rats have been shown to harbor another organism in addition to P. carinii, Pneumocystis wakefieldiae sp. nov., formerly known as Pneumocystis carinii f. sp. ratti, which is described here. Although often found together and morphologically similar, P. carinii and P. wakefieldiae are phenotypically and genetically divergent. We used the phylogenetic species recognition approach to distinguish these organisms as two distinct species and estimated the evolutionary time of their separation. Nucleotide sequence comparisons of seven homologous genes showed 4-7% divergence between the P. wakefieldiae and P. carinii sequences, which was in contrast to the 0-0.8% divergence observed within P. carinii species. Even greater divergence (30%) occurred in sequences located between genes. The MSG (major surface glycoprotein) gene families of P. carinii and P. wakefieldiae are 35% divergent from one another and differ with respect to sequence elements associated with regulation of their transcription. Differences in reactivity of monoclonal antibodies and polyclonal antisera reflected these genetically distinct surface antigens. Karyotypic analysis of P. wakefieldiae produced a single profile that was distinct from all 12 profiles known for P. carinii. Eight homologous genes were localized to chromosomes of different sizes in the two species. The cumulative genotypic and phenotypic data support a species distinction between these two organisms.

Formation of carcinogenic chromosomal rearrangements in human thyroid cells after induction of double-strand DNA breaks by restriction endonucleases.

Ionizing radiation (IR) exposure increases the risk of thyroid cancer and other cancer types. Chromosomal rearrangements, such as RET/PTC, are characteristic features of radiation-associated thyroid cancer and can be induced by radiation in vitro. IR causes double-strand breaks (DSBs), suggesting that such damage leads to RET/PTC, but the rearrangement mechanism has not been established. To study the mechanism, we explored the possibility of inducing RET/PTC by electroporation of restriction endonucleases (REs) into HTori-3 human thyroid cells. We used five REs, which induced DSB in a dose-dependent manner similar to that seen with IR. Although all but one RE caused DSB in one or more of the three genes involved in RET/PTC, rearrangement was detected only in cells electroporated with either PvuII (25 and 100  U) or StuI (100 and 250  U). The predominant rearrangement type was RET/PTC3, which is characteristic of human thyroid cancer arising early after Chernobyl-related radioactive iodine exposure. Both enzymes that produced RET/PTC had restriction sites only in one of the two fusion partner genes. Moreover, the two enzymes that produced RET/PTC had restriction sites present in clusters, which was not the case for RE that failed to induce RET/PTC. In summary, we establish a model of DSB induction by RE and report for the first time the formation of carcinogenic chromosomal rearrangements, predominantly RET/PTC3, as a result of DSB produced by RE. Our data also raise a possibility that RET/PTC rearrangement can be initiated by a complex DSB that is induced in one of the fusion partner genes.