Asymmetric diversification of mating pheromones in fission yeast.

In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4-8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation-perhaps as a first step toward prezygotic isolation in S. pombe.

The asymmetric chemical structures of two mating pheromones reflect their differential roles in mating of fission yeast.

In the fission yeast Schizosaccharomyces pombe, the mating reaction is controlled by two mating pheromones, M-factor and P-factor, secreted by M- and P-type cells, respectively. M-factor is a C-terminally farnesylated lipid peptide, whereas P-factor is a simple peptide. To examine whether this chemical asymmetry in the two pheromones is essential for conjugation, we constructed a mating system in which either pheromone can stimulate both M- and P-cells, and examined whether the resulting autocrine strains can mate. Autocrine M-cells responding to M-factor successfully mated with P-factor-lacking P-cells, indicating that P-factor is not essential for conjugation; by contrast, autocrine P-cells responding to P-factor were unable to mate with M-factor-lacking M-cells. The sterility of the autocrine P-cells was completely restored by expressing the M-factor receptor. These observations indicate that the different chemical characteristics of the two types of pheromone, a lipid and a simple peptide, are not essential; however, a lipid peptide might be required for successful mating. Our findings allow us to propose a model of the differential roles of M-factor and P-factor in conjugation of S. pombeThis article has an associated First Person interview with the first author of the paper.

Molecular coevolution of a sex pheromone and its receptor triggers reproductive isolation in Schizosaccharomyces pombe.

The diversification of sex pheromones is regarded as one of the causes of prezygotic isolation that results in speciation. In the fission yeast Schizosaccharomyces pombe, the molecular recognition of a peptide pheromone by its receptor plays an essential role in sexual reproduction. We considered that molecular coevolution of a peptide-mating pheromone, M factor, and its receptor, Map3, might be realized by experimentally diversifying these proteins. Here, we report the successful creation of novel mating-type pairs by searching for map3 suppressor mutations that rescued the sterility of M-factor mutants that were previously isolated. Several strong suppressors were found to also recognize WT M factor. The substituted residues of these Map3 suppressors were mapped to F204, F214, and E249, which are likely to be critical residues for M-factor recognition. These critical residues were systematically substituted with each of the other amino acids by in vitro mutagenesis. Ultimately, we successfully obtained three novel mating-type pairs constituting reproductive groups. These novel mating-type pairs could not conjugate with WT maters. Furthermore, no flow of chromosomally integrated drug-resistance genes occurred between the novel and the WT mating pairs, showing that each experimentally created reproductive group [e.g., M factor(V5H) and Map3(F214H)] was isolated from the WT group. In conclusion, we have succeeded in creating an artificial reproductive group that is isolated from the WT group. In keeping with the biological concept of species, the artificial reproductive group is a new species.

The fission yeast synaptobrevin ortholog Syb1 plays an important role in forespore membrane formation and spore maturation.

Synaptobrevin, also called vesicle-associated membrane protein (VAMP), is a component of the plasma membrane N-methylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which plays a key role in intracellular membrane fusion. Previous studies have revealed that, similar to synaptobrevin in other organisms, the fission yeast synaptobrevin ortholog Syb1 associates with post-Golgi secretory vesicles and is essential for cytokinesis and cell elongation. Here, we report that Syb1 has a role in sporulation. After nitrogen starvation, green fluorescent protein (GFP)-Syb1 is found in intracellular dots. As meiosis proceeds, GFP-Syb1 accumulates around the nucleus and then localizes at the forespore membrane (FSM). We isolated a syb-S1 mutant, which exhibits a defect in sporulation. In syb1-S1 mutants, the FSM begins to form but fails to develop a normal morphology. Electron microscopy shows that an abnormal spore wall is often formed in syb1-S1 mutant spores. Although most syb1-S1 mutant spores are germinated, they are less tolerant to ethanol than wild-type spores. The syb1-S1 allele carries a missense mutation, resulting in replacement of a conserved cysteine residue adjacent to the transmembrane domain, which reduces the stability and abundance of the Syb1 protein. Taken together, these results indicate that Syb1 plays an important role in both FSM assembly and spore wall formation.

Two fission yeast rab7 homologs, ypt7 and ypt71, play antagonistic roles in the regulation of vacuolar morphology.

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe (S. pombe) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71(+) exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7Delta cells. Like ypt7Delta cells, overexpression of ypt71(+) caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/alpha3-L7.

Schizosaccharomyces pombe calmodulin, Cam1, plays a crucial role in sporulation by recruiting and stabilizing the spindle pole body components responsible for assembly of the forespore membrane.

Calmodulin in Schizosaccharomyces pombe is encoded by the cam1(+) gene, which is indispensable for both vegetative growth and sporulation. Here, we report how Cam1 functions in spore formation. We found that Cam1 preferentially localized to the spindle pole body (SPB) during meiosis and sporulation. Formation of the forespore membrane, a precursor of the plasma membrane in spores, was blocked in a missense cam1 mutant, which was viable but unable to sporulate. Three SPB proteins necessary for the onset of forespore membrane formation, Spo2, Spo13, and Spo15, were unable to localize to the SPB in the cam1 mutant although five core SPB components that were tested were present. Recruitment of Spo2 and Spo13 is known to require the presence of Spo15 in the SPB. Notably, Spo15 was unstable in the cam1 mutant, and as a result, SPB localization of Spo2 and Spo13 was lost. Overexpression of Spo15 partially alleviated the sporulation defect in the cam1 mutant. These results indicate that calmodulin plays an essential role in forespore membrane formation by stably maintaining Spo15, and thus Spo2 and Spo13, at the SPB in meiotic cells.

The sixth transmembrane region of a pheromone G-protein coupled receptor, Map3, is implicated in discrimination of closely related pheromones in Schizosaccharomyces pombe.

Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycete fungi including yeasts, mating between cells of opposite mating type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focused on the stringency of pheromone receptors. The fission yeast Schizosaccharomyces pombe has two mating types, Plus (P) and Minus (M). Here, we investigated the stringency of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs between S. pombe and the closely related species Schizosaccharomyces octosporus, which showed that SoMam2 (Mam2 of S. octosporus) is partially functional in S. pombe, whereas SoMap3 (Map3 of S. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the activation by the pheromone. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are key residues important for discrimination of closely related M-factors. Thus, the differences in these two GPCRs might reflect the significantly distinct stringency/flexibility of their respective pheromone/receptor systems; nevertheless, species-specific pheromone recognition remains incomplete.

Geranylgeranyl diphosphate synthase in fission yeast is a heteromer of farnesyl diphosphate synthase (FPS), Fps1, and an FPS-like protein, Spo9, essential for sporulation.

Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1(+) and spo9(+), whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1(+), but not spo9(+), suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1(+) nor spo9(+) expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9Delta cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9Delta cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation.

Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins.

Yeast cells producing mammalian-type N-linked oligosaccharide show severe growth defects and the decreased protein productivity because of the disruption of yeast-specific glycosyltransferases. This decreased protein productivity in engineered yeast strains is an obstacle to the development of efficient glycoprotein production in yeast. For economic and effective synthesis of such therapeutic glycoproteins in yeast, the development of appropriate strains is highly desirable. We applied a novel mutagenesis technique that utilized the proofreading-deficient DNA polymerase delta variant encoded by the pol3-01 gene of Saccharomyces cerevisiae or the cdc6-1 gene of Schizosaccharomyces pombe to the engineered S. cerevisiae TIY20 strain and S. pombe KT97 strain, respectively. TIY20, which is deficient in the outer chain of mannan due to the disruption of three genes (och1Delta, mnn1 Delta, mnn4 Delta), and KT97, which is an och1 disruptant, are impractical as hosts for the production of therapeutic glycoproteins since they show a temperature-sensitive (ts) phenotype, a growth defect phenotype, and decreased protein productivity. We successfully isolated YAB mutants that alleviated the growth defect of the TIY20 strain. Surprisingly, these mutants generally secreted foreign proteins better than the wild-type strain. Furthermore, we successfully isolated YPAB mutants that alleviated the growth defect of the KT97 strain, too. The development of these new mutants by the combination of genetic engineering of yeast and this mutagenesis technique are major breakthroughs for the production of therapeutic glycoproteins in engineered yeast cells.

The Schizosaccharomyces pombe syntaxin 1 homolog, Psy1, is essential in the development of the forespore membrane.

Syntaxin is a component of t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), which is responsible for docking membrane vesicles at the target membrane and is highly conserved among eukaryotes. In the fission yeast Schizosaccharomyces pombe, the psy1(+) gene encoding a syntaxin 1 homolog was originally isolated as a multicopy suppressor of the sporulation-deficient mutant, spo3, but little is known about the way Psy1 is involved in sporulation. Here we report the isolation of a sporulation-defective mutant, psy1-S1, generated by random PCR mutagenesis. psy1-S1 also exhibited temperature sensitivity in growth. In psy1-S1 cells, assembly of the forespore membrane (FSM) initiated near the spindle pole bodies during meiosis II, but subsequent expansion of the membrane was severely impaired. Overproduction of the cognate SNARE proteins, Syb1 and Sec9, suppressed both the temperature sensitivity and sporulation defects of psy1-S1. These results indicate that Psy1 plays an essential role in FSM formation coordinated by Syb1 and Sec9.

Role of pheromone recognition systems in creating new species of fission yeast.

Many species, from mammals to microorganisms, release sex pheromones to attract a potential partner of the opposite sex. The combination of a pheromone and its corresponding receptor determines the species-specific ability of males and females to recognize each other, and therefore causes reproductive isolation. This barrier, which has arisen to restrict gene flow between mating pairs, might facilitate reproductive isolation leading to incipient speciation, but how do new combinations of pheromone and receptor evolve? Our recent study demonstrated an "asymmetric" pheromone recognition system in the fission yeast Schizosaccharomyces pombe: among the two pheromone/receptor pairs in this yeast, recognition between one pair is stringent, while that between the other pair is rather relaxed. We speculate that the asymmetric properties of these pheromone recognition systems are beneficial for gradual evolution resulting in reproductive isolation in yeasts.

Novel fission yeast Cdc7-Dbf4-like kinase complex required for the initiation and progression of meiotic second division.

Cdc7, a conserved serine/threonine protein kinase, controls initiation of DNA replication. A regulatory subunit, Dbf4, stimulates the kinase activity of Cdc7 and recruits it to the replication origins. Schizosaccharomyces pombe has a homologous kinase complex, composed of Hsk1 and Dfp1/Him1. Here, we report a novel protein kinase of S. pombe, Spo4, which shares common structural features with the Cdc7 kinases. In spite of the structural similarities, Spo4 is dispensable for mitotic growth and premeiotic DNA replication. Intriguingly, spo4 null mutants are defective in initiation and progression of the second meiotic division. Spindles for meiosis II are often fragmented. Spo4 kinase activity is markedly enhanced when the enzyme is associated with its regulatory subunit, Spo6, a Dbf4-like protein. Expression of Spo4 is specifically induced during meiosis. Spo4 is preferentially present in nuclei, but this nuclear localization does not require Spo6. These results suggest that Spo4 is a Cdc7 kinase whose primary role is in meiosis, not in DNA replication. This is the first report of an organism which has two Cdc7-related kinase complexes with different biological functions.

The fission yeast spo14+ gene encoding a functional homologue of budding yeast Sec12 is required for the development of forespore membranes.

The Schizosaccharomyces pombe spo14-B221 mutant was originally isolated as a sporulation-deficient mutant. However, the spo14(+) gene is essential for cell viability and growth. spo14(+) is identical to the previously characterized stl1(+) gene encoding a putative homologue of Saccharomyces cerevisiae Sec12, which is essential for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. In the spo14 mutant cells, ER-like membranes were accumulated beneath the plasma membrane and the ER/Golgi shuttling protein Rer1 remained in the ER. Sec12 is a guanine nucleotide exchange factor for the Sar1 GTPase. Overproduction of psr1(+) coding for an S. pombe Sar1 homologue suppressed both the sporulation defect of spo14-B221 and cold-sensitive growth of newly isolated spo14-6 and spo14-7 mutants. These results indicate that Spo14 is involved in early steps of the protein secretory pathway. The spo14-B221 allele carries a single nucleotide change in the branch point consensus of the fifth intron, which reduces the abundance of the spo14 mRNA. During meiosis II, the forespore membrane was initiated near spindle pole bodies; however, subsequent extension of the membrane was arrested before its closure into a sac. We conclude that Spo14 is responsible for the assembly of the forespore membrane by supplying membrane vesicles.

Localization of type I myosin and F-actin to the leading edge region of the forespore membrane in Schizosaccharomyces pombe.

Myo1, a heavy chain of type I myosin of the fission yeast Schizosaccharomyces pombe, is essential for sporulation. Here we have analyzed the expression, localization and cellular function of the type I myosin light chain calmodulin, Cam2, encoded by cam2(+). Transcription of cam2(+) was constitutive and markedly enhanced in meiosis. The cam2 null mutant was viable and completed sporulation normally at 28 degrees C, but formed four-spored asci poorly at 34 degrees C. In those sporulation-defective cells, the forespore membrane was formed abnormally. A Cam2-GFP fusion protein accumulated at the cell poles in interphase cells and at the medial septation site in postmitotic cells, colocalizing with Myo1 and F-actin patches. During the mating process, a single Cam2-GFP dot was detected at the tip of the mating projection. During meiosis-I, the Cam2-GFP dots dispersed into the cell periphery and the cytoplasm. At metaphase-II, intense Cam2-GFP signals appeared near Meu14 rings which were formed at the leading edge of expanding forespore membranes. This localization of Cam2 was dependent upon Myo1; and sporulation defect of cam2Delta at 34 degrees C was alleviated by overexpressing Myo1DeltaIQ. These results suggest a close relationship between Cam2 and Myo1. In addition, both F-actin and Myo1 localized with Cam2 in the leading edge region. In summary, type I myosin and F-actin accumulate at the leading edge area of the forespore membrane and may play a pivotal role in its assembly.

Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae.

Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.

A fission yeast SNAP-25 homologue, SpSec9, is essential for cytokinesis and sporulation.

The soluble NSF attachment protein 25 (SNAP-25) is a component of the SNARE complex that is essential for regulated exocytosis in diverse cell types. Here, we identified a fission yeast SNAP-25 homologue, SpSec9. The sec9+ gene was essential for vegetative growth. sec9 mRNA was detected in vegetative cells and further increased during sporulation. This increase during sporulation was dependent on Mei4, a meiosis-specific transcription factor. A sporulation-deficient sec9 mutant was isolated by random PCR mutagenesis (sec9-10). The sec9-10 mutant also exhibited temperature sensitivity for growth and cell division was found to arrest before completion of cell separation at restrictive temperatures. In sec9-10 cells, the forespore membrane was normally initiated near spindle pole bodies during meiosis II. However, subsequent extension of the membrane was severely impaired. These results indicate that SpSec9 plays an important role both in cytokinesis and in sporulation.

A role for fission yeast Rab GTPase Ypt7p in sporulation.

Ypt7p, a fission yeast (Schizosaccharomyces pombe) homologue of Rab7 GTPase, mediates fusion of endosomes to vacuoles and homotypic vacuole fusion. Here, we report that Ypt7p plays important roles in sporulation. Most ypt7Delta asci produced less than four spores, which were apparently immature and germinated at low frequency. Furthermore, ypt7Delta cells were defective in development of the forespore membranes. Vacuoles in sporulating cells were found to undergo extensive homotypic vacuole fusion to form a few large compartments occupying the entire cytoplasm of asci. This extensive vacuole fusion depended on Ypt7p.

The Schizosaccharomyces pombe cdt2(+) gene, a target of G1-S phase-specific transcription factor complex DSC1, is required for mitotic and premeiotic DNA replication.

We have defined five sev genes by genetic analysis of Schizosaccharomyces pombe mutants, which are defective in both proliferation and sporulation. sev1(+)/cdt2(+) was transcribed during the G1-S phase of the mitotic cell cycle, as well as during the premeiotic S phase. The mitotic expression of cdt2(+) was regulated by the MCB-DSC1 system. A mutant of a component of DSC1 affected cdt2(+) expression in vivo, and a cdt2(+) promoter fragment containing MCB motifs bound DSC1 in vitro. Cdt2 protein also accumulated in S phase and localized to the nucleus. cdt2 null mutants grew slowly at 30 degrees and were unable to grow at 19 degrees. These cdt2 mutants were also medially sensitive to hydroxyurea, camptothecin, and 4-nitroquinoline-1-oxide and were synthetically lethal in combination with DNA replication checkpoint mutations. Flow cytometry analysis and pulsed-field gel electrophoresis revealed that S-phase progression was severely retarded in cdt2 mutants, especially at low temperatures. Under sporulation conditions, premeiotic DNA replication was impaired with meiosis I blocked. Furthermore, overexpression of suc22(+), a ribonucleotide reductase gene, fully complemented the sporulation defect of cdt2 mutants and alleviated their growth defect at 19 degrees. These observations suggest that cdt2(+) plays an important role in DNA replication in both the mitotic and the meiotic life cycles of fission yeast.

The cation-transporting P-type ATPase Cta4 is required for assembly of the forespore membrane in fission yeast.

A novel sporulation-deficient mutant, sev4-L5, was isolated in a genetic screen of a collection of temperature-sensitive mutants of Schizosaccharomyces pombe. The wild-type sev4 gene was identified as cta4+, which encodes a putative cation-transporting P-type ATPase. The sev4-L5 allele harbored a single missense mutation that caused replacement of Gly615 with a glutamate at the putative ATP-binding site. Similar to cta4-null mutants, sev4-L5 exhibited defects in growth at high and low temperatures, and sensitivity to high and extremely low concentrations of Ca2+. The cta4+ mRNA level was considerably enhanced during meiosis. When sev4-L5 cells were incubated in sporulation medium at the permissive temperature, meiotic nuclear divisions proceeded with normal kinetics, but spores were not formed. Structural alteration of the spindle pole body, which is prerequisite to construction of the forespore membrane in wild type, was incomplete. Consequently, formation of the forespore membrane was severely impaired. These observations show that perturbation of Ca2+ homeostasis by mutation of cta4/sev4 blocks sporulation mainly by interfering with forespore membrane assembly.

Expression and radiation-induced phosphorylation of histone H2AX in mammalian cells.

The mouse histone H2AX (H2AX) has unique C-terminal Ser residues, which are phosphorylated in response to DNA double-strand breaks (DSBs) by ionizing radiation, suggesting that it plays a role in the maintenance of genomic stability. Here, we show that the H2AX protein was detected in most cells in various tissues, and was abundant in the S phase of the cell cycle. Following X-ray irradiation, H2AX was phosphorylated (gamma-H2AX) in the thymus, small intestine and testis. However, H2AX in epithelial cells in the villi of the small intestine were not strongly phosphorylated, even after X-irradiation. Thus, H2AX was expressed in almost all cells. However, the cells that expressed H2AX were not always phosphorylated by X-irradiation, suggesting a different mechanism of kination in those cells.