Involvement of Rev1 in alkylating agent-induced loss of heterozygosity in Oryzias latipes.

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C-terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C-terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild-type fish. There was no significant difference in the frequency of DENA-induced mutations between mutant with reduced dCMP transferase activity and wild-type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.

Coulomb and CH-π interactions in (6-4) photolyase-DNA complex dominate DNA binding and repair abilities.

(6-4) Photolyases ((6-4)PLs) are flavoenzymes that repair the carcinogenic UV-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts ((6-4)PPs), in a light-dependent manner. Although the reaction mechanism of DNA photorepair by (6-4)PLs has been intensively investigated, the molecular mechanism of the lesion recognition remains obscure. We show that a well-conserved arginine residue in Xenopus laevis (6-4)PL (Xl64) participates in DNA binding, through Coulomb and CH-π interactions. Fragment molecular orbital calculations estimated attractive interaction energies of -80-100 kcal mol-1 for the Coulomb interaction and -6 kcal mol-1 for the CH-π interaction, and the loss of either of them significantly reduced the affinity for (6-4)PP-containing oligonucleotides, as well as the quantum yield of DNA photorepair. From experimental and theoretical observations, we formulated a DNA binding model of (6-4)PLs. Based on the binding model, we mutated this Arg in Xl64 to His, which is well conserved among the animal cryptochromes (CRYs), and found that the CRY-type mutant exhibited reduced affinity for the (6-4)PP-containing oligonucleotides, implying the possible molecular origin of the functional diversity of the photolyase/cryptochrome superfamily.

Viable neuronopathic Gaucher disease model in Medaka (Oryzias latipes) displays axonal accumulation of alpha-synuclein.

Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.

Functional Conversion of CPD and (6-4) Photolyases by Mutation.

Ultraviolet (UV) light from the sun damages DNA by forming a cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) PP]. Photolyase (PHR) enzymes utilize near-UV/blue light for DNA repair, which is initiated by light-induced electron transfer from the fully reduced flavin adenine dinucleotide chromophore. Despite similar structures and repair mechanisms, the functions of PHR are highly selective; CPD PHR repairs CPD, but not (6-4) PP, and vice versa. In this study, we attempted functional conversion between CPD and (6-4) PHRs. We found that a triple mutant of (6-4) PHR is able to repair the CPD photoproduct, though the repair efficiency is 1 order of magnitude lower than that of wild-type CPD PHR. Difference Fourier transform infrared spectra for repair demonstrate the lack of secondary structural alteration in the mutant, suggesting that the triple mutant gains substrate binding ability while it does not gain the optimized conformational changes from light-induced electron transfer to the release of the repaired DNA. Interestingly, the (6-4) photoproduct is not repaired by the reverse mutation of CPD PHR, and eight additional mutations (total of 11 mutations) introduced into CPD PHR are not sufficient. The observed asymmetric functional conversion is interpreted in terms of a more complex repair mechanism for (6-4) repair, which was supported by quantum chemical/molecular mechanical calculation. These results suggest that CPD PHR may represent an evolutionary origin for photolyase family proteins.

Human papillomavirus and p53 mutations in head and neck squamous cell carcinoma among Japanese population.

We aimed to reveal the prevalence and pattern of human papillomavirus (HPV) infection and p53 mutations among Japanese head and neck squamous cell carcinoma (HNSCC) patients in relation to clinicopathological parameters. Human papillomavirus DNA and p53 mutations were examined in 493 HNSCCs and its subset of 283 HNSCCs. Oropharyngeal carcinoma was more frequently HPV-positive than non-oropharyngeal carcinoma (34.4% vs 3.6%, P < 0.001), and HPV16 accounted for 91.1% of HPV-positive tumors. In oropharyngeal carcinoma, which showed an increasing trend of HPV prevalence over time (P < 0.001), HPV infection was inversely correlated with tobacco smoking, alcohol drinking, p53 mutations, and a disruptive mutation (P = 0.003, <0.001, <0.001, and <0.001, respectively). The prevalence of p53 mutations differed significantly between virus-unrelated HNSCC and virus-related HNSCC consisting of nasopharyngeal and HPV-positive oropharyngeal carcinomas (48.3% vs 7.1%, P < 0.001). Although p53 mutations were associated with tobacco smoking and alcohol drinking, this association disappeared in virus-unrelated HNSCC. A disruptive mutation was never found in virus-related HNSCC, whereas it was independently associated with primary site, such as the oropharynx and hypopharynx (P = 0.01 and 0.03, respectively), in virus-unrelated HNSCC. Moreover, in virus-unrelated HNSCC, G:C to T:A transversions were more frequent in ever-smokers than in never-smokers (P = 0.04), whereas G:C to A:T transitions at CpG sites were less frequent in ever-smokers than in never-smokers (P = 0.04). In conclusion, HNSCC is etiologically classified into virus-related and virus-unrelated subgroups. In virus-related HNSCC, p53 mutations are uncommon with the absence of a disruptive mutation, whereas in virus-unrelated HNSCC, p53 mutations are common, and disruptive mutagenesis of p53 is related with oropharyngeal and hypopharyngeal carcinoma.

p53-Dependent suppression of genome instability in germ cells.

Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2(-/-) males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2(-/-) and wild-type fish. By contrast, irradiated p53(-/-) fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2(-/-) fish, but negligible levels in p53(-/-) fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

Involvement of stress kinase mitogen-activated protein kinase kinase 7 in regulation of mammalian circadian clock.

The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes, such as cell proliferation, apoptosis, differentiation, and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of an MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1ε. Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.

Mitotic slippage underlies the relationship between p53 dysfunction and the induction of large micronuclei by colcemid.

Micronuclei induced by aneugens are larger than those induced by clastogens in both in vitro and in vivo micronucleus (MN) assays. p53 dysfunction increases the formation of large micronuclei following treatment with aneugens; this study sought to identify the mechanisms responsible for this. Treatment with colcemid, both a mitotic inhibitor and an aneugen, induced MN containing two or more chromosomes more frequently in NH32 cells, in which p53 function is compromised, than in TK6 cells, in which p53 is functional. This indicates that p53 dysfunction enhances aneugen-induced chromosome loss or perturbs apoptosis, resulting in the formation of large MN. To examine the former hypothesis, the incidence of chromosome malsegregation in colcemid-treated TK6 and NH32 cells was compared using the cytokinesis-block MN assay. The incidence of chromosome non-disjunction was higher in NH32 cells than in TK6 cells, whereas the incidence of MN containing two or more chromosomes was similar between the two cell lines. To address the involvement of apoptosis in cell cycle progression, examination of chromosome 8 distribution revealed that more mononuclear NH32 than TK6 cells were tetraploid after prolonged mitotic inhibition, which indicated that the more number of NH32 cells may have bypassed the spindle assembly checkpoint via mitotic slippage and progression into the next interphase. Cells that underwent mitotic slippage were likely to contain lagging chromosomes formed via chromosome malsegregation, resulting in MN separated from the main nucleus. The number of TK6 cells containing large MN following colcemid treatment was increased by treatment with a caspase inhibitor in a dose-dependent manner, indicating that TK6 cells with MN normally undergo apoptosis. In conclusion, these findings indicate that mitotic slippage and perturbed apoptosis contribute to the induction of large MN in p53-compromised cells following treatment with colcemid.

Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka.

DMRT1, which is found in many vertebrates, exhibits testis-specific expression during the sexual differentiation period, suggesting a conserved function of DMRT1 in the testicular development of vertebrate gonads. However, functional analyses have been reported only in mammals. The current study focused on the Dmrt1 function in the teleost medaka, Oryzias latipes, which has an XX-XY sex determination system. Although medaka sex is determined by the presence or absence of the Y chromosome-specific gene Dmy, we demonstrated that in one Dmrt1 mutant line, which was found by screening a gene-driven mutagenesis library, XY mutants developed into normal females and laid eggs. Histological analyses of this mutant revealed that the XY mutant gonads first developed into the normal testis type. However, the gonads transdifferentiated into the ovary type. The mutant phenotype could be rescued by transgenesis of the Dmrt1 genomic region. These results show that Dmrt1 is essential to maintain testis differentiation after Dmy-triggered male differentiation pathway.

Circadian clock-controlled diurnal oscillation of Ras/ERK signaling in mouse liver.

Accumulating evidence indicates that ERK MAP kinase signaling plays an important role in the regulation of the circadian clock, especially in the clock-resetting mechanism in the suprachiasmatic nucleus (SCN) in mammals. Previous studies have also shown that ERK phosphorylation exhibits diurnal variation in the SCN. However, little is known about circadian regulation of ERK signaling in peripheral tissues. Here we show that the activity of Ras/ERK signaling exhibits circadian rhythms in mouse liver. We demonstrate that Ras activation, MEK phosphorylation, and ERK phosphorylation oscillate in a circadian manner. As the oscillation of ERK phosphorylation is lost in Cry1/Cry2 double-knockout mice, Ras/ERK signaling should be under the control of the circadian clock. Furthermore, expression of MAP kinase phosphatase-1 (Mkp-1) shows diurnal changes in liver. These results indicate that Ras/ERK signaling is strictly regulated by the circadian clock in liver, and suggest that the circadian oscillation of the activities of Ras, MEK, and ERK may regulate diurnal variation of liver function and/or homeostasis.(Communicated by Shigekazu NAGATA, M.J.A.).

Infrared laser-mediated gene induction in targeted single cells in vivo.

We developed infrared laser-evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock-mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses.

Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes.

Homologous flavoproteins from the photolyase (PHR)/cryptochrome (CRY) family use the FAD cofactor in PHRs to catalyze DNA repair and in CRYs to tune the circadian clock and control development. To help address how PHR/CRY members achieve these diverse functions, we determined the crystallographic structure of Arabidopsis thaliana (6-4) PHR (UVR3), which is strikingly (>65%) similar in sequence to human circadian clock CRYs. The structure reveals a substrate-binding cavity specific for the UV-induced DNA lesion, (6-4) photoproduct, and cofactor binding sites different from those of bacterial PHRs and consistent with distinct mechanisms for activities and regulation. Mutational analyses were combined with this prototypic structure for the (6-4) PHR/clock CRY cluster to identify structural and functional motifs: phosphate-binding and Pro-Lys-Leu protrusion motifs constricting access to the substrate-binding cavity above FAD, sulfur loop near the external end of the Trp electron-transfer pathway, and previously undefined C-terminal helix. Our results provide a detailed, unified framework for investigations of (6-4) PHRs and the mammalian CRYs. Conservation of key residues and motifs controlling FAD access and activities suggests that regulation of FAD redox properties and radical stability is essential not only for (6-4) photoproduct DNA repair, but also for circadian clock-regulating CRY functions. The structural and functional results reported here elucidate archetypal relationships within this flavoprotein family and suggest how PHRs and CRYs use local residue and cofactor tuning, rather than larger structural modifications, to achieve their diverse functions encompassing DNA repair, plant growth and development, and circadian clock regulation.

3D reconstructed brain images reveal the possibility of the ogg1 gene to suppress the irradiation-induced apoptosis in embryonic brain in medaka (Oryzias latipes).

The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

FTIR study of light-dependent activation and DNA repair processes of (6-4) photolyase.

The UV component of sunlight threatens all life on the earth by damaging DNA. The photolyase (PHR) DNA repair proteins maintain genetic integrity by harnessing blue light to restore intact bases from the major UV-induced photoproducts, cyclobutane pyrimidine dimers (CPD), and (6-4) photoproducts ((6-4) PPs). The (6-4) PHR must catalyze not only covalent bond cleavage between two pyrmidine bases but also hydroxyl or amino group transfer from the 5'- to 3'-pyrimidine base, requiring a more complex mechanism than that postulated for CPD PHR. In this paper, we apply Fourier transform infrared (FTIR) spectroscopy to (6-4) PHR and report difference FTIR spectra that correspond to its photoactivation, substrate binding, and light-dependent DNA repair processes. The presence of DNA carrying a single (6-4) PP uniquely influences vibrations of the protein backbone and a protonated carboxylic acid, whereas photoactivation produces IR spectral changes for the FAD cofactor and the surrounding protein. Difference FTIR spectra for the light-dependent DNA damage repair reaction directly show significant DNA structural changes in the (6-4) lesion and the neighboring phosphate group. Time-dependent illumination of samples with different enzyme:substrate stoichiometries successfully distinguished signals characteristic of structural changes in the protein and the DNA resulting from binding and catalysis.

Light-induced conformational change and product release in DNA repair by (6-4) photolyase.

Proteins of the cryptochrome/photolyase family share high sequence similarities, common folds, and the flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse physiological functions. Mammalian cryptochromes are essential regulatory components of the 24 h circadian clock, whereas (6-4) photolyases recognize and repair UV-induced DNA damage by using light energy absorbed by FAD. Despite increasing knowledge about physiological functions from genetic analyses, the molecular mechanisms and conformational dynamics involved in clock signaling and DNA repair remain poorly understood. The (6-4) photolyase, which has strikingly high similarity to human clock cryptochromes, is a prototypic biological system to study conformational dynamics of cryptochrome/photolyase family proteins. The entire light-dependent DNA repair process for (6-4) photolyase can be reproduced in a simple in vitro system. To decipher pivotal reactions of the common FAD cofactor, we accomplished time-resolved measurements of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6-4) photolyase on DNA carrying a single UV-induced damage. The (6-4) photolyase by itself showed significant volume changes after blue-light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change was observed only in the presence of both (6-4) photolyase and damaged DNA, and not for (6-4) photolyase alone or with undamaged DNA. Thus, we propose that this diffusion change reflects the rapid (50 µs time constant) dissociation of the protein from the repaired DNA product. Conformational changes with such fast turnover would likely enable DNA repair photolyases to access the entire genome in cells.

Loss of circadian rhythm and light-induced suppression of pineal melatonin levels in Cry1 and Cry2 double-deficient mice.

Cryptochrome 1 and 2 (Cry1 and Cry2) are considered essential for generating circadian rhythms in mammals. The role of Cry1 and Cry2 in circadian rhythm expression and acute light-induced suppression of pineal melatonin was assessed using Cry1 and Cry2 double-deficient mice (Cry1(-/-) /Cry2(-/-) ) developed from the C3H strain that synthesizes melatonin. We examined the circadian variation of pineal melatonin under a 12:12-h light-dark (LD) cycle and constant darkness (DD). Light suppression of pineal melatonin concentration was analyzed by subjecting a 30-min light pulse at the peak phase of melatonin concentration. Wild-type mice showed significant rhythmicity in pineal melatonin concentration with the highest level at Zeitgeber time 22 (ZT22, where time of light on was defined as ZT0) under LD or ZT18 on the first day of DD. In contrast, Cry1(-/-) /Cry2(-/-) mice did not show significant circadian rhythmicity, with only a small peak observed at ZT22 in LD. Nevertheless, a significant daily variation could be observed under DD, with a small increase at ZT6 and ZT18 h. Melatonin concentration was significantly suppressed by acute light pulse at ZT22 in wild-type mice but not in Cry1(-/-) /Cry2(-/-) mice. The present results suggest that Cry genes are required for regulating pineal melatonin synthesis via circadian and photic signals from the suprachiasmatic nucleus of the hypothalamus (SCN).

Diatom PtCPF1 is a new cryptochrome/photolyase family member with DNA repair and transcription regulation activity.

Members of the cryptochrome/photolyase family (CPF) are widely distributed throughout all kingdoms, and encode photosensitive proteins that typically show either photoreceptor or DNA repair activity. Animal and plant cryptochromes have lost DNA repair activity and now perform specialized photoperceptory functions, for example, plant cryptochromes regulate growth and circadian rhythms, whereas mammalian and insect cryptochromes act as transcriptional repressors that control the circadian clock. However, the functional differentiation between photolyases and cryptochromes is now being questioned. Here, we show that the PtCPF1 protein from the marine diatom Phaeodactylum tricornutum shows 6-4 photoproduct repair activity and can act as a transcriptional repressor of the circadian clock in a heterologous mammalian cell system. Conversely, it seems to have a wide role in blue-light-regulated gene expression in diatoms. The protein might therefore represent a missing link in the evolution of CPFs, and act as a novel ultraviolet/blue light sensor in marine environments.

A rapid and easy method for the qualitative detection of intracellular deposition of inhaled nanoparticles.

In recent years, nanoparticle exposure risk has drawn increasing attention from the research community and the general public. However, analysis of nanoparticles is hindered by their small size, which prevents the development of methods for their detection in cells and tissues. For risk assessment of nanoparticle exposure, it is important to measure the exact amount of deposited material in pulmonary tissue. Using a nanoparticle exposure device, A/JJmsSlc mice were chronically exposed transtracheally to anatase-type titanium dioxide particles. A microscope-integrated laser Raman spectrometer was used to detect differentially stained macrophages in a pulmonary wash obtained from the mice exposed to the particles. This detection method allowed rapid and easy sample collection and qualitative analysis, and the method may be useful for conducting large-scale evaluations in workers exposed to environments heavily contaminated with nanoparticles. FROM THE CLINICAL EDITOR: This paper discusses a microscope-integrated laser Raman spectrometer method to measure the exact amount of nanoparticles deposited in pulmonary tissue. This method allows rapid sample collection, qualitative analysis, and may be useful for large-scale evaluations.

Estrogen receptor 2b is the major determinant of sex-typical mating behavior and sexual preference in medaka.

Male and female animals typically display innate sex-specific mating behaviors, which, in vertebrates, are highly dependent on sex steroid signaling. While estradiol-17ß (E2) signaling through estrogen receptor 2 (ESR2) serves to defeminize male mating behavior in rodents, the available evidence suggests that E2 signaling is not required in teleosts for either male or female mating behavior. Here, we report that female medaka deficient for Esr2b, a teleost ortholog of ESR2, are not receptive to males but rather court females, despite retaining normal ovarian function with an unaltered sex steroid milieu. Thus, contrary to both prevailing views in rodents and teleosts, E2/Esr2b signaling in the brain plays a decisive role in feminization and demasculinization of female mating behavior and sexual preference in medaka. Further behavioral testing showed that mutual antagonism between E2/Esr2b signaling and androgen receptor-mediated androgen signaling in adulthood induces and actively maintains sex-typical mating behaviors and preference. Our results also revealed that the female-biased sexual dimorphism in esr2b expression in the telencephalic and preoptic nuclei implicated in mating behavior can be reversed between males and females by altering the sex steroid milieu in adulthood, likely via mechanisms involving direct E2-induced transcriptional activation. In addition, Npba, a neuropeptide mediating female sexual receptivity, was found to act downstream of E2/Esr2b signaling in these brain nuclei. Collectively, these functional and regulatory mechanisms of E2/Esr2b signaling presumably underpin the neural mechanism for induction, maintenance, and reversal of sex-typical mating behaviors and sexual preference in teleosts, at least in medaka.

High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library.

BACKGROUND: During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective. RESULTS: In this study, we developed a high resolution melting (HRM) assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature. CONCLUSIONS: These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.