Continuous Cell Characterization and Separation by Microfluidic Alternating Current Dielectrophoresis.

A novel alternating current (ac)-dielectrophoretic (DEP) microfluidic chip for continuous cell characterization and separation is presented in this paper. To generate DEP forces, two electrode-pads are embedded in a set of asymmetric orifices on the opposite sidewalls to produce the nonuniform electric fields. In the vicinity of a small orifice, the cells experience the strongest nonuniform gradient and are drawn toward it by the positive DEP forces, while the cells experiencing a negative DEP force are repelled away and move toward the large orifice. The DEP behaviors of yeast cells in suspending media with different ionic concentrations, i.e., different electrical conductivities, and over a large range of the ac electric field frequency were investigated. Furthermore, the lateral migrations of yeast cells as a function of the ac frequency were measured. The trends of measured lateral migrations of yeast cells are similar to the corresponding Clausius-Mossotti (CM) factors. In addition, by adjusting the frequency and strength of the ac electric field, the continuous separation of live and dead yeast cells as well as the yeast cells with targeted diameter and dielectric property can be easily achieved. This is the first time that the measurement of ac-DEP lateral migration of yeast cells in solutions with different electrical conductivities as a function of the applied frequency in a microfluidic chip was reported. This ac-DEP system provides a method to characterize the crossover frequency of the specific cells and manipulate the targeted cells.

A novel non-canonical forkhead-associated (FHA) domain-binding interface mediates the interaction between Rad53 and Dbf4 proteins.

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.

Dbf4 and Cdc7 proteins promote DNA replication through interactions with distinct Mcm2-7 protein subunits.

The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2-7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2-7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2-7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2ΔDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4ΔDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4ΔDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.

An acidic loop in the forkhead-associated domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at double-strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover-specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a 5-amino acid sequence, RPSKR, located between the DNA-binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full-length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a noncanonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt 2-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint and, in certain circumstances, exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

An acidic loop in the FHA domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at the double strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a five amino acid sequence, RPSKR, located between the DNA binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a non-canonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt two-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint, and in certain circumstances exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

Saccharomyces cerevisiae Dbf4 has unique fold necessary for interaction with Rad53 kinase.

Dbf4 is a conserved eukaryotic protein that functions as the regulatory subunit of the Dbf4-dependent kinase (DDK) complex. DDK plays essential roles in DNA replication initiation and checkpoint activation. During the replication checkpoint, Saccharomyces cerevisiae Dbf4 is phosphorylated in a Rad53-dependent manner, and this, in turn, inhibits initiation of replication at late origins. We have determined the minimal region of Dbf4 required for the interaction with the checkpoint kinase Rad53 and solved its crystal structure. The core of this fragment of Dbf4 folds as a BRCT domain, but it includes an additional N-terminal helix unique to Dbf4. Mutation of the residues that anchor this helix to the domain core abolish the interaction between Dbf4 and Rad53, indicating that this helix is an integral element of the domain. The structure also reveals that previously characterized Dbf4 mutants with checkpoint phenotypes destabilize the domain, indicating that its structural integrity is essential for the interaction with Rad53. Collectively, these results allow us to propose a model for the association between Dbf4 and Rad53.

Proteomic profiles of white sucker (Catostomus commersonii) sampled from within the Thunder Bay Area of Concern reveal up-regulation of proteins associated with tumor formation and exposure to environmental estrogens.

White sucker (Catostomus commersonii) sampled from the Thunder Bay Area of Concern were assessed for health using a shotgun approach to compile proteomic profiles. Plasma proteins were sampled from male and female fish from a reference location, an area in recovery within Thunder Bay Harbour, and a site at the mouth of the Kaministiquia River where water and sediment quality has been degraded by industrial activities. The proteins were characterized using reverse-phase liquid chromatography tandem to a quadrupole-time-of-flight (LC-Q-TOF) mass spectrometer and were identified by searching in peptide databases. In total, 1086 unique proteins were identified. The identified proteins were then examined by means of a bioinformatics pathway analysis to gain insight into the biological functions and disease pathways that were represented and to assess whether there were any significant changes in protein expression due to sampling location. Female white sucker exhibited significant (p = 0.00183) site-specific changes in the number of plasma proteins that were related to tumor formation, reproductive system disease, and neurological disease. Male fish plasma had a significantly different (p < 0.0001) number of proteins related to neurological disease and tumor formation. Plasma concentrations of vitellogenin were significantly elevated in females from the Kaministiquia River compared to the Thunder Bay Harbour and reference sites. The protein expression profiles indicate that white sucker health has benefited from the remediation of the Thunder Bay Harbour site, whereas white sucker from the Kaministiquia River site are impacted by ongoing contaminant discharges.

Differential chromatin proteomics of the MMS-induced DNA damage response in yeast.

BACKGROUND: Protein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. Comparisons of whole-cell extracts and chromatin fractions were used to provide a measure of the degree of chromatin association for individual proteins, which could be compared across sample treatments. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins. RESULTS: Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity using either DAmP (decreased abundance by mRNA perturbation) or knockout strains, including Acf2, Arp3, Bmh1, Hsp31, Lsp1, Pst2, Rnr4, Rpa1, Rpa2, Ste4, Ycp4 and Yrb1. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress. CONCLUSION: The described method was effective at unveiling chromatin-associated proteins that are less likely to be detected in the absence of fractionation. Several novel proteins with altered chromatin abundance were identified including Yrb1, pointing to a role for this nuclear import associated protein in DNA damage response.

Crystallization and preliminary X-ray diffraction analysis of motif N from Saccharomyces cerevisiae Dbf4.

The Cdc7-Dbf4 complex plays an instrumental role in the initiation of DNA replication and is a target of replication-checkpoint responses in Saccharomyces cerevisiae. Cdc7 is a conserved serine/threonine kinase whose activity depends on association with its regulatory subunit, Dbf4. A conserved sequence near the N-terminus of Dbf4 (motif N) is necessary for the interaction of Cdc7-Dbf4 with the checkpoint kinase Rad53. To understand the role of the Cdc7-Dbf4 complex in checkpoint responses, a fragment of Saccharomyces cerevisiae Dbf4 encompassing motif N was isolated, overproduced and crystallized. A complete native data set was collected at 100 K from crystals that diffracted X-rays to 2.75 A resolution and structure determination is currently under way.

A mutation in Dbf4 motif M impairs interactions with DNA replication factors and confers increased resistance to genotoxic agents.

Dbf4/Cdc7 is required for DNA replication in Saccharomyces cerevisiae and appears to be a target in the S-phase checkpoint. Previously, a 186-amino-acid Dbf4 region that mediates interactions with both the origin recognition complex and Rad53 was identified. We now show this domain also mediates the association between Dbf4 and Mcm2, a key Dbf4/Cdc7 phosphorylation target. Two conserved sequences, the N and M motifs, have been identified within this Dbf4 region. Removing motif M (Dbf4DeltaM) impairs the ability of Dbf4 to support normal cell cycle progression and abrogates the Dbf4-Mcm2 association but has no effect on the Dbf4-Rad53 interaction. In contrast, deleting motif N (Dbf4DeltaN) does not affect the essential function of Dbf4, disrupts the Dbf4-Rad53 interaction, largely preserves the Dbf4-Mcm2 association, and renders the cells hypersensitive to genotoxic agents. Surprisingly, Dbf4DeltaM interacts strongly with Orc2, while Dbf4DeltaN does not. The DBF4 allele dna52-1 was cloned and sequenced, revealing a single point mutation within the M motif. This mutant is unable to maintain interactions with either Mcm2 or Orc2 at the semipermissive temperature of 30 degrees C, while the interaction with Rad53 is preserved. Furthermore, this mutation confers increased resistance to genotoxic agents, which we propose is more likely due to a role for Dbf4 in the resumption of fork progression following checkpoint-induced arrest than prevention of late origin firing. Thus, the alteration of the M motif may facilitate the role of Dbf4 as a checkpoint target.

The characterization of γH2AX and p53 as biomarkers of genotoxic stress in a rainbow trout (Oncorhynchus mykiss) brain cell line.

Rainbow trout cell cultures were exposed to three genotoxicants and examined for effects on γH2AX and p53 levels by western blotting and on cell viability using the indicator dyes Alamar Blue (AB) for energy metabolism and 5'-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for plasma membrane integrity. Bleomycin induced γH2AX and p53 in a dose- and time-dependent manner and had little cytotoxic effect. However, induction was first seen at 0.3 µM for γH2AX but not until 16.5 µM for p53. Methyl methanesulfonate (MMS) increased H2AX phosphorylation but diminished p53 levels as the dose was increased from 908 µM up to 2724 µM. Over this dose range cell viability was progressively lost. 4-nitroquinoline N-oxide (NQO) induced both γH2AX and p53, beginning at 62.5 nM, which was also the concentration at which cell viability began to decline. As the NQO concentration increased further, elevated γH2AX was detected at up to 2.0 µM, while p53 was elevated up to 1.0 µM. Therefore, H2AX phosphorylation was superior to p53 levels as a marker of DNA damage caused by genotoxicants that act by introducing double-stranded DNA breaks (bleomycin), alkyl groups (MMS), and quinoline adducts (NQO).

Mechanisms Governing DDK Regulation of the Initiation of DNA Replication.

The budding yeast Dbf4-dependent kinase (DDK) complex-comprised of cell division cycle (Cdc7) kinase and its regulatory subunit dumbbell former 4 (Dbf4)-is required to trigger the initiation of DNA replication through the phosphorylation of multiple minichromosome maintenance complex subunits 2-7 (Mcm2-7). DDK is also a target of the radiation sensitive 53 (Rad53) checkpoint kinase in response to replication stress. Numerous investigations have determined mechanistic details, including the regions of Mcm2, Mcm4, and Mcm6 phosphorylated by DDK, and a number of DDK docking sites. Similarly, the way in which the Rad53 forkhead-associated 1 (FHA1) domain binds to DDK-involving both canonical and non-canonical interactions-has been elucidated. Recent work has revealed mutual promotion of DDK and synthetic lethal with dpb11-1 3 (Sld3) roles. While DDK phosphorylation of Mcm2-7 subunits facilitates their interaction with Sld3 at origins, Sld3 in turn stimulates DDK phosphorylation of Mcm2. Details of a mutually antagonistic relationship between DDK and Rap1-interacting factor 1 (Rif1) have also recently come to light. While Rif1 is able to reverse DDK-mediated Mcm2-7 complex phosphorylation by targeting the protein phosphatase glycogen 7 (Glc7) to origins, there is evidence to suggest that DDK can counteract this activity by binding to and phosphorylating Rif1.

The p53 inhibitor, pifithrin-α, disrupts microtubule organization, arrests growth, and induces polyploidy in the rainbow trout gill cell line, RTgill-W1.

Pifithrin-α (PFT-α) blocks p53-dependent transcription and is an example of the many drugs being developed to target the p53 pathway in humans that could be released into the environment with potential impacts on aquatic animals if they were to become successful pharmaceuticals. In order to understand how p53 drugs might act on fish, the effects of PFT-α on rainbow trout gill epithelial cell line, RTgill-W1, were studied. PFT-α was not cytotoxic to RTgill-W1 in cultures with or without fetal bovine serum (FBS), but at 5.25µg/ml, PFT-α completely arrested proliferation. When FBS was present, PFT-α increased the number of polyploid cells over 12days. Those results suggest that like in mammals, p53 appears to regulate ploidy in fish. However, several effects were seen that have not been observed with mammalian cells. PFT-α caused a transient rise in the mitotic index and a disruption in cytoskeletal microtubules. These results suggest that in fish cells PFT-α affects microtubules either directly through an off-target action on tubulin or indirectly through an on-target action on p53-regulated transcription.

'AND' logic gates at work: Crystal structure of Rad53 bound to Dbf4 and Cdc7.

Forkhead-associated (FHA) domains are phosphopeptide recognition modules found in many signaling proteins. The Saccharomyces cerevisiae protein kinase Rad53 is a key regulator of the DNA damage checkpoint and uses its two FHA domains to interact with multiple binding partners during the checkpoint response. One of these binding partners is the Dbf4-dependent kinase (DDK), a heterodimer composed of the Cdc7 kinase and its regulatory subunit Dbf4. Binding of Rad53 to DDK, through its N-terminal FHA (FHA1) domain, ultimately inhibits DDK kinase activity, thereby preventing firing of late origins. We have previously found that the FHA1 domain of Rad53 binds simultaneously to Dbf4 and a phosphoepitope, suggesting that this domain functions as an 'AND' logic gate. Here, we present the crystal structures of the FHA1 domain of Rad53 bound to Dbf4, in the presence and absence of a Cdc7 phosphorylated peptide. Our results reveal how the FHA1 uses a canonical binding interface to recognize the Cdc7 phosphopeptide and a non-canonical interface to bind Dbf4. Based on these data we propose a mechanism to explain how Rad53 enhances the specificity of FHA1-mediated transient interactions.

Multiple roles of replication forks in S phase checkpoints: sensors, effectors and targets.

There is mounting evidence that replication defects are the major source of spontaneous genomic instability in the cell and that S phase checkpoints are the principle defense against such instability. In Saccharomyces cerevisiae, S phase checkpoints can be provoked by either depletion of dNTPs or DNA damage. In both cases the checkpoint kinases Mec1 and Rad53 act to suppress late origin firing, stabilize slowed or stalled replication forks and prevent S phase progression until conditions are appropriate for the resumption of DNA replication. The present review highlights recent work emphasizing the central importance of replication forks, not just as targets, but also as sensors and primary effectors of checkpoint responses, and identifies the roles played by specific fork-associated factors in these processes.

Omics for aquatic ecotoxicology: control of extraneous variability to enhance the analysis of environmental effects.

There are multiple sources of biological and technical variation in a typical ecotoxicology study that may not be revealed by traditional endpoints but that become apparent in an omics dataset. As researchers increasingly apply omics technologies to environmental studies, it will be necessary to understand and control the main source(s) of variability to facilitate meaningful interpretation of such data. For instance, can variability in omics studies be addressed by changing the approach to study design and data analysis? Are there statistical methods that can be employed to correctly interpret omics data and make use of unattributed, inherent variability? The present study presents a review of experimental design and statistical considerations applicable to the use of omics methods in systems toxicology studies. In addition to highlighting potential sources that contribute to experimental variability, this review suggests strategies with which to reduce and/or control such variability so as to improve reliability, reproducibility, and ultimately the application of omics data for systems toxicology.

ORC-associated replication factors as biomarkers for cancer.

Early detection and treatment of cancer are of central importance to improving patient prognoses. Traditional biomarkers of cell proliferation, such as Ki-67 and PCNA, have had a mixed clinical track record, proving to be good indicators of certain types of cancers but of limited use for many others. Recently, human counterparts of replication factors originally identified in budding yeast have shown great promise as new cancer biomarkers. Each of these factors has been shown to interact with the origin recognition complex (ORC) in yeast, and each has an essential role in the initiation of DNA replication. Studies with minichromosome maintenance (MCM) family proteins show that their levels are upregulated in tumor cells and are much better indicators of a wide variety of cancers than traditional biomarkers. Similarly encouraging results have been obtained in preliminary studies examining Cdc6 protein and Cdc7 kinase transcript levels in normal and cancerous cells.

Cdc7 kinases (DDKs) and checkpoint responses: lessons from two yeasts.

Principally characterized for its requirement in the initiation of DNA replication, compelling evidence from two yeast model organisms now points to a central role for the Dbf4/Cdc7 kinase complex in S-phase checkpoint responses. Among the key findings supporting this view are observations that orthologs Dfp1 (Schizosaccharomyces pombe) and Dbf4 (Saccharomyces cerevisiae) interact with equivalent checkpoint kinases Cds1 and Rad53, respectively, and that mutants for Dbf4 and Cdc7 in these species are sensitive to genotoxic agents. Recently, these findings have been extended through mutational analyses of conserved regions in both Dfp1 and Dbf4, leading to the identification of distinct motifs which mediate cellular responses to DNA damage and replication fork arrest. The present review is a comparative survey of data obtained from studies conducted with S. pombe and S. cerevisae, and a consideration of models for the role played by Dbf4/Cdc7 in checkpoint responses.

The p53/HSP70 inhibitor, 2-phenylethynesulfonamide, causes oxidative stress, unfolded protein response and apoptosis in rainbow trout cells.

The effect of 2-phenylethynesulfonamide (PES), which is a p53 and HSP70 inhibitor in mammalian cells, was studied on the rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1, in order to evaluate PES as a tool for understanding the cellular survival pathways operating in fish. As judged by three viability assays, fish cells were killed by 24h exposures to PES, but cell death was blocked by the anti-oxidant N-acetylcysteine (NAC). Cell death had several hallmarks of apoptosis: DNA laddering, nuclear fragmentation, Annexin V staining, mitochondrial membrane potential decline, and caspases activation. Reactive oxygen species (ROS) production peaked in several hours after the addition of PES and before cell death. HSP70 and BiP levels were higher in cultures treated with PES for 24h, but this was blocked by NAC. As well, PES treatment caused HSP70, BiP and p53 to accumulate in the detergent-insoluble fraction, and this too was prevented by NAC. Of several possible scenarios to explain the results, the following one is the simplest. PES enhances the generation of ROS, possibly by inhibiting the anti-oxidant actions of p53 and HSP70. ER stress arises from the ROS and from PES inhibiting the chaperone activities of HSP70. The ER stress in turn initiates the unfolded protein response (UPR), but this fails to restore ER homeostasis so proteins aggregate and cells die. Despite these multiple actions, PES should be useful for studying fish cellular survival pathways.

The rad1 gene in Rainbow Trout (Oncorhynchus mykiss) is highly conserved and may express proteins from non-canonical spliced isoforms.

Cell-cycle checkpoint proteins maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. RAD1 is a checkpoint protein involved in the sensing of damaged DNA and is a part of the 9-1-1 complex. In this project rainbow trout rad1 (rtrad1) was cloned, sequenced, expressed as a recombinant protein and anti-rtRAD1 antibodies were developed. RAD1 protein levels were characterized in various rainbow trout tissues. It was determined that an 840 bp open-reading frame encodes 279 aa with a predicted protein size of 31 kDa. The rtRAD1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify three non-canonical splice variants of rtrad1, two of which are capable of forming functional proteins. The rad1 splice variant that encodes an 18 kDa protein appears to be abundant in rainbow trout spleen, heart and gill tissue and in the RTgill-W1 cell-line. Based on the genomic rtrad1 sequence the splice variants contain only partial exons which are consistent with the splicing of rad1 variants in mammals. This is the first time that rad1 has been fully characterized in a fish species.