Saccharomyces cerevisiae, we performed a comparative study and found that Tsa1 was distinguished from the other Prxs in that by itself it played a key role in maintaining genome stability and in sustaining aerobic viability of rad51 mutants that are deficient in recombinational repair. Tsa2 and Dot5 played minor but distinct roles in suppressing the accumulation of mutations in cooperation with Tsa1. Tsa2 was capable of largely complementing the absence of Tsa1 when expressed under the control of the Tsa1 promoter. The presence of peroxidatic cysteine (Cys47) was essential for Tsa1 activity, while Tsa1C170S lacking the resolving Cys was partially functional. In the absence of Tsa1 activity (tsa1 or CCStsa1 lacking the peroxidatic and resolving Cys) and recombinational repair (rad51), dying cells displayed irregular cell size/shape, abnormal cell cycle progression, and significant increase of phosphatidylserine externalization, an early marker of apoptosis-like cell death. The CCS rad51tsa1– or tsa1 rad51–induced cell death did not depend on the caspase Yca1 and Ste20 kinase, while the absence of the checkpoint protein Rad9 accelerated the cell death processes. These results indicate that the peroxiredoxin Tsa1, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect S. cerevisiae cells against toxic levels of DNA damage that occur during aerobic growth.Peroxiredoxins (Prxs) constitute a family of thiol-specific peroxidases that utilize cysteine (Cys) as the primary site of oxidation during the reduction of peroxides. To gain more insight into the physiological role of the five Prxs in budding yeast  S. cerevisiae, we performed a comparative study and found that one, Tsa1, played a key role in preventing DNA damage and assuring genome stability. Tsa1 also cooperated with other peroxiredoxins in antioxidant defense. These functions of Tsa1 required the presence of a cysteine at the catalytic site of this enzyme. Additional studies revealed that Tsa1 activity, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect cells against toxic levels of DNA damage that occur during aerobic growth.Aerobically growing cells are continuously challenged by potent oxidants produced during normal cellular metabolism. These oxidants, including hydrogen peroxide and organic peroxides, are important components mediating various cell functions. However, they can also cause cell damage when present at toxic levels. Aerobic organisms possess extensive antioxidant systems to regulate oxidant levels. Among these, peroxiredoxins have received considerable attention in recent years as an expanding protein family involved in the enzymatic degradation of hydrogen peroxide and organic peroxides. To better understand the physiological role of the five peroxiredoxins in budding yeast  Saccharomyces cerevisiae and six in human cells 2O2 or alkyl hydroperoxides Escherichia coli AhpC, which uses another protein, AhpF, for the regeneration of the reduced Prx 2O2, some Prxs suffer an oxidation of the peroxidatic Cys to sulfinic acid, which temporarily inactivates these enzymes. This hyperoxidation of 2-Cys Prx enzymes is reversible in cells by sulfiredoxin, an ATP-dependent reductases for Cys-sulfinic acid in Prxs Peroxiredoxins (Prxs), previously termed the thioredoxin peroxidases, have received considerable attention in recent years as a new and expanding family of thiol-specific antioxidant proteins S. cerevisiae Tsa1 was the first characterized Prx, followed by Aph1 (cTpxIII) S. cerevisiae Prxs have different sub-cellular localizations: Tsa1, Tsa2 and Ahp1 are localized in the cytoplasm; Prx1 is localized in mitochondria; and Dot5 is localized in the nucleus 2O2 rather than alkyl hydroperoxide, whereas Ahp1 and Dot5 showed the reverse selectivity The five PRX genes are also quite different from each other. There are discrepancies in the reported relative order of mRNA abundance; however, most studies show that TSA1 mRNA is the most abundant PRX mRNA at different growth stages, followed by AHP15 molecules per log phase cell, higher than Ahp1 which is present at approximately 1.6×104 molecules per log phase cell and much higher than Tsa2, Prx1 and Dot5 which are present at about 1.8–4.8×103 molecules per log phase cell PRX genes are usually upregulated to various degrees in response to treatment with various oxidants and TSA2 is the most strikingly upregulated of the PRX genes TSA1 gene The transcriptional levels of the PRX genes only play a minor role in the suppression of genome instability, but their deletion increases the spontaneous mutation frequency of a tsa1 mutant rad51 or rad52, or the Rad6-mediated post-replication repair pathway including rad6, cause tsa1 cells to be very sick or to die rapidly under aerobic conditions but not anaerobic conditions, emphasizing the importance of Tsa1 for genome protection tsa1 mutants exhibit reduced clearance of reactive oxygen species (ROS) induced by exogenous treatment with H2O2S. cerevisiae cells begin to use fatty acids as energy sources S. cerevisiae Prxs are only known to have stress-protective functions in contrast to the unique Schizosaccharomyces pombe Prx, Tpx1, which is also involved in H2O2 signaling TSA1, TSA2, AHP1, PRX1 and DOT5 are all deleted are still viable S. pombe mutant lacking Tpx1 S. cerevisiae Prx plays a specific role, defined by its cellular location, substrate specificity and expression pattern. On the other hand, different Prxs might cooperate with each other in antioxidant defense.The physiological role of each Prx has been explored. Notably, Tsa1 has been shown to be a significant contributor to genome stability; its absence leads to a broad spectrum of spontaneous mutations and genome rearrangements that are thought to result from increased oxidative damage to the DNA S. cerevisiae Prxs, we performed a comparative study of Prxs for their respective role in maintaining the genome stability and in sustaining aerobic viability of cells deficient in recombinational repair. Tsa1 played a key role in this regard while Tsa1 and Dot5 cooperated to suppress DNA damage that leads to gross chromosomal rearrangements (GCRs). Tsa2 shared functional similarity with Tsa1 and was capable of largely complementing the absence of Tsa1 when expressed under the control of Tsa1 promoter. Peroxidase active site was essential for Tsa1 to function in suppressing genome instability and cell death. Finally we addressed the fate of mutant cells with compromised Tsa1 activity and deficient recombinational repair.To gain more insight into the physiological role of the TSA1 resulted in elevated mutation rates as determined by the Canr assay that detects all gene inactivating mutations that can occur in the 1.8-kb CAN1 gene, which are most frequently base substitution and frameshift mutations, the hom3-10 assay that detects reversion of a +1 insertion in the HOM3 gene, the lys2-Bgl assay that detects reversion of a +4 insertion in the LYS2 gene, and the GCR assay that measures gross chromosomal rearrangements, indicating the importance of Tsa1 in preventing a broad spectrum of types of genomic instability. Deletion of TSA2, AHP1, PRX1 or DOT5 did not cause a mutator phenotype that could be detected with these mutator assays, confirming the unique role of Tsa1 in maintaining genome stability , whereas the GCR rate of the double mutant appeared to be similar compared to the tsa1 mutant. However, the Canr, hom3-10 and lys2-Bgl rates of the tsa1 tsa2 double mutant were not much higher than the sum of the rates of each single mutant (95% confidence limits). Simultaneous elimination of TSA1 and AHP1, or TSA1 and PRX1 did not appear to change the mutation rates in the four assays compared to tsa1 single mutant. The Canr, hom3-10 and lys2-Bgl mutation rates of the tsa1 dot5 double mutant appeared to be only slightly higher compared to tsa1 single mutant. In contrast, the GCR rate of the tsa1 dot5 double mutant showed a significant increase relative to the tsa1 and dot5 single mutants  was replaced by the TSA2 coding sequence was constructed as illustrated in TSA2 mRNA was 5% of that of TSA1 mRNA in log phase wild-type cells but increased to 70% of the wild-type TSA1 mRNA levels in the tsa1::TSA2 strain  epitope tag, enabling the immunodetection of these proteins with the same anti-HA antibody. The protein level of Tsa1 was much higher than that of Tsa2 in wild-type cells while Tsa2 produced in the tsa1::TSA2 strain was comparable to the physiologic level of Tsa1 , but were higher than the mutation rates of the wild-type strain  were sporulated and dissected, and the spore dissection plates were incubated under aerobic conditions for four days. As previously shown, a tsa1 mutation in combination with a rad51 mutation resulted in lethality  and resolving Cys (Cys170). Both Cys47 and Cys170 were shown to be necessary for the maintenance of the dimeric structure of oxidized Tsa1, and Cys47, but not Cys170, is essential for antioxidant activity measured in vitroC47Stsa1, C170Stsa1 and CCStsa1 that express mutant forms of Tsa1 in which Cys47, Cys170 or both were substituted with serines  but were higher than that of the wild-type strain. We then analyzed the meiosis products of the heterozygous diploid cells containing a tsa1 allele of interest and a rad51 mutation. Tetrad dissection and genotyping of viable spore colonies revealed that a rad51 mutation was lethal in combination with the C47Stsa1 or CCStsa1 alleles  obtained under anaerobic conditions were inoculated into liquid cultures and grown under the same anaerobic conditions for 3 to 4 days. Cultures were diluted into fresh medium to a density of 2×106 cells/ml under aerobic conditions and samples were taken for flow cytometry (FACS) and microscopic analysis at various time points after shifting to aeration. At time 0, the CCS rad51tsa1 cells exhibited cell cycle profiles and cell morphology that were similar to that of wild-type cells .The pe cells  and to tpe cells . At thisragments . HoweverCCS rad51tsa1 cells displayed a cell morphology and FACS profile suggestive of a protracted G2/M checkpoint arrest , late apoptosis eventually leading to secondary necrosis (annexin V+/PI+) and primary necrosis (PI+) 2O2 for 200 min as previously described CCStsa1 single mutant and rad51 single mutant cells maintained low level annexin V staining at 8 hr and 24 hr after shifting to aerobic conditions . Taken together, these results suggest that the CCS rad51tsa1 and tsa1 rad51 double mutant cells undergo a DNA damage induced apoptosis-like cell death.The nditions . In contme point . CultureCCS rad51tsa1 cells. Deletion of YCA1 or STE20 makes cells more resistant to H2O2 induced apoptosis CCStsa1/TSA1 rad51/RAD51 yca1/YCA1 and CCStsa1/TSA1 rad51/RAD51 ste20/STE20 were sporulated and dissected. Tetrads were analyzed and the genotypes were determined or inferred from the mutation segregation patterns. The colony size of CCS rad51 yca1tsa1 and CCS rad51 ste20tsa1 triple mutants was similar to that of CCS rad51tsa1 double mutants (data not shown). We then tested whether the accumulation of annexin V+ cells in aerobic cultures of CCS rad51tsa1 mutants was Yca1- or Ste20- dependent. Anaerobic culture condition restores cellular viability of these double and triple mutants, allowing us to obtain sufficient number of cells in liquid cultures for analysis. As shown in CCS rad51 yca1tsa1 and CCS rad51 ste20tsa1 cells exhibited annexin V and PI staining features that were similar to those of CCS rad51tsa1 cells at different time points after shifting to aeration. Therefore, neither deletion of YCA1 nor deletion of STE20 reduced the apoptotic like phenotype of CCS rad51tsa1 cells. Thus, the Yca1 and Ste20 mediated apoptosis programs do not by themselves contribute to the lethal effects caused by loss of both Tsa1 and Rad51 function. In addition, a cell-permeant pan caspase inhibitor Z-VAD-FMK failed to improve growth and survival of CCS rad51tsa1 cells (data not shown).We next investigated whether the two independent cell death processes mediated by the proapoptotic caspase Yca1 and the Ste20 kinase, respectively, play a role in the death of 2O2 scavengers, redox signal transducers and molecular chaperones is of significant interest S. cerevisiae results in a complex protein network that may reflect the particular physiological roles of each isoform. In this study, our comparative analysis of the five Prxs demonstrated that Tsa1 was distinguished from other Prxs in that by itself it played a key role in suppressing the accumulation of mutations and genome rearrangements, and maintaining cellular survival and growth in the absence of recombinational repair. The predominant role of Tsa1 in this regard may mask the potential phenotype of other prx mutants. Indeed, analysis of double mutants combining tsa1 and mutation of one of the other four PRXs revealed distinct roles of Tsa2 and Dot5 in suppressing the accumulation of mutations and in preventing killing by exogenous oxidative stress.Prxs have generated considerable of interest during recent years, and understanding their distinct roles as Htsa1 mutation, a tsa2 mutation did not cause sensitivity to H2O2, a mutator phenotype or synthetic lethality when combined with a rad51 mutation. However, the tsa1 tsa2 double mutant exhibited much higher H2O2 sensitivity than tsa1 single mutant. The mutator phenotype of tsa1 tsa2 double mutant was more pronounced than that of tsa1 single mutant due to the increase in base substitution and frameshift mutations as revealed by different mutator assays performed in this study. Furthermore, overexpression of Tsa2 under the control of TSA1 promoter largely restored the genetic defects caused by a tsa1 mutation. These observations indicate that Tsa1 and Tsa2 share functional similarity in removing low endogenous concentrations of H2O2 and in dealing with more aggressive exogenous oxidant challenge. This view is consistent with the fact that Tsa2 is unique in sharing striking homology with Tsa1 among the five Prxs in S. cerevisiae, that Tsa1 and Tsa2 exhibit comparable antioxidant activity in vitro and that both have heat shock protective chaperone activity TSA2 is much lower than that of TSA1, transcription of TSA2 shows greater induction after treating cells with different oxidants, including H2O22O2 sensitivity of the tsa1 single mutant  or by the thioredoxin system, whereas Tsa1C170S is active in the presence of DTT and inactive in the presence of the thioredoxin system C170S was only partially functional in vivo, as shown by the partial complementation of a tsa1 strain expressing this protein. The basis for this activity is unclear as it is not clear what provides the needed redox system in vivo. Consistent with these observations, the only 2-Cys Prx of S. pombe, Tpx1, which is essential for cell viability, is able to support aerobic growth when the resolving Cys is eliminated in the Tpx1C169S mutant protein in vivo evidence suggesting that Tsa1 may be the primary scavenger of endogenous H2O2 in S. cerevisiae. Several aspects of observations are consistent with this view. First, Tsa1's affinity for H2O2 with a Km of ∼12 µM 2O22O2 concentrations are high. Third, and more important, Tsa1 is functionally unique with regard to maintenance of genome stability and to interaction with other aspects of DNA metabolism, as high rates of accumulating Canr mutations and chromosomal rearrangements were found only in tsa1 mutants and synthetic lethal interactions with rad51 mutations were only seen with a tsa1 mutation The peroxidase active site is essential for Tsa1 to function in suppressing genome instability and cell death. Tsa1S. cerevisiae prx mutants did not have growth defects and a prx null mutant in which TSA1, TSA2, AHP1, PRX1 and DOT5 were all deleted was still viable S. pombe mutant lacking Tpx1 S. cerevisiae cells do not generate enough H2O2 or other reactive species to produce lethal levels of damage, another cellular antioxidant activity compensates for the loss of the Prxs, or other pathways help limit the damage and prevent cell death. Recombinational repair is such a pathway critical for cell survival and growth of tsa1 mutants, including deletion mutants and mutants with a compromised peroxidase active site (C47Stsa1 and CCStsa1). These results confirm the view that aerobic organisms generate enough H2O2 to damage their own DNA. Scavenging enzymes and appropriate DNA repair are required for growth under aerobic conditions and Tsa1 is the dominant peroxidase activity that keeps the steady-state concentration of H2O2 at non-toxic levels. Consistent with this, E. coli fur, ubiH or ubiE mutations most likely lead to an increased level of ROS and cause cell death in the absence of RecA-dependent recombinational repair fur recA synthetic lethality has been ascribed to elevated chromosomal fragmentation. We investigated the cell death processes of CCS rad51tsa1 and tsa1 rad51 under aerobic conditions. Several hours after shifting from anaerobic culture to aeration, CCS rad51tsa1 cell cultures were characterized by the accumulation of cells with irregular cell size/shape, abnormal nuclear staining, and a broader S/G2/M DNA content peak. At longer time after shifting to aeration, dying cells were characterized by a significant increase in the levels of typical early markers of S. cerevisiae apoptosis-like cell death such as phosphatidylserine externalization. This apoptosis-like phenotype did not depend on the caspase Yca1 and Ste20 kinase, two gene products each thought to control a different apoptosis-like cell death process S. cerevisiae and demonstrates enzymatic peptidase activity analogous to mammalian caspase activity S. cerevisiae2O2 treatment S. cerevisiae that triggers the death of CCS rad51tsa1 mutants. Similarly, it is known that endogenous DNA damage, replication failure and perturbation of transcription induces an apoptosis-like death in S. cerevisiaeIndividual The results reported here and in previous studies can be interpreted as follows. Endogenous oxidative stress due to the absence of appropriate and sufficient Tsa1 activity induces a variety of types of DNA damage, including mutagenic, replication blocking and potential lethal DNA lesions. These lesions are repaired by diversity of mechanisms including base excision repair, nucleotide excision repair, post-replication repair, recombination and potentially other mechanisms. Unrepaired mutagenic DNA lesions cause mutations and GCRs but do not significantly affect cell survival. In contrast, in the absence of appropriate repair, a growing number of potential lethal DNA lesions persist or are converted to lethal double-strand break (DSB), activating the Rad9-dependent G2/M checkpoint. This checkpoint activation facilitates repair of at least some of the DNA damage by back-up repair mechanisms allowing checkpoint proficient cells to proliferate to a greater extent than checkpoint deficient cells. Finally, the presence of irreparable or excessive DSBs appears to initiate a DNA damage-induced apoptotic program, although how such damage signals the apoptotic program is not well understood.S. cerevisiae strains were grown in standard media including yeast extract peptone dextrose medium (YPD) or synthetic complete medium (SC) lacking appropriate amino acids as indicated. Cycloheximide resistant strains (CyhR) containing mutations in the CYH2 gene were selected on YPD agar plates containing 10 µg/ml cycloheximide. Canavanine resistant mutants (Canr) caused by inactivation of the CAN1 gene were selected on SC–arginine dropout plates containing 60 mg/l canavanine. Hom3+ revertants were selected for on SC–threonine dropout plates and Lys2+ revertants were selected for on SC–lysine dropout plates. Canavanine- and 5-fluoroorotic acid (5FOA)- resistant mutants (Canr-5FOAr) resulted from the loss of the region including CAN1 and URA3 on chromosome V were selected on SC–arginine and –uracil dropout plates containing 60 mg/l canavanine, 1 g/l 5FOA and 50 mg/l uracil 2O2 was assayed on solid media by spotting 10-fold serial dilutions of mid-logarithmic phase cells onto SC plates containing H2O2 at indicated concentrations.MATa, ura3-52, leu2Δ1, trp1Δ63, his3Δ200, lys2ΔBgl, hom3-10, ade2Δ1, ade8, hxt13::URA3tsa1::TSA2 mutant in which the entire TSA1 gene was replaced by TSA2 coding sequence under control of the native TSA1 promoter is illustrated schematically in + CyhS) in which the entire TSA1 gene was replaced by the TRP1-CYH2 cassette as previously described TSA2 flanked by sequences homologous to the upstream and downstream sequence of TSA1, were used to transform MEHY1539 (Trp+ CyhS) to replace TRP1-CYH2 at the TSA1 locus yielding the strain MEHY1710 (Trp− CyhR) in which the TSA2 is under the control of the native TSA1 promoter. Correct integration of TSA2 at TSA1 locus was confirmed by genomic PCR and DNA sequencing.The strains used for this study were all isogenic to the S288c based parental strain RDKY3615 TSA1  were created as follows. The TSA1 gene with 5′ and 3′ flanking DNA sequence was amplified from genomic DNA using primers containing, respectively, the restriction sites XhoI and HindIII. The resulting PCR product was digested with XhoI and HindIII and cloned in XhoI and HindIII sites of the plasmid pRS416 TCTCCAACCGAAATCATTGC-3′5′-GCCTTCACTTTCGTC and AGACGAAAGTGAAGGC-3′5′-GCAATGATTTCGGTTGGAG (sequences for Cys47 to Ser substitution are underlined), and mutagenic primers TCTAACTGGACTCCAGGTGC-3′5′-GGTACTGTCTTGCCA and AGATGGCAAGACAGTACC-3′5′-GCACCTGGAGTCCAGTT (sequences for Cys170 to Ser substitution are underlined). The mutated TSA1 gene was sequenced to verify the presence of the desired substitutions and the absence of other mutations. PCR-generated fragments containing mutant alleles of TSA1 flanked by sequences homologous to the upstream and downstream sequence of TSA1, were used to transform MEHY1539 (Trp+ CyhS) to replace TRP1-CYH2 at the TSA1 locus yielding the strains MEHY1702 (C47Stsa1), MEHY1593 (C170Stsa1) and MEHY1590 (CCStsa1), expressing a given mutant allele of TSA1 under the control of the native TSA1 promoter  according to an established procedure TSA1, TSA2, and ADH1 . Serial dilutions of cDNA from each strain were amplified using the appropriate primers and iQ SYBR Green Supermix (Bio-Rad). A single melt curve peak was observed for each sample used in data analysis, thus confirming the purity and specificity of all amplified products. The relative ratio of TSA1 or TSA2 expression to ADH1 was calculated using the relative quantity (ΔΔCT) analysis formula in the Bio-Rad iQ5 Gene Expression Optical System Software.Wild-type and For Western blots, strains were grown to mid-logarithmic phase in YPD medium and whole cell extracts isolated by trichloroacetic acid extraction r mutations, Hom3+ revertants, Lys2+ revertants, and GCRs in cell populations was determined by fluctuation analysis as described previously 8 cells/ml, harvested, washed and resuspended in sterile water. Sufficient quantities of cells were plated on appropriate selection media to identify Canr mutations, Hom3+ revertants, Lys2+ revertants and GCR events, and appropriate dilutions were plated on YPD for total cell counts. Colonies were counted after four days of growth at 30°C. The number of mutant cells per culture was calculated and the median value from at least 15 independent cultures for each strain was used to determine the mutation rate as described http://www.math.unb.ca/~knight/utility/MedInt95.htm.The rate of accumulation of CanYPD medium with or without agar was supplemented with Tween 80 and ergosterol to a final concentration of 1.32 ml/l and 6.75 mg/l, respectively. Anaerobic liquid cultures or anaerobic growth of dissected spores in plates were placed in an airtight jar containing a disposable hydrogen- and carbon dioxide-generating envelope (BBL GasPak Plus) and grown anaerobically at 30°C for 3–5 days to yield enough cells for analysis. Anaerobic conditions were monitored with the redox indicator Anaerotest (Merck) placed inside of the airtight jar.6 cells/ml and samples were taken at various time points after shifting to aeration. Cells were collected, resuspended in 70% (vol/vol) ethanol for fixation. The cells were washed in 50 mM Tris buffer (pH 8.0), incubated 24 hr with 5 mg/ml DNase-free RNase, centrifuged and labeled with 50 µg/ml PI, sonicated and submitted to FACS measurements of DNA content with a FACScalibur cytometer (Beckton-Dickinson). Same fixed cells were also stained with DAPI Cells of the desired genotype were grown in liquid culture under same anaerobic condition for 3 to 4 days. Cultures were diluted in fresh medium to a density of 2×106 intact yeast living cells were treated by 5% glusulase (Perkin Elmer) and 5 U/ml lyticase (Bio-Rad) at 28°C for 2 hr in a buffer consisting of 50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2, pH 7.4. The labeled cells were then analyzed by FACS using an argon-ion laser (excitation wavelength 488 nm and emission of 530 nm) and the data were processed using BD Cell Quest Pro software (version 5.2).Annexin V/PI co-labeling was performed using the Vybrant Apoptosis Assay kit #3 (Invitrogen) according to the manufacturer's instructions, excepted that about 1×10


The composition and expression of vertebrate gene families is shaped by species specific gene loss in combination with a number of gene and genome duplication events  and depends on the ecological and evolutionary context. In this study we analyzed the evolutionary history of the solute carrier 1 (SLC1) gene family. These genes are supposed to be under strong selective pressure (purifying selection) due to their important role in the timely removal of glutamate at the synapse.slc1 genes in ray-finned fishes originated from R3, the increased number of SLC1 genes in prototheria, sauropsida, and amphibia genomes originates from specific genes retained in these lineages.In a genomic survey where we manually annotated and analyzing sequences from more than 300 SLC1 genes (from more than 40 vertebrate species), we found evidence for an interesting evolutionary history of this gene family. While human and mouse genomes contain 7 SLC1 genes, in prototheria, sauropsida, and amphibia genomes up to 9 and in actinopterygii up to 13 SLC1 genes are present. While some of the additional slc1 gene family which we named slc1a8/eaat6 and slc1a9/eaat7.Phylogenetic comparison and microsynteny analyses of the SLC1 genes indicate, that theria genomes evidently lost several SLC1 genes still present in the other lineage. The genes lost in theria group into two new subfamilies of the The phylogeny of the SLC1/EAAT gene family demonstrates how multiple genome reorganization and duplication events can influence the number of active genes. Inactivation and preservation of specific SLC1 genes led to the complete loss of two subfamilies in extant theria, while other vertebrates have retained at least one member of two newly identified SLC1 subfamilies. Genomes of extant species are shaped by extensive gene loss and duplication events. In the radiation of vertebrate species, two whole genome duplication events at the base of the vertebrate lineage (approximately 500 million years (Mya) ago) -3 and a slc1 gene family of neutral and excitatory amino acid transporters (EAATs), whose presence on glia cells or neurons is indispensable for precise and sustained synaptic activity [In the context of lineage specific gene loss and duplication events, we studied members of the vertebrate activity -22. EAATactivity . Moreoveactivity . Based oIn mammals, five EAAT genes EAAT 1-5) together with two neutral amino acid transporters form the 'solute carriers 1' (SLC1) gene family , an increased number of SLC1 genes are expected in their genomes. We indeed identified up to 13 SLC1s in fish genomes. While some of these additional SLC1 genes clearly are the consequence of the teleost specific R3, others are evidently members of two additional subfamilies , that were lost in the lineage leading to human and mouse. Interestingly members of these two subfamilies can also be found in prototherian, sauropsidan, and amphibian genomes, suggesting that these genes are specifically retained in these lineages.slc1 genes in the zebrafish genome. While in the human and mouse 7 different members of the slc1 gene family have been described, we identified and annotated 13 slc1 family genes in the zebrafish genome. Cloning from whole embryo cDNA indicated that all of the 13 annotated slc1 sequences are indeed transcribed. Sequencing of the amplified cDNA fragments revealed no significant deviation from our predicted sequences, except for some sites of single nucleotide polymorphisms.As a basis to study the evolutionary history of a given gene family, we analyzed the abundance of slc1 gene family in zebrafish consists of nearly twice as many genes as present in genomes of human and mouse suggests that at least some of the zebrafish slc1 orthologs have originated from the teleost specific whole-genome duplication. Physical and virtual mapping indicated that none of the sequences within a subgroup of slc1 are located within the same chromosomal cluster . Even teleost genomes only distantly related to zebrafish displayed an almost identical number and phylogeny of slc1 family members as those identified in the zebrafish genome , we decided to extend our analysis to encompass the whole vertebrate tree Fig. . TherefoXenopus tropicalis, as a representative of the amphibians on the other hand has retained members of both subfamilies. Interestingly, additional gene losses occurred in a variety of lineages. For instance, chicken  has lost SLC1A5 and the green anole lizard (Anolis carolinensis) has lost SLC1A6  display various gene losses and retentions Fig. . Monotre1A6 Fig. .Phylogenetic reconstruction including now SLC1 sequences from teleosts, amphibia, sauropsida and mammalia clearly indicates that the newly assigned SLC1A8 and SLC1A9 each form a separate clade Fig. .Callorhinchus milii) and the sea lamprey (Petromyzon marinus). The elephant shark, belonging to the cartilaginous fish, represents an independent evolutionary branch of the vertebrates, whose genomes are thought to have undergone the same number of genome duplications as the mammalian branch [slc1 cDNA sequences. Nevertheless, our comparison revealed only exons for one ortholog per subfamily, with the notable exception of the SLC1A2/A9 subgroup, where we detected a retained SLC1A9 ortholog. Interestingly we also identified two paralogs of the SLC1A2/A9 subgroup in the lamprey, a jawless vertebrate at the base of the vertebrate tree .slc1a3 and slc1a2 subfamily genes.As an example to compare the spatial and temporal expression pattern of duplicated genes, we investigated the expression patterns of the zebrafish in situ hybridization experiments show that slc1a3a transcripts can be found in glia cells of the larval zebrafish brain at 3 days post fertilization  genes in zebrafish, we found as expected evidence for many duplicated genes. However, some zebrafish slc1 genes turned out to be not the result of the teleost specific R3. Further analyses ruled out the possibility that these SLC1 genes originated from the teleost specific genome duplication. One possible explanation for these additional genes is lineage and species specific gene loss. This has long been recognized in unicellular organisms [Modern genomes are shaped by frequent duplication and deletion events. Most dramatic are whole genome duplications that were prominently stated by Susumo Ohno as the most important driving force in the evolution of metazoans . This idrganisms ,28. Howerganisms ,29-31, wAmbystoma tigrinum retina reports the cloning of a SLC1A8/EAAT6, as well as a SLC1A9/EAAT7 gene  [In order to reconstruct the phylogenetic history of this gene family we studied a number of vertebrate species from all major vertebrate lineages. We found representatives of both new subfamilies in all analyzed teleost species, showing that the existence of these genes is common to teleost species and is not restricted to zebrafish. Analysis of amphibian genomes reveal the existence of one member of each SLC1A8/EAAT6 and SLC1A9/EAAT7 subfamily, arguing that the tetrapod ancestral species still contained the full complement of SLC1 subfamilies. In this respect it is interesting to note that a study of glutamate transporters in the tiger salamander ctively) ,33, whicAnolis carolinensis) have retained SLC1A9 but lost SLC1A8. The situation is reversed in the archosauromorpha (represented by the bird species zebrafinch and chicken), where SLC1A9 was lost but SLC1A8 was retained. This suggests that the genome of the last common ancestor of the sauropsida still contained both subfamilies, followed by independent gene loss in the two branches of the sauropsida. Following this logic the genome of the amniote ancestors should have contained members of both subfamilies.A clue to when the first subfamily was lost is provided by the sauropsida. Here the Lepidsauromorpha (represented by the green anole lizard Ornithorhynchus anatinus) have retained the member of SLC1A8, while all other remaining mammals have lost members of both subfamilies. In the most parsimonious scenario the last common mammalian ancestor has already lost SLC1A9 but has retained SLC1A8. Subsequently SLC1A8 was lost at the base of the therian lineage, whereas a member of this subfamily was retained in the branched off protheria species. Hence the SLC1A8 and SLC1A9 subfamilies have been lost independently in a number of lineages. While for instance SLC1A8 has been lost at least twice independently in the archosauromorpha and therian lineage, SLC1A9 has been lost independently in the therian and lepidosauromorpha lineage. In order to infer the ancestral situation we assessed the SLC1/EAAT repository in species that are at the base of the gnathosomes.In mammals only the protherian species  and the sea lamprey (Petromyzon marinus), is available. While the former represents the cartilaginous fish, an independent branch of the vertebrates that has split before the division of the tetrapod and the teleost lineage, the later belongs to a clade of skulled chordate animals lacking jaws. Since both lineages branched off before R3, cartilaginous fish as well as lampreys are expected to have undergone the same number of genome duplications as the mammalian branch. Consequently the gene repertoire between lamprey, sharks, and mammals should be comparable [Genome information for two species of interest, the elephant shark , the most likely position of the inactivated SLC1A9 is on the long arm at position 19q13.3. For this human chromosome extensive rearrangements within the short arm and many intrachromosomal breaks in the long arm have been reported, even since the time of primate rodent divergence . TherefoThe phylogenetic analysis of a gene family spanning multiple taxa can yield important information about their evolutionary history. In the case of the solute carrier family 1, consistent with the duplication-complementation-degeneration model, most of the 7 human or mouse orthologs have one or two teleost orthologs. However, for two teleost SLC1 subfamilies we found no corresponding therian counterpart. This indicates that therians must have lost these two subfamilies, now named SLC1A8/EAAT6 and SLC1A9/EAAT7.These two subfamilies are not teleost specific, but can be found throughout different vertebrates. An analysis of the major vertebrate lineages revealed an intriguing pattern of lineage specific gene losses, shaping the phylogenetic history of SLC1 genes. In all non-therian vertebrate species we found at least one member of the SLC1A8/9 subfamilies. Amphibians have neither lost SLC1A8 nor SLC1A9, lepidosauria  on the one hand have lost the SLC1A8 ortholog, while birds on the other hand have lost SLC1A9. Interestingly egg laying mammals (platypus) are the only mammals that have also retained a SLC1A8 ortholog.By studying one gene family across a number of vertebrate taxa, inferences can be made about the evolutionary history of these genes. Such an analysis is needed to guide the interpretation of functional data obtained for those genes and provides a fascinating scenario to study the evolution of vertebrate gene families.2, 0.33 mM MgSO4) at 28°C. Stages refer to the development in days post fertilization (dpf).Wild-type fish of the Tübingen (Tü) inbred strain were bred and crossed as previously described . Embryosslc1 genes were annotated manually. Sequences from the species listed in Additional File http://www.ncbi.nlm.nih.gov; Ensembl, http://www.ensembl.org/index.html; version 50/51, 2008) and WGS http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&BLAST_SPEC=TraceArchive&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch databases. Human and mouse sequences were used as initial query. Genomic regions coding for SLC1 family genes were identified by using the tblastx alignment algorithm . Gaps islc1 genes  were translated into proteins using the EditSeq software  and obtained protein sequences were used to generate a combined sequence file in FASTA format  configured for highest accuracy (MUSCLE with default settings). After alignment, ambiguous regions  were removed with Gblocks  using t, according to the manufacturer's instructions. Oligo dT primers were used to reverse-transcribe the total RNA with reverse transcriptase . Polymerase chain reaction (PCR) was performed using sequence-specific oligonucleotide primers. Amplified fragments were ligated into the pCR-II vector  and at least three independently amplified cDNA fragments per gene were sequenced to confirm our previously annotated zebrafish sequences. Accession numbers for the zebrafish SLC1/EAAT genes are: SLC1A1/EAAT3 (HM138690), SLC1A2a/EAAT2a (HM138691), SLC1A2b/EAAT2b (HM138692), SLC1A3a/EAAT1a (HM138693), SLC1A3b/EAAT1b (HM138694), SLC1A4 (HM138695), SLC1A5 (HM138696), SLC1A6/EAAT4 (HM138697), SLC1A7a/EAAT5a (HM138698), SLC1A7b/EAAT5b (HM138699), SLC1A8a/EAAT6a (HM138700), SLC1A8b/EAAT6b (HM138701), SLC1A9/EAAT7 (HM138702).slc1 genes except slc1a6/eaat4 were localized in the zebrafish genome by radiation hybrid mapping [All zebrafish  mapping  using thslc1 locus of human or zebrafish were used as initial queries for a tblastx [For microsynteny analyses, genes flanking the  tblastx  search aIn vitro transcription of DIG-labeled probes was performed using the Roche RNA Labeling Kit . RNA probes were hydrolyzed to obtain fragments of about 300 bp length. Whole-mount in situ hybridization on 3 dpf zebrafish larvae was performed as follows. PTU treated larvae were anesthetized on ice and immediately fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4) for 40 min at ambient temperature (RT). The in situ hybridization was performed according to the Zebrafish Book protocol [in situ hybridization machine .protocol  using anslc1 genes, determined the intron/exon pattern of the slc1 genes and performed the synteny analysis. AL cloned the slc1 genes used in this study, wrote the first draft of the manuscript and contributed to expression analysis, CMM performed the expression analysis and HBS contributed to cloning and expression analysis. SCFN and MG wrote the manuscript in its present form, and conceived the project and designed experiments. Al authors have read and approved of the final manuscript.MG contributed to cloning, performed the database searches and the phylogenetic analysis, annotated the slc1 genes in the zebrafish genomeGenomic localizations of . Chromosomal localizations were identified both physically, using radiation hybrid mapping, and in silico. All zebrafish slc1 genes localize to different chromosomal regions. For details on radiation hybrid mapping, see Materials and Methods. *The localization of slc1a6/eaat4 could not unambiguously be determined by radiation hybrid mapping and has been mapped in silico only.Click here for filePhylogenetic analysis of SLC1 genes using a Bayesian algorithm. Bayesian phylogeny of members of the zebrafish (dr), mouse (mm) and human (hs) SLC1 family. The phylogenetic tree was build using 370 representative amino acids determined by the program Gblocks after sequence alignment using MUSCLE. The tree was reconstructed using the bayesian inference method implemented in the MrBayes program (v3.1.2). The number of substitution types was fixed to 6. The Poisson model was used for amino acid substitution, while rates variation across sites was fixed to "invgamma". Four Markov Chain Monte Carlo (MCMC) chains were run for 10000 generations, sampling every 10 generations, with the first 250 sampled trees discarded as "burn-in". Finally, a 50% majority rule consensus tree was constructed. Note that in comparison to the maximum likelihood calculated tree, roots in the Bayesian tree are slightly different. While in the Bayesian build tree it appears that there is a common ancestor of SLC1A1, SLC1A2, SLC1A3 and SLC1A6, the tree deriving from maximum likelihood phylogeny suggests that there is a common ancestor between SLC1A3, SLC1A6 and SLC1A7/8  and yellow (low coverage) belong to species that have been used to generate the phylogenetic tree covering the major linages. Coverage's shown in red belong to species whose sequences have been annotated but not included in the phylogenetic tree.Click here for fileLinks to transcript and genome information of the annotated sequences. The identity of the predicted and the manually annotated sequences is indicated by different colours. Identical predicted and annotated sequences are shown in green, whereas sequences with different predicted open reading frames are highlighted in red. Sequences covering the whole open reading frame are indicated as "Full" sequences and sequences lacking one or more exons are marked as partial sequences. Sequences for which neither a genomic nor a transcript link have been found are given by the letters n.d. (not detected).Click here for fileIntron/Exon sizes of SLC1 genes. The different SLC1 gene families are colour coded. Exons serving as identifiers for the corresponding gene family are highlighted in the identical colour as the gene family. Exons containing the start and the stop codon are highlighted in red. Intron sequences containing multiple place holders (Ns) are indicated.Click here for fileThe SLC1A9 subfamily is also present in cartilaginous fish. Database analysis on the elephant shark  and the sea lamprey (Petromyzon marinus) genome identified sequences that represent parts of the SLC1A9 gene. For the exon 234 , two corresponding slc1a2 exons could be found, one of them representing a retained slc1a9 gene. The phylogenetic tree was build using the maximum likelihood method on the entire nucleotide sequence of corresponding 234 exons. Bootstrap values above 50% (0.5) are shown. Elephant shark sequences are highlighted in dark red, lamprey sequences are shown in claret red and teleost genes are depicted in light red. Note that the FSGD generated two retained SLC1A2 genes and most likely also two SLC1A9 genes of which in modern teleosts only one is still present.Click here for fileProtein fastas of species used in the phylogenetic analysis covering the main vertebrate linages.Click here for fileNucleotide fastas of the sequences used in the exon 234 analysis.Click here for fileOligonucleotide Primers Used for Genomic Mapping.Click here for file


Infection without disease may occur under natural conditions after contact with infected birds. Avian influenza A virus subtype H5N1 was transmitted to domestic cats by close contact with infected birds. Virus-specific nucleic acids were detected in pharyngeal swabs from 3 of 40 randomly sampled cats from a group of 194 animals (day 8 after contact with an infected swan). All cats were transferred to a quarantine station and monitored for clinical signs, virus shedding, and antibody production until day 50. Despite unfamiliar handling, social distress and the presence of other viral and nonviral pathogens that caused illness and poor health and compromised the immune systems, none of the cats developed clinical signs of influenza. There was no evidence of horizontal transmission to other cats because only 2 cats developed antibodies against H5N1 virus. Avian influenza has attracted worldwide attention because highly pathogenic avian influenza virus subtype H5N1 can cause fatal infections in humans . PCR and egg culture identified avian influenza virus (H5N1) in the swan and in 13 of 38 other culled birds  (day 4) housed with the swan at the same time. Only the swan developed clinical signs of disease. On day 4, the poultry area was disinfected after all 38 birds were removed.In the same shelter were 194 cats; most had access to an outdoor enclosure near the poultry area and were separated from the birds by a wire-mesh fence. On several occasions, 1 or 2 unidentified cats were observed climbing the fence and entering the poultry area. Ingestion of birds by cats was not observed. Austrian authorities ordered random sampling of the cat population at the shelter because of spatial proximity of poultry and cats and the possible exposure of cats to infective debris of the birds. The bird area was left unoccupied while the cats were under observation. The purpose of this study was to monitor health status and possible transmission within a large cat population with proven natural exposure to H5N1 influenza virus.Pharyngeal swabs of 40 cats were sampled  were still available for further observations. Three cats had died and 24 other cats had been placed in private households. Before discharge from the shelter and within 1 week thereafter, all of these cats were examined and no abnormal health status was observed.2. The larger group contained 139 cats (including cats 1 and 2); the smaller group contained 28 cats. Cat 3 was not available for further examination because it was healthy before leaving the shelter and, to our knowledge, did not die. The smaller group was always separated from the larger group and was kept indoors at the animal shelter in Graz. In the quarantine area, the 167 cats were housed in 2 closed rooms, without any activity restriction, and had free access to food and water. Routine physical examination, including auscultation of the chest, was done on days 22, 29, and 50 for all cats at the quarantine station. In case of an obvious health problem, clinical signs were monitored by daily physical examination and serologic testing. The litter pans of the cats and floors of the quarantine areas were cleaned every day and disinfected every other day.A total of 167 cats were transported in small groups in ≈50 containers for 12 h from the animal shelter to a quarantine area and housed in 2 separate groups from day 22 until day 50. Average floor space for each cat was ≈1.4mOn days 22 and 29, pharyngeal and rectal swabs were obtained and transported in phosphate-buffered saline containing antimicrobial drugs .agtggcatgaccgccct-3′, reverse primer: 5′-cgttagcgcaggttgagca-3′), and 5 pmol/L of probe (5′-FAM-cactgtgatgtgttcgaagtttgagca-TAMRA-3′). The cycler scheme consisted of 2 pre-PCR steps of 50°C for 15 min and 95°C for 2 min, followed by 45 cycles of 95°C for 15 s and 60°C for 30 s. Cycle threshold values were calculated by using PCR 7300 software (Applied Biosystems). FHV-1 nucleic acid was detected by PCR as described by Reubel et al.  according to the method of Spackman et al. (Plasma samples were tested for feline leukemia virus (FeLV) antigen and antibodies against influenza virus A H5N1, feline immunodeficiency virus (FIV), and feline coronavirus (FCoV). Antibodies to influenza virus were detected with a hemgglutination inhibition test according to the procedures of the World Organisation for Animal Health (Two cats that seroconverted for H5N1 virus (cats 1 and 4) were humanely killed on day 50. Necropsy was performed on these 2 cats and on 12 other cats that had died during the observation period; organ homogenates  were tested for influenza virus–specific nucleic acids for each cat.H5N1 virus–positive cats (1 and 2) and H5N1 virus antibody–positive cats (1 and 4) did not show any signs of influenza virus–associated illness after the swan had been placed in the animal shelter (days 1–50). Upper respiratory symptoms  were evident in 30 cats, bronchopneumonia in 40 cats, diarrhea in 7 cats, mucosal lesions in 37 cats, and traumatic wounds and lesions in 10 cats. However, for each cat with clinical symptoms that might have been associated with influenza infection, another specific etiologic reason for illness could be documented. Pathomorphologic examination showed no lesions associated with respiratory infection in cats 1 and 4 or in any other cat that had died before day 50. Influenza A virus–specific nucleic acids were not detected in any organ sample tested by PCR. Likewise, all pharyngeal and rectal swabs obtained at the quarantine station were negative for influenza A virus by PCR. Antibodies against influenza virus A H5N1 were detected in 2 cats  with titers 256 on day 50 in both cats.Cats 1, 2, and 4 had negative test results for FeLV and FIV, but all 3 cats had high antibody titers against FCoV. FCV was detected in the swab from cat 2, and a double infection with FCV and FHV-1 was detected in cat 4. Clinical, bacteriologic, and virologic tests identified infection with FeLV in 15 cats, FIV in 12 cats, and antibodies against FCoV in all but 1 cat. A total of 44 swabs showed positive results for FCV-specific nucleic acids, 4 for FHV-1; 13 samples showed a double infection with FCV and FHV-1. Some pathologic bacterial infections of the respiratory and digestive system were confirmed by swab cultures.All veterinarians and staff members at the animal shelter and at the quarantine area were clinically monitored for any influenzalike symptoms. Because results of this monitoring were unremarkable and virus excretion by the cats was not detected, serologic tests were not conducted for these persons.This is the first description of an asymptomatic infection with highly pathogenic H5N1 influenza virus in domestic cats. Although infection was detected in a group of cats by positive PCR results for pharyngeal swabs in 3 cats and seroconversion in 2 cats, there was no evidence for influenza-associated disease. This finding contrasts with reports documenting cats with rapidly and developing and fatal disease caused by influenza A virus subtype H5N1 , oral mucosal lesions, and diarrhea were observed in cats in both groups in the animal shelter and in the quarantine station. A long (12 hours) and uncomfortable transport to the quarantine area, social distress caused by high population density, repeated restraint for examinations and sample collection, and multiple infectious agents may have caused such a high level of illness. Twelve cats died or were humanely killed while in a moribund state between days 22 and 50. All showed signs of disease other than infection with influenza virus A and died of feline infectious peritonitis, cardiomyopathy, enteritis, or nephropathy; none tested positive for H5N1 virus.During the observation period from days 22 to 50, excretion of virus was not detected in the pharynx or feces. Positive results were observed only on day 8 for 3 of the randomly sampled swabs. Therefore, viral shedding is assumed to have lasted <2 weeks in cats 1 and 2. In 1 study, no information was reported on the duration of virus shedding because only severe illness with a lethal outcome was reported or the cats were killed 7 days after experimental infection  or whether the cat did not produce sufficient amount of antibodies. Little information is available on immune responses after infection with influenza virus H5N1 in cats.H5N1 virus can cross species barriers (Until recently, the avian flu situations in Asia and Europe appeared to differ. In Asia, large numbers of poultry have been infected and culled. Human and feline cases are mainly associated with close contact with infected poultry or ingestion of contaminated meat that was not sufficiently cooked. In Europe, mainly wild aquatic birds were infected, and only a few turkey farms were affected by H5N1 infection. Because direct contact with poultry is more limited in Europe than in Asian countries and the main source of food for cats in Europe is either commercial cat food or wild rodents and small birds, virus uptake during hunting and ingestion of poultry and aquatic birds is unlikely. Large aquatic birds are normally not a major source of food for cats, although infected birds may have caused the deaths of 3 cats found on the island of Ruegen, Germany (We have shown that under natural conditions infection of cats with influenza virus H5N1 may occur after contact with infected birds or their excrement without inducing clinical disease. However, horizontal transmission between cats was not observed, although infected cats had been introduced into a large cat population that had other viral and bacterial infections and lived under stressful conditions. Avian flu infection in cats is rarely documented and there is no evidence to date that cats are responsible for transmitting the virus to humans. Although this study does not rule out H5N1 infection leading to disease and possible transmission to other mammals and birds by domestic cats under natural conditions, without ingestion of infected birds, cats do not represent a major risk in the epidemiology of H5N1 influenza. The risk posed by cats could change because the virus can rapidly undergo genetic mutation and reassortment, and efforts should be made to minimize contact of domestic cats with infected birds. To have better insights into whether cats represent a potential risk in the epidemiology of H5N1 influenza, more detailed knowledge is needed about the role of viral load, virus uptake, and immune mechanisms of the host on the outcome of infection with H5N1 influenza virus.


Strong genetic interactions of TSA1 with DNA damage checkpoint components DUN1, SML1, and CRT1 were found when mutant cells were analyzed for either sensitivity to DNA damage or rate of spontaneous base substitutions. An elevation in intracellular dNTP production was observed in tsa1Δ cells. This was associated with constitutive activation of the DNA damage checkpoint as indicated by phosphorylation of Rad9/Rad53p, reduced steady-state amount of Sml1p, and induction of RNR and HUG1 genes. In addition, defects in the DNA damage checkpoint did not modulate intracellular level of reactive oxygen species, but suppressed the mutator phenotype of tsa1Δ cells. On the contrary, overexpression of RNR1 exacerbated this phenotype by increasing dNTP levels. Taken together, our findings uncover a new role of TSA1 in preventing the overproduction of dNTPs, which is a root cause of genome instability.Peroxiredoxins are a family of antioxidant enzymes critically involved in cellular defense and signaling. Particularly, yeast peroxiredoxin Tsa1p is thought to play a role in the maintenance of genome integrity, but the underlying mechanism is not understood. In this study, we took a genetic approach to investigate the cause of genome instability in  Peroxiredoxins are a family of antioxidant enzymes highly conserved from yeast to human. Loss of peroxiredoxin in mice can lead to severe anemia and malignant tumors, but the underlying cause is not understood. One way to derive new knowledge of peroxiredoxins is through genetic analysis in yeast. We have shown that loss of peroxiredoxins in yeast is associated with an increase in mutation rates. Here, we demonstrate that this elevation of mutation rates in yeast cells lacking a peroxiredoxin is due to increased production of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Our findings suggest a new model in which compromised antioxidant defense causes accumulation of damaged DNA and activation of the DNA damage checkpoint. For yeast cells to survive DNA damage, dNTP production is increased to facilitate DNA replication, but at the price of high mutation rates. This new model could lead to a better understanding of human diseases including cancer. Peroxiredoxins belong to a family of thiol-specific peroxidases widely and abundantly expressed in most living organisms TSA1 was a strong suppressor of gross chromosomal rearrangements and spontaneous mutations TSA1 in the maintenance of genome stability, many genetic interaction partners of TSA1 identified through synthetic genetic array analysis were components of DNA repair machinery or DNA checkpoints TSA1 was found to interact genetically with REV1/REV3 and OGG1, which are critically involved in translesion synthesis (TLS) and the repair of oxidative DNA damage, respectively Loss-of-function studies in mice implicated an essential role of peroxiredoxins in antioxidant defense and tumor suppression Intracellular dNTP levels are one important determinant of cellular response to DNA damage tsa1Δ cells, additional events subsequent to the disruption of TSA1 might also be influential in the induction of genome instability. In this study we asked whether perturbation of dNTP pools might contribute to the mutator phenotype observed in tsa1Δ cells. We then investigated the cause of dNTP pool expansion. Our findings suggested that constitutive activation of the DNA damage checkpoint and consequent overproduction of dNTPs are the root cause of genome instability in tsa1Δ cells.We previously demonstrated that yeast Tsa1p is a house-keeping peroxiredoxin which sufficiently suppressed the mutator phenotype TSA1 was found to be a strong suppressor of mutations and gross chromosomal rearrangements tsa1Δ cells TSA1 with the DNA damage checkpoint and particularly the machinery of dNTP synthesis, in order to understand the role of TSA1 in the maintenance of genome stability.Yeast peroxiredoxin tsa1Δ cells to various DNA damaging agents. tsa1Δ cells were sensitive to hydroxyurea (HU), 4-nitroquinoline 1-oxide (4NQO) and ultraviolet (UV) irradiation  inhibitor Sml1p DUN1, SML1 or CRT1 in tsa1Δ cells exerted a significant impact on their sensitivity to HU, 4NQO and UV irradiation. Loss of DUN1 further sensitized tsa1Δ cells to H2O2, HU, 4NQO and UV  and 5FC-resistant (5FCR) assays  cells  and frameshifts (13.3%). Large deletions were very rare in tsa1Δ cells (3.3%) when compared with WT (13.3%) cells. In addition, all of the mutations found in tsa1Δ sml1Δ cells with high dNTP levels .Consistent with the activation of Rad53p and Rad9p, the levels of Rad53p target Sml1-3HAp were diminished in RNR genes is transcriptionally activated in response to DNA damage RNR1/2/3/4 transcripts in the presence and absence of HU. In this analysis we included an additional control termed HUG1, a target of Mec1p induced highly by DNA damage RNR transcripts were induced to higher levels in tsa1Δ cells than in WT and sod1Δ cells. The induction of RNR1 and RNR3 was greatest in both untreated and HU-treated tsa1Δ cells. The level of HUG1 transcript was also elevated in tsa1Δ cells and this was more pronounced in the presence of HU  rate of spontaneous 5FCR mutations over tsa1Δ RAD53 cells  as described Rates of spontaneous forward mutations to confer CANTotal RNA was extracted by phenol/freeze RNA preparation method as described Western blot analysis was performed essentially as described


Investigations following stroke first of all require information about the spatio-temporal dimension of the ischemic core as well as of perilesional and remote affected tissue. Here we systematically evaluated regions differently impaired by focal ischemia.Wistar rats underwent a transient 30 or 120 min suture-occlusion of the middle cerebral artery (MCAO) followed by various reperfusion times  or a permanent MCAO . Brains were characterized by TTC, thionine, and immunohistochemistry using MAP2, HSP72, and HSP27. TTC staining reliably identifies the infarct core at 1 d of reperfusion after 30 min MCAO and at all investigated times following 120 min and permanent MCAO. Nissl histology denotes the infarct core from 2 h up to 30 d after transient as well as permanent MCAO. Absent and attenuated MAP2 staining clearly identifies the infarct core and perilesional affected regions at all investigated times, respectively. HSP72 denotes perilesional areas in a limited post-ischemic time (1 d). HSP27 detects perilesional and remote impaired tissue from post-ischemic day 1 on. Furthermore a simultaneous expression of HSP72 and HSP27 in perilesional neurons was revealed.TTC and Nissl staining can be applied to designate the infarct core. MAP2, HSP72, and HSP27 are excellent markers not only to identify perilesional and remote areas but also to discriminate affected neuronal and glial populations. Moreover markers vary in their confinement to different reperfusion times. The extent and consistency of infarcts increase with prolonged occlusion of the MCA. Therefore interindividual infarct dimension should be precisely assessed by the combined use of different markers as described in this study. Focal cerebral ischemia results in typical pathophysiological events that evolve in time and space. These processes are not limited to the lesion itself but differentially affect perilesional and even remote areas , that is known to mainly determine the degree of injury Nowadays non-invasive techniques like MRI - that are routinely applied in humans - can be also used to define ischemic lesions in rodents over time Hereafter differently impaired regions were separated into (1) the infarct core [necrotic damage], (2) perilesional areas [non-necrotic areas affected by a decreased blood flow mainly in territories of the MCA and branches of the ICA] and (3) remote areas [non-ischemic but indirectly affected regions].Male Wistar rats weighing 270–300 g were used. All animal procedures were approved by the local government  and conformed to international guidelines on the ethical use of animals.2O/O2 (70%∶30%). A commercial 4-0 monofilament nylon suture coated with silicone rubber on the tip  was introduced through an incision of the right common carotid artery into the ICA to occlude the origin of the right MCA for 30 min (n = 32), 120 min (n = 32) or permanently (n = 11). During surgery body temperature was maintained at a physiological level. Cerebral blood flow was measured by laser Doppler flowmetry (LDF)  in cortical areas supplied by the MCA  from before the onset of MCAO until 15 min after reperfusion. Sham operated animals (n = 12) underwent the same procedure but without occluding the MCA.Transient MCAO was induced according to Nagasawa and Kogure Brains were removed at various times after reperfusion . Using a rat brain slicer  3 mm coronal sections were dissected, cutting at distances of 2, 5, 8, and 11 mm from frontal pole. Section between 5 and 8 mm  for further use.Slices from the anterior and posterior sections (previously stained in TTC) where mounted onto slides and stained with 0.2% thionine.2O2 before the antibodies  were applied in TBS containing 3% normal donkey serum and 0.2% Triton X-100. Sections were incubated with the antibodies at 4°C overnight and further processed by the Vectastain Elite ABC Kit  using either a donkey anti-mouse or a donkey anti-goat biotinylated secondary antibody . Finally, immunoreactivity was developed in 3,3′-diaminobenzidine tetrahydrochloride . For co-labeling of HSP72 and HSP27 the primary antibodies were simultaneously applied and detected by fluorescence-labeled secondary antibodies . Slices were analysed via confocal microscopy .Free floating sections were treated with 0.24% HAll animals with a dropdown of LDF signal to ≤40% were included in this study. The LDF signals were 27,7%±8,9% of control values (before insertion of the filament) following occlusion of the MCA and recovered to 80,7%±13,1 during reperfusion. There was a very low mortality rate following 30 min MCAO [9% overall including all reperfusion times (3 out of 32 rats)]. Following 120 min MCAO the mortality rates increased over time . Because of the still higher mortality rate in the 120 min MCAO group within 30 days this reperfusion time was omitted. Until 1 d following permanent MCAO 27% (3 out of 11) of operated rats died.All MCAO rats displayed an infarct whereby the infarct size varied. Following mild MCAO (30 min) the infarct always included the striatum and frequently the neocortex. After a severe MCAO (120 min) the necrotic area extended over the striatum to large portions of the adjacent neocortex, amygdala, and hypothalamus. Permanent MCAO produced the largest and most consistent infarcts with an infarct core always including striatum, neocortex, amygdala, and hypothalamus. The formation of edema was strongest under these conditions.Following 30 min MCAO TTC staining did reliably delineate the infarct  at 1 d after reperfusion . No cleaIntact cells  could beThe extent of neuronal injury could be identified by an absence as well as an attenuation of MAP2 immunoreactivity. After 30 min MCAO a diminished MAP2 expression could be detected as early as 2 h after reperfusion in striatum and neocortex. From 1 d of reperfusion on lack of MAP2 (necrotic areas) could be observed mainly in the striatum and occasionally in parts of the neocortex , 3. FollIn agreement with many other studies HSP27 was detectable at 1 d of permanent MCAO and from 1 d of reperfusion on after mild as well as severe ischemia . RegionsSlices from sham operated animals were adequately stained. We and others found HSP27 constitutively expressed in few astrocytes (mainly in fibre tracts) The TTC method rests on the functioning of mitochondrial enzymes By using immunohistochemical markers not only the infarct core but also perilesional and remote affected regions could be discriminated . BesidesMAP2 not only identifies the infarct core but also reveals ischemic damage in perilesional zones (defined by attenuated MAP2 staining) very sensitively. After mild MCAO in general a relatively broad area of partial MAP2 degradation was revealed. Following severe as well as permanent ischemia the infarct core was extended whereas perilesional regions were narrow. Since MAP2 is involved in maintaining the structural integrity of the neuronal cytoskeleton Transient post-ischemic HSP72 expression might start as early as 3 h after brain injury persisting for about 7 d Our results confirmed a post-ischemic upregulation of HSP27 after focal ischemia mainly in reactive astrocytes For the bilateral hippocampal induction of HSP27 one might discuss several causes. Though a bilateral transient impairment of cerebral blood flow has to be considered we think that secondary responses to ischemia are more relevant  Additionally to the HSP27 expressing astrocytes HSP27 positive neurons were detected. Double immunofluorescence stainings of HSP27 and HSP72 revealed a co-staining in the majority of labeled neurons. The induction of two heat shock proteins strengthens the possibility of these perilesional affected neurons to survive. All in all HSP27 is a useful marker to sensitively detect perilesional and far remote affected regions over a large period of time.Though a standardized protocol for infarct induction was used individual deviations occur inter alia due to varying cerebrovascular anatomy. The variability of infarcts as well as the involvement of perilesional and remote affected regions was most pronounced after mild MCAO . With pr


BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). 72 cell lines from affected women in high-risk breast ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS). BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status, with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82–94%), poor for BRCAX with an LCS (40–50%), and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71–100%). This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity.The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between  BRCA1 and BRCA2 increase risk of breast cancer and contribute to a proportion of breast cancer families. However, more than half of the reported sequence alterations in BRCA1 and BRCA2 are currently of unknown clinical significance. We analysed gene expression in lymphoblastoid cell lines derived from blood of patients with sequence alterations in BRCA1 and BRCA2 and compared these to lymphoblastoid cells from familial breast cancer patients without such alterations. We then classified these lymphoblastoid cells based on their gene profiles. We found that BRCA1 and BRCA2 samples were more similar to each other than to familial breast cancer patients without BRCA1/2 mutations, and that the type of sequence change in BRCA1 and BRCA2 (missense or truncating) influenced gene expression. We included in the study ten familial breast cancer samples, which carried sequence changes in BRCA1 or BRCA2, that are believed to be of little clinical significance. Interestingly these samples were distinct from other familial breast cancer cases without any sequence alteration in BRCA1 or BRCA2, indicating that further work needs to be performed to determine the possible association of these “low clinical significance” sequence changes with a low to moderate risk of cancer.Inherited mutations in the genes  BRCA1 and BRCA2 account for a considerable proportion of these familial breast cancer cases, with the average cumulative risk in BRCA1 and BRCA2 mutation carriers by age 70 years estimated at 65% and 45%, respectively http://research.nhgri.nih.gov/bic/) currently has more than 1400 and 1800 unique sequence variants listed in the BRCA1 and BRCA2 genes, respectively. These include frameshift, nonsense, missense, splice site alterations and polymorphisms. Greater than a third of the BRCA1 and greater than half of the BRCA2 unique variants are “unclassified variants” without compelling evidence of pathogenicity or functional significance. The majority of unclassified variants recorded in the BIC database are predicted missense changes (more than 400 BRCA1 and 800 BRCA2). However other variants which may be categorised as unclassified variants are in-frame deletions or duplications, variants that may disrupt splicing, or variants in the 3′UTR that may affect RNA stability (www.kconfab.org). BRCA1/2 unclassified variants represent a problem in the clinical setting as it is not known which variants are associated with the high risk of disease reported for classical truncating mutations.Approximately 7% of breast cancer cases occur in women with a strong family history of the disease Several functional assays may be used to determine the significance of unclassified variants, including transcription activation and complementation assays BRCA1 mutation carriers BRCA1 or BRCA2 genes, compared to healthy women undergoing reduction mammoplastic surgery with no family or personal history of any cancer or sporadic breast-cancer-affected controls ATM mutations, some of which are known to be associated with an increased risk in breast cancer BRCA1 or BRCA2 mutation status and to assist in assessing the clinical significance of BRCA1 and BRCA2 unclassified variants.Gene expression profiling has increased our understanding of the molecular events in breast tumor development, has been used to predict prognosis, and has characterised breast tumors into subtypes BRCA1 or BRCA2, to familial breast cancer cases with no known BRCA1/2 mutations (BRCAX). We also considered the possibility that BRCAX individuals with a BRCA1 or BRCA2 sequence variant classified to be neutral/low clinical significance (LCS) using multifactorial likelihood analysis may differ in gene expression profile from BRCAX individuals without such sequence variants. In addition, since truncating alterations comprise the majority of known pathogenic mutations but most BRCA1 and BRCA2 unclassified variants are predicted missense alterations, we compared profiles from individuals with known missense or truncating mutations to determine if mutation effect will affect the mutation-associated expression profile for each gene. We derived gene lists to predict mutation status defined by gene and mutation effect, and then tested the efficacy of these gene lists to predict the gene mutation status of LCLs. We provide evidence that gene lists differ according to gene and mutation effect, and according to the presence of sequence variants of low clinical significance. We also demonstrate that the use of appropriately-derived gene lists improves the prediction of pathogenicity of known mutations.In this study we compared LCL gene expression signatures of breast cancer cases carrying pathogenic mutations in BRCA1 or BRCA2 pathogenic mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). BRCAX breast cancer families are likely to result from mutations in several other genes, and thus represent a heterogeneous group. Moreover, included in the BRCAX group were a subset of 10 BRCAX individuals who carried a BRCA1/2 variant previously classified to be of low clinical significance using multifactorial likelihood approaches BRCAX LCLs containing a BRCA1 or BRCA2 variant of low clinical significance clustered away from the majority of remaining BRCAX samples . For this reason, BRCAX samples were stratified according to the presence of an LCS variant for further analyses.The ultimate aim of this experiment was to establish if gene expression profiles could distinguish between  samples . A t-tesBRCA1 and BRCA2 truncating mutations and rare sequence variants of low clinical significance, but differs from BRCA1 and BRCA2 missense mutations and BRCAX non-BRCA1/2 familial cases.Gene expression is similar for carriers of BRCA1 and BRCA2 samples were more similar to each other than BRCAX samples without an LCS variant. This result suggests that germline effects of heterozygous mutations in BRCA1 and BRCA2 cannot easily be separated using the experimental conditions used in this study. Although BRCAX samples tended to cluster distinctly from BRCA1/2 samples, nine of ten BRCAX individuals who carried a BRCA1/2 variant previously classified to be of low clinical significance fell within the major BRCA1 or BRCA2 mutation cluster. In contrast, six of the nine pathogenic missense mutations of BRCA1 or BRCA2 fell into a BRCA1/2 outlier group, which clustered closer to the BRCAX samples.Unsupervised hierarchical clustering  of LCL eBRCA1/2 pathogenic carriers and BRCAX individuals, we used a Gaussian Process Classifier (GPC). GPC analysis was used previously in an analysis of microarray profiles from irradiated short-term fibroblasts of BRCA1/2 mutation carriers BRCA1 truncating mutation carriers to BRCA2 truncating mutation carriers, and to BRCAX samples without an LCS variant. Samples with BRCA1 or BRCA2 pathogenic missense mutations or classified as BRCAX with an LCS variant were then included to determine their affect on the prediction accuracy. A summary of the prediction accuracy is shown in BRCAX with an LCS, and samples with BRCA1 or BRCA2 missense mutations. This prediction accuracy is above the expected performance, as a random prediction with three classes comprised of a similar sample number would be 33% accuracy. When BRCA1 and BRCA2 samples were compared to only BRCAX samples with an LCS variant, the prediction dropped to 43.46%, and the addition of the BRCAX samples without an LCS variant improved the accuracy. In all comparisons the inclusion of the pathogenic non-truncating mutations of BRCA1 and BRCA2 lowered the prediction accuracy.To determine the accuracy of using gene expression data from LCLs to predict BRCA1 or BRCA2 are generally identified after full sequencing of both genes. Therefore the most common clinical question is whether a variant in or BRCA2BRCA1  is pathogenic or not. We thus performed pair wise analyses to determine if BRCAX samples could be distinguished from those with pathogenic mutations in BRCA1 or BRCA2. Based on observations from hierarchical clustering analyses and the GPC analysis, we also considered the possibility that the effect of pathogenic BRCA1/2 mutations (truncating or missense) affected LCL gene expression. T-tests were performed using the 20,874 detected probes to elucidate gene differences between i) BRCA1 or BRCA2 truncating mutations vs BRCAX without an LCS variant; ii) BRCA1 or BRCA2 missense mutations vs BRCAX without an LCS variant. The number of genes which passed these restrictions and the overlap between them is outlined in BRCAX with an LCS variant  appears to affect the genes that are differentially expressed in the carriers after IR  vs BRCAX (noLCS)] and [BRCA1 (truncating) vs BRCAX (noLCS)] was 16 transcripts, with no overlap between the top 200 genes from [BRCA2 (missense) vs BRCAX (noLCS)] and [BRCA2 (truncating) vs BRCAX (noLCS)]. A total of 715 different genes were represented in the four lists of top 200 gene-lists from comparison of BRCAX (no LCS) to the different BRCA1/2 groups above. The results are summarised in BRCA2 truncating pathogenic carriers were consistently predicted with higher accuracy compared to BRCA1 truncating pathogenic carriers. The accuracy of prediction was improved when the gene list used for prediction was appropriate to the mutation effect (truncating or missense) being tested. When the missense-associated gene list was used, pathogenic truncating mutations were predicted with 35% and 68% accuracy for BRCA1 and BRCA2, respectively. Predictions increased to 71% and 84% for BRCA1 and BRCA2, respectively, using the truncating-associated genes. Similarly, the pathogenic missense mutation carriers were predicted with 83% and 100% accuracy when the missense-associated gene list is used, but this accuracy was lower or remained the same when the truncating-specific gene list was used (83% and 0%). Prediction of BRCAX samples that did not carry an LCS variant was high in all comparisons (82–94%). In contrast, prediction of BRCAX samples that did carry an LCS variant was poor (40–50%).SVM is a widely accepted classification approach for assessing differences in mRNA expression, and was used to compare after IR , we asseto BRCAX . The gentriction . The topBRCA1 (or BRCA2) and BRCAX samples. The significance of the predictions for the BRCA1 pathogenic missense mutations is summarised in When using SVM, the significance of the predictions can also be represented by the distance the prediction is from the plane, where predictions called with greater confidence are further from the plane that separates the BRCA1 or BRCA2 compared to BRCAX samples without an LCS variant was performed to determine the potential functional relevance of the differentially expressed genes. All BRCA1 and BRCA2 pathogenic mutations resulted in gene expression changes relating to cell cycle, cancer and cellular growth and development, while BRCA1 and BRCA2 missense mutations shared some additional similarities (cell death and cell development pathways). There were also alterations in several pathways that were unique to BRCA1 truncating mutations, BRCA2 truncating mutations, BRCA1 missense mutations, or BRCA2 missense mutations .It is difficult to counsel patients with a strong family history of breast cancer who are found to carry an unclassified variant in BRCA1 or BRCA2 genes from sporadic breast-cancer-affected controls BRCA1 or BRCA2 mutation status, with the ultimate aim of predicting the significance of unclassified variants of BRCA1 or BRCA2. We chose LCLs as a minimally invasive source of germline material that can be maintained as long term cultures, and because previous studies have shown that LCL array profiling is robust to sourcing of LCLs established in different laboratories BRCA1 and BRCA2 carriers to those of non-BRCA1/2 BRCAX familial breast cancer patients, an appropriate reference group for the proposed evaluation of unclassified variants identified in familial breast cancer patients. A relatively early time-point of 30 minutes post-irradiation was chosen to capture gene expression initiation, and minimize possible downstream compensation effects. It has previously been shown that 10Gy IR treatment of normal LCLs has an effect on the transcriptional response, with greatest change in mRNA levels for most genes within one hour post-treatment Expression profiles of short-term fibroblasts have previously been reported to separate carriers of a heterozygous mutation in the BRCAX cases carried BRCA1 or BRCA2 sequence variants that had been previously classified using multifactorial likelihood modelling methods to be neutral or of low clinical significance-that is, these rare variants are extremely unlikely to be a high-risk mutation in either of these genes, but the modelling methods used cannot assess whether they are truly neutral or associated with a much lower risk of disease. We found that the BRCAX samples with such LCS variants were separated from the majority of BRCAX samples without such LCS variants using unsupervised hierarchical clustering. This result indicates that LCS samples differ in expression profile as a result of their BRCA1 or BRCA2 sequence variant, and was substantiated by the class prediction methods: GPC prediction of the BRCAX samples decreased in accuracy when BRCAX samples with an LCS were included. In addition, SVM to detect BRCA1 or BRCA2 mutation-related gene lists yielded differences in the significant genes for comparisons to BRCAX samples without an LCS variant, compared to BRCAX samples with an LCS variant. Accordingly, prediction of BRCAX subgroup status based on the more robust gene list derived from comparisons to BRCAX individuals without an LCS variant was generally poorer for BRCAX samples with an LCS (40–50%) compared to those without an LCS (82%–94%). These rather provocative results indicate that the possible effect of all rare variants should be considered in development of assays to assess which variants have features of high-risk mutations. Moreover, the similarity in expression profile of these variants to other BRCA1/2 pathogenic mutations suggests that at least some of these LCS variants may confer small-moderate risks of breast cancer, presumably acting in concert with alterations in other genes in the BRCA1/2 pathway to lead to breast cancer. Given the rarity of these variants, alternative statistical approaches will be required to assess the risk of cancer associated with them.A number of BRCA1 mutations and those with pathogenic BRCA2 mutations. Ionising radiation has previously been show to separate fibroblast cells which carry BRCA1 or BRCA2 mutations from sporadic cases with 100% accuracy BRCA1 and BRCA2 cases to familial BRCAX cases as an appropriate reference group for familial breast cases likely to be identified as carriers of BRCA1/2 mutations or unclassified variants, we used LCLs instead of fibroblasts, we selected a lower IR exposure (10Gy vs 15Gy), and we chose a relatively early time point of 30 mins after exposure to IR in order to gain a better understanding of the functional differences in response to IR between the BRCA1, BRCA2 and BRCAX cell lines. Some or all of these factors may explain the difference in the ability of this study to distinguish BRCA1 from BRCA2, both of which are involved in DNA damage repair. However, differences in post-irradiation response between BRCAX individuals and BRCA1/2 mutation carriers are supported by alternative analysis we have conducted of the subset of genes reported to be involved in post-irradiation response, comparing mutation-negative normal female controls to BRCAX individuals without an LCS variant, or to BRCA1 or BRCA2 truncating mutation carriers. Our results indicate substantial differences in radiation response between normal controls and the patient groups, and also considerable differences between the BRCAX group and BRCA1 and BRCA2 carriers The assay conditions used in this study could not distinguish between samples with pathogenic BRCA1 or BRCA2. In the clinical setting, individuals generally present with full sequencing of both genes, and presence of a variant in one gene or the other. We thus assessed the ability to distinguish BRCA1 or BRCA2, separately, from BRCAX individuals. Importantly, since most unclassified variants are predicted to cause amino acid substitutions, we also assessed the relevance of mutation effect for expression profiles. We found that the genes which significantly differed between BRCA1 or BRCA2 and BRCAX LCLs were dependent on mutation effect. Accordingly, the SVM prediction for each mutation effect was best if the appropriate gene list was used, in terms of both accuracy of prediction (BRCA1 or BRCA2 vs BRCAX) and confidence in the classification as determined by the distance of the prediction from the SVM plane. Thus we strongly urge that mutation effect is taken into account if this type of assay is to be developed for use in predicting the clinical significance of BRCA1/2 variants. The current challenge is that few missense variants have been classified with respect to their clinical significance, with the only 23 individual missense variants termed clinically important by BIC, 17 in BRCA1 and six in BRCA2. Moreover, these are restricted in terms of the domains/regions in which they occur, residing in the BRCA1 start site (n = 2), ring finger (n = 4) or transactivation domains (n = 11), and the BRCA2 CDK2 phosphorylation site (n = 3) or at one codon  in a region of unknown function. It will thus be difficult to accrue a panel of known pathogenic missense variants for use in such predictive assays, and will require a concerted collaborative effort. Assuming sufficient pathogenic variants are identified, the successful execution of such a study may eventually distinguish missense-associated gene expression patterns that are generic to missense mutations, and/or those that are specific to the domain location of missense mutations. In addition, a possibly greater challenge will be identifying assay conditions  that can also identify gene expression differences between patients with rare variants of low clinical significance in BRCA1 and/or BRCA2 and those with truly high-risk pathogenic mutations (truncating or missense) in these genes. Our study, using conditions that were not optimal for separating BRCA1 and BRCA2 mutations nevertheless identified gene expression differences between BRCA1/2 pathogenic mutations and LCS variants, suggesting that larger sample sizes and further experimentation may identify a more robust gene list to separate pathogenic mutations, variants of low clinical significance, and individuals with no sequence alterations in BRCA1/2.The ultimate aim of this experiment was to identify array profiles that would be useful for the classification of unclassified sequence variants of BRCA1 and BRCA2 mutation carrier groups is consistent with the known functions of BRCA1 and BRCA2Pathway analysis confirming altered expression of cancer, cell proliferation and cell cycle pathways in BRCA1 and BRCA2 variants considered to be of low clinical significance have array profiles distinct from other non-BRCA1/2 familial cases, but resembling profiles of pathogenic BRCA1/2 cases, indicating that further work will be required to evaluate their possible association with a low-moderate risk of cancer. We have also shown that it will be important to consider mutation effect when developing array-based assays for predicting the clinical significance of BRCA1 or BRCA2 unclassified variants. Lastly, our findings demonstrate the ability of array profiling of immortalized lines derived from lymphoblastoid cells to detect germline mutations in genes that result in breast and ovarian cancer, and thus have relevance to the investigation of other genetic diseases irrespective of the organs or tissues they affect.In conclusion, we have provided evidence that carriers of BRCA1 or BRCA2 mutation have been identified, and families in which no predisposing mutation has been identified (BRCAX). The recruitment criteria for BRCAX families are: 1) at least one member of the family at high-risk according to the National Breast Cancer Centre Category III guidelines (http://www.nbcc.org.au), and four or more cases of breast or ovarian cancer (on one side of the family), and two or more living affecteds with breast or ovarian cancer, and four or more living first or second degree unaffected female relatives of affected cases, over the age of 18 ; 2) two or three cases of breast or ovarian cancer (on one side of the family) in same or adjacent generations, if at least one of these cases is ‘high risk’ , and two or more living affected cases with breast or ovarian cancer, and four or more living first or second degree unaffected female relatives of affected cases, over the age of 18.LCLs were derived from breast cancer-affected women recruited into the Kathleen Cuningham Foundation for Research into Breast Cancer (kConFab), a consortium which ascertains multiple-case breast cancer families BRCA1 and BRCA2 pathogenic mutations and variants of low clinical significance (LCS) are described on http://www.kconfab.org/Progress/Classification.shtml. Briefly, LCS variants include BRCA1 or BRCA2 variants described in trans with a deleterious mutation in the same gene in an individual and occur at a frequency of less than 1% in unaffected controls, or considered neutral/low clinical significance as measured using multifactorial likelihood approaches Classifications for A cohort of 72 LCLs were used in this study. The full listing of mutation details for LCLs is shown in BRCA1, 17 of which are predicted to lead to a truncated protein, and six of which were missense mutations ;23 LCLs from women carrying a pathogenic mutation in BRCA2, 19 of which are predicted to lead to a truncated protein, and three of which were missense mutations ;22 LCLs from women carrying a pathogenic mutation in BRCA1 or BRCA2 (BRCAX) after complete sequencing and multiplex ligation-dependent probe amplification gene dosage assay (MLPA) large deletion testing of BRCA1 and BRCA2. Ten samples, carried either BRCA1 or BRCA2 sequence germline variants considered from multifactorial likelihood classification to be LCS ; BRCA2 353 A>G Y42C, 2834 C>T S869L, 3031 G>A D935N (3 samples), 8795 A>C E2856A) BRCA1 or BRCA2 sequence variants other than common polymorphisms.27 LCLs from women from breast cancer families that have tested negative for pathogenic mutations in t tests were performed to determine the significance of gene expression changes. Expression differences were validated for 8/10 genes tested.LCLs were grown in RPMI 1640 media with 15% fetal bovine serum, 1% penicillin-streptomycin and 1% L-glutamine. The cell number was normalised and fresh medium was added to cells 24hr prior to irradiation with 10Gy, using a calibrated Cs137 c-source delivering 1 Gy/1.5 min. Total RNA was harvested 30min later using an RNeasy kit . The Illumina Totalprep RNA amplification kit  was used to amplify and biotinylate 450ng of total RNA. Biotinylated RNA was hybridised overnight at 55°C to Illumina Human-6 version 1 BeadChips containing >46,000 probes . The microarrays were washed, stained with streptavidin-Cy3, and then scanned with an Illumina BeadArray Scanner. Duplicate arrays were performed for eight cell lines across the different groups for quality control purposes, with duplicates performed on different days. All duplicate arrays showed highest correlation with each other (correlation >0.98). Duplicate samples were not included in analysis. Comparative real-time PCR was performed for ten genes on 6–8 samples, using GAPDH to normalise all data, and the comparative cycle threshold method for analysis. Paired student BRCA1/BRCA2 mutation status of irradiated fibroblasts Raw data was imported into Illumina Beadstudio and then exported into Genespring v7.3  for further analysis. Data was normalised  and filtered using an Illumina detection score of >0.99 in at least one sample, which yielded 20,874 probes that were used in all further analyses. The majority of these probes used in the analysis were designed by Illumina to assay the curated portion of the NIH Ref sequence database-16,923 were present in the Ref sequence database, comprising 65% of all Ref sequence-listed probes on the array. Transcripts which had a >2-fold change versus the mean were visualised using unsupervised Hierarchical Clustering  and 2. Twww.ingenuity.com) was used for biological interpretation of gene lists. Analysis of the transcripts found to be up- and down-regulated in irradiated LCLs as identified for the different mutation categories identified those biochemical networks most likely to be affected by a BRCA1 and BRCA2 truncating and missense mutation, relative to BRCAX. Those pathways with multiple hits or a significance score ≥20 were then compared.Ingenuity Pathway Analysis Click here for additional data file.Table S1Detailed Mutation Status of LCLs.(0.03 MB XLS)Click here for additional data file.Table S2BRCA1 Missense vs BRCAX without an LCS.The top 200 significant genes from the comparison of (0.05 MB XLS)Click here for additional data file.Table S3BRCA1 Truncating vs BRCAX without an LCS.The top 200 significant genes from the comparison of (0.05 MB XLS)Click here for additional data file.Table S4BRCA2 Missense vs BRCAX without an LCS.The top 200 significant genes from the comparison of (0.06 MB XLS)Click here for additional data file.Table S5BRCA2 Truncating vs BRCAX without an LCS.The top 200 significant genes from the comparison of (0.05 MB XLS)Click here for additional data file.


We have developed a mouse monoclonal antibody 5C11 (IgG2a) against cell surface antigen of Ewing's sarcoma (ES). 5C11 specifically reacted with ESs but not with other small round cell tumours in childhood, i.e. neuroblastomas, primitive neuroectodermal tumours (PNETs), rhabdomyosarcomas and malignant lymphomas. 5C11 did not react with any other tumours in children except for hepatoblastomas. No reactivity has been identified in normal tissues with the exception of fetal hepatocytes. Immunoelectron microscopically, 5C11 reactive antigen was located on cell membrane of ES cells. Biochemically, 5C11 immunoprecipitated a cell surface protein having molecular weight of 81,000 Da. 5C11 is the first antibody which can clearly distinguish ES from neurogenic tumours, especially from PNETs which were recently reported to have common features to ESs regarding chromosal abnormality and proto-oncogene expression but show evident differentiation into neurogenic direction. The results strongly indicate the usefulness of 5C11 not only for diagnostic purpose when no specific marker is available but also for studying the histogenesis of ES. In addition, no reactivity in normal tissue implies its potential application as a therapeutic reagent when the management of ES patients is still a great problem in clinical field.


To establish whether loss of heterozygosity (LOH) for chromosome 16q in Wilms' tumours confers an adverse prognosis, DNA from 40 Wilms' tumour/normal pairs were analysed using highly polymorphic microsatellite markers along the length of 16q. Fifteen per cent of tumours showed LOH for 16q. Although the common region of allele loss spanned the 16q24-qter region, a second distinct region of LOH was identified in 16q21. Five out of six tumours showing LOH were either (1) high stage or (2) low stage with unfavourable histology. In addition, there was a higher mortality rate in patients showing LOH for 16q than those that did not. These data strongly support the suggestion that LOH for 16q is associated with an adverse prognosis.