Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.     

Genetic heterogeneity of residual variance in broiler chickens

Aims were to estimate the extent of genetic heterogeneity in environmental variance. Data comprised 99 535 records of 35-day body weights from broiler chickens reared in a controlled environment. Residual variance within dam families was estimated using ASREML, after fitting fixed effects such as genetic groups and hatches, for each of 377 genetically contemporary sires with a large number of progeny (> 100 males or females each). Residual variance was computed separately for male and female offspring, and after correction for sampling, strong evidence for heterogeneity was found, the standard deviation between sires in within variance amounting to 15–18% of its mean. Reanalysis using log-transformed data gave similar results, and elimination of 2–3% of outlier data reduced the heterogeneity but it was still over 10%. The correlation between estimates for males and females was low, however. The correlation between sire effects on progeny mean and residual variance for body weight was small and negative (-0.1). Using a data set bigger than any yet presented and on a trait measurable in both sexes, this study has shown evidence for heterogeneity in the residual variance, which could not be explained by segregation of major genes unless very few determined the trait.     

Predicted expansion of the claudin multigene family

Claudins (Cldn) are essential membrane proteins of tight junctions (TJs), which form the paracellular permselective barrier. They are produced by a multi-gene family of 24 reported members in mouse and human. Based on a comprehensive search combined with phylogenetic analyses, we identified three novel claudins (claudin-25, -26, and -27). Quantitative RT-PCR revealed that the three novel claudins were expressed in a tissue- and/or developmental stage-dependent manner. Claudins-25 and -26, but not claudin-27, were immunofluorescently localized to TJs when exogenously expressed in cultured MDCK and Eph epithelial cell lines. These findings expand the claudin family to include at least 27 members.     

The mechanism of fibril formation of a non-inhibitory serpin ovalbumin revealed by the identification of amyloidogenic core regions

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.     

Mechanism of oxidative DNA damage induced by carcinogenic 4-aminobiphenyl

DNA adduct formation is thought to be a major cause of DNA damage by carcinogenic aromatic amines. We investigated the ability of an aromatic amine, 4-aminobiphenyl (4-ABP) and its N-hydroxy metabolite (4-ABP(NHOH)) to cause oxidative DNA damage, using (32)P-labeled human DNA fragments from the p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. 4-ABP(NHOH) was found to cause Cu(II)-mediated DNA damage, especially at thymine residues. Addition of the endogenous reductant NADH led to dramatic enhancement of this process. Catalase and bathocuproine, a Cu(I)-specific chelator, reduced the amount of DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). 4-ABP(NHOH) dose-dependently induced 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in the presence of Cu(ll) and NADH. 4-ABP(NHOH) conversion to nitrosobiphenyl, as measured by UV-visible spectroscopy, occurred rapidly in the presence of Cu(II), suggesting Cu(II)-mediated autoxidation. Increased amounts of 8-OHdG were found in HL-60 cells compared to the H(2)O(2)-resistant clone HP100 following 4-ABP(NHOH) treatment, further supporting the involvement of H(2)O(2). The present study demonstrates that an N-hydroxy derivative of 4-ABP induces oxidative DNA damage through H(2)O(2) in both a cell-free system and in cultured human cells. We conclude that, in addition to DNA adduct formation, oxidative DNA damage may play an important role in the carcinogenic process of 4-ABP.     

Glutamine synthetase I-deficiency in Mesorhizobium loti differentially affects nodule development and activity in Lotus japonicus

In this study, we focused on the effect of glutamine synthetase (GSI) activity in Mesorhizobium loti on the symbiosis between the host plant, Lotus japonicus, and the bacteroids. We used a signature-tagged mutant of M. loti (STM30) with a transposon inserted into the GSI (mll0343) gene. The L. japonicus plants inoculated with STM30 had significantly more nodules, and the occurrence of senesced nodules was much higher than in plants inoculated with the wild-type. The acetylene reduction activity (ARA) per nodule inoculated with STM30 was lowered compared to the control. Also, the concentration of chlorophyll, glutamine, and asparagine in leaves of STM30-infected plants was found to be reduced. Taken together, these data demonstrate that a GSI deficiency in M. loti differentially affects legume–rhizobia symbiosis by modifying nodule development and metabolic processes.     

Extraordinarily low evolutionary rates of short wavelength-sensitive opsin pseudogenes

Aquatic organisms such as cichlids, coelacanths, seals, and cetaceans are active in UV–blue color environments, but many of them mysteriously lost their abilities to detect these colors. The loss of these functions is a consequence of the pseudogenization of their short wavelength-sensitive (SWS1) opsin genes without gene duplication. We show that the SWS1 gene (Bden_S1ψ) of the deep-sea fish, pearleye (Benthalbella dentata), became a pseudogene in a similar fashion about 130 million years ago (Mya) yet it is still transcribed. The rates of nucleotide substitution (~ 1.4 × 10^− 9/site/year) of the pseudogenes of these aquatic species as well as some prosimian and bat species are much smaller than the previous estimates for the globin and immunoglobulin pseudogenes.     

Enhancement of VOC without Loss of JSC in Organic Solar Cells by Modification of Donor/Acceptor Interfaces

Organic solar cells (OSCs) are promising low‐cost devices for generating electricity. In addition to fill factor, the short circuit current density (J_SC) and the open circuit voltage (V_OC) are two key factors that have critical influence on the device performance. The energy levels of the donor and acceptor materials are crucial for achieving a high J_SC and V_OC. However, the interfacial structures between the organic materials substantially affect the J_SC and V_OC through the energy of the charge transfer (CT) states and the charge separation and recombination reaction kinetics. Here, it is reported that separating the donor and acceptor layer in bilayer OSCs with a thin insulating layer increases the energy of the CT state by weakening the Coulomb interaction at the interface and this also suppresses photoinduced CT and recombination. Although these effects usually increase V_OC and decrease J_SC, the trade‐off is avoided by doping the insulating layer with a dye to utilize the energy transfer process. The increase in V_OC without the reduction in J_SC enhances the conversion efficiency of the OSCs by 30%.