Our study has implicated post-transcriptional regulation playing a role in upregulating DNA repair gene expression upon UV radiation exposure. Consistent with ROS serving as a key regulatory signal for ddb2 expression in both zebrafish and cavefish cells, endogenous ddb2 gene expression was robustly induced in both zebrafish PAC-2 (blue traces) and cavefish EPA (orange traces) cells upon treatment with H2O2 (Fig 3A). Still looking? (B) Below: representative real-time bioluminescence assays from zebrafish PAC-2 cells transfected with the E2F/D-boxddb2-Luc and D-boxddb2-Luc reporter respectively, and treated in DD with 300 μM H2O2 at the time points indicated by the blue arrows. Our characterization of ddb2 regulation in zebrafish and blind cavefish provides new insight into the mechanisms whereby light exposure regulates gene expression in fish. DNA repair of UV induced damage in constant darkness. Finally, like P. andruzzii, the zebrafish is a member of the Cyprinidae family and so gene sequences are well conserved between the two species. However, countering this argument, in a comparative study using cave and surface populations of the amphipod crustacean Gammarus minus, reduced expression of both photolyase and the DNA repair endonuclease XPG (ERCC5, a component of the NER pathway) was observed in the cave population, suggesting that all these genes are under relaxed selection in the cave populations [59]. Specifically, zebrafish and cavefish cells were cultured according to former reports. We previously showed that in the case of the zebrafish photolyase genes, the functionality of two D-box enhancer elements is necessary and sufficient to activate transcription [27]. Copyright: © 2021 Zhao et al. Given that NER is able to repair UV damaged DNA and also that expression of the NER recognition factor, ddb2 is light inducible, we questioned whether NER might also exhibit loss of function in a similar fashion to photoreactivation? ... We take great pride in equipping our customers to do their own blind repair… cDNA was synthesized from total RNA extracts using M-MLV reverse transcriptase (Thermo Scientific). You can also change some of your preferences. Interestingly, we observed a similar increase in transcript stability of the other key NER regulatory factor XPC in both zebrafish and cavefish cells (Fig 5G and 5H), pointing to a general role for increased transcript stability in the upregulation of NER function following UV exposure. Reverse transcription was performed using M-MLV reverse transcriptase (Thermo Scientific) and then the cDNAs were cloned into the pGEM-T Easy Vector (Promega). [ view less ], Affiliations: Nathalie Geyer, Two-way ANOVA followed by Sidak’s multiple comparisons test results are reported in S1 Table. Interestingly this enhancer element represents the binding site for a complex repertoire of homo and heterodimers of PAR bZip factors and E4BP4 / nfil3 bZip factors [20,46–48]. For example, previous studies have analysed changes in mRNA stability in human cell lines which had been subjected to stress-inducing agents. Detailed information on each construct is described in Fig 2 and S3 Table. Instructions show you step by step how to fix blinds, shades, shutters. Consistent with the notion that E2F may serve a similar function in fish cells, E2F/D-boxddb2-Luc reporter (including the E2F site) expression levels were significantly higher than those of the D-boxddb2-Luc reporter (lacking the E2F site) during exposure to light-dark cycles (S3 Fig, panel A). However, the prediction that a single regulatory mechanism based on the D-box may direct light responsive gene expression is clearly challenged by our observation that P. andruzzii ddb2 expression is still strongly light inducible. After 24h, cells were exposed to specific doses of UV radiation (160 and 640 J/m2), using a UV-strata linker (Stratagene). ddb2 gene regulation contrasts with that of the photolyase genes, where both UV-induced transcription and transcript stability observed in the zebrafish are completely absent in P. andruzzii cells. Hongxiang Li, Blue arrows above the D-boxes indicate their orientation. NER is a major DNA repair system which removes a broad spectrum of DNA helix-distorting lesions, including UV-induced photoproducts as well as bulky base adducts induced by other types of genotoxic stress such as various chemical carcinogens [7–11]. Therefore, we performed real-time qPCR analysis of ddb2 expression in zebrafish and P. andruzzii cells over a 60-hour period in constant darkness following acute exposure (13 seconds) to a UV-C pulse of 20 J/m2, assaying 6–4 photolyase mRNA expression as a control. Below, within the context of the minimal, light-responsive promoter construct LRRddb2-Luc are indicated the E2F site, D-box and ATF1/CREB enhancer elements by red, green and blue rectangles, respectively. 8 AM – 3 PM Our findings provide valuable additional insight into the general mechanisms whereby light, UV and ROS exposure trigger changes in gene expression. Contrary to what we previously reported for the photoreactivation DNA repair mechanism, the levels of fragmented DNA following UV exposure (from 40 J/m2 to 640 J/m2) in both zebrafish [36] and cavefish [25] cells increased, reaching a peak 2 hours after UV exposure and then decreasing at 4 hours in a UV dose dependent manner, with similar induced levels of fragmented DNA in the two cell types (Fig 1A–1D). After 24hrs, 0.5 mM beetle luciferin, potassium salt (Promega) was added to the culture medium. Shutter & Blind Repair Experts. While significant progress has been made in understanding how these mechanisms recognize and repair … Furthermore, we reveal complexity in the transcriptional and posttranscriptional mechanisms regulating the repair of UV-induced DNA damage. BLINDPARTS.COM SALE! Furthermore, we have extensively studied the transcriptional mechanisms underlying light induced gene expression in this genetic model species [22,27,29–33]. A non-exposed plate served as negative control. (B,C) Representative real-time bioluminescence assays in PAC-2 (B) or EPA (C) cells transfected with the ddb2-Luc luciferase reporter. As in the case of the photolyase genes, D-boxes in the ddb2 promoter are sufficient to induce transcription in zebrafish. Daniela Vallone, Thus, our results may be consistent with the presence of a non-transcriptional mechanism mediating the UV-induced expression of ddb2 in the cavefish P. andruzzii. 4.3 out of 5 stars 174. Pacific Standard Time. Upgrade any RollEase roller shade with a 1.5 inch outside diameter tube and convert it to motorized functionality. This is the result of a set of point mutations which generate truncated photolyase proteins as well as significant changes in the regulation of photolyase gene expression. The expression of P. andruzzii ddb2, a key recognition factor in NER DNA repair, displays the normal visible light, UV and ROS-inducible expression pattern observed in zebrafish. Interestingly, eutherians including human and mouse, completely lack photoreactivation repair, an additional, efficient and highly conserved mechanism which is catalysed by photolyases and harnesses visible light to repair UV-induced cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproduct (6-4PPs) lesions [4,6]. Furthermore, high basal levels of expression of CPD photolyase have also been reported in the blind cave forms of this species [21]. The integrity of genetic information is frequently challenged by environmental factors such as sunlight which induce mutations in DNA. Bromelain is a group of enzymes found in pineapple juice and in the pineapple stem. Furthermore, this mechanism appears to demonstrate species and gene-specific flexibility, with loss of UV induced mRNA stability in the case of the photolyase genes but not the genes encoding the NER factors ddb2 and xpc in the blind cavefish P. andruzzii. We offer thousands of repair parts & string for window blinds & shades. We have previously revealed that the Somalian cavefish Phreatichthys andruzzii, lacks photoreactivation repair via the loss of light, UV and ROS-induced photolyase gene transcription mediated by D-box enhancer elements. ... Hunter Douglas Restring KIT 0.9mm Cord for Shades: Cellular, Honeycomb, Pleated, Roman. ... 1-1/2 Inch RollEase Cellular & Pleated Shade Clutch Kit $ 24.99 SKU: RE-VRS-1.5. Therefore, DNA damage repair mechanisms are ubiquitous and highly conserved. For promoter analysis, fragments from the zebrafish ddb2 gene promoters were amplified by genomic PCR based on database information (ENSDARG00000041140) and subcloned into the luciferase reporter pGL3-basic vector (Promega).