Genome analysis of DNA repair genes in the alpha proteobacterium <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Background  

The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria.     

Histone gene expression and histone mRNA 3' end structure in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

Histone protein synthesis is essential for cell proliferation and required for the packaging of DNA into chromatin. In animals, histone proteins are provided by the expression of multicopy replication-dependent histone genes. Histone mRNAs that are processed by a histone-specific mechanism to end after a highly conserved RNA hairpin element, and lack a poly(A) tail. In vertebrates and Drosophila, their expression is dependent on HBP/SLBP that binds to the RNA hairpin element. We showed previously that these cis and trans acting regulators of histone gene expression are conserved in C. elegans. Here we report the results of an investigation of the histone mRNA 3' end structure and of histone gene expression during C. elegans development.     

Search for computational modules in the <Emphasis Type="Italic">C. elegans</Emphasis>brain

Background  

Does the C. elegans nervous system contain multi-neuron computational modules that perform stereotypical functions? We attempt to answer this question by searching for recurring multi-neuron inter-connectivity patterns in the C. elegans nervous system's wiring diagram.     

The <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>CDT-2 ubiquitin ligase is required for attenuation of EGFR signalling in vulva precursor cells

Background  

Attenuation of the EGFR (Epidermal Growth Factor Receptor) signalling cascade is crucial to control cell fate during development. A candidate-based RNAi approach in C. elegans identified CDT-2 as an attenuator of LET-23 (EGFR) signalling. Human CDT2 is a component of the conserved CDT2/CUL4/DDB1 ubiquitin ligase complex that plays a critical role in DNA replication and G2/M checkpoint. Within this complex, CDT2 is responsible for substrate recognition. This ubiquitin ligase complex has been shown in various organisms, including C. elegans, to target the replication-licensing factor CDT1, and the CDK inhibitor p21. However, no previous link to EGFR signalling has been identified.     

Liquid-holding and recovery from UV-induced damage in Eudorina elegans

Summary Eudorina elegans does not respond to liquid-holding or to postirradiation medium effects by changes in recovery.A decrease in survival ability is observed if a culture is starved prior to irradiation, or is incubated at 22°C rather than 32°C following UV irradiation. Eudorina loses the ability to photoreactivate UV damage within 10 to 48 h following irradiation, depending upon the pre-and post-UV culture conditions.The results are interpreted as indicating a failure of Eudorina elegans to carry out specific dark repair of UV damage. Some reactivation may occur during cellular DNA synthesis.Abbreviations used PR photoreactivation - LHR liquid holding recovery - LHP liquid holding protection - ERR excision-resynthesis-repair - BC complete medium - BM minimal medium - cfa colony forming ability - cfu colony forming units Supported by grants from the National Research Council of Canada # A4431.     

Chemically defined medium and <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

C. elegans has been established as a powerful genetic system. Use of a chemically defined medium (C. elegans Maintenance Medium (CeMM)) now allows standardization and systematic manipulation of the nutrients that animals receive. Liquid cultivation allows automated culturing and experimentation and should be of use in large-scale growth and screening of animals.     

Regulation of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> body size and male tail development by the novel gene <Emphasis Type="Italic">lon-8</Emphasis>

Background  

In C. elegans and other nematode species, body size is determined by the composition of the extracellular cuticle as well as by the nuclear DNA content of the underlying hypodermis. Mutants that are defective in these processes can exhibit either a short or a long body size phenotype. Several mutations that give a long body size (Lon) phenotype have been characterized and found to be regulated by the DBL-1/TGF-β pathway, that controls post-embryonic growth and male tail development.     

An imaging system for standardized quantitative analysis of <Emphasis Type="Italic">C. elegans</Emphasis> behavior

Background  

The nematode Caenorhabditis elegans is widely used for the genetic analysis of neuronal cell biology, development, and behavior. Because traditional methods for evaluating behavioral phenotypes are qualitative and imprecise, there is a need for tools that allow quantitation and standardization of C. elegans behavioral assays.     

The twisted pharynx phenotype in <Emphasis Type="Italic">C. elegans</Emphasis>

Background  

The pharynx of C. elegans is an epithelial tube whose development has been compared to that of the embryonic heart and the kidney and hence serves as an interesting model for organ development. Several C. elegans mutants have been reported to exhibit a twisted pharynx phenotype but no careful studies have been made to directly address this phenomenon. In this study, the twisting mutants dig-1, mig-4, mnm-4 and unc-61 are examined in detail and the nature of the twist is investigated.     

A proteomic view of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> caused by short-term hypoxic stress

Background  

The nematode Caenorhabditis elegans is both sensitive and tolerant to hypoxic stress, particularly when the evolutionarily conserved hypoxia response pathway HIF-1/EGL-9/VHL is involved. Hypoxia-induced changes in the expression of a number of genes have been analyzed using whole genome microarrays in C. elegans, but the changes at the protein level in response to hypoxic stress still remain unclear.     

Pioglitazone retrieves hepatic antioxidant DNA repair in a mice model of high fat diet

Background  

Pioglitazone was reported to improve hepatic steatosis and necroinflammation in human studies. To investigate whether the hepato-protective effect of pioglitazone was associated with an improvement of antioxidant defense mechanism, oxidative DNA damage and repair activity were determined in a high fat diet model. Male C57BL/6 mice were respectively fed with a 30% fat diet, the same diet with pioglitazone 100 mg/kg/day, or a chow diet as control for 8 weeks. Tissue oxidative stress was indicated by malondialdehyde concentration. Oxidative DNA damage was detected by immunohistochemical 8-oxoG staining. Enzymatic antioxidant defense was detected by the real-time PCR of superoxide dismutase (Sod1, Sod2) and DNA glycosylase (Ogg1, MutY). Oxidative DNA repair was detected by immunohistochemical staining and western blotting of OGG1 expression.     

Genome-wide investigation reveals pathogen-specific and shared signatures in the response of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>to infection

Background  

There are striking similarities between the innate immune systems of invertebrates and vertebrates. Caenorhabditis elegans is increasingly used as a model for the study of innate immunity. Evidence is accumulating that C. elegans mounts distinct responses to different pathogens, but the true extent of this specificity is unclear. Here, we employ direct comparative genomic analyses to explore the nature of the host immune response.     

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background

DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.

Aim

To investigate the effect of red blood cells on H₂O₂-induced DNA damage and repair in human peripheral blood mononuclear cells.

Methods

DNA breaks were induced in peripheral blood mononuclear cells by H₂O₂ in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by ³H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.

Results

Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.

Conclusion

Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.     

<Emphasis Type="Italic">N</Emphasis>-(3-oxo-acyl) homoserine lactone induced germ cell apoptosis and suppressed the over-activated RAS/MAPK tumorigenesis via mitochondrial-dependent ROS in <Emphasis Type="Italic">C. elegans</Emphasis>

As a quorum-sensing molecule for bacteria–bacteria communication, N-(3-oxododecanoyl)-homoserine lactone (C12) has been found to possess pro-apoptotic activities in various cell culture models. However, the detailed mechanism of how this important signaling molecule function in the cells of live animals still remains largely unclear. In this study, we systematically investigated the mechanism for C12-mediated apoptosis and studied its anti-tumor effect in Caenorhabditis elegans (C. elegans). Our data demonstrated that C12 increased C. elegans germ cell apoptosis, by triggering mitochondrial outer membrane permeabilization (MOMP) and elevating the reactive oxygen species (ROS) level. Importantly, C12-induced ROS increased the expression of genes critical for DNA damage response (hus-1, clk-2 and cep-1) and genes involved in p38 and JNK/MAPK signaling pathway (nsy-1, sek-1, pmk-1, mkk-4 and jnk-1). Furthermore, C12 failed to induce germ cell apoptosis in animals lacking the expression of each of those genes. Finally, in a C. elegans tumor-like symptom model, C12 significantly suppressed tumor growth through inhibiting the expression of RAS/MAPK pathway genes (let-23/EGFR, let-60/RAS, lin-45/RAF, mek-2/MEK and mpk-1/MAPK). Overall, our results indicate that DNA damage response and MAPK activation triggered by mitochondrial ROS play important roles in C12-induced apoptotic signaling in C. elegans, and RAS/MAPK suppression is involved in the tumor inhibition effect of C12. This study provides in vivo evidence that C12 is a potential candidate for cancer therapeutics by exerting its pro-apoptotic and anti-tumor effects via elevating mitochondria-dependent ROS production.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background  

Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair.     

Oocyte production and sperm utilization patterns in semi-fertile strains of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

Caenorhabditis elegans hermaphrodites are capable of producing hundreds of progeny. However, genetic and environmental factors can keep many animals from attaining their full reproductive potential. In these situations, efficient use of any functional gametes becomes more important for reproductive success. To learn about this aspect of C. elegans reproductive biology, we examined oocyte production and sperm utilization patterns in a unique collection of semi-fertile sperm function mutants.