Repair and tolerance of DNA damage in higher plants

DNA repair mechanisms constitute an essential cellular response to DNA damage arising either from metabolic processes or from environmental sources such as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by processes such as nucleotide excision repair (NER), a coordinated pathway in which lesions and the surrounding nucleotides are excised and replaced via DNA resynthesis. The importance of repair is illustrated by human disease states such as xeroderma pigmentosum and Cockayne's syndrome which result from defects in the NER system arising from mutations in XP- genes or XP- and CS- genes respectively Little detail is known of DNA damage repair processes in plants, despite the economic and ecological importance of these organisms. This study aimed to expand our knowledge of the process of NER in plants, largely via a polymerase chain reaction (PCR)-based approach involving amplification, cloning and characterisation of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1 and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from the model dicotyledonous plant Arabidopsis thaliana. The sequence of the 3'-untranslated region of atXPD was also determined. Comparison of genomic and cDNA sequences allowed a detailed analysis of gene structures, including details of intron/exon processing. Variable transcript processing to produce three distinct transcripts was found in the case of atXPF. In an attempt to validate the proposed homologous function of these cDNAs, assays to test complementation of resistance to ultraviolet radiation in the relevant yeast mutants were performed. Despite extensive amino acid sequence conservation, neither plant cDNA was able to restore UV-resistance. As the yeast RAD3 gene product is also involved in vivo in transcription, and so is required for viability, the atXPD cDNA was tested in a complementation assay for this function in an appropriate yeast mutant. The plant cDNA was found to substantially increase the viability of the yeast mutant. The structural and functional significance of these results is discussed comparatively with reference to yeast, human and other known homologues. Other putative NER homologues were identified in A. thaliana database sequences, including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects of ongoing investigations. This study also describes preliminary investigations of putative REVS and RAD30 translesion synthesis genes from A. thaliana.

Investigation of abalone mitochondrial DNA and new applications

The work developed and applied new mitochondrial DNA markers to abalone aquaculture and wild fisheries. The number of markers and their success in the areas applied were all firsts in the abalone industry. The work will contribute to the future sustainability of this resource in culture and in the wild.

Influence of age and DNA damage on expression of damage-processing genes

The effect of DNA damaging agents and age on expression of damage-processing genes was examined in plants and mice. Treatment with these agents increased expression of some genes. The effect of gene expression in the absence of treatment decreased with age, suggesting links between ageing and genetic instability.

DNA fingerprinting the Powerful Owl (Ninox strenua)

Genetic markers to DNA fingerprint the Powerful Owl were developed. These markers enable individual owls to be identified through the collection of shed feathers. Individual identification allowed for crucial aspects of the Powerful Owl's ecology and biology to be investigated.

Conservation genetics of the New Holland mouse Pseudomys novaehollandiae, Waterhouse, 1843 (Rodentia: Muridae) : an analysis of mitochondrial DNA control region variation

An investigation of the genetic diversity of New Holland mouse populations using DNA. Ten distinct restriction enzyme fragment patterns or haplotypes were detected. From the fragment patterns, estimates of genetic divergence between the haplotypes revealed a degree of genetic structuring within New Holland mouse with four population assemblages apparent.

Integrated DNA extraction and amplification using electrokinetic pumping in a microfluidic device

An integrated system employing anion exchange for the extraction of DNA from biological samples prior to polymerase chain reaction DNA amplification has been developed, based on microfluidic methodology utilising electrokinetic pumping. In this system, the biological samples were added directly to chitosan-coated silica beads to facilitate DNA immobilisation. The purified, pre-concentrated DNA was then eluted using a combination of electro-osmotic flow enhanced with electrophoretic mobility, which enable DNA to be transported by both mechanisms into the DNA amplification chamber. Through optimisation of the DNA elution conditions, average DNA extraction efficiencies of 69.1% were achievable. Subsequent DNA amplification performed on the microfluidic system demonstrated not only the ability to use electrokinetic movement to integrate the two processes on a single device, but also that the quality and quantity of DNA eluted was suitable for downstream analysis. This work offers an attractive real-world to chip interface and a route to simpler Lab-on-a-Chip technology which eliminates the need for moving parts.

Validation of DNA methylation biomarkers for diagnosis of acute lymphoblastic leukemia

DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing.

Effect of self-collection of HPV DNA offered by community health workers at home visits on uptake of screening for cervical cancer (the EMA study): a population-based cluster-randomised trial

Control of cervical cancer in developing countries has been hampered by a failure to achieve high screening uptake. HPV DNA self-collection could increase screening coverage, but implementation of this technology is difficult in countries of middle and low income. We investigated whether offering HPV DNA self-collection during routine home visits by community health workers could increase cervical screening.

DNA damage in rats after treatment with methylphenidate.

Methylphenidate (MPH) is a widely prescribed psychostimulant for the treatment of attention-deficit hyperactivity disorder (ADHD). Recently, some studies have addressed the genotoxic potential of the MPH, but the results have been contradictory. Hence, the present study aimed to investigate the index of cerebral and peripheral DNA damage in young and adult rats after acute and chronic MPH exposure.

YM155 down-regulates survivin and XIAP, modulates autophagy and induces autophagy-dependent DNA damage in breast cancer cells

The aim of this study was to determine the potency and molecular mechanism of action of YM155, a first-in-class survivin inhibitor that is currently under phase I/II clinical investigations, in various drug-resistant breast cancers including the oestrogen receptor positive (ER(+) ) tamoxifen-resistant breast cancer and the caspase-3-deficient breast cancer.

Geographical variation and population structure in the White-rumped Sandpiper Calidris fuscicollis as shown by morphology, mitochondrial DNA and carbon isotope ratios

We studied the population structure of a high arctic breeding wader bird species, the White-rumped Sandpiper Calidris fuscicollis. Breeding adults, chicks and juveniles were sampled at seven localities throughout the species' breeding range in arctic Canada in 1999. The mitochondrial control region was analysed by DNA sequencing, feathers were analysed for carbon isotope ratios (C13/C12) by isotope ratio mass spectrometry, and morphological measurements were analysed using principal component analyses, taking the effect of sex into account (identified by molecular genetic methods). In general, our results support the notion that the White-rumped Sandpiper is a monotypic species with no subspecies, and most of the morphological and genetic variation occurs within sites. Nevertheless, some differences between sites were found. Birds from the two northernmost sites (Ellesmere and Devon Islands) had relatively longer bill and wing and shorter tarsus than birds sampled further south, possibly reflecting genetic differences between populations. The carbon isotope ratios were higher at the easternmost site (Baffin Island), revealing differences in the isotope content of the food. The mtDNA sequences showed no significant differentiation between sites and no pattern of isolation-by-distance was found. Based on the mtDNA variation, the species was estimated to have a long-term effective population size of approximately 9,000 females. The species shows no clear evidence of any population expansion or decline. Our results indicate that carbon isotope ratios, and possibly also certain mtDNA haplotypes, may be useful as tools for identifying the breeding origin of White-rumped Sandpipers on migration and wintering sites.

DNA transfer by examination tools--a risk for forensic casework?

The introduction of profiling systems with increased sensitivity has led to a concurrent increase in the risk of detecting contaminating DNA in forensic casework. To evaluate the contamination risk of tools used during exhibit examination we have assessed the occurrence and level of DNA transferred between mock casework exhibits, comprised of cotton or glass substrates, and high-risk vectors (scissors, forceps, and gloves). The subsequent impact of such transfer in the profiling of a target sample was also investigated. Dried blood or touch DNA, deposited on the primary substrate, was transferred via the vector to the secondary substrate, which was either DNA-free or contained a target sample (dried blood or touch DNA). Pairwise combinations of both heavy and light contact were applied by each vector in order to simulate various levels of contamination. The transfer of dried blood to DNA-free cotton was observed for all vectors and transfer scenarios, with transfer substantially lower when glass was the substrate. Overall touch DNA transferred less efficiently, with significantly lower transfer rates than blood when transferred to DNA-free cotton; the greatest transfer of touch DNA occurred between cotton and glass substrates. In the presence of a target sample, the detectability of transferred DNA decreased due to the presence of background DNA. Transfer had no impact on the detectability of the target profile, however, in casework scenarios where the suspect profiles are not known, profile interpretation becomes complicated by the addition of contaminating alleles and the probative value of the evidence may be affected. The results of this study reiterate the need for examiners to adhere to stringent laboratory cleaning protocols, particularly in the interest of contamination minimisation, and to reduce the handling of items to prevent intra-item transfer.

Deploying aptameric sensing technology for rapid pandemic monitoring

The genome of virulent strains may possess the ability to mutate by means of antigenic shift and/or antigenic drift as well as being resistant to antibiotics with time. The outbreak and spread of these virulent diseases including avian influenza (H1N1), severe acute respiratory syndrome (SARS-Corona virus), cholera (Vibrio cholera), tuberculosis (Mycobacterium tuberculosis), Ebola hemorrhagic fever (Ebola Virus) and AIDS (HIV-1) necessitate urgent attention to develop diagnostic protocols and assays for rapid detection and screening. Rapid and accurate detection of first cases with certainty will contribute significantly in preventing disease transmission and escalation to pandemic levels. As a result, there is a need to develop technologies that can meet the heavy demand of an all-embedded, inexpensive, specific and fast biosensing for the detection and screening of pathogens in active or latent forms to offer quick diagnosis and early treatments in order to avoid disease aggravation and unnecessary late treatment costs. Nucleic acid aptamers are short, single-stranded RNA or DNA sequences that can selectively bind to specific cellular and biomolecular targets. Aptamers, as new-age bioaffinity probes, have the necessary biophysical characteristics for improved pathogen detection. This article seeks to review global pandemic situations in relation to advances in pathogen detection systems. It particularly discusses aptameric biosensing and establishes application opportunities for effective pandemic monitoring. Insights into the application of continuous polymeric supports as the synthetic base for aptamer coupling to provide the needed convective mass transport for rapid screening is also presented.

Associations between DNA damage, DNA base excision repair gene variability and Alzheimer's disease risk

BACKGROUND: Increased oxidative damage to DNA is one of the pathways involved in Alzheimer's disease (AD). Insufficient base excision repair (BER) is in part responsible for increased oxidative DNA damage. The aim of the present study was to assess the effect of polymorphic variants of BER-involved genes and the peripheral markers of DNA damage and repair in patients with AD. MATERIAL AND METHODS: Comet assays and TaqMan probes were used to assess DNA damage, BER efxFB01;ciency and polymorphic variants of 12 BER genes in blood samples from 105 AD patients and 130 controls. The DNA repair efficacy (DRE) was calculated according to a specific equation. RESULTS: The levels of endogenous and oxidative DNA damages were higher in AD patients than controls. The polymorphic variants of XRCC1 c.580C>T XRCC1 c.1196A>G and OGG1 c.977C>G are associated with increased DNA damage in AD. CONCLUSION: Our results show that oxidative stress and disturbances in DRE are particularly responsible for the elevated DNA lesions in AD. The results suggest that oxidative stress and disruption in DNA repair may contribute to increased DNA damage in AD patients and risk of this disease. In addition, disturbances in DRE may be associated with polymorphisms of OGG1 and XRCC1.

Folic acid supplementation in postpolypectomy patients in a randomized controlled trial increases tissue folate concentrations and reduces aberrant DNA biomarkers in colonic tissues adjacent to the former polyp site

BACKGROUND: Low folate status is associated with an increased risk of colorectal carcinogenesis. Optimal folate status may be genoprotective by preventing uracil misincorporation into DNA and DNA hypomethylation. Adenomatous polyps have low folate status compared with normal colonic mucosa, and they are surrounded by histologically normal mucosa that also is of low folate status. OBJECTIVE: In a randomized controlled trial conducted at a single Dublin hospital between April 2002 and March 2004, we assessed the effect of folic acid supplementation on tissue folate, uracil misincorporation into DNA, and global DNA hypomethylation in colonocytes isolated from sites of adenomatous polyps and from histologically normal tissue adjacent and 10-15 cm distal to them. METHODS: Twenty patients with adenomatous polyps on initial colonoscopy and polypectomy were randomly assigned to receive either 600 μg folic acid/d [n = 12, 38% men, mean age 64.3 y, and body mass index (BMI, in kg/m(2)) 26.6] or placebo (n = 8, 50% men, mean age 68.4 y, and BMI 27.2) for 6 mo, and then repeat the colonoscopy. Blood and colonocyte tissue folate concentrations were measured with the use of a microbiological assay. Uracil misincorporation and global DNA hypomethylation were measured in colonocytes with the use of modified comet assays. RESULTS: Over time, folic acid supplementation, compared with placebo, increased tissue folate (mean ± SEM) from 15.6 ± 2.62 pg/10(5) cells to 18.1 ± 2.12 pg/10(5) cells (P < 0.001) and decreased the global DNA hypomethylation ratio from 1.7 ± 0.1 to 1.0 ± 0.1 (P < 0.001). The uracil misincorporation ratio decreased by 0.5 ± 0.1 for the site adjacent to the polyp over time (P = 0.05). CONCLUSION: A response to folic acid supplementation, which increased colonocyte folate and improved folate-related DNA biomarkers of cancer risk, was seen in the participants studied. Exploratory analysis points toward the area formerly adjacent to polyps as possibly driving the response. That these areas persist after polypectomy in the absence of folate supplementation is consistent with a potentially carcinogenic field's causing the appearance of the polyp.