Non-destructive sampling of maternal DNA from the external shell of bird eggs

The use of non-destructive sampling methods to collect genetic material from wildlife allows researchers to minimize disturbance. Most avian studies employ capturing and handling of young and parents to draw blood for DNA analysis. In some cases adult female birds are difficult to catch, so maternal genotyping has required collection of contour feathers from nests, or destructive sampling of eggs. Many species do not leave contour feathers in the nest, and destructive sampling has been unreliable due to contamination with embryonic DNA. Alternative field sampling techniques for collection of maternal DNA from birds are therefore desirable. Here we demonstrate that avian maternal DNA can be isolated in a non-invasive and non-destructive way from the external surface of eggs. We used cotton swabs to collect maternal DNA from the external shells of herring gull (Larus argentatus) and Caspian tern (Sterna caspia) eggs. DNA was then amplified by the polymerase chain reaction (PCR) for microsatellite genotyping. We verified that the DNA samples were maternal by comparing microsatellite profiles to those obtained from adults and chicks from the same nests. In 100% of Caspian tern (n=16) and herring gull families (n=12), the egg swabs that amplified matched the maternal microsatellite genotype. In a screening of many nests of both species, we successfully amplified microsatellite markers from 101/115 (88%) egg swabs. Swabs from eggs with blood stains on the shell were more likely to amplify successfully than those from clean eggs. The advantages of this new method include increased parentage assignment/exclusion power, and increased availability of maternal DNA for genotyping of species that do not deposit contour feathers in nests.     

The oxidative DNA lesion 8,5'-(S)-cyclo-2'-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.     

Aluminum resistance mechanisms in oat (Avena sativa L.)

Background and aims

Enhanced aluminum (Al) resistance has been observed in dicots over-expressing enzymes involved in organic acid synthesis; however, this approach for improving Al resistance has not been investigated in monocots. Among the cereals, oat (Avena sativa L.) is considered to be Al resistant, but the basis of resistance is not known.

Methods

A hydroponic assay and hematoxylin staining for Al accumulation in roots were used to evaluate Al resistance in 15 oat cultivars. Malate and citrate release from roots was measured over a 24?h period. A malate dehydrogenase gene, neMDH, from alfalfa (Medicago sativa L.) was used to transform oat.

Results

Oat seedlings were highly resistant to Al, as a concentration of 325?μM AlK(SO₄)₂ was needed to cause a 50% decrease in root growth. Most oat cultivars tested are naturally resistant to high concentrations of Al and effectively excluded Al from roots. Al-dependent release of malate and Al-independent release of citrate was observed. Al resistance was enhanced in a transgenic oat line with the highest accumulation of neMDH protein. However, overall root growth of this line was reduced and expression of neMDH in transgenic oat did not enhance malate secretion.

Conclusions

Release of malate from oat roots was associated with Al resistance, which suggests that malate plays a role in Al resistance of oat. Over-expression of alfalfa neMDH enhanced Al resistance in some lines but was not effective alone for crop improvement.     

Histological Study of Seed Coat Development in Arabidopsis thaliana

Arabidopsis seed coat development using light and transmission electron microscopy revealed major morphological changes associated with the transition of the integuments into the mature seed coat. By the use of a metachromatic staining procedure, cytological events such as the production of phenolic compounds and acidic polysaccharides were followed. Immediately after fertilization, the cells of the inner epidermis of the inner integument became vacuolated and subsequently accumulated pigment within them. This pigment started to disappear from the cytoplasm at the torpedo stage of the embryo, as it became green. During the torpedo stage, mucilage began to accumulate in the cells of the external epidermis of the outer integument. Furthermore, starch grains accumulated against the central part of the inner periclinal wall of these cells, resulting in the formation of small pyramidal domes that persisted until seed maturity. At the maturation stage, when the embryo became dormant and colourless, a new pigment accumulation was observed in an amorphous layer derived from remnants of crushed integument layers. This second pigment layer was responsible for the brown seed colour. These results show that seed coat formation may proceed in a coordinated way with the developmental phases of embryogenesis. Received 25 May 1999/ Accepted in revised form 10 February 2000     

Differential metabolism of sodium azide in maize callus and germinating embryos

Sodium azide is a potent mutagen of maize (Zea mays L.) kernels that may have potential as a point mutagen for inducing biochemical mutations in maize tissue cultures. Azide mutagenicity was evaluated in friable, embryogenic maize callus and a nonregenerable maize suspension culture by determining the number of resistant variant cell lines able to grow on media containing inhibitory concentrations of lysine plus threonine (LT). The number of LT-resistant variants selected from either culture type did not increase in response to azide treatment. In addition, there was no increase in somatic mutations in more than 100 plants regenerated from azide treated LT-resistant lines. The levels of mutagenic metabolite of azide (presumably azidoalanine), were determined by bioassay in the two azide-treated maize callus types and compared to levels of mutagenic metabolite in embryos isolated from azide-treated kernels. The two types of maize tissue cultures and isolated embryos contained similar levels of mutagenic metabolite 4 h after azide treatment indicating similar uptake and conversion of azide to mutagenic metabolite in the three tissues. Mutagenic metabolite in azide-treated embryos did not significantly decrease after 40 h. However, mutagenic metabolite levels in both azide-treated tissue cultures decreased to near background levels within 20 h providing evidence for rapid metabolism of the azide mutagenic metabolite. The lack of evidence for azide mutagenicity in maize callus and its known potent mutagenicity in kernels appears to be associated with specific differences in azide metabolism between callus tissues and kernel embryos.     

Neuropsychological and behavioural correlates of CSF biomarkers in dementia

To improve clinical, neuropsychological and behavioural characterisation of the cerebrospinal fluid (CSF) biomarkers beta-amyloid((1-42)) protein (Abeta42), protein tau (tau) and tau phosphorylated at threonine 181 (P-tau181) across diagnostic dementia categories, a prospective study was set up. Patients with probable Alzheimer's disease (AD) (n=201), AD with cerebrovascular disease (CVD) (AD+CVD) (n=33), frontotemporal dementia (FTD) (n=27), dementia with Lewy bodies (DLB) (n=22) and healthy controls (n=148) were included. All patients underwent neuropsychological examination and behavioural assessment by means of a battery of behavioural assessment scales. CSF was obtained by lumbar puncture and levels of Abeta42, tau and P-tau181 were determined with commercially available ELISA kits. Negative correlations between CSF Abeta42 levels and aggressiveness (Spearman: r=-0.223; p=0.002) and positive correlations with age at inclusion (r=0.195; p=0.006), age at onset (r=0.205; p=0.003) and MMSE scores (r=0.198; p=0.005) were found in AD. In AD+CVD, CSF Abeta42 levels were correlated with MMSE (r=0.482; p=0.006), Hierarchic Dementia Scale (r=0.503; p=0.017) and Boston Naming Test (r=0.516; p=0.012) scores. In controls, age was positively correlated with CSF tau (r=0.465; p<0.001) and P-tau181 levels (r=0.312; p<0.001). CSF tau and P-tau181 levels correlated significantly in all groups, whereas CSF Abeta42 correlated with tau and P-tau181 levels in healthy controls only. Negative correlations between CSF Abeta42 levels and aggressiveness were found in AD patients. CSF Abeta42 seems to be a stage marker for AD (+/-CVD) given the positive correlations with neuropsychological test results suggesting that CSF Abeta42 might be of help for monitoring disease progression. Different correlations between age and CSF biomarker levels were obtained in healthy controls compared to AD patients, indicating that AD-induced pathophysiological processes change age-dependent regulation of CSF biomarker levels.     

An ensemble biclustering approach for querying gene expression compendia with experimental lists

MOTIVATION: Query-based biclustering techniques allow interrogating a gene expression compendium with a given gene or gene list. They do so by searching for genes in the compendium that have a profile close to the average expression profile of the genes in this query-list. As it can often not be guaranteed that the genes in a long query-list will all be mutually coexpressed, it is advisable to use each gene separately as a query. This approach, however, leaves the user with a tedious post-processing of partially redundant biclustering results. The fact that for each query-gene multiple parameter settings need to be tested in order to detect the 'most optimal bicluster size' adds to the redundancy problem. RESULTS: To aid with this post-processing, we developed an ensemble approach to be used in combination with query-based biclustering. The method relies on a specifically designed consensus matrix in which the biclustering outcomes for multiple query-genes and for different possible parameter settings are merged in a statistically robust way. Clustering of this matrix results in distinct, non-redundant consensus biclusters that maximally reflect the information contained within the original query-based biclustering results. The usefulness of the developed approach is illustrated on a biological case study in Escherichia coli. Availability and implementation: Compiled Matlab code is available from http://homes.esat.kuleuven.be/~kmarchal/Supplementary_Information_DeSmet_2011/.