Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea–agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of ∼1–20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.     

Influencing factors on the foot health of captive Asian elephants (Elephas maximus) in European zoos

Pathological lesions of feet occur frequently in captive elephant populations. To improve foot health, it is important to identify risk factors associated with such pathologies. Several previous studies have analyzed potentially influencing factors but were limited, for example, by small sample sizes. This study analyzed the relationship between 87 independent variables and the foot health score of 204 Asian elephants (Elephas maximus) in European zoos using bivariate correlation, multivariable regression models, and principal component analysis (PCA). Correlation and regression tests revealed significant results for 30 different variables, mainly with small effect sizes. Only three variables were significant in more than one test: sex, time spent indoors, and time spent on hard ground, with lower scores (i.e. less or less severe pathological lesions) in females, and when less time is spent indoors or on hard ground. Due to small effect sizes and differing results of the statistical tests, it is difficult to determine which risk factors are most important. Instead, a holistic consideration appears more appropriate. A biplot of the PCA shows that factors representing more advanced husbandry conditions (e.g. large areas, high proportions of sand flooring) were associated with each other and with decreased foot scores, whereas indicators of more limited conditions (e.g. high proportions of hard ground, much time spent indoors) were also associated with each other but increased the foot score. In conclusion, instead of resulting from just one or two factors, reduced foot health might be an indicator of a generally poorer husbandry system.     

Conserved structure of the chloroplast-DNA encoded D1 protein is essential for effective photoprotection via non-photochemical thermal dissipation in higher plants

Naturally selected atrazine-resistant (AR) weeds possessing a Ser₂₆₄ → Gly D1 protein encoded by a mutant psbA allele in the chloroplast-DNA have increased photosensitivity and lower fitness. The D1 mutant lines of S. nigrum revealed impaired regulation of photosystem II (PSII) activity as compared with the wild-type plants resulting in a less effective photochemical light utilization and in addition, a lower capacity of non-photochemical thermal dissipation (NPQ), one of the main photoprotective mechanisms in oxygenic photosynthetic organisms. In this work, comparative chlorophyll fluorescence analysis in attached leaves of wild-type and AR Solanum nigrum L. and in their reciprocal crosses has been used to establish how the lower NPQ is inherited. Both a 50% reduction in steady-state NPQ and a 60–70% reduction in the rapidly reversible, energy-dependent (qE) component of NPQ were common phenomena in the parent and hybrid lines of D1 mutant S. nigrum. The nuclear hybrid status of the F2 plant material was confirmed by morphological observations on fully developed leaves. No alteration was found in the nucleotide sequence and the deduced amino acid sequences of the nuclear psbS gene isolated from different biotypes of S. nigrum, and there were no differences in the expressions of both the PsbS and the D1 proteins. All things considered, co-inheritance of the lower photoprotective NPQ capacity and the Ser₂₆₄ → Gly D1 protein mutation was clearly observed, suggesting that the evolutionarily conserved D1 structure must be indispensable for the efficient NPQ process in higher plants.     

New Silver-Gold Intensification Method of Diaminobenzidine for Double-Labeling Immunoelectron Microscopy

The available methods for double-labeling preembedding immunoelectron microscopy are highly limited because not only should the ultrastructure be preserved, but also the different antigens should be visualized by reaction end products that can be clearly distinguished in gray-scale images. In these procedures, one antigen is detected with 3,3′-diaminobenzidine (DAB) chromogen, resulting in a homogeneous deposit, whereas the other is labeled with either a gold-tagged immunoreagent, or DAB polymer, on the surface of which metallic silver is precipitated. The detection of the second antigen is usually impeded by the first, leading to false-negative results. The authors aimed to diminish this hindrance by a new silver intensification technique of DAB polymer, which converts the deposit from amorphous to granular. The method includes three major postdevelopmental steps: (1) treatment of nickel-enhanced DAB with sulfide, (2) silver deposition in the presence of hydroquinone under acidic conditions, and (3) precious metal replacement with gold thiocyanate. This new sulfide-silver-gold intensification of DAB (SSGI) allows a subsequent detection of other antigens using DAB. In conclusion, the new technique loads fine gold particles onto the DAB deposit at a very low background level, thereby allowing a reliable discernment between the elements stained for the two antigens at the ultrastructural level.