Metabolic fingerprinting reveals differences between northern and southern strains of the cryptic diatom Chaetoceros socialis

Morphology and molecular phylogeny constitute the structural elements of diatom taxonomy. These approaches do not, however, give information on the functioning of taxa. Additional methods to serve a more integrated and wide-ranging taxonomy have therefore been called for. Metabolic fingerprinting is one approach used within the field of metabolomics, often applied in classification of samples. Here we apply metabolic fingerprinting in a taxonomic study of a cryptic diatom species. Strains of the cosmopolitan diatom Chaetoceros socialis from two geographical areas; the north-east Atlantic and Arctic and the Gulf of Naples, were cultivated at three different temperatures; 2.5, 8 and 13°C. The strains from the two different geographical areas exhibited different growth rates as well as different photosynthetic efficiencies. Algal extracts, collected at the end of the growth experiments, were analysed by Ultra-Performance Liquid Chromatography High Resolution Mass Spectrometry. The two groups of strains were separated by principal component analysis of their metabolic fingerprints. Analysis of the data revealed both qualitative and quantitative differences in metabolite markers. These phenotypic differences reinforce differences also found for morphology, phylogenetic markers and growth rates, and point at different adaptive characteristics in organisms living under different temperature regimes.     

比色法结合HPLC指纹图谱法用于诃子质量控制研究

采用比色法与HPLC指纹图谱法对诃子进行质量控制,建立了诃子中的主要药效成分总鞣质含量测定方法以及诃子的HPLC指纹图谱质量控制方法。结果诃子的总鞣质含量达到20.6%,生成的对照指纹图谱与各图谱相似度≥0.94。本实验建立的评价方法能够快速、准确、简便、全面地评价中药诃子质量,在一定程度上弥补药典中对诃子的质量控制不足之处。     

Assessment of artificial substrates for evaluating groundwater microbial quality

Protection of groundwater resources requires the development of reliable ecological indicators. Microorganisms involved in ecological services or being associated with particular hosts or habitats could be used for this purpose. Nevertheless, their tracking remains limited because of sampling issues, and a lack of devices for their long term monitoring. In the present study, three artificial substrates (glass and clay beads, and gravel particles) were tested in terms of efficacy at favoring bacterial growth, and at capturing bacterial diversity of waters (i.e., groundwater, surface water and wastewater). Total proteins, total carbohydrates, dehydrogenase and hydrolytic activities were used to monitor biofilm development on these artificial substrates. Fingerprinting analyses based on rrs (16S rRNA) − rrl (23S rRNA) spacer analyses (ARISA) and next generation sequencing (NGS) of partial rrs DNA segments (V5-V6) were used to compare operating taxonomic units (OTUs), and infer bacterial genera trapped on these substrates. Glass beads were found less efficient than the other two artificial substrates at increasing protein contents and microbial activities (hydrolytic and dehydrogenase activities). ARISA showed a discrimination of bacterial communities developing on artificial substrates that was matching water types. An incubation period of 7 days allowed a reliable assessment of bacterial diversity. From this incubation period, around 75% of water genera with more than four V5-V6 rrs DNA sequences detected in a water type were recovered from biofilms growing on artificial substrates. Based on relative abundances of genera, clay beads and gravel particles were more efficient than glass beads to capture and obtain bacterial communities matching those of the initial waters. Between 45–67% of similarities were found for these artificial substrates while it was between 36 and 43% for glass beads. This study demonstrated clay beads and gravel particles as being efficient tools for capturing bacterial diversity and monitoring bacterial growth. Overall, clay beads appeared the best choice for field monitoring because of the ease of their size standardization in comparison with gravel particles.     

Progress and Challenges for Live-cell Imaging of Genomic Loci Using CRISPR-based Platforms

Chromatin conformation,localization,and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states,the requirement for cell fix-ation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activ-ities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review,we describe currently-available approaches for imaging single genomic loci in cells,with special focus on clus-tered regularly interspaced short palindromic repeats (CRISPR)-based imaging approaches. In addition,we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches,and potential solutions to address these challenges. We anticipate that,with continued refinement of CRISPR-based imaging techniques,significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.     

Stable integration and conditional expression of electroporated transgenes in chicken embryos

The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals.     

p53 suppression overwhelms DNA polymerase eta deficiency in determining the cellular UV DNA damage response

Xeroderma pigmentosum variant (XP-V) cells lack the damage-specific DNA polymerase eta and have normal excision repair but show defective DNA replication after UV irradiation. Previous studies using cells transformed with SV40 or HPV16 (E6/E7) suggested that the S-phase response to UV damage is altered in XP-V cells with non-functional p53. To investigate the role of p53 directly we targeted p53 in normal and XP-V fibroblasts using short hairpin RNA. The shRNA reduced expression of p53, and the downstream cell cycle effector p21, in control and UV irradiated cells. Cells accumulated in late S phase after UV, but after down-regulation of p53 they accumulated earlier in S. Cells in which p53 was inhibited showed ongoing genomic instability at the replication fork. Cells exhibited high levels of UV induced S-phase gammaH2Ax phosphorylation representative of exposed single strand regions of DNA and foci of Mre11/Rad50/Nbs1 representative of double strand breaks. Cells also showed increased variability of genomic copy numbers after long-term inhibition of p53. Inhibition of p53 expression dominated the DNA damage response. Comparison with earlier results indicates that in virally transformed cells cellular targets other than p53 play important roles in the UV DNA damage response.     

Genetic paternity analyses in Little Owls (Athene noctua): does the high rate of paternal care select against extra-pair young?

Summary Previous studies have shown that extra-pair fertilizations are much less frequent in Non-Passeriformes, especially in raptors, than in Passeriformes. Low breeding densities, high breeding synchrony and high rates of paternal effort have been discussed as possible causes of these low extra-pair fertilization rates. Using DNA fingerprinting, we studied the mating system of Little Owls (Athene noctua) in a population of relatively high breeding density and comparatively low breeding synchrony. We found no cases of extra-pair fertilization among 53 nestlings of 16 breeding pairs. We conclude that paternal effort is probably the most important factor in preventing extra-pair fertilizations in Little Owls.

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Genetische Vaterschaftsanalysen bei Steinkäuzen (Athene noctua): Beeinflußt der hohe elterliche Aufwand der Männchen das Auftreten von Vaterschaften außerhalb des Paarbundes?

Zusammenfassung Zahlreiche Untersuchungen haben gezeigt, dass Befruchtungen außerhalb des Paarbundes bei Nicht-Singvogelarten wesentlich seltener vorkommen als bei Singvögeln. Dies gilt insbesondere auch für Greifvögel. Als Ursache für das seltene Auftreten von Befruchtungen außerhalb des Paarbundes in dieser Gruppe werden niedrige Brutpaardichten, eine hohe Brutsynchronisation und ein hoher elterlicher Aufwand auf Seiten der Männchen diskutiert. In der vorliegenden Studie haben wir das Paarungssystem des Steinkauzes (Athene noctua) in einer Population im Kreis Viersen (Niederrhein) mit Hilfe des DNA-Fingerprinting untersucht. Diese Population wies eine relativ hohe Brutpaardichte und eine vergleichsweise niedrige Brutsynchronisation auf. Bei der Analyse von 16 Bruten, die insgesamt 53 Nestlinge enthielten, konnte kein einziger Fall einer Befruchtung außerhalb des Paarbundes nachgewiesen werden. Dies führt uns zu dem Schluss, dass der wichtigste Faktor für die genetische Monogamie — zumindest beim Steinkauz — das hohe Maß des väterlichen Aufwandes bei der Brutversorgung ist.

     

Isolation and characterization of the Δ12-fatty acid desaturase in peanut (Arachis hypogaea L.) and search for polymorphisms for the high oleate trait in Spanish market-type lines

An understanding of the molecular mechanisms that are responsible for increased oleic acid accumulation would open avenues to alter peanut fatty acid composition and allow detection of polymorphic regions which can be used for marker assisted selection (MAS). Δ¹²-Fatty acid desaturase (FAD) was isolated and characterized from genotypes having a low or high oleic to linoleic acid O/L ratio – genotypes, Tamspan 90 (T-90) and F435–2-2 (F435), respectively. Southern blots showed three to four copies per haploid genome, and no major differences in organization between the two parental lines. Approximately 3525 bp was isolated from both genotypes, including a genomic walk toward the promoter region. The Δ¹²-Fad contains a putative intron, the coding region at the 3′ end, and an open reading frame (ORF) of 1140 bp encoding 379 amino acids. Comparisons of the coding sequences from the high and low oleic acid genotypes revealed several single nucleotide polymorphisms (SNPs). Two polymorphisms appear to be associated with the high O/L trait. The first is an ”A” insertion 442 bp after the start codon. The ”A” insertion shifts the amino acid reading frame, probably resulting in a truncated, inactive protein and the loss of one of three histidine boxes believed to be involved in metal ion complexation required for the reduction of oxygen. Another polymorphism at 448 bp from the start codon results in an amino acid change. The region containing the polymorphisms was amplified from leaf tissue of several independently derived backcross lines (IDBLs). Most high O/L lines had either the ”A” insertion or the amino acid substitution. Received: 1 February 2000 / Accepted: 20 March 2000