Sven Becker

PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities

ABSTRACT Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69–75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 104-fold excess of phylogenetically similar DNA and of an up to 107-fold excess of dissimilar DNA.

Simple approach to reduce PCR artefact formation leads to reliable genotyping of MHC and other highly polymorphic loci — Implications for evolutionary analysis

Genetic variation in coding regions is of strong interest for biologists as it represents an important factor that drives evolution. To analyse polymorphic loci, researchers usually rely on commonly used typing techniques such as cloning, SSCP, DGGE or RSCA. However, there are potential pitfalls in screening multi-allelic templates, which are mainly the formation of sequence chimeras during PCR amplification, and mosaic sequences during cloning. One of the most challenging genomic regions to explore is the Major Histocompatibility Complex (MHC), which codes for peptide-binding proteins of the vertebrates adaptive immune system and is well known for its exceptional polymorphism. We compared the effect of two different PCR amplification approaches in a study of the MHC class IIB genes of the three-spined stickleback (Gasterosteus aculeatus). One approach used standard PCR conditions and the other a combination of several measures to eliminate PCR artefacts. In both approaches, the amplicons obtained were cloned and sequenced. In the first, established approach, 24% of the clones represented artefacts, while in the second approach the number of artefacts were reduced ten-fold. Furthermore, it enabled easy differentiation between real alleles and artificial sequences. We also analysed the potential effects of such artefacts in genetic analysis and evolutionary interpretation, and found a slight reduction in the signature of positive selection and an increase in recombination events. Consequently, we strongly recommend to apply the new PCR approach described in this study when genotyping MHC or other polymorphic genes.

Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

BackgroundDuring the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG), depending on the tissue and experimental treatment, the gene has been used as a reference gene in such studies. In the three-spined stickleback, Gasterosteus aculeatus, other HKG than the one for beta-actin have not been established so far.ResultsTo establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT) PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and NormFinder. Our results showed that in most of the tissues and treatments HKG that could not be used so far due to unknown sequences, proved to be more stably expressed than the one for beta-actin.ConclusionAs they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.

Quantitative Tracing, by Taq Nuclease Assays, of a Synechococcus Ecotype in a Highly Diversified Natural Population

ABSTRACT Quantitative Taq nuclease assays (TNAs) (TaqMan PCR), nested PCR in combination with denaturing gradient gel electrophoresis (DGGE), and epifluorescence microscopy were used to analyze the autotrophic picoplankton (APP) of Lake Constance. Microscopic analysis revealed dominance of phycoerythrin (PE)-rich Synechococcus spp. in the pelagic zone of this lake. Cells passing a 3-μm-pore-size filter were collected during the growth period of the years 1999 and 2000. The diversity of PE-rich Synechococcus spp. was examined using DGGE to analyze GC-clamped amplicons of a noncoding section of the 16S-23S intergenic spacer in the ribosomal operon. In both years, genotypes represented by three closely related PE-rich Synechococcus strains of our culture collection dominated the population, while other isolates were traced sporadically or were not detected in their original habitat by this method. For TNAs, primer-probe combinations for two taxonomic levels were used, one to quantify genomes of all known Synechococcus-type cyanobacteria in the APP of Lake Constance and one to enumerate genomes of a single ecotype represented by the PE-rich isolate Synechococcus sp. strain BO 8807. During the growth period, genome numbers of known Synechococcus spp. varied by 2 orders of magnitude (2.9 × 103 to 3.1 × 105 genomes per ml). The ecotype Synechococcus sp. strain BO 8807 was detected in every sample at concentrations between 1.6 × 101 and 1.3 × 104 genomes per ml, contributing 0.02 to 5.7% of the quantified cyanobacterial picoplankton. Although the quantitative approach taken in this study has disclosed several shortcomings in the sampling and detection methods, this study demonstrated for the first time the extensive internal dynamics that lie beneath the seemingly arbitrary variations of a population of microbial photoautotrophs in the pelagic habitat.

Phenotypical and ultrastructural features of Oct4‐positive cells in the adult mouse lung

Octamer binding trascription factor 4 (Oct4) is a transcription factor of POU family specifically expressed in embryonic stem cells (ESCs). A role for maintaining pluripotency and self‐renewal of ESCs is assigned to Oct4 as a pluripotency marker. Oct4 can also be detected in adult stem cells such as bone marrow‐derived mesenchymal stem cells. Several studies suggest a role for Oct4 in sustaining self‐renewal capacity of adult stem cells. However, Oct4 gene ablation in adult stem cells revealed no abnormalities in tissue turnover or regenerative capacity. In the present study we have conspicuously found pulmonary Oct4‐positive cells closely resembling the morphology of telocytes (TCs). These cells were found in the perivascular and peribronchial areas and their presence and location were confirmed by electron microscopy. Moreover, we have used Oct4‐GFP transgenic mice which revealed a similar localization of the Oct4‐GFP signal. We also found that Oct4 co‐localized with several described TC markers such as vimentin, Sca‐1, platelet‐derived growth factor receptor‐beta C‐kit and VEGF. By flow cytometry analyses carried out with Oct4‐GFP reporter mice, we described a population of EpCAMneg/CD45neg/Oct4‐GFPpos that in culture displayed TC features. These results were supported by qRT‐PCR with mRNA isolated from lungs by using laser capture microdissection. In addition, Oct4‐positive cells were found to express Nanog and Klf4 mRNA. It is concluded for the first time that TCs in adult lung mouse tissue comprise Oct4‐positive cells, which express pluripotency‐related genes and represent therefore a population of adult stem cells which might contribute to lung regeneration.

Characterization of the platelet-derived growth factor receptor-α-positive cell lineage during murine late lung development.

A reduced number of alveoli is the structural hallmark of diseases of the neonatal and adult lung, where alveoli either fail to develop (as in bronchopulmonary dysplasia), or are progressively destroyed (as in chronic obstructive pulmonary disease). To correct the loss of alveolar septa through therapeutic regeneration, the mechanisms of septa formation must first be understood. The present study characterized platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cell populations during late lung development in mice. PDGFRα(+) cells (detected using a PDGFRα(GFP) reporter line) were noted around the proximal airways during the pseudoglandular stage. In the canalicular stage, PDGFRα(+) cells appeared in the more distal mesenchyme, and labeled α-smooth muscle actin-positive tip cells in the secondary crests and lipofibroblasts in the primary septa during alveolarization. Some PDGFRα(+) cells appeared in the mesenchyme of the adult lung. Over the course of late lung development, PDGFRα(+) cells consistently expressed collagen I, and transiently expressed markers of mesenchymal stem cells. With the use of both, a constitutive and a conditional PDGFRα(Cre) line, it was observed that PDGFRα(+) cells generated alveolar myofibroblasts including tip cells of the secondary crests, and lipofibroblasts. These lineages were committed before secondary septation. The present study provides new insights into the time-dependent commitment of the PDGFRα(+) cell lineage to lipofibroblasts and myofibroblasts during late lung development that is needed to better understand the cellular contribution to the process of alveolarization.

Impact of Arachidonic Acid and the Leukotriene Signaling Pathway on Vasculogenesis of Mouse Embryonic Stem Cells

Embryonic stem (ES) cells can differentiate into various kinds of cells, such as endothelial and hematopoietic cells. In addition, some evidence suggests that inflammatory mediators such as leukotrienes (LTs), which include the 5-lipoxygenase (LOX) family, can regulate endothelial cell differentiation. In the present study, the eicosanoid precursor arachidonic acid (AA) stimulated vasculogenesis of ES cells by increasing the number of fetal liver kinase-1+ vascular progenitor cells as well as vascular structures positive for platelet endothelial cell adhesion protein-1 and vascular endothelial cadherin. The stimulation of vasculogenesis and expression of the rate-limiting enzyme in the LT signaling pathway, 5-LOX-activating protein (FLAP), was blunted upon treatment with the FLAP inhibitors AM643 and REV5901. Vasculogenesis was significantly restored upon exogenous addition of LTs. Downstream of FLAP, the LTB4 receptor (BLT1) blocker U75302, the BLT2 receptor blocker LY255283 as well as the cysteinyl LT blocker BAY-u9773 inhibited vasculogenesis of ES cells. AA treatment of differentiating ES cells increased reactive oxygen species (ROS) generation, which was not affected upon either FLAP or cyclooxygenase-2 inhibition. Prevention of ROS generation by either the free radical scavengers vitamin E and N-(2-mercaptopropionyl)glycine or the NADPH oxidase inhibitor VAS2870 downregulated vasculogenesis of ES cells and blunted the provasculogenic effect of AA. In summary, our data demonstrate that proinflammatory AA stimulates vasculogenesis of ES cells via the LT pathway by mechanisms involving ROS generation.