Reevaluating the hype: four bacterial metabolites under scrutiny.

With microbiome research being a fiercely contested playground in science, new data are being published at tremendous pace. The review at hand serves to critically revise four microbial metabolites widely applied in research: butyric acid, flagellin, lipoteichoic acid, and propionic acid. All four metabolites are physiologically present in healthy humans. Nevertheless, all four are likewise involved in pathologies ranging from cancer to mental retardation. Their inflammatory potential is equally friend and foe. The authors systematically analyze positive and negative attributes of the aforementioned substances, indicating chances and dangers with the use of pre- and probiotic therapeutics. Furthermore, the widespread actions of microbial metabolites on distinct organs and diseases are reconciled. Moreover, the review serves as critical discourse on scientific methods commonly employed in microbiome research and comparability as well as reproducibility issues arising thereof.

Arabidopsis thaliana: a source of candidate disease-resistance genes for Brassica napus.

Common structural and amino acid motifs among cloned plant disease-resistance genes (R genes), have made it possible to identify putative disease-resistance sequences based on DNA sequence identity. Mapping of such R-gene homologues will identify candidate disease-resistance loci to expedite map-based cloning strategies in complex crop genomes. Arabidopsis thaliana expressed sequence tags (ESTs) with homology to cloned plant R genes (R-ESTs), were mapped in both A. thaliana and Brassica napus to identify candidate R-gene loci and investigate intergenomic collinearity. Brassica R-gene homologous sequences were also mapped in B. napus. In total, 103 R-EST loci and 36 Brassica R-gene homologous loci were positioned on the N-fo-61-9 B. napus genetic map, and 48 R-EST loci positioned on the Columbia x Landsberg A. thaliana map. The mapped loci identified collinear regions between Arabidopsis and Brassica which had been observed in previous comparative mapping studies; the detection of syntenic genomic regions indicated that there was no apparent rapid divergence of the identified genomic regions housing the R-EST loci.

Molecular mapping of resistance to Leptosphaeria maculans in Australian cultivars of Brassica napus.

Doubled haploid (DH) lines together with a cotyledon bioassay were employed for the molecular analysis of resistance to the blackleg fungus Leptosphaeria maculans in the Australian Brassica napus cultivars Shiralee and Maluka. We used bulked segregant analysis to identify 13 RAPD and two RFLP markers linked to the resistance phenotype and mapped these markers in the segregating DH population. Our data suggest the presence of a single major locus controlling resistance in the cultivar Shiralee, confirming our previous results obtained from Mendelian genetic analyses. In addition, preliminary mapping data for the cultivar Maluka also support a single locus model for resistance and indicate that the resistance genes from 'Shiralee' and 'Maluka' are either linked or possibly identical. The molecular markers identified in this study should be a useful tool for breeding blackleg resistant varieties using marker-assisted selection, and are the essential first step towards the map-based cloning of this resistance gene.

Monitoring of spatial expression of firefly luciferase in transformed zebrafish.

The firefly luciferase gene attached to the cytomegalovirus promoter was transferred into zebrafish (Brachydanio rerio) by microinjection of fertilized eggs. Light emission could be monitored in vivo in eggs and throughout the early development of the fish by low-light video-image analysis. Gene expression was transient in most of the cases lasting for about 2 weeks. This gene cassette proved to be a very convenient and non-destructive transformation marker and the firefly luciferase gene appears to be a powerful tool for real-time imaging of tissue-specific gene expression in transgenic fish.

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana.

A recessive pale mutation, designated as cs, was identified by transferred-DNA (T-DNA)-mediated insertional mutagenesis in Arabidopsis thaliana. The pale mutation, cosegregating with the hygromycin resistance marker of the T-DNA, was mapped to the position of the ch-42 (chlorata) locus on chromosome 4. Lack of genetic complementation between cs and ch-42 mutants indicated allelism. Plant boundaries of the T-DNA insert rescued from the pale mutant were used as probes for the isolation of genomic and full-length cDNA clones of the wild-type cs gene. Transformation of the pale mutant with T-DNA vectors carrying these clones resulted in a normal green phenotype, thus demonstrating positive complementation of the T-DNA induced mutation. DNA sequence comparison of the cs mutant and its wild-type allele revealed that the T-DNA insertion occurred 11 bp upstream of the stop codon. A fusion protein, seven amino acids longer than its wild-type counterpart of Mr 46,251, is therefore synthesized in the pale mutant. Transcript analysis during dark-light transition, in vitro protein transport assay, and the absence of DNA sequence homology between cs and known genes indicates that the light regulated expression of the cs gene results in the synthesis of a novel chloroplast protein.

The Marfan syndrome--analysis of growth and cardiovascular manifestation.

Forty-eight children and adolescents (mean age 10.5 years, range 1.25-18 years) with clinical evidence of Marfan syndrome were studied. Height and weight percentiles were established. Cardiac dimensions and morphology were studied by M-mode and 2D-echocardiography. At diagnosis left atrial and left ventricular end-diastolic diameter and left ventricular posterior wall thickness were within normal limits except in a few adolescent patients. Interventricular septum was thickened in about 20% and aortic diameter increased in 56% of the patients. An additional 13% of patients developed aortic dilation during the study period. At diagnosis regression analysis revealed a significant (P less than 0.05) correlation of the aortic diameter, septal thickness and the posterior left ventricular wall thickness and body surface area. Follow up studies of 19 patients allowed documentation of the development of aortic root dilation.

High-frequency T-DNA-mediated gene tagging in plants.

An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')II (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')II enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusions and construction of chimeric genes, callus- and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3'-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.