Electroporation-mediated DNA delivery to seedling tissues ofPhaseolus vulgaris L. (common bean)

Summary DNA was delivered to intact embryonic axes of the legumePhaseolus vulgaris L. through electroporation. Expression of the ß-glucuronidase reporter gene was observed in hypocotyl and epicotyl tissue in a spot-like manner. Transgene expression was high when a single pulse of 260 ms at a field strength of 225 V·cm^–1 was applied but could be achieved within a wide range of electrical conditions. Linearization of plasmid DNA greatly enhanced transient expression levels. The procedure was successful for embryonic axes of all testedP. vulgaris cultivars, for similar explants of several large-seeded leguminous species, as well as for some other tissues ofP. vulgaris.     

A novel colorimetric yeast bioassay for detecting trichothecene mycotoxins.

A novel colorimetric microbial bioassay for toxicity has been developed; it shows particular sensitivity to trichothecene mycotoxins. The assay uses inhibition of expression of beta-galactosidase activity within the yeast Kluyveromyces marxianus as a sensitive toxicity indicator, cultures remaining yellow, rather than turning deep green-blue, in the presence of X-gal, a chromogenic substrate. The assay is conducted in standard microtitre plates, permitting small volumes (160 microl) and many replicates, and can be scored either automatically by a plate-reader, or by eye. Factors likely to affect the efficacy of the bioassay, including carbon source, solvents, inoculum cell density, and the use of membrane-modulating agents (MMAs), were assessed. Polymyxin B nonapeptide was the most effective toxicity-enhancing MMA tested, enabling the trichothecene mycotoxin, verrucarin A, to be detected at a concentration of about 1 ng/ml. The assay's reproducibility was examined using polymyxin B sulfate, a cheaper MMA, and another trichothecene mycotoxin, T2 toxin: reproducibility and sensitivity were better for the beta-galactosidase X-gal endpoint than for an alternative chromogenic toxicity indicator, the respiratory substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).     

Hybridizationin situ of whole-mount messenger RNA in plants

A procedure is presented for the detection of mRNA in whole-mount preparations of youngArabidopsis seedlings using digoxigenin (DIG)-labeled RNA probes. It includes tissue preservation with formaldehyde, permeabilization with polyoxyethy lenesorbitan (Tween 20), DMSO, and heptane. Hybridization signal is detected using colloidal gold or alkaline-phosphatase-conjugated anti-DIG antibodies.     

Dental topographic and three-dimensional geometric morphometric analysis of carnassialization in different clades of carnivorous mammals (Dasyuromorphia,Carnivora, Hyaenodonta)

The evolution of carnassial teeth in mammals, especially in the Carnivora, has been subject of many morphometric and some dental topographic studies. Here, we use a combination of dental topographic analysis (Dirichlet normal energy) and 3D geometric morphometrics of less and high carnassialized lower teeth of carnivoran, dasyuromorph and hyaenodont taxa. Carnassial crown curvature, as indicated by Dirichlet normal energy, is high in lesser carnassialized teeth and low in higher carnassialized teeth, where it is influenced by the reduction of crown features such as cusps and crests. PC1 of the geometric morphometric analysis is linked to enlargement of the carnassial blade, reduction of the talonid crushing basin and an increasingly asymmetric cervix line with an enlarged mesial flexure in more carnassialized teeth. Distribution of PC1 values further indicates that along the tooth row of dasyuromorphs (m2–m4) and hyaenodonts (m1–m3) the most distal carnassial is the most carnassialized (principal carnassial), and in most taxa with overall higher carnassialized teeth, carnassialization successively increases from the anterior to the posterior tooth position along the tooth row. PC2 indicates that a longitudinal elongated carnassial is present in caniforms and in unspecialized feliforms, which separates these taxa in morphospace from all dasyuromorphs, hyaenodonts and specialized feliforms. An ancestral state reconstruction shows that this longitudinal elongation may be a plesiomorphic ancestral state for the Carnivora, which is different from the Dasyuromorphia and the Hyaenodonta. This elongation, enabling the presence of a longitudinally aligned carnassial blade as well as a complete talonid basin, might have provided the Carnivora with an advantage in terms of adaptive versatility.     

Differential splicing of thymosin beta 4 mRNA

A cDNA clone was isolated from a mouse pre-B cell line, the sequence of which has a very high homology with rat and human thymosin beta 4 genes. However, the mouse clone has an insertion of 98 bp relative to the published rat and human sequences upstream of the coding region. By isolation of a second set of clones from a different cDNA library and by cloning a PCR amplified region of mouse genomic DNA it was confirmed that the insertion is not a cloning artifact. Furthermore, it was shown by RNase protection assays with RNA from the pre-B cell line that two sizes of thymosin beta 4 mRNA exist, a long form containing the 98 nucleotide insertion, and a short form that corresponds to the known rat and human mRNA. The short form is about 50 times more abundant than the long form. Analysis of genomic DNA by sequencing and Southern blotting revealed that both forms are encoded by a single gene in the mouse. The two forms of mRNA arise by differential RNA splicing; the long mRNA contains three separate exons, whereas the short mRNA is missing exon 2. The long mRNA is present in two different pre-B cell lines, spleen and thymus, but could not be detected in brain, liver, and kidney. It is possible that the longer mRNA, which encodes a hydrophobic NH2-extension of six additional amino acids, plays a role in lymphocyte function or development. In contrast to the mouse which has a single thymosin beta 4 gene, rat and human have multiple homologs. Most or all of these also contain sequences that cross-hybridize with the newly discovered exon 2. A polymorphic thymosin beta 4 gene has been found in human DNA.     

Correspondenz

Ohne Zusammenfassung     

Distribution of the rDNA and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana

The distribution of the ribosomal RNA (rRNA) genes and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana was studied for the first time through non-isotopic in situ hybridization and luminescence digital imaging microscopy. Each of the three classes of highly repetitive DNA exhibited a characteristic hybridization pattern, and one class was seen to be primarily localized on two chromocentres, which would allow it to distinguish a particular chromosome. The rDNA was consistently localized on the two largest chromocentres and on one or two smaller chromocentres. A limited number of nuclei exhibited more than four labelled chromocentres, indicative of either polypoidy or differential amplification of the rDNA. In nuclei where the nucleolus could be clearly observed, the nucleolar associated chromocentres (NACs) were seen to be labelled by the ribosomal DNA (rDNA) probe.by W. Hennig     

The nucleotide sequence and protein-coding capability of the transposable element IS5

The nucleotide sequence of IS5, a bacterial insertion sequence, has been determined. It is 1195 bp long and contains an inverted terminal repetition of 16 bp with one mismatch. One open reading frame, spanning nearly the entire length of the element, could encode a polypeptide of 338 amino acids. Upon insertion into a DNA segment, IS5 causes a duplication of 4 bp. Based on seven examples, this site of insertion appears to be nonrandom, and the consensus target site sequence is C . T/A . A . G/A (or C/T . T . A/T . G on the opposite strand). The nucleotide sequences of IS5 insertions into the B and cim genes of bacteriophage Mu have allowed tentative identification of the protein-coding frames of B and cim.