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.     

α2 High molecular mass cysteine proteinase inhibitor: HMrα2-CPI: An inhibitor of human liver cathepsin H as probed by kinetic study

HMrα₂CPI was found to be an inhibitor of human liver cathepsin H by the measurement of the dissociation constant (K_i), the association rate constant (k₁) and the dissociation rate constant (k_?1) between the enzyme and the inhibitor. These data suggest that this protein-proteinase inhibitor can play a physiological role in the regulation of free cathepsin H.     

Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.     

Adhesion strength and contractility enable metastatic cells to become adurotactic

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AtGRP5, a vacuole-located glycine-rich protein involved in cell elongation

Although several glycine-rich protein (GRP) genes were isolated and characterized, very little is known about their function. The primary structure of AtGRP5 from Arabidopsis thaliana has a signal peptide followed by a region with high glycine content. In this work, green fluorescent protein fusions were obtained in order to characterize the sub-cellular localization of the AtGRP5 protein. The results indicated that this protein is the first described vacuolar GRP. Sense, antisense and RNAi transgenic A. thaliana plants were generated and analyzed phenotypically. Plants overexpressing AtGRP5 showed longer roots and an enhanced elongation of the inflorescence axis, while antisense and RNAi plants demonstrated the opposite phenotype. The analysis of a knockout T-DNA line corroborates the phenotypes obtained with the antisense and RNAi plants. Altogether, these results suggest that this vacuolar GRP could be involved in organ growth by promoting cell elongation processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Amanda Mangeon and Claudia Magioli contributed equally to this work.     

Mesenchymal stem cell durotaxis depends on substrate stiffness gradient strength

Mesenchymal stem cells (MSCs) respond to the elasticity of their environment, which varies between and within tissues. Stiffness gradients within tissues can result from pathological conditions, but also occur through normal variation, such as in muscle. MSC migration can be directed by shallow stiffness gradients before differentiating. Gradients with fine control over substrate compliance – both in range and rate of change (strength) – are needed to better understand mechanical regulation of MSC migration in normal and diseased states. We describe polyacrylamide stiffness gradient fabrication using three distinct systems, generating stiffness gradients of physiological (1 Pa/μm), pathological (10 Pa/μm), and step change (≥ 100Pa/μm) strength. All gradients spanned a range of physiologically relevant elastic moduli for soft tissues (1–12 kPa). MSCs migrated to the stiffest region on each gradient. Time-lapse microscopy revealed that migration velocity correlated directly with gradient strength. Directed migration was reduced in the presence of the contractile agonist lysophosphatidic acid (LPA) and cytoskeleton-perturbing drugs nocodazole and cytochalasin. LPA- and nocodazole-treated cells remained spread and protrusive on the substrate, while cytochalasin-treated cells did not. Nocodazole-treated cells spread in a similar manner to untreated cells, but exhibited greatly diminished traction forces. These data suggest that a functional actin cytoskeleton is required for migration whereas microtubules are required for directed migration. The data also imply that, in vivo, MSCs may preferentially accumulate in regions of high elastic modulus and make a greater contribution to tissue repairs in these locations.     

Exploration of the late stages of the tomato-Phytophthora parasitica interactions through histological analysis and generation of expressed sequence tags.

The oomycete Phytophthora parasitica is a soilborne pathogen infecting numerous plants. The infection process includes an initial biotrophic stage, followed by a necrotrophic stage. The aim here was to identify genes that are involved in the late stages of infection. Using the host tomato and a transformed strain of P. parasitica expressing the green fluorescent protein (GFP), the various infection steps from recognition of the host to the colonization of plant tissues were studied. This late stage was selected to generate 4000 ESTs (expressed sequence tags), among which approx. 80% were from the pathogen. Comparison with an EST data set created previously from in vitro growth of P. parasitica allowed the identification of several genes, the expression of which might be regulated during late stages of infection. Changes in gene expression of several candidate genes predicted from in silico analysis were validated by quantitative RT-PCR experiments. These results give insights into the molecular bases of the necrotrophic stage of an oomycete pathogen.     

Botanische Gesellschaften,Vereine, Congresse etc.

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