Interaction of ribonuclease T1 with DNA,mononucleotides and oligonucleotides

The interaction of ribonuclease T₁ with DNA and nucleotides was investigated by fluorescence titration to establish whether or not this enzyme is a helix-destabilizing protein. Binding of the enzyme to DNA, ribonucleotides and oligodeoxyribonucleotides of chain length ten or more leads to enhancement of fluorescence emission of the enzyme as a function of increasing nucleotide/protein ratio. For deoxyribonucleotides of chain length less than ten, only quenching is observed. Energy transfer from the bases is postulated to be the source of the enhancement of fluorescence, while the decrease can be ascribed to changes in the distribution of charged groups in or near the binding site.     

Efficacy of RNase inhibitors during brain polysome isolation

We have investigated the efficiency of heparin, polyvinyl sulfate and yeast RNA (as competitive RNase inhibitors), liver extract (as crude preparation of liver RNase inhibitors) and DEPC (as irreversible non-competitive inhibitor) for the preparation of rat brain polysomes. Sucrose gradient sedimentation profiles, obtained from PMS, were used to determine the optimal concentration of each inhibitor. Diethylpyrocarbonate, whatever the composition of isolation buffer, was found detrimental for brain polysomes. Most of the other inhibitors where found useless or even harmful. A slight positive effect was observed with heparin 0.75 mg/mL both for total yield and sedimentation pattern. It is concluded that the utilisation of most of the widely used RNase inhibitors is of questionnable effectiveness for brain polysome preparation.Abbreviations DEPC diethylpyrocarbonate - PVS polyvinyl sulfate - PMS post-mitochondrial supernatant - HSS high-speed supernatant     

Definition of the Th/ To ribonucleoprotein by RNase P and RNase MRP

We show that the Th/ To ribonucleoprotein is defined by (i) the co-immunoprecipitation of two RNAs, (ii) the co-immunoprecipitation of four major polypeptides and (iii) the quantitative immune recognition of both RNase P and RNase MRP. No serum was found that recognizes either one of these two enzymes exelusively. The specific co-immunoprecipitation of RNase MRP and RNase P by all Th/ To ribonucleoprotein autoantibodies indicates that the anti-Th/ To autoimmune response is directed against both enzymes in a quantitatively indistinguishable manner. Thus the Th/ To ribonucleoprotein is defined by RNase P and RNase MRP.     

Antibiotics present and future

Here we make a brief survey of the present state of antibiotic research and use. We also describe a novel antibiotic that contains a basic peptide covalently bound to a morpholino oligonucleotide.     

Identification of a plant-specific Zn<Superscript>2+</Superscript>-sensitive ribonuclease activity

Ribonucleases (RNases) play a variety of cellular and biological roles in all three domains of life. In an attempt to perform RNA immuno-precipitation assays of Arabidopsis proteins, we found an EDTA-dependent RNase activity from Arabidopsis suspension tissue cultures. Further investigations proved that the EDTA-dependent RNase activity was plant specific. Characterization of the RNase activity indicated that it was insensitive to low pH and high concentration of NaCl. In the process of isolating the activity with cation exchange chromatography, we found that the EDTA dependency of the activity was lost. This led us to speculate that some metal ions, which inhibited the RNase activity, may be removed during cation exchange chromatography so that the nuclease activity was released. The EDTA dependency of the activity could be due to the ability of the EDTA chelating those metal ions, mimicking the effect of the cation exchange chromatography. Indeed, Zn²⁺ strongly inhibited the activity, and the inhibition could be released by EDTA based on both in-solution and in-gel assays. In-gel assays identified two RNase activity bands. Mass spectrometry assays of those activity bands revealed more than 20 proteins. However, none of them has an apparent known nuclease domain, suggesting that one or more of those proteins might possess a currently uncharacterized nuclease domain. Our results may shed light on RNA metabolism in plants by introducing a novel plant-specific RNase activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Embryonic fibronectins are up-regulated following peripheral nerve injury in rats

Fibronectin mRNAs that include the alternatively spliced exons EIIIA, EIIIB, and V are prevalent during embryogenesis, and EIIIA and EIIIB reappear during wound healing. Using ribonuclease protection analyses, we found an up-regulation of V120 (containing the α₄β₁ integrin binding site), as well as EIIIA, and EIIIB in fibronectin mRNAs in the crush-injured adult rat sciatic nerve. In situ hybridization using splice variant-specific probes revealed that cells within endoneurial tubes of the injured nerve synthesize these embryonic forms of fibronectin. Our results suggest that embryonic fibronectins synthesized within the nerve contribute to the permissiveness of the peripheral nervous system to axon regrowth and a mechanism by which alternative splicing of the V region in fibronectin mRNA could enhance nervous system regeneration. © 1995 John Wiley & Sons, Inc.