Cauliflower mosaic virus gene VI causes growth suppression, development of necrotic spots and expression of defence-related genes in transgenic tobacco plants

Summary In order to study possible functions of the inclusion body matrix protein (IBMP) encoded by gene VI of cauliflower mosaic virus (CaMV), the XbaI fragment containing the gene VI of a Japanese strain of CaMV (CaMV S-Japan) was transferred to tobacco plants by Ti mediated transformation. Eight out of 18 kanamycin resistant plants (40%) expressed detectable levels of IBMP. Those transgenic plants expressing IBMP produced leaves with light green color, and their growth was suppressed as compared with control plants. Symptom-like necrotic spots also appeared on the leaves and stems of the mature transgenic plants. Furthermore, in these transgenic plants, pathogenesis-related proteins 1a, 1b and 1c were highly expressed and the activity of 1,3--glucanase was increased up to eightfold. From these results, we concluded that expression of the IBMP is associated with symptom development.     

Specificity of the interactions between the Rep proteins and the origins of replication of Staphylococcus aureus plasmids pT181 and pC221

Summary pT181 and pC221 are closely relatedStaphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, Rep, essential for plasmid replication. Former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both Rep proteins to act on either origin. One possible explanation for this difference was based on a previous analysis of the incompatibility expressed by the origin of replication of these plasmids, showing that the origin embedded in therep gene competes for Rep utilization with the origin of a test plasmid and that changes in the sequence of the origin reduce its ability to compete. To avoid this problem, in the present work special hybrids were constructed in which the origin of replication overlapping therep gene was mutationally inactivated, without changing the amino acid sequence of the encoded protein. The level of Rep expression by these hybrids could be varied by taking advantage of what is presently known about the control of Rep synthesis in plasmid pT181. The results of complenentation studies conducted using these hybrids have shown that: (i) at the usual level of expression for a wild-type plasmid each Rep protein can initiate replication strictly from its corresponding origin; (ii) when overproduced, the pT181 RepC protein could also act efficiently on the pC221 origin; a functional pT181 origin present in the same host completely prevented this complementation; (iii) in excess, the RepD protein encoded by pC221 could replicate a plasmid carrying the pT181 origin but could not ensure the hereditary stability of such a plasmid in the absence of another active replication system; (iv) when overproduced both RepC and RepD could act on the origin of replication of three other related plasmids pS194, pC223 and pUB112.     

Different routes for integral protein insertion into Ricinus communis protein-body and glyoxysome membranes

Total polyadenylated RNA from ripening or germinating Ricinus communis L. endosperm was translated in rabbit reticulocyte lysate in the absence or presence of canine pancreatic microsomes. The products were immunoprecipitated using antibodies raised againts Triton X-114-extracted integral membrane proteins of protein bodies or glyoxysomes. While the proteins of proteinbody membranes were found to insert co-translationally into added microsomes, this was not observed in the case of glyoxysomal proteins. This observation was confirmed using antibodies raised against a purified glyoxysome membrane protein, alkaline lipase. These results indicate that different routes exist for the insertion of membrane proteins into the two organelles. In both cases membrane-protein insertion does not appear to be accompanied by proteolytic processing.Abbreviations anti-PB antiserum to integral protein-body membrane proteins - anti-G antiserum to integral glyoxysomal membrane proteins - anti-L antiserum to alkaline lipase - ER endoplasmic reticulum - M_r relative molecular mass - mRNA poly(A)-rich messenger RNA - PAGE polyacrylamide gel electrophoresis - poly(A) polyadenylic acid - SDS sodium dodecyl sulphate     

Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.     

Investigation of histone proteins in plant nuclei possessing different ultrastructural organization

Summary According to Nagl and Fusenig (1979) the structure and ultrastructure of plant nuclei is species-specific and is determined by the DNA (2C) value and the amount of the repetitive DNA. Light and electron microscopic observations ofZea mays L.,Pisum sativum L., andPhaseolus vulgaris L. nuclei led us to define their organization as chromonematic, chronomeric and chromocentric, respectively. Nuclear proteins, soluble in 0.4N H₂SO₄ and 0.74M HC1O₄, were extracted from isolated nuclei and resolved according to their solubility and mobility in SDS and acetic acid-urea PAGE and 2D-Triton X 100 PAGE. Differences in the variants (and modifications) of the H 1 histone class and the nucleosomal H 2 A, H 2 B, and H 3 isoforms probably reflect that species-specific nuclear ultrastructure is based, not only on the heterogeneity and the quantity of DNA, but also on the diversity of the protein component of chromatin.Abbreviations MES Morpholinoethane sulfonic acid - PMSF phenylmethylsulphonyl fluoride - DMSO dimethylsulfoxid - SDS sodium dodecylsulfate - TEMED N, N, N N-tetramethylethylen-diamin - PAGE polyacrylamide gel electrophoresis     

Heat shock proteins in Sorghum bicolor and Pennisetum americanum I. genotypic and developmental variation during seed germination

Abstract The capacity to synthesize heat shock proteins (HSPs) during seed germination of sorghum (Sorghum bicolor) and pearl millet (Pennisetum americanum) has been examined. HSP synthesis is detectable in a thermotolerant genotype of sorghum during the first hour of imbibition of the seed under high temperature stress. A non-coordinate control of HSP synthesis during germination was revealed. Genotypic differences were manifest in the stage of germination at which the ability to synthesize HSPs was first apparent and this related to the thermosensitivity of that genotype.     

Resonance electroconformational coupling: A proposed mechanism for energy and signal transductions by membrane proteins

Recent experiments show that membrane ATPases are capable of absorbing free energy from an applied oscillating electric field and converting it to chemical bond energy of ATP or chemical potential energy of concentration gradients. Presumably these enzymes would also respond to endogenous transmembrane electric fields of similar intensity and waveform. A mechanism is proposed in which energy coupling is achieved via Coulombic interaction of an electric field and the conformational equilibria of an ATPase. Analysis indicates that only an oscillating or fluctuating electric field can be used by an enzyme to drive a chemical reaction away from equilibrium.In vivo, the stationary transmembrane potential of a cell must be modulated to become locally oscillatory if it is to derive energy and signal transduction processes.     

Non-histone chromatin proteins in beef thyroid: distinct phosphorylation patterns of several protein kinases

Summary Non-histone chromatin protein (NHCP) fractions were extracted from purified beef thyroid nuclear preparations and tested for the presence of protein kinase activities using several known mediators of thyroid regulation, as well as potential phosphotransferase substrates using purified or partially purified protein kinase activities. The addition of cAMP/3-isobutyl-l-methylxanthine had no effect on NHCP historic kinase activity; the addition of 10 g of the heat-stable cAMP-dependent protein kinase A inhibitor, however, resulted in a 47% reduction in histone H₂ kinase activity. Nuclear casein kinase II activity was present in the NHCP fractions as evidenced by the capacity of spermine to stimulate (ED₅₀ = 0.19 mM) and heparin to inhibit (ID₅₀ = 0.09 g/ml) the phosphorylation of casein; further, the phosphotransferase activity could be purified by sequential casein-agarose and spermine-agarose affinity chromatography. Neither calcium-calmodulin nor calcium/phosphatidylserine/diolein had an effect on NHCP casein kinase or histone kinase activities, respectively. The addition of cAMP-dependent protein kinase A catalytic subunit, nuclear casein kinase II, calcium-activated calmodulin-dependent protein kinase and diacylglycerol-activated calcium/phospholipid-dependent protein kinase C activities exhibited distinct phosphorylation patterns when NHCP were used as substrates and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. We conclude that NHCP fraction from beef thyroid: 1) contains both cAMP-dependent protein kinase A catalytic subunit and nuclear casein kinase II and 2) substrates for cAMP-dependent protein kinase A, calcium-activited calmodulin-dependent protein kinase, protein kinase C, and nuclear casein kinase II.Abbreviations NHCP Non-Histone Chromatin Proteins - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - PK-C Diacylglycerol-Activated Calcium/phospholipid-dependent Protein Kinase - NK-11 Nuclear Casein Kinase 11 - CK-G Cytosolic Casein Kinase G or 11 - PMSF Phenylmethyl Sulfonyl Fluoride - PKI the Heat Stable PK-A Inhibitor (Walsh inhibitor) - SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis - EDTA Ethylenediamine Tetraacetic Acid - EGTA Ethyleneglycol bis- (B-aminoethyl ether) N,N,N,N,-Tetraacetic Acid - PS Phosphatidylserine - DO 1,2-Diolein