Cloning and characterization of the cDNA coding for the catalytic alpha subunit of CK2 from tobacco.

We have previously reported the participation of the protein kinase CK2 in the mechanism by which salicylic acid activates transcription of genes, such as those coding for glutathion S-transferases, in tobacco. With the purpose of further studying the participation of CK2 in this signal transduction pathway, we isolated and sequenced the cDNA from the NtCK2A gene, coding for the catalytic alpha subunit of CK2 from tobacco. The NtCK2A cDNA was isolated by screening of a tobacco cDNA library with a heterologous probe from Arabidopsis thaliana, followed by 3' RACE to obtain the 3' region. Sequence analysis of the NtCK2A cDNA showed a high level of identity between this CK2alpha protein sequence and the corresponding sequences of other plant species such as Arabidopsis and maize (92-95% identity), or those of animal species such as human and Xenopus laevis (75% identity). The expression of the NtCK2A gene in different tissues from tobacco plants was analyzed by Northern blot. High levels of expression of this gene were observed in proliferating tissues such as shoot and root apical meristems. A recombinant CK2alpha protein was obtained after expression of the NtCK2A cDNA in Escherichia coli. The ability of this recombinant CK2alpha subunit to phosphorylate casein was inhibited by heparin and stimulated by the CK2beta subunit from Xenopus laevis.

Direct evidence for ribonucleolytic activity of a PR-10-like protein from white lupin roots.

An abundant 17 kDa protein which was isolated and characterized from 10-day old healthy root tissue of white lupin (Lupinus albus) proved to have a high sequence similarity to pathogenesis-related proteins found in other species. Subsequently, a corresponding clone (LaPR-10) was identified in a cDNA library prepared from the same tissue that exhibited a high amino acid sequence similarity to a number of the PR-10 family proteins. The clone contains an open reading frame encoding a polypeptide of 158 amino acids, with a predicted molecular mass of 16,905 Da and an isoelectric point of 4.66. Southern blot analysis indicates that LaPR-10 is likely a single-copy gene, or a member of a small gene family. The clone was expressed in Escherichia coli, and its protein product was purified to near homogeneity. Both the native and the recombinant proteins were immunorecognized by antibodies raised against pea PR-10 proteins, and exhibited a ribonucleolytic activity against several RNA preparations, including lupin root total RNA. Characterization of its enzymatic properties indicates that the LaPR-10 protein belongs to the class II ribonucleases. We present evidence that the white lupin 17 kDa protein is constitutively expressed during all stages of root development and, to a lesser extent, in other plant parts. In addition, we demonstrate the presence, in the LaPR-10 amino acid sequence, of a number of motifs that are common to most PR-10 proteins, as well as a RGD motif that is shared only with the alfalfa SRG1 sequence.

Plant O-methyltransferases: molecular analysis, common signature and classification.

Comparative analysis of the predicted amino acid sequences of a number of plant O-methyltransferase cDNA clones show that they share some 32-71% sequence identity, and can be grouped according to the different compounds they utilise as substrates. Five highly conserved regions are proposed as a signature for plant O-methyltransferases, two of which (regions I and IV) are believed to be involved in S-adenosyl-L-methionine and metal binding, respectively. The glycine-rich signature regions include a 36 amino acid domain which is located in the mid-terminal section of the carboxy terminus of most O-methyltransferase sequences. Cladistic analysis of the amino acid sequences suggests that plant O-methyltransferases may have arisen from common ancestral genes that were driven by different structural and/or functional requirements, and whose descendants segregated into different biochemical species. A comprehensive classification of plant O-methyltransferases is proposed following the guidelines of the Commission of Plant Gene Nomenclature.

Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures.

A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. The enzyme formed only geranyl diphosphate as a product. In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected. The enzyme showed a native molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation. On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 [plus or minus] 2 kD. Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 [mu]M, respectively. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme.