Molecular cloning and characterization of rat LC3A and LC3B--two novel markers of autophagosome

Rat microtubule-associated protein light chain 3 (LC3) is a homologue of yeast Atg8, an essential component of autophagy. Following synthesis, the C-terminus of rat LC3 is cleaved by a cysteine protease-Atg4, to produce LC3-I, which is located in cytosolic fraction. LC3-I can be converted to LC3-II through the processing by Atg7 (E1-like enzyme) and Atg3 (E2-like enzyme). LC3-II is modified by phosphatidylethanolamine on C-terminus and binds tightly to autophagosomal membrane. Here we reported the cloning of two novel variants of rat LC3, named LC3A and LC3B, respectively, and LC3B is an alternative splicing variant of LC3. LC3A, LC3B, and LC3 showed different expression patterns in rat tissues, suggesting a functional divergence among these proteins. When LC3A and LC3B were overexpressed, both exhibited two forms (18 and 16 kDa, representing types of I and II, separately), which might be due to post-translational modification including the characteristic C-terminal cleavage at these two proteins as similar to that found in rat LC3 and yeast Atg8. Subcellular localization demonstrated that both LC3A and LC3B are colocalized with LC3 and associated with the autophagic membranes. Mutation analysis further revealed that the conserved Gly120 residues of LC3A and LC3B are essential for their characteristic C-terminal cleavage and localization to autophagic membranes. Present data suggested that LC3A and LC3B could also be used as two novel autophagosomal markers.     

A description of the Mei2-like protein family; structure,phylogenetic distribution and biological context

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a "mei2-dot" in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.     

Characterization of a stress-responsive ankyrin repeat-containing zinc finger protein of Capsicum annuum (CaKR1)

We isolated many genes induced from pepper cDNA microarray data following their infection with the soybean pustule pathogen Xanthomonas axonopodis pv. glycines 8ra. A full-length cDNA clone of the Capsicum annuum ankyrin-repeat domain C(3)H(1) zinc finger protein (CaKR1) was identified in a chili pepper using the expressed sequence tag (EST) database. The deduced amino acid sequence of CaKR1 showed a significant sequence similarity (46%) to the ankyrin-repeat protein in very diverse family of proteins of Arabidopsis. The gene was induced in response to various biotic and abiotic stresses in the pepper leaves, as well as by an incompatible pathogen, such as salicylic acid (SA) and ethephon. CaKR1 expression was highest in the root and flower, and its expression was induced by treatment with agents such as NaCl and methyl viologen, as well as by cold stresses. These results showed that CaKR1 fusion with soluble, modified green fluorescent protein (smGFP) was localized to the cytosol in Arabidopsis protoplasts, suggesting that CaKR1 might be involved in responses to both biotic and abiotic stresses in pepper plants.     

Signalling molecules and blast pathogen attack activates rice OsPR1a and OsPR1b genes: A model illustrating components participating during defence/stress response

Recently the rice (Oryza sativa L.) OsPR1a and OsPR1b genes were primarily characterized against jasmonic acid, ethylene and protein phosphatase 2A inhibitors. The dicot PR1 are recognized as reliable marker genes in defence/stress responses, and we also propose OsPR1 as marker genes in rice, a model monocot crop genus. Therefore, to gain further insight into the expression/regulation of OsPR1 genes, we characterized their activation against signalling molecules such as salicylic acid (SA), abscisic acid (ABA) and hydrogen peroxide (H₂O₂), and the blast pathogen Magnaporthe grisea. Here, we report that SA and H₂O₂ strongly induced the mRNA level of both OsPR1 genes, whereas ABA was found to be moderately effective. These inductions were specific in nature and required a de novo synthesized protein factor. A potential interaction amongst the signalling molecules in modulating the expression of OsPR1 genes was observed. Moreover, a specific induction of OsPR1 expression in an incompatible versus compatible host-pathogen interaction was also found. Finally, based on our present and previous results, a model of OsPR1 expression/regulation has been proposed, which reveals their essential role in defence/stress responses in rice and use as potent gene markers.     

Novel cry gene from Paenibacillus lentimorbus strain Semadara inhibits ingestion and promotes insecticidal activity in Anomala cuprea larvae

A positive clone was selected from a library of total cell DNA of Paenibacillus lentimorbus strain Semadara that reacted with an antiserum that was raised against parasporal crystal proteins produced by this strain. The positive clone had a DNA insert containing two whole cry genes (cry43Aa1, cry43Ba1), one partial cry gene (cry43-like), and three smaller genes located upstream. Eight blocks that are conserved in the Cry proteins of Bacillus thuringiensis [Microbiol. Mol. Biol. Rev. 62 (1998) 775] were detected in their deduced amino acid sequences. The Escherichia coli transformant expressing cry43Aa1 caused inhibition of ingestion and 90% mortality in the first stadium larvae of Anomala cuprea. A low concentration of sporangia mixed with the transformant expressing cry43Aa1 easily infected the larvae of A. cuprea. The protein of approximately 150 kDa produced by the transformants expressing the cry genes reacted with antiserum specific for the parasporal crystal proteins. Southern hybridization analysis demonstrated that the cry genes were located on the chromosomal DNA of this strain, which possessed at least four cry genes.     

Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea

Mago nashi (Mago) and Y14 proteins, highly conserved among eukaryotes, participate in mRNA localization and splicing, and as such play important roles in oogenesis, embryogenesis and germ-line sex determination during animal development. Here we identified mago (Acmago) and Y14 (AcY14) homologues derived from Antrodia cinnamomea. Acmago encodes 149 amino acids and AcY14 encodes 168 amino acids. Multiple amino acid sequence alignment as well as secondary and tertiary structure prediction showed that AcMago and AcY14 have similar protein structure to the reported crystal structures of other Mago and Y14 proteins. During fungal development both Acmago and AcY14 genes were abundantly expressed in natural basidiomes. This is the first report of the molecular characterization and expression analysis of the mago and Y14 genes from fungi.     

The phylogenetic significance of peptidoglycan types: Molecular analysis of the genera Microbacterium and Aureobacterium based upon sequence comparison of gyrB, rpoB, recA and ppk and 16SrRNA genes

The type strains of 27 species of the genus Microbacterium, family Microbacteriaceae, were analyzed with respect to the phylogeny of the housekeeping genes coding for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA) and polyphosphate kinase (ppk). The resulting gene trees were compared to the 16S rRNA gene phylogeny of the same species. The topology of neighbour-joining and maximum parsimony phylogenetic trees based upon nucleic acid sequences and protein sequences of housekeeping genes differed among each other and no gene tree was identical to that of the 16S rRNA gene tree. Only some species showed consistent clustering by all genes analyzed, but the majority of species branched with different neighbours in most gene trees. The failure to phylogenetically cluster type strains into two groups based upon differences in the amino acid composition of peptidoglycan on the basis of 16S rRNA gene sequence similarity, once leading to the union of the genera Microbacterium and Aureobacterium, was also seen in the analysis of recA, rpoB and gyrB gene and protein phylogenies. Analysis of the pkk gene and protein as well as of a concatenate tree, combining sequences of all five genes (total of 3.700 nucleotides), sees members of the former genus Aureobacterium and other type strains with lysine as diagnostic diamino acid to form a coherent cluster that branches within the radiation of Microbacterium species with ornithine in the peptidoglycan.     

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.     

Iodus 40, salicylic acid, heptanoyl salicylic acid and trehalose exhibit different efficacies and defence targets during a wheat/powdery mildew interaction

Prophylactic efficacies of Iodus 40 and salicylic acid (SA) against wheat powdery mildew caused by Blumeria graminis f. sp. tritici have been shown and compared with those of heptanoyl salicylic acid (HSA) and trehalose. Plantlets treated once exhibited 55%, 50%, 95%, and 38% protection levels, respectively. Two sprayings increased these levels up to 60%, 65%, 100%, and 60%, respectively. Biological effects of these resistance inducers on reactive oxygen species (ROS) metabolism and lipid peroxidation were also investigated. We found clear differences in the extent and the type of induced responses, with HSA exhibiting both the most numerous and the highest effects. HSA and SA induced a 5.5-fold increase of whole cell DAB staining due to hydrogen peroxide accumulation, whereas Iodus 40 and trehalose increased staining intensity at the penetration sites only. However, these effects were not correlated with any modification of catalase (CAT), oxalate oxidase (OXO) or lipoxygenase (LOX) activities, except for HSA which decreased CAT in non-inoculated conditions and increased LOX in infectious conditions. HSA also induced an increase in the rate of lipid peroxidation, whereas Iodus 40 induced a decrease. The effects of the inducers on germinating conidia and wheat epidermal cells responding to fungal penetration were also investigated. Papilla-linked autofluorescence was affected by SA and Iodus 40 whereas germination was slightly altered by Iodus 40. The newly described protective efficacies and the partial, distinct and non-overlapping activities of these inducers on the wheat/powdery mildew interaction are discussed.     

Induction of tomato stress protein mRNAs by ethephon, 2,6-dichloroisonicotinic acid and salicylate

To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - PR pathogenesis-related - SA salicylic acid - SAR systemic acquired resistance     

Effect of salicylic acid on protein composition of Tatar buckwheat Fagopyrum tataricum calluses with different ability for morphogenesis

The effect of salicylic acid on the content of soluble proteins and individual polypeptides in Tatar buckwheat Fagopyrum tataricum calluses differing in ability for morphogenesis was studied. Changes in the protein composition of the calluses cultivated in the dark and in the light indicated the higher sensitivity of the non-morphogenic callus. Different response of callus cultures to salicylic acid and conditions of cultivation (light, darkness) is suggested to be associated with the antioxidant defense system, which is, in particular, characterized by the hydrogen peroxide content in the calluses. Salicylic acid increased the H₂O₂ content in non-morphogenic calluses more strongly than in morphogenic calluses, and the difference was more significant for the calluses cultivated in the light.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 390–396.Original Russian Text Copyright © 2005 by Maksyutova, Galeeva, Rumyantseva, Viktorova.