Isolation and expression analysis of two rice genes encoding the major intrinsic protein

We isolated two rice cDNAs (rMip1 and rTip1) which are homologous to the genes encoding the major intrinsic protein (Mip) (soybean nod-26 and Arabidopsis -Tip), respectively. Expression of rTip1 in shoots and roots of rice seedlings was enhanced by water stress, salt stress and exogenous ABA. rMip1 was expressed only in shoots. Although mRNA level of rMip1 in shoots was induced to a small extent by exogenous ABA, it did not show any increase under water or salt stress over the course of 12 h. On the basis of the differential expression patterns and evolutional distinctions, it is suggested that the possible channel proteins encoded by rMip1 and rTip1 genes may function in different transport systems.     

Genome-wide analysis of genes encoding FK506-binding proteins in rice

The FK506-binding proteins (FKBPs) are a class of peptidyl-prolyl cis/trans isomerase enzymes, some of which can also operate as molecular chaperones. FKBPs comprise a large ubiquitous family, found in virtually every part of the cell and involved in diverse processes from protein folding to stress response. Higher plant genomes typically encode about 20 FKBPs, half of these found in the chloroplast thylakoid lumen. Several FKBPs in plants are regulators of hormone signalling pathways, with important roles in seed germination, plant growth and stress response. Some FKBP isoforms exists as homologous duplicates operating in finely tuned mechanisms to cope with abiotic stress. In order to understand the roles of the plant FKBPs, especially in view of the warming environment, we have identified and analysed the gene families encoding these proteins in rice using computational approaches. The work has led to identification of all FKBPs from the rice genome, including novel high molecular weight forms. The rice FKBP family appears to have evolved by duplications of FKBP genes, which may be a strategy for increased stress tolerance.     

Transcription dynamics of Saltol QTL localized genes encoding transcription factors,reveals their differential regulation in contrasting genotypes of rice

Functional & Integrative Genomics - Salinity is one of the major environmental factors affecting the growth and yield of rice crop. Salinity stress response is a multigenic trait and numerous...     

Phylogenetic analysis of the rplA genes encoding ribosomal protein L1

Previously we have identified therplA gene encoding ribosomal protein L1 inStreptomyces aureofaciens. Sequence comparison of ribosomal protein L1 among several bacterial genera revealed a high level of conservation. Based on this conservation, these proteins were used as a phylogenetic tool to compare evolutionary relationships among eubacteria and archaebacteria. This phylogenetic analysis of L1 ribosomal proteins including theS. aureofaciens rplA gene product revealed, except similar bacterial groupings, some new evolutionary relationships.     

Comprehensive analysis of the helix-X-helix motif in soluble proteins

Alpha-helices are the most common secondary structures found in globular proteins. In this report, we analyze the stereochemical and sequence properties of helix-X-helix (HXH) motifs in which two alpha-helices are linked by a single residue, in search of characteristic structures and sequence signals. The analysis is carried out on a database of 837 nonredundant HXH motifs. The kinks are characterized by the bend angle between the axes of the N-terminal and C-terminal helices and the wobble angle corresponding to the rotation of C-terminal helix axis on the plane perpendicular to the N-terminal one. The phi-psi dihedral angles of the linker residue are clustered in six distinct areas of the Ramachandran plot: two areas are located in the additional allowed alpha region (alpha(1) and alpha(2)), two areas are in the additional allowed beta region (beta(1) and beta(2)) and two areas have positive phi values (alpha(L) and beta(M)). Each phi/psi region corresponds to characteristic bend and wobble angles and amino acid distributions. Bend angles can vary from 0 degrees to 160 degrees. Most wobble angles correspond to a counter-clockwise rotation of the C-terminal helix. Proline residues are rigorously excluded from the linker position X but have a high propensity at position X+1 of the beta(1) and beta(2) motifs (12 and 7, respectively) and at position X+3 of the alpha(1) motifs (9). Glycine linkers are located either in the alpha(L) region (20%) or in the beta(M) region (80%). This latter conformation is characterized by a marked bend angle (124 degrees +/- 18 degrees) and a clockwise wobble. Among other amino acids, Asn is remarkable for its high propensity (>3) at the linker position of the alpha(2), beta(1), and beta(2) motifs. Stabilization of HXH motifs by H-bonds between polar side chains of the linker and polar groups of the backbone is determined. A method based on position-specific scoring matrices is developed for conformational prediction. The accuracy of the predictions reaches 80% when the method is applied to proline-induced kinks or to kinks with bend angles in the 50 degrees-100 degrees range.     

Hypoxia-inducible genes encoding small EF-hand proteins in rice and tomato

Rice has evolved metabolic and morphological adaptations to low-oxygen stress to grow in submerged paddy fields. To characterize the molecular components that mediate the response to hypoxia in rice, we identified low-oxygen stress early response genes by microarray analysis. Among the highly responsive genes, five genes, OsHREF1 to OsHREF5, shared strong homology. They encoded small proteins harboring two EF-hands, typical Ca(2+)-binding motifs. Homologous genes were found in many land plants, including SlHREF in tomato, which is also strongly induced by hypoxia. SlHREF induction was detected in both roots and shoots of tomato plants under hypoxia. With the exception of OsHREF5, OsHREF expression was unaffected by drought, salinity, cold, or osmotic stress. Fluorescent signals of green fluorescent protein-fused OsHREFs were detected in the cytosol and nucleus. Ruthenium red, an inhibitor of intracellular Ca(2+) release, repressed induction of OsHREF1-4 under hypoxia. The HREFs may be related to the Ca(2+) response to hypoxia.     

Molecular cloning of a family of plant genes encoding a protein homologous to plasma membrane H+-translocating ATPases

cDNA clones from Nicotiana plumbaginifolia have been isolated by hybridization to a yeast H+-ATPase gene. The largest one encodes a polypeptide (PMA2) of 956 amino acid residues which exhibits a homology of 73% with a limited protein sequence obtained from purified oat plasma membrane H+-ATPase (Schaller and Sussman, Plant Physiol. 86, 512-516, 1988) and an 82% homology with the Arabidopsis thaliana pma gene (Harper et al., Proc. Natl. Acad. Sci. USA 86, 1234-1238). It is therefore concluded that the N. plumbaginifolia pma2 gene encodes a plasma membrane H+-ATPase. Southern blot hybridization indicates that the plant pma2 gene belongs to a multigene family. Partial sequences of cDNA clones show that at least three pma genes are expressed in root cells.     

Cloning and characterization of three genes encoding Qb-SNARE proteins in rice

Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H₂O₂, but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H₂O₂, but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H₂O₂ and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of nuclear genes encoding mitochondrial proteins in Saccharomyces cerevisiae.

Selection for mutants which release glucose repression of the CYB2 gene was used to identify genes which regulate repression of mitochondrial biogenesis. We have identified two of these as the previously described GRR1/CAT80 and ROX3 genes. Mutations in these genes not only release glucose repression of CYB2 but also generally release respiration of the mutants from glucose repression. In addition, both mutants are partially defective in CYB2 expression when grown on nonfermentable carbon sources, indicating a positive regulatory role as well. ROX3 was cloned by complementation of a glucose-inducible flocculating phenotype of an amber mutant and has been mapped as a new leftmost marker on chromosome 2. The ROX3 mutant has only a modest defect in glucose repression of GAL1 but is substantially compromised in galactose induction of GAL1 expression. This mutant also has increased SUC2 expression on nonrepressing carbon sources. We have also characterized the regulation of CYB2 in strains carrying null mutation in two other glucose repression genes, HXK2 and SSN6, and show that HXK2 is a negative regulator of CYB2, whereas SSN6 appears to be a positive effector of CYB2 expression.     

Comprehensive cloning of Schizosaccharomyces pombe genes encoding translation elongation factors

In the course of the Schizosaccharomyces pombe cDNA project, we succeeded in cloning all the genes encoding translation elongation factors EF-1α, EF-1β, EF-1γ, EF-2 and EF-3. With the exception of the EF-1γ gene, the nucleotide (nt) sequence of S. pombe elongation factors has not been previously reported. For EF-1α, we found three genes whose amino acid (aa) sequences are quite homologous each other (99.5%), but whose 3′ untranslated regions (UTRs) are completely different. Southern blot indicated that those three EF-1α genes are located at different loci. Northern analysis indicated that one of three EF-1α genes was inducible with UV-irradiation, while the level of expression for another of three EF-1α genes was repressed by UV and heat-shock (HS) treatments. The aa sequence predicted from the nt sequence of the S. pombe EF-1β cDNA clone covered almost all the coding sequence (CDS) of EF-1β except the first methionine which has 55.4% identity with that of S. cerevisiae. We also identified two copies of S. pombe EF-2 genes. Their aa sequences deduced from nt sequences are identical (100%), but they have different 3′ UTRs. The location of these two EF-2 genes in different loci was proved by Southern analysis. The S. pombe EF-3 cDNA clone encoded only a third of the CDS from the C-terminal and its deduced aa sequence has a 76% identity with those of other yeasts and fungi.     

Compartmentalized linkage of genes encoding interacting protein pairs

Emulsion technology has been successfully applied to the fields of next-generation high-throughput sequencing, protein engineering and clinical diagnostics. Here, we extend its scope to proteomics research by developing and characterizing a method, termed iCLIP (in vitro compartmentalized linkage of interacting partners), which enables genes encoding interacting protein pairs to be linked in a single segment of DNA. This will facilitate archiving of the interactomes from library versus library two-hybrid screens as libraries of linked DNAs. We further demonstrate the ability to interrogate a model yeast two-hybrid iCLIP library for interactants by "PCR-pulldown," using a primer specific to a gene of interest along with a universal primer. iCLIP libraries may also be subjected to high-throughput sequencing to generate interactome information. The applicability of the technique is also demonstrated in the related context of the bacterial two-hybrid system.