Overexpression of OsRab7B3, a small GTP-binding protein gene, enhances leaf senescence in transgenic rice

Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the OsRab genes, OsRab7B3, closely associated with senescence in expression pattern, was chosen for functional analysis. Expression of sGFP under the control of the OsRab7B3 promoter increased in leaves when ABA and NaCl were applied or when kept in dark. In transgenic rice overexpressing OsRab7B3, the senescence-related genes were upregulated and leaf senescence was significantly enhanced under dark conditions. Moreover, leaf yellowing occurred earlier in the transgenic plants than in the wild type at the ripening stage. Hence it is suggested that OsRab7B3 act as a stress-inducible gene that plays an important role in the leaf senescence process.     

Genetic mapping of the mouse Rab7 gene and pseudogene and of the human RAB7 homolog

Rab proteins are small GTP-ases localized to distinct membrane compartments in eukaryotic cells and regulating specific steps of intracellular vesicular membrane traffic. The Rab7 protein is localized to the late endosomal compartment and controls late steps of endocytosis. We have isolated, by library screening, the 5′ region, including the promoter, of the mouse Rab7 gene and a Rab7 pseudogene. We have mapped, by genetic linkage analysis, the mouse Rab7 gene on Chromosome (Chr) 6 and the Rab7-ps1 pseudogene on Chr 9, where the Rab7 gene has been previously reported to map. By radiation hybrid mapping, we have located the human RAB7 gene on Chr 3, in a region homologous to the mouse Chr 6, where the Rab7 gene maps. Received: 27 October 1997 / Accepted: 1 January 1998     

Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.     

Molecular cloning and characterization of a cDNA for a rice Sec31p homolog

A cDNA for a putative Sec31p in rice has been cloned and sequenced. In yeast, Sec31p is a component of a protein-coated vesicle, COPII, which functions in the transport of cargo proteins from the endoplasmic reticulum to the cis-Golgi network. Structural similarities between yeast Sec31p and the rice putative homolog are discussed.     

Molecular, biochemical and immunological analyses of porcine pancreatic DNase I.

Deoxyribonuclease I (DNase I) was purified 26500-fold in 39% yield from porcine pancreas to electrophoretic homogeneity using three-step column chromatography. The purified enzyme was inhibited by an antibody specific to the purified enzyme but not by G-actin. A 1303 bp cDNA encoding porcine DNase I was constructed from total RNA from porcine small intestine using a rapid amplification of cDNA ends method, followed by sequencing. Mature porcine DNase I protein was found to consist of 262 amino acids. Unlike all other mammalian DNase I enzymes that are inhibited by G-actin, porcine DNase I has H65 and S114 instead of Y65 and A114, which presumably results in the lack of inhibition. Porcine DNase I was more sensitive to low pH than rat or bovine enzymes. Compared with their primary structures, the amino acid at position 110 was N in porcine enzyme, but S in rat and bovine enzymes. A porcine mutant enzyme in which N was substituted by S alone at position 110 (N110S) became resistant to low pH to a similar extent as the rat and bovine enzymes.     

Molecular characterization and expression analysis of a highly conserved rice mago nashil homolog.

Mago Nashi, a protein initially shown to be essential in the development of the Drosophila oocyte, is highly conserved among species and shows no homology to any other known cellular proteins. Here we report the nucleotide sequence of a cDNA and a partial gene that encode rice Mago Nashi protein homologs. In addition, we present the tissue-specific expression pattern of mago nashi at the level of RNA and protein. The rice Mago Nashi protein shares at least 73% amino acid identity with all known animal homologs. Genomic DNA gel blot analysis indicates that two copies of the mago nashi gene exist in the rice genome, one of which has identical intron positions to those found in an Arabidopsis homolog. mago nashi is expressed in root, leaf and developing seed tissue as determined by RNA and protein gel blot analysis. Evidence from Drosophila, Caenorhabditis elegans and human studies of Mago Nashi suggests that a major function of this protein is its involvement in RNA localization. The highly conserved amino acid sequence of all Mago Nashi protein homologs across kingdoms suggests that the plant version of this protein may similarly be involved in RNA localization.     

The Rab-interacting lysosomal protein, a Rab7 and Rab34 effector, is capable of self-interaction

Rab-interacting lysosomal protein (RILP) has been identified as an interacting partner of the small GTPases Rab7 and Rab34. Active Rab7 recruits RILP on the late endosomal/lysosomal membrane and RILP then functions as a Rab7 effector controlling transport to degradative compartments. Indeed, RILP induces recruitment of dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Recently, Rab7 and RILP have been found to be key proteins also for the biogenesis of phagolysosomes. Therefore, RILP represents probably an important factor for all endocytic routes to lysosomes. In this study, we show, using the yeast two-hybrid system, that RILP is able to interact with itself. The data obtained with the two-hybrid system were confirmed using co-immunoprecipitation in HeLa cells. The data together indicate that RILP, as already demonstrated for several other Rab effector proteins, is capable of self-association, thus probably forming a homo-dimer.     

OsRab5a regulates endomembrane organization and storage protein trafficking in rice endosperm cells

Rice glutelins are synthesized at the endoplasmic reticulum (ER) as precursors (pro-glutelins), and are transported to protein storage vacuoles, where they are processed into mature proteins. The molecular basis of this process is largely unknown. Here, we report the isolation of a rice mutant, gpa1, that accumulates 57 kDa pro-glutelins in seeds and whose endosperm has a floury appearance. Transmission electron microscopy analysis showed that the gpa1 endosperm cells have an enlarged ER lumen and a smaller protein body II (PBII), and accumulated three types of newly generated subcellular structures. Moreover, a proportion of glutelins in the gpa1 endosperm cells were not delivered to PBII, and instead were mis-targeted to two of the newly generated structures or secreted. The gene corresponding to the gpa1 mutation was found to be OsRab5a, which encodes a small GTPase. In Arabidopsis protoplasts, OsRab5a protein was found to co-localize predominantly with AtVSR2, a molecular marker for the pre-vacuolar compartments (PVC). We conclude that OsRab5a plays an essential role in trafficking of storage protein to PBII, possibly as part of its function in organizing the endomembrane system in developing endosperm cells of rice.     

Molecular and biochemical characterization of a proteasome subunit from rice and carrot cells

Proteasomes function mainly in the ATP-dependent degradation of proteins that have been conjugated with ubiquitin. We isolated a cDNA clone for a rice protein that exhibited high homology to subunit C2 of proteasomes. Southern blot analysis revealed that the corresponding gene was present as a single copy in the rice genome. After fractionation of a crude extract from cultured cells, a 35-kDa protein that cross-reacted with antibodies against the C2 subunit was recovered in the peak fraction of both 20 S and 26 S complexes. The same antibodies cross-reacted with two proteins in seedlings, one of which was the same as that detected in cultured cells. The level of the protein was reduced in roots under conditions of high salinity. The 35-kDa protein was not detected in the nuclei of rice or carrot cells. However, during somatic embryogenesis of carrot cells, the C2 subunit was found in the nucleus at the globular stage, and it gradually disappeared in the period from the heart to the torpedo stage. Cells at the globular stage are proliferating rapidly, thus, it is possible that proteasomes are associated with the proliferation of plant cells. Received: 11 November 1996 / Accepted: 28 January 1997     

Molecular Cloning and Characterization of Rab27a and Rab27b,Novel Human Rab Proteins Shared by Melanocytes and Platelets

Rabs are prenylated, membrane-bound proteins involved in vesicular fusion and trafficking. We isolated the complete cDNAs of two rab isoforms, rab27a and rab27b, from human melanoma cells and melanocytes. Rab27a is the human homolog of a rat megakaryocyte rab called ram p25. Rab27b corresponds to a small GTP-binding protein, c25KG, which was previously purified from platelets but whose cDNA had not been cloned. Sequence comparisons with known rabs indicate that rab27a and rab27b comprise a melanocyte/platelet subfamily within the rab family. In addition, rab27a was expressed in a large variety of cell and tissue types, excluding brain, and rab27b manifested itself primarily in testis. Bacterially expressed and purified rab27a and rab27b exhibited GTP-binding activity and can now be used for antibody production and studies of the substrate specificities of geranylgeranyl transferase. In addition, the expression of rab27a and rab27b in both melanocytes and platelets makes them candidates for involvement in mouse and human disorders characterized by the combination of pigment dilution and a platelet storage pool defect.     

Molecular cloning, bacterial expression and properties of Rab31 and Rab32.

GTP-binding proteins of the Rab family were cloned from human platelets using RT-PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in platelets previously, were isolated. The coding sequence of Rab31 (GenBank accession no. U59877) corresponded to a 194 amino-acid protein of 21.6 kDa. The Rab32 sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but the 5' coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino-acid protein of 25.0 kDa with the unusual GTP-binding sequence DIAGQE in place of DTAGQE. Northern blots for Rab31 and Rab32 identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti-peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 microg x mg of platelet protein(-1)) and contained substantial amounts of Rab32 (0.11 microg x mg protein(-1)). Little Rab31 was present (0.005 microg x mg protein(-1)). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28-kDa Rab32 was replaced by a 52-kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione S-transferase (GST)-fusion proteins, did not bind [alpha-(32)P]GTP on nitrocellulose blots but did bind [(35)S]GTP[S] in a Mg(2+)-dependent manner. Binding of [(35)S]GTP[S] was optimal with 5 microm Mg(2+)(free) and was markedly inhibited by higher Mg(2+) concentrations in the case of GST-Rab31 but not GST-Rab32. Both proteins displayed low steady-state GTPase activities, which were not inhibited by mutations (Rab31(Q64L) and Rab32(Q85L)) that abolish the GTPase activities of most low-M(r) GTP-binding proteins.     

Rabring7, a novel Rab7 target protein with a RING finger motif

Rab7, a member of the Rab family small G proteins, has been shown to regulate intracellular vesicle traffic to late endosome/lysosome and lysosome biogenesis, but the exact roles of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins that function in the exocytic/endocytic pathway. We have isolated a new Rab7 target protein, Rabring7 (Rab7-interacting RING finger protein), using a CytoTrap system. It contains an H2 type RING finger motif at the C termini. Rabring7 shows no homology with RILP, which has been reported as another Rab7 target protein. GST pull-down and coimmunoprecipitation assays demonstrate that Rabring7 specifically binds the GTP-bound form of Rab7 at the N-terminal portion. Rabring7 is found mainly in the cytosol and is recruited efficiently to late endosomes/lysosomes by the GTP-bound form of Rab7 in BHK cells. Overexpression of Rabring7 not only affects epidermal growth factor degradation but also causes the perinuclear aggregation of lysosomes, in which the accumulation of the acidotropic probe LysoTracker is remarkably enhanced. These results suggest that Rabring7 plays crucial roles as a Rab7 target protein in vesicle traffic to late endosome/lysosome and lysosome biogenesis.     

Phylogeny and evolution of Rab7 and Rab9 proteins

Background  

An important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network.     

Antibody identification, chromosome map assignment, and sequence analysis of a Rab escort protein homolog in Drosophila1

Using a polyclonal antiserum a cDNA encoding a Rab escort protein (REP) homolog in Drosophila has been identified and sequenced. The gene encodes a 511 residue protein with a predicted molecular mass of 56855 Da. Antibody labeling demonstrates that Drosophila REP protein is present in the early embryo and that it is being apportioned uniformly throughout the embryo in a process likely to be linked to the syncytial nuclear divisions. In situ hybridization to polytene chromosomes reveals that the Drosophila REP gene is located in the 56E region on the second chromosome. Drosophila REP is the first invertebrate REP homolog to be identified and characterized.     

Overexpression of OsRab7B3, a Small GTP-Binding Protein Gene,Enhances Leaf Senescence in Transgenic Rice

Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the OsRab genes, OsRab7B3, closely associated with senescence in expression pattern, was chosen for functional analysis. Expression of sGFP under the control of the OsRab7B3 promoter increased in leaves when ABA and NaCl were applied or when kept in dark. In transgenic rice overexpressing OsRab7B3, the senescence-related genes were upregulated and leaf senescence was significantly enhanced under dark conditions. Moreover, leaf yellowing occurred earlier in the transgenic plants than in the wild type at the ripening stage. Hence it is suggested that OsRab7B3 act as a stress–inducible gene that plays an important role in the leaf senescence process.     

Molecular,biochemical and bioassay based evidence of lower allelopathic potential in genetically modified rice

This study examines allelopathic potential of genetically modified rice. The experiment was conducted on two isogenic lines Bacillus thuringiensis (Bt) and non-Bacillus thuringiensis (non-Bt). Both isogenic lines have same allelopathic ability before insect feeding and after limited insect feeding (Spodoptera litura) non-Bt rice genotype demonstrates more allelopathic potential. The S. litura cannot feed Bt rice genotype. The role of shoot herbivory in allelopathic induction is further supported when Bt plants also exhibited higher allelopathic potential after insect regurgitant application to the damaged leaves. Allelopathic potential was assessed through several methods after treatments of mechanical damage, insect feeding and insect regurgitant application to damaged rice leaves. Rhizosphere soil and leaf leachates of non-Bt rice cultivar exhibited higher allelopathic potential on lettuce and barnyard grass after herbivore feeding. Enzyme activities (PAL and C4H) responsible for biosynthesis of phenolic compounds and their concentration were significantly higher in non-Bt plant after herbivore feeding and attain the same level in Bt plants after insect regurgitant application to damaged leaves. Similarly, genes (OsPAL and OsCYC1) responsible for biosynthesis of allelopathic compounds showed high expression in non-Bt plants after herbivore feeding. Our results indicate that herbivore feeding enhance rice allelopathic potential and no insect feeding as incase of Bt plants may reduce allelopathic potential of genetically modified rice.     

Molecular and biochemical analyses of spontaneous and X-ray-induced mutants in human lymphoblastoid cells

We have isolated a series of 14 spontaneously arising and 28 X-ray-induced mutants at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in human lymphoblastoid cells. Among the spontaneous mutants, 5/14 (36%) had detectable alterations in their restriction fragment pattern after hybridization with a human cDNA probe for hgprt. Of the 10 remaining mutants, 4 had partial HGPRT enzyme activity, which suggested that they contained point mutations. Among the 28 mutants induced by 150 rad of X-rays, 15 (54%) had deletions of part or all of the hgprt gene. 5 of the remaining 13 (18% overall) had partial HGPRT enzyme activity, which suggested that they contained point mutations. These data imply that in this human cell system, X-rays induce both point mutants which have residual enzyme activity as well as mutations involving relatively large deletions of DNA.