A novel ras-related rgp1 gene encoding a GTP-binding protein has reduced expression in 5-azacytidine-induced dwarf rice.

Summary Exposure of normal, tall rice (Oryza sativa) seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by treatment were identified by differential screening of a cDNA library, and a ras-related gene, rgp1, was subsequently isolated. The cDNA of rgp1 was found to encode a deduced protein sequence of 226 amino acids with a relative molecular mass of 24850, which was most closely related to the ras-related ypt3 protein of fission yeast, Shizosaccharomyces pombe. The rgp1 protein, expressed in transformed Escherichia coli, clearly showed GTP-binding activity. During seedling growth, rgp1 expression was first observed 14 days after germination, reaching a maximum level between 28 and 42 days, and gradually decreased thereafter until 63 days when it attained the same level of expression as in 14-day-old seedlings. Expression of rgp1 was found to be markedly reduced throughout the growth period of both 5-azacytidine-induced dwarf plants and their progenies, relative to levels in untreated tall control plants. These results suggest that expression of rgp1 may be influenced, either directly or indirectly, by DNA methylation, and that the rgp1 protein may play an important role in plant growth and development.     

Transgenic tobacco plants expressing rgp1, a gene encoding a ras-related GTP-binding protein from rice,show distinct morphological characteristics1

The rgp1 gene, originally Isolated from rice seedlings, encodes a small GTP-binding protein which is related to the product of the human proto-oncogene, ras-p21. To determine the physiological role of the rgp1 protein, rgp1-p25, the coding region of rgp1 was introduced into tobacco plants in both sense and antisense orientations. Transformants, which were found to contain the rgp1 gene at up to three loci, showed distinct phenotypic changes. The most notable was a reduction in apical dominance with increased tillering, together with dwarfism or abnormal flower development or both. These effects were similarly observed in both sense and antisense transformants. Northern hybridization analysis showed that rgp1 was expressed only in phenotypically abnormal transformants and not in the apparently normal phenotypes. Furthermore, the R₁ progenies from most transformants co-segregated into a 3:1 ratio for both kanamycin resistance and tillering. The expression of tgp1, a presumed tobacco homologue of rgp1, was markedly reduced in transformants expressing the antisense rgp1, whereas it was apparently unaffected in transformants with sense rgp1. These observations suggest that the phenotypic changes in antisense transformants may be mediated by an effect on native tgp1 mRNA, whereas in sense transformants the changes may be induced by over-production of rgp1-p25. The possibility that the increased tillering may be related to abnormal phytohormone metabolism or response pathways, and that rgp1-p25 may mediate the transmission of signals in these pathways is discussed.     

Characterization of transgenic rice plants that express rgp1, the gene for a small GTP-binding protein from rice

 The rgp1 gene, which encodes a small GTP-binding protein from rice, was introduced into rice protoplasts by electroporation. Transformed protoplasts were cultured on liquid protoplast-culture medium for 1 month, and then cells that had proliferated were transferred to a selection medium that contained 50 mg/l hygromycin B. Among 50 colonies that were selected and transferred to regeneration medium, 3 colonies generated shoots. However, two of the three shoots failed to form roots and ceased growing. A single regenerated shoot that formed roots was planted in soil and transferred to a greenhouse. Southern hybridization showed that the regenerated plant harbored a single copy of the introduced gene. The transformant (T₀) plant was shorter than the controls, it developed three times as many tillers as controls, it developed three times as many tillers as control plants but it produced mostly sterile seeds. In a test of hygromycin resistances, viable seeds segregated into resistant and sensitive seedings at a ratio of approximately 1 : 3. The progeny (T₁) plants were short with many tillers, and some produced seeds normally. The T₂ seedlings grew more rapidly than control seedlings for the first 28 days after germination, but control plants subsequently outgrew the T₂ plants. Northern blotting analysis revealed that the rgp1 gene in T₂ plants was expressed consitutively throughout all developmental stages. The results suggest that the observed phenotypic changes were due to expression of the exogenous rgp1 gene. Received: 21 September 1997/Accepted: 31 March 1998     

The ras-related YPT1 gene product in yeast: A GTP-binding protein that might be involved in microtubule organization

The 23.5 kd protein product of the ras-related YPT1 gene of S. cerevisiae was found to be essential for cell growth. The loss of YPT1 function, studied in cells with the YPT1 gene on chromosome VI regulated by the galactose-inducible GAL10 promoter, led to arrested cells that were multibudded and exhibited a complete disorganization of microtubules and an apparent loss of nuclear integrity. The YPT protein binds GTP specifically. GTP binding of the protein is essential for its intracellular function. The Asn¹²¹→lle substitution, generated by site-directed mutagenesis, had a dominant lethal phenotype, the expression of the mutant protein led to binucleated cells and abnormal spindles. In contrast to the S. cerevisiae RAS1 and RAS2 gene products, the YPT protein seems to be involved, directly or indirectly, in microtubule organization and function.     

小麦中小GTP结合蛋白基因TaRop2克隆及其表达分析

小GTP结合蛋白属于Rho家族,是植物特有的一类蛋白,在调控植物生长发育、抗逆和抗病过程中发挥着重要的作用。本研究通过筛选非亲和条锈菌小种CYR32侵染诱导的抗条锈病基因Yr5近等基因系(Taichung29*6/Yr5)c DNA文库,分离获得1个Rop家族基因的全长c DNA序列,命名为TaRop2(Triticum aestivum Rop2)。TaRop2包含1个591 bp的开放阅读框,预测编码含197个氨基酸残基的蛋白质,分子量为21.52 k D,理论等电点为9.49。通过在烟草表皮细胞瞬时表达,发现TaRop2分布于细胞核内和细胞膜上。RT-PCR分析结果表明,在高盐处理、非亲和条锈菌小种CYR32和亲和混合白粉菌菌株侵染时,TaRop2基因的表达水平升高,但被干旱、高温、低温和ABA处理抑制。说明TaRop2可能参与小麦防卫和抗逆反应过程。     

Isolation of a cDNA encoding a novel GTP-binding protein of Arabidopsis thaliana

A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68). This clone is a member of a gene family that codes for a class of large GTP-binding proteins. This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins. The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein. The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels. Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron.     

Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice

Summary We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family.     

Cell-cycle-dependent expression of the STK-1 gene encoding a novel murine putative protein kinase

We have cloned a novel putative serine/threonine kinase-encoding gene, designated STK-1, from murine embryonic stem (ES) cell and testis cDNA libraries. The kinase most closely related to STK-1 is Xenopus laevis XLP46 protein kinase which shows 71% amino-acid identity to STK-1 between their kinase domains. Nevertheless, STK-1 is conserved throughout phylogeny with hybridizing sequences being detected in DNA from mammals, amphibians, insects and yeast. STK-1 mRNA is detected in testis, intestine and spleen, tissues that contain a large number of proliferating cells, but not in other tissues. All cell lines tested expressed STK-1 mRNA with levels being dependent upon proliferation rates. In NIH 3T3 cells, STK-1 is expressed in a cell-cycle-dependent fashion. These findings suggest a role for STK-1 in cell growth     

A novel plant nuclear gene encoding chloroplast ribosomal protein S9 has a transit peptide related to that of rice chloroplast ribosomal protein L12

We have cloned a novel nuclear gene for a ribosomal protein of rice and Arabidopsis that is like the bacterial ribosomal protein S9. To determine the subcellular localization of the gene product, we fused the N-terminal region and green fluorescent protein and expressed it transiently in rice seedlings. Localized fluorescence was detectable only in chloroplasts, indicating that this nuclear gene encodes chloroplast ribosomal protein S9. The N-terminal region of rice ribosomal protein S9 was found to have a high sequence similarity to the transit peptide region of the rice chloroplast ribosomal protein L12, suggesting that these transit peptides have a common lineage.     

Analysis of a Nicotiana plumbaginifolia cDNA encoding a novel small GTP-binding protein.

Small GTP-binding proteins belonging to the Ras superfamily have been found in evolutionarily divergent organisms. Here, we report the isolation and analysis of a cDNA encoding a putative small GTP-binding protein, designated Rhn1, from the plant, Nicotiana plumbaginifolia. The 21.8-kDa protein has 60% amino acid similarity with the mammalian Rab5 proteins. The Rhn1 protein is encoded by a small multigene family. Northern analysis shows the highest steady-state mRNA levels to be in roots and flowers. Furthermore, the Rhn1 protein has 80% amino acid similarity with an Arabidopsis small GTP-binding protein, designated Rha1.     

Disruption of a novel gene for a NAC-domain protein in rice confers resistance to Rice dwarf virus

Rice dwarf virus (RDV) is a serious viral pest that is transmitted to rice plants ( Oryza sativa L.) by leafhoppers and causes a dwarfism in infected plants. To identify host factors involved in the multiplication of RDV, we screened Tos17 insertion mutant lines of rice for mutants with reduced susceptibility to RDV. One mutant, designated rim1-1 , did not show typical disease symptoms upon infection with RDV. The accumulation of RDV capsid proteins was also drastically reduced in inoculated rim1-1 mutant plants. Co-segregation and complementation analyses revealed that the rim1-1 mutation had been caused by insertion of Tos17 in an intron of a novel NAC gene. The rim1-1 mutant remained susceptible to the two other viruses tested, one of which is also transmitted by leafhoppers, suggesting that the multiplication rather than transmission of RDV is specifically impaired in this mutant. We propose that RIM1 functions as a host factor that is required for multiplication of RDV in rice.     

Global gene expression analysis of a rice high-tillering dwarf mutant

Plant height and tiller number are indispensible for the establishment of grain production in rice (Oryza sativa L.). A new rice mutant high-tillering dwarf 3 (htd3) exhibiting more tiller number and shorter culm length than the wild-type Guichao 2 (GC2, an indica cultivar) was used to investigate the global gene expression patterns at days after germination 25 (DAG25) and DAG60. In this study, we identified 305 and 987 genes with at least twofold change in gene expression level at DAG25 and DAG60 respectively using the rice microarray chip. Gene ontology enrichment analysis of these twofold change regulated genes revealed that large numbers of genes were involved in binding activity, catalytic activity and metabolic process. The chip results also showed that some of the regulated genes involved in diverse molecular pathways, including gibberellin pathway, brassinosteroid pathway and auxin signal, had significant differences in gene expression abundance at DAG60. This genome-wide gene expression analysis could provide a new opportunity to uncover the regulation mechanisms of the development of culm and tiller, two important components of yields in rice.     

Sequence of a plant cDNA from Vicia faba encoding a novel Ran-related GTP-binding protein

A clone obtained from a broad bean (Vicia faba) developing cotyledon cDNA library contained the complete coding sequence of a polypeptide with very high homology to the small GTP-binding proteins Ran from human cells and Spi1 from yeast. These proteins belong to the ras superfamily of proteins involved in different basic cellular processes. The Ran/Spi1 proteins interact with a protein bound to DNA (RCC1) and are thought to function in the regulation of the cell cycle. The amino acid sequence of the obtained plant Ran-homologue, designated Vfa-ran, is 74% and 76% identical to Ran and Spi1, respectively. The five functional, conserved domains of ras-related proteins are present in the Vfa-ran sequence. However, as in Ran/Spi1 the C-terminus of Vfa-ran is very acidic and lacks the Cys motif for isoprenylation.Northern blotting revealed a corresponding mRNA expression in broad bean roots, leaves, and cotyledons with the highest level in roots.     

NABP1, a novel RORgamma-regulated gene encoding a single-stranded nucleic-acid-binding protein

RORgamma2 (retinoid-related orphan receptor gamma2) plays a critical role in the regulation of thymopoiesis. Microarray analysis was performed in order to uncover differences in gene expression between thymocytes of wild-type and RORgamma-/- mice. This analysis identified a novel gene encoding a 22 kDa protein, referred to as NABP1 (nucleic-acid-binding protein 1). This subsequently led to the identification of an additional protein, closely related to NABP1, designated NABP2. Both proteins contain an OB (oligonucleotide/oligosaccharide binding) motif at their N-terminus. This motif is highly conserved between the two proteins. NABP1 is highly expressed in the thymus of wild-type mice and is greatly suppressed in RORgamma-/- mice. During thymopoiesis, NABP1 mRNA expression is restricted to CD4+CD8+ thymocytes, an expression pattern similar to that observed for RORgamma2. These observations appear to suggest that NABP1 expression is regulated either directly or indirectly by RORgamma2. Confocal microscopic analysis showed that the NABP1 protein localizes to the nucleus. Analysis of nuclear proteins by size-exclusion chromatography indicated that NABP1 is part of a high molecular-mass protein complex. Since the OB-fold is frequently involved in the recognition of nucleic acids, the interaction of NABP1 with various nucleic acids was examined. Our results demonstrate that NABP1 binds single-stranded nucleic acids, but not double-stranded DNA, suggesting that it functions as a single-stranded nucleic acid binding protein.