Identification and isolation of differentially expressed genes in osmotically stressed human oral keratinocytes

Complementary DNA fragments which showed differential expression relative to unstressed controls were identified and isolated from human oral keratinocytes exposed to hyperosmotic stress. The up- or downregulation of the expression of nine of these cDNAs in response to osmotic stress was determined by Northern blotting. Sequence analysis showed that clones K-5 and K-46 contained identical sequences. Homology searches revealed that K-13 and K-33 were fragments of unknown genes. Among the upregulated cDNAs, K-16 and K-32 were 94 and 83% identical to chromosome 16 bacterial artificial chromosome (CIT987K-A-418G10) and a cDNA (ai49b01.s1) clone, respectively. Another clone, K-34, encoded a protein 73% identical to Bare. Among the downregulated genes, K-5/46 and K-45 were 99% identical to the og24d08.s1 cDNA clone and to mitochondrial genes for tRNAs and 12S and 16S ribosomal RNAs, respectively, while K-50 was 100% identical to KIAA0905 protein. The gene expression induced by osmotic stress occurred in parallel with the induction of apoptosis and a reduction in protein biosynthesis. This observation, together with the characteristics of the some of the differentially expressed genes, suggests that among the major events induced in oral keratinocytes by hyperosmotic stress are the induction of apoptosis and a decrease in protein biosynthesis, brought about by upregulation of pro-apoptotic genes and downregulation of genes involved in protein biosynthesis. (C) 2001 Elsevier Science Ltd. All rights reserved.X114sciescopu

Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids

Dynamin-related proteins are high molecular weight GTPase proteins found in a variety of eukaryotic cells from yeast to human. They are involved in diverse biological processes that include endocytosis in animal cells and vacuolar protein sorting in yeast. We isolated a new gene, ADL2, that encodes a dynamin-like protein in Arabidopsis. The ADL2 cDNA is 2.68 kb in size and has an open reading frame for 809 amino acid residues with a calculated molecular mass of 90 kDa. Sequence analysis of ADL2 revealed a high degree of amino acid sequence similarity to other members of the dynamin superfamily. Among those members ADL2 was most closely related to Dnm1p of yeast and thus appears to be a member of the Vps1p subfamily. Expression studies showed that the ADL2 gene is widely expressed in various tissues with highest expression in flower tissues. In vivo targeting experiments showed that ADL2:smGFP fusion protein is localized to chloroplasts in soybean photoautroph cells. In addition experiments with deletion constructs revealed that the N-terminal 35 amino acid residues were sufficient to direct the smGFP into chloroplasts in tobacco protoplasts when expressed as a fusion protein.X1148sciescopu

Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress

Plant responses to high salt stress have been studied for several decades. However, the molecular mechanisms underlying these responses still elude us. In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis. Here, we report the cloning of a cDNA encoding a novel Ca2+-binding protein, named AtCP1, which shares sequence similarities with calmodulins. AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca2+-binding loops of the EF hands of calmodulin. However, unlike calmodulin, AtCP1 appears to have only three Ca2+-binding loops. We examined Ca2+ binding of the protein by a Ca2+-dependent electrophoretic mobility shift assay. A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca2+-dependent electrophoretic mobility shift. To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out. The AtCP1 gene had a tissue-specific expression pattern: high levels of expression In flower and root tissues and nearly undetectable levels in leaves and siliques. Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment. Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein.X1136sciescopu

A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes

Dynamin, a GTP-binding protein found in rat brain, plays a role in endocytosis. Suborganellar fractionation studies of Arabidopsis leaf tissue revealed that a dynamin-like protein, ADL1, is localized in the thylakoid membranes of chloroplasts. This notion was supported further by lit vivo targeting experiments using an ADL1-green fluorescent fusion protein and immunogold labeling with the anti-ADL1 antibody. Transgenic plants harboring various deletion mutant genes of ADL1 had a yellow leaf phenotype where the cells had very few chloroplasts, In addition, the remaining chloroplasts appeared morphologically not fully developed, The detailed structure of the chloroplasts revealed by electron microscopy showed a greatly reduced amount of thylakoid membranes. Also, the level of thylakoid membrane proteins such as the light-harvesting complex II and CP29 was greatly reduced in these transgenic plants, When we examined the expression of the ADL1 deletion mutant genes, these genes were highly expressed at the transcriptional level, However, the mutant ADL1s were not detectable at the protein level by Western blot analysis, Moreover, the endogenous ADL1 protein level was greatly reduced in these transgenic plants, probably due to a posttranscriptional silencing effect of the transgenes, We propose, therefore, that ADL1 is involved in the biogenesis of thylakoid membranes.X1148sciescopu

AN ARABIDOPSIS GSK3/SHAGGY-LIKE GENE THAT COMPLEMENTS YEAST SALT STRESS-SENSITIVE MUTANTS IS INDUCED BY NACL AND ABSCISIC ACID

GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stress-sensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl. Also, the AtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress. To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at the MCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant. The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1. Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants. Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant. To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression of AtGSK1. Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues. In addition, the AtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCI. Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis.X1169sciescopu

Characterization of two new channel protein genes in Arabidopsis

Aquaporins, small channel proteins, found in a variety of organisms are members of the major intrinsic protein (MIP) superfamily and have been shown to facilitate water transport when expressed in Xenopus oocytes, We isolated two Arabidopsis cDNAs, SIMIP and SITIP, that encode protein homologues of the MIP superfamily, SIMIP exhibits a high degree of sequence homology to PIP3 and MIP1, and thus may belong to the plasmamembrane intrinsic protein (PIP) subfamily, whereas salt-stress inducible tonoplast intrinsic protein (SITIP) is highly homologous to VM23 and gamma-TIP, and therefore may belong to the TIP subfamily Expression studies revealed that the two genes showed a different expression pattern. The SIMIP gene was expressed in a tissue-specific manner, for example, its highest transcript level is found in flowers, relatively low levels in siliques, and very low level in leaves and roots. In contrast, SITIP was expressed in nearly equal amounts in all the tissues we examined, Also, the expression of SIMIP and SITIP showed a temporal regulation pattern. For example, the highest expression level was at 1 week after germination. In addition, the transcript levels of SIMIP and SMTIP were increased upon NaCl and ABA treatments. The biological function of the 2 genes were investigated using two NaCl stress-sensitive yeast mutant strains. The mutant yeast cells expressing these 2 genes were more resistant to high NaCI conditions. The results suggest that the proteins encoded by these genes may be involved in the osmoregulation in plants under high osmotic stress such as under a high salt condition.X1116sciescopu

A dynamin-like protein, ADL1, is present in membranes as a high-molecular-mass complex in Arabidopsis thaliana

Dynamin, a GTP-binding protein, is involved in endocytosis in animal cells. We found that a dynamin-like protein, ADL1, is present in multiple forms in Arabidopsis leaf tissue. Subcellular fractionation experiments, together with gel-filtration and nondenaturing-gel electrophoresis revealed that most of ADL1 is present as a high-molecular-mass complex of 400 to 600 kD in the membrane or pellet fraction, whereas ADL1 is present in the soluble fraction as a monomer. The subcellular distribution of ADL1 is affected by various agents such as Ca2+, cyclosporin A, GTP, and ATP. Ca2+ increases the amount of ADL1 present in the membrane fraction, whereas cyclosporin A inhibits the membrane association. Furthermore, Ca2+ and GTP change the migration pattern of ADL1 in nondenaturing polyacrylamide gels, indicating that these chemicals influence either the complex formation and/or the conformation of the ADL1 complex. Our results demonstrate that ADL1 has characteristics that are similar to Dynamin I, which is found in animal cells. Therefore, it is possible that ADL1 is also involved in biological processes that require vesicle formation.close343

Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells

Very limited information is available on the role of phosphatidytinositol 3-phosphate (P1[3]P) in vesicle trafficking in plant cells. To investigate the role of PI(3)P during the vesicle trafficking in plant cells, we exploited the Pt(3)P-specific binding property of the endosome binding domain (EBD) (amino acids 1257 to 1411) of human early endosome antigen 1, which is involved in endosome fusion. When expressed transiently in Arabidopsis protoplasts, a green fluorescent protein (GFP):EBD fusion protein exhibited PI(3)P-dependent localization to various compartments-such as the trans-Golgi network, the prevacuolar compartment, the tonoplasts, and the vesicles in the vacuolar lumen-that varied with time. The internalized GFP:EBD eventually disappeared from the lumen. Deletion experiments revealed that the P1(3)P-dependent localization required the Rab5 binding motif in addition to the zinc finger motif. Overexpression of GFP:EBD inhibited vacuolar trafficking of sporamin but not trafficking of Hf-ATPase to the plasma membrane. On the basis of these results, we propose that the trafficking of GFP:EBD reflects that of P1(3)P and that P1(3)P synthesized at the trans-Golgi network is transported to the vacuole through the prevacuolar compartment for degradation in plant cells.X11185sciescopu

A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes

Dynamin, a GTP-binding protein found in rat brain, plays a role in endocytosis. Suborganellar fractionation studies of Arabidopsis leaf tissue revealed that a dynamin-like protein, ADL1, is localized in the thylakoid membranes of chloroplasts. This notion was supported further by lit vivo targeting experiments using an ADL1-green fluorescent fusion protein and immunogold labeling with the anti-ADL1 antibody. Transgenic plants harboring various deletion mutant genes of ADL1 had a yellow leaf phenotype where the cells had very few chloroplasts, In addition, the remaining chloroplasts appeared morphologically not fully developed, The detailed structure of the chloroplasts revealed by electron microscopy showed a greatly reduced amount of thylakoid membranes. Also, the level of thylakoid membrane proteins such as the light-harvesting complex II and CP29 was greatly reduced in these transgenic plants, When we examined the expression of the ADL1 deletion mutant genes, these genes were highly expressed at the transcriptional level, However, the mutant ADL1s were not detectable at the protein level by Western blot analysis, Moreover, the endogenous ADL1 protein level was greatly reduced in these transgenic plants, probably due to a posttranscriptional silencing effect of the transgenes, We propose, therefore, that ADL1 is involved in the biogenesis of thylakoid membranes.close464