Sequence database of 1172 T-DNA insertion sites in Arabidopsis activation-tagging lines that showed phenotypes in T1 generation

Plant genomic resources harbouring gain-of-function mutations remain rare, even though this type of mutation is believed to be one of the most useful for elucidating the function of unknown genes that have redundant partners in the genome. An activation-tagging T-DNA was introduced into the genome of Arabidopsis creating 55,431 independent transformed lines. Of these T1 lines, 1,262 showed phenotypes different from those of wild-type plants. We called these lines 'AT1Ps' (activation T1 putants). The phenotypes observed include abnormalities in morphology, growth rate, plant colour, flowering time and fertility. Similar phenotypes re-appeared either in dominant or semi-dominant fashion in 17% of 177 AT2P plants tested. Plasmid rescue or an adaptor-PCR method was used to identify 1172 independent genomic loci of T-DNA integration sites in the AT1P plants. Mapping of the integration sites revealed that the chromosomal distribution of these sites is similar to that observed in conventional T-DNA knock-out lines, except that the intragenic type of integration is slightly lower (27%) in the AT1P plants compared to that observed in other random knock-out populations (30-35%). Ten AT2P lines that showed dominant phenotypes were chosen to monitor expression levels of genes adjacent to the T-DNA integration sites by RT-PCR. Activation was observed in 7 out of 17 of the adjacent genes detected. Genes located up to 8.2 kb away from the enhancer sequence were activated. One of the seven activated genes was located close to the left-border sequence of the T-DNA, having an estimated distance of 5.7 kb from the enhancer. Surprisingly, one gene, the first ATG of which is located 12 kb away from the enhancer, showed reduced mRNA accumulation in the tagged line. Application of the database generated to Arabidopsis functional genomics research is discussed. The sequence database of the 1172 loci from the AT1P plants is available (http://pfgweb.gsc.riken.go.jp/index.html).     

Production of lupin acid phosphatase in transgenic rice for use as a phytate-hydrolyzing enzyme in animal feed

The acid phosphatase gene from lupin was expressed in transgenic rice plants under the control of the maize ubiquitin promoter or rice chlorophyll a/b binding protein (Cab) promoter. Transgenic rice leaves exhibited up to an 18-fold increase in phytate-hydrolyzing activity. Based on the phytate-hydrolyzing activity at pH 5.5, more than 85% this activity was retained after heat-treatment at 80 degrees C for 15 min, and the heterologous enzyme in leaf sections and leaf extracts was relatively stable during storage. A distinct increase in released phosphate was observed when the heterologous enzyme was mixed with the feed extract. These results suggest that the heterologous enzyme in rice plants may maintain its desired characteristics as a phytate-hydrolyzing enzyme when added to animal feed.     

Signal transduction of betacellulin in growth and migration of vascular smooth muscle cells

Epidermal growth factor (EGF) family ligands have been implicated in cardiovascular diseases because of their enhanced expression in vascular lesions and their promoting effects on growth and migration of vascular smooth muscle cells (VSMCs). Betacellulin (BTC), a novel EGF family ligand, has been shown to be expressed in atherosclerotic lesions and to be a potent growth factor of VSMCs. However, the molecular mechanisms downstream of BTC involved in mediating vascular remodeling remain largely unknown. Therefore, the aim of this study was to examine the effects of BTC on signal transduction, growth, and migration in VSMCs. We found that BTC stimulated phosphorylation of EGF receptor (EGFR) at Tyr1068, which was completely blocked by an EGFR kinase inhibitor, AG-1478. BTC also phosphorylated ErbB2 at Tyr877, Tyr1112, and Tyr1248 and induced association of ErbB2 with EGFR, suggesting their heterodimerization in VSMCs. In postreceptor signal transduction, BTC stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and p38 mitogen-activated protein kinase (MAPK). Moreover, BTC stimulated proliferation and migration of VSMCs. ERK and Akt inhibitors suppressed migration markedly and proliferation partially, whereas the p38 inhibitor suppressed migration partially but not proliferation. In addition, we found the presence of endogenous BTC in conditioned medium of VSMCs and an increase of BTC on angiotensin II stimulation. In summary, BTC promotes growth and migration of VSMCs through activation of EGFR, ErbB2, and downstream serine/threonine kinases. Together with the expression and processing of endogenous BTC in VSMCs, our results suggest a critical involvement of BTC in vascular remodeling. epidermal growth factor receptors; ErbB2; migration; signal transduction     

Site-directed mutagenesis establishes aspartic acids-227 and -342 as essential for enzyme activity in an isomalto-dextranase from Arthrobacter globiformis

Isomalto-dextranase, from Arthrobacter globiformis T6, is a member of the glycoside hydrolase family 27. However, the alignments of the whole amino acid sequence are distinct from other members of this family. The enzymes cleave the glycosidic bond of the substrate in two different manners: either retaining or inverting the anomeric configuration. We believe that a retaining enzyme is involved in a two-step, double-displacement mechanism utilizing active site carboxylic acids as the nucleophile and general acid/base catalysts in the hydrolytic reaction. The critical amino acid residues at the isomalto-dextranase active site that catalyzes the hydrolysis reaction of dextran have been identified and the roles of nine amino acid residues (D107, D163, D227, D295, D340, D342, D373, D396, and E420) in the isomalto-dextranase from A. globiformis analyzed by site-directed mutagenesis. Of 15 mutant enzymes that were prepared, eight had reduced activities for dextran hydrolysis. Aspartic acids-227 and -342, which are part of the apparent catalytic dyad, were essential for hydrolase activity toward dextran.     

Enzymatic synthesis of two novel non-reducing oligosaccharides using transfructosylation activity with β-fructofuranosidase from Arthrobacter globiformis

Two novel non-reducing oligosaccharides together with tri- and tetra-saccharides were synthesized by transfructosylation activity from sucrose as a donor and cellobiose or cellotriose as an acceptor with a purified beta-fructofuranosidase from Arthrobacter globiformis IFO 3062, and these oligosaccharides were identified as O-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside and O-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside by spectrometric analyses. Both oligosaccharides were stable under condition at 100 degrees C for 30 min, and showed no degradation at pH 2.     

Mechanism of gene expression of Arabidopsis glutathione S-transferase, AtGST1, and AtGST11 in response to aluminum stress

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5'-upstream region of each gene was fused to a beta-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress.     

Origin of apparent negative cooperativity of F(1)-ATPase

In order to get insight into the origin of apparent negative cooperativity observed for F(1)-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F(1)-ATPase, alpha((W463F)3)beta((Y341W)3)gamma and alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma. For alpha((W463F)3)beta((Y341W)3)gamma, apparent K(m)'s of ATPase kinetics (4.0 and 233 microM) did not agree with apparent K(m)'s deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 microM). On the other hand, in case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, which lacks noncatalytic nucleotide binding sites, the apparent K(m) of ATPase activity (10 microM) roughly agreed with the highest K(m) of fluorescence measurements (27 microM). The results indicate that in case of alpha((W463F)3)beta((Y341W)3)gamma, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K(m) of ATPase activity does not represent the ATP binding to a catalytic site. In case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, the K(m) of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma was rather linear compared with that of alpha((W463F)3)beta((Y341W)3)gamma, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F(1)-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K(m)'s of 100-300 microM and 1-30 microM range for wild-type F(1)-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.     

Impact of genetic background and ablation of insulin receptor substrate (IRS)-3 on IRS-2 knock-out mice

Although we and others have generated IRS-2 knock-out (IRS-2(-/-)) mice, significant differences were seen between the two lines of IRS-2(-/-) mice in the severity of diabetes and alterations of beta-cell mass. It has been reported that although IRS-1 and IRS-3 knock-out mice showed normal blood glucose levels, IRS-1/IRS-3 double knock-out mice exhibited marked hyperglycemia. Thus, IRS-1 and IRS-3 compensate each other's functions in maintaining glucose homeostasis. To assess the effect of genetic background and also ablation of IRS-3 on IRS-2(-/-), we generated IRS-2/IRS-3 double knock-out (IRS-2(-/-)IRS-3(-/-)) mice by crossing IRS-3(-/-) mice (129/Sv and C57Bl/6 background) with our IRS-2(-/-) mice (CBA and C57Bl/6 background). Intercrosses of IRS-2(+/-)IRS-3(+/-) mice yielded nine genotypes, and all of them including IRS-2(-/-)IRS-3(-/-) mice were apparently healthy and showed normal growth. However, at 10-20 weeks of age, 20-30% mice carrying a null mutation for the IRS-2 gene, irrespective of the IRS-3 genotype, developed diabetes. When mice with diabetes were excluded from the analysis of glucose and insulin tolerance test, IRS-2(-/-)IRS-3(-/-) showed a degree of glucose intolerance and insulin resistance similar to those of IRS-2(-/-) mice. Both IRS-2(-/-) and IRS-2(-/-)IRS-3(-/-) mice had moderately reduced beta-cell mass despite having insulin resistance. Insulin-positive beta-cells were decreased to nearly zero in IRS-2(-/-) mice with diabetes. Although Pdx1 and glucose transporter 2 expressions were essentially unaltered in islets from IRS-2(-/-) mice without diabetes, they were dramatically decreased in IRS-2(-/-) mice with diabetes. Taken together, these observations indicate that IRS-3 does not play a role compensating for the loss of IRS-2 in maintaining glucose homeostasis and that the severity of diabetes in IRS-2(-/-) mice depends upon genetic background, suggesting the existence of modifier gene(s) for diabetes in mice of the 129/Sv genetic strain.