DFL2, a new member of the Arabidopsis GH3 gene family, is involved in red light-specific hypocotyl elongation

A new GH3-related gene, designated DFL2, causes a short hypocotyl phenotype when overexpressed under red and blue light and a long hypocotyl when antisensed under red light conditions. Higher expression of this gene was observed in continuous white, blue and far-red light but the expression level was low in red light and darkness. DFL2 gene expression was induced transiently with red light pulse treatment. DFL2 transgenic plants exhibited a normal root phenotype including primary root elongation and lateral root formation, although primary root elongation was inhibited in antisense transgenic plants only under red light. The adult phenotypes of sense and antisense transgenic plants were not different from that of wild type. DFL2 promoter activity was observed in the hypocotyl. Our results suggest that DFL2 is located downstream of red light signal transduction and determines the degree of hypocotyl elongation.     

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).     

Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence

Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of beta2-microgobulin (beta2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent beta2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid beta-peptide.     

Kinetic modeling and determination of reaction constants of Alzheimer's beta-amyloid fibril extension and dissociation using surface plasmon resonance

To establish the kinetic model of the extension and dissociation of beta-amyloid fibrils (f(A)beta) in vitro, we analyzed these reactions using a surface plasmon resonance (SPR) biosensor. Sonicated f(A)beta were immobilized on the surface of the SPR sensor chip as seeds. The SPR signal increased linearly as a function of time after amyloid beta-peptides (Abeta) were injected into the f(A)beta-immobilized chips. The extension of f(A)beta was confirmed by atomic force microscopy. When flow cells were washed with running buffer, the SPR signal decreased with time after the extension reaction. The curve fitting resolved the dissociation reaction into the fast exponential and slow linear decay phases. Kinetic analysis of the effect of Abeta/f(A)beta concentrations on the reaction rate indicated that both the extension reaction and the slow linear phase of the dissociation were consistent with a first-order kinetic model; i.e., the extension/dissociation reactions proceed via consecutive association/dissociation of Abeta onto/from the end of existing fibrils. On the basis of this model, the critical monomer concentration ([M](e)) and the equilibrium association constant (K) were calculated, for the first time, to be 20 nM and 5 x 10(7) M(-1), respectively. Alternatively, [M](e) was directly measured as 200 nM, which may represent the equilibrium between the extension reaction and the fast phase of the dissociation. The SPR biosensor is a useful quantitative tool for the kinetic and thermodynamic study of the molecular mechanisms of f9A)beta formation in vitro.     

Chemotactic Adaptation Is Altered by Changes in the Carboxy-Terminal Sequence Conserved among the Major Methyl-Accepting Chemoreceptors

In Escherichia coli and Salmonella typhimurium, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless E. coli strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.     

A New Monoclonal Antibody that Recognizes 180 kDa Polypeptide Expressed on Early Mouse Embryos and Mouse Embryonal Carcinoma Cells

A hybridoma clone 1F7 producing a monoclonal antibody against OTF9-63 mouse embryonal carcinoma cell line was isolated with the aid of the intrasplenic primary immunization technique. This antibody was capable of recognizing embryonal carcinoma cell lines originated from certain mouse strains, including 129/Sv, but not those which were established from other strains as well as human and other murine cell lines except FM3A, a mouse mammary carcinoma cell line. Indirect immunofluorescence study revealed that 1F7 antigen was expressed on early mouse embryos in a stage specific manner. In order to characterize the 1F7 antigenic molecules, we analyzed both glycoshingolipids and glycoproteins recovered from OTF9-63 by means of immunostaining after thin layer chromatography and SDS-polyacrylamide gel electrophoresis followed by Western blotting, respectively. It was concluded that 1F7 antigenic determinants were carried by 180 kDa polypeptides. One of the most interesting characteristics of 1F7 antigen is complete failure to express on the embryonic ectoderm of 5.5d and 6.5d embryos, one of cell types most closely related to embryonal carcinoma cells. 1F7 antibody may help analyse the process of teratocarcinogenesis in 129/Sv mice.     

A new purification procedure for bovine C-reactive protein and serum amyloid P component.

1. A new purification procedure was started with salting-out fractionation of serum proteins at 45-75% saturated ammonium sulfate concentration, followed by HE agarose affinity chromatography by which calcium-dependently bound CRP and SAP were purely eluted with EDTA-containing buffer. 2. Pure CRP and SAP were finally separated by DEAE-5PW HPLC. 3. This procedure gave recovery of 15 and 26%, and fold purification of 2650 and 2400 for CRP and SAP, respectively. 4. Each subunit of CRP and SAP had one intrasubunit disulfide bond, determined by reduction and carboxymethylation.     

ydk1-D, an auxin-responsive GH3 mutant that is involved in hypocotyl and root elongation

To study the GH3 gene family of Arabidopsis, we investigated a flanking sequence database of Arabidopsis activation-tagged lines. We found a dwarf mutant, named yadokari 1-D (ydk1-D), that had a T-DNA insertion proximal to a GH3 gene. ydk1-D is dominant and has a short hypocotyl not only in light but also in darkness. Moreover, ydk1-D has a short primary root, a reduced lateral root number, and reduced apical dominance. A GH3 gene, named YDK1, was upregulated in ydk1-D, and YDK1 transgenic plants showed the ydk1-D phenotype. YDK1 gene expression was induced by exogenously applied auxin and regulated by auxin-response factor (ARF)7. In addition, YDK1 gene expression was downregulated by blue and far-red (FR) lights. Strong promoter activity of YDK1 was observed in roots and flowers. These results suggest that YDK1 may function as a negative component in auxin signaling by regulating auxin activity.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.