Photosystem 2 photochemistry and pigment composition of a yellow mutant of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) under different irradiances

A yellow leaf colouration mutant (named ycm) generated from rice T-DNA insertion lines was identified with less grana lamellae and low thylakoid membrane protein contents. At weak irradiance [50 μmol(photon) m⁻² s⁻¹], chlorophyll (Chl) contents of ycm were ≈20 % of those of WT and Chl a/b ratios were 3-fold that of wild type (WT). The leaf of ycm showed lower values in the actual photosystem 2 (PS2) efficiency (Φ_PS2), photochemical quenching (q_P), and the efficiency of excitation capture by open PS2 centres 1 (F_v′/F_m′) than those of WT, except no difference in the maximal efficiency of PS2 photochemistry (F_v/F_m). With progress in irradiance [100 and 200 μmol(photon) m⁻² s⁻¹], there was a change in the photosynthetic pigment stoichiometry. In ycm, the increase of total Chl contents and the decrease in Chl a/b ratio were observed. Φ_PS2, q_P, and F_v′/F_m′ of ycm increased gradually along with the increase of irradiance but still much less than in WT. The increase of xanthophyll ratio [(Z+A)/(V+A+Z)] associated with non-photochemical quenching (q_N) was found in ycm which suggested that ycm dissipated excess energy through the turnover of xanthophylls. No significant differences in pigment composition were observed in WT under various irradiances, except Chl a/b ratio that gradually decreased. Hence the ycm mutant developed much more tardily than WT, which was caused by low photon energy utilization independent of irradiance.     

GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

Here, we report the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal RING finger domain, which is mostly conserved with those of the lAP family members and is required for GIDE＇s E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases, since caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockage of JNK activation inhibits GIDE-induced release of cytochrome c and Smac as well as apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE- induced apoptosis. These pro-apoptotic properties of GIDE require its E3 ligase activity. When somewhat over-or underexpressed, GIDE slows or accelerates cell growth, respectively. These pro-apoptotic or growth inhibition effects of GIDE may account for its absence in tumor cells.     

p190RhoGEF Binds to a destabilizing element in the 3' untranslated region of light neurofilament subunit mRNA and alters the stability of the transcript.

Stabilization of neurofilament (NF) mRNAs plays a major role in regulating levels of NF expression and in establishing axonal size and rate of axonal conduction. Previous studies have identified a 68-nucleotide destabilizing element at the junction of the coding region and 3' untranslated region of the light NF subunit (NF-L) mRNA. The present study has used the destabilizing element (probe A) to screen a rat brain cDNA library for interactive proteins. A cDNA clone encoding 1068 nucleotides in the C-terminal domain of p190RhoGEF (clone 39) was found to bind strongly and specifically to the RNA probe. The interaction was confirmed using a glutathione S-transferase/clone 39 fusion protein in Northwestern, gel-shift, and cross-linkage studies. The glutathione S-transferase/clone 39 fusion protein also enhanced the cross-linkage of a major 43-kDa protein in brain extract to the destabilizing element. Functional studies on stably transfected neuronal cells showed that p190RhoGEF expression increased the half-life of a wild-type NF-L mRNA but did not alter the half-life of a mutant NF-L mRNA lacking the destabilizing element. The findings reveal a novel interactive feature of p190RhoGEF that links the exchange factor with NF mRNA stability and regulation of the axonal cytoskeleton.     

Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton

We have previously shown that tyrosine phosphorylation of the actin-regulatory protein villin is accompanied by the redistribution of phosphorylated villin and a concomitant decrease in the F-actin content of intestinal epithelial cells. The temporal and spatial correlation of these two events suggested that tyrosine phosphorylation of villin may be involved in the rearrangement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetics of actin polymerization by reconstituting in vitro the tyrosine phosphorylation of villin and its association with actin. Full-length recombinant human villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding properties of villin were examined using a co-sedimentation assay. Phosphorylation of villin did not change the stoichiometry (1:2) but decreased the binding affinity (4.4 microm for unphosphorylated versus 0.6 microm for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we demonstrated that tyrosine phosphorylation had a negative effect on actin nucleation by villin. In contrast, tyrosine phosphorylation enhanced actin severing by villin. Electron microscopic analysis showed complementary morphological changes. Phosphorylation inhibited the actin bundling and enhanced the actin severing functions of villin. Taken together our data show that tyrosine phosphorylation of villin decreases the amount of villin bound to actin filaments, inhibits the actin-polymerizing properties of villin, and promotes the actin-depolymerizing functions instead. These observations suggest a role for tyrosine phosphorylation in modulating the microvillar cytoskeleton in vivo by villin in response to specific physiological stimuli.     

C3G, a guanine nucleotide exchange factor bound to adapter molecule c-Crk, has two alternative splicing forms

Two types of C3G cDNA were isolated from mouse 3T3-L1 adipocyte cDNA library. A 114-bp sequence in the middle of C3G cDNA is deleted in the short type cDNA. By RT-PCR analysis, it was found that these two types of C3G mRNA existed in all the mouse tissues. Sequence comparison revealed 88% nucleotide sequence identity between mouse and human C3G cDNA. Comparison of mouse C3G cDNA with the human genome database suggested that this 114-bp sequence comprised an entire exon, and it is confirmed by PCR analysis using mouse genomic DNA and cDNA template. These results indicate that two C3G mRNAs and proteins result from alternative RNA splicing.     

Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1

The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.     

Chromosome landing at the bacterial blight resistance gene<Emphasis Type="Italic"> Xa4</Emphasis> locus using a deep coverage rice BAC library

Xa4 is a dominantly inherited rice gene that confers resistance to Philippine race 1 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae in rice. In order to isolate the gene by positional cloning, a bacterial artificial chromosome (BAC) library was constructed from genomic DNA isolated from an Xa4-harboring accession, IRBB56. The library contains 55,296 clones with an average insert size of 132 kb, providing 14 rice genome equivalents. Three DNA markers closely linked to Xa4 were used to screen the library. The marker RS13, a resistance gene analogue that co-segregates with Xa4, identified 18 clones, of which four and six, respectively, were simultaneously detected by the other two markers, G181 and L1044. Fingerprinting and Southern analysis indicated that these clones overlapped and define an interval spanning 420 kb. In an F2 population derived from an indica variety, IR24, and its Xa4-containing near isogenic line (NIL), IRBB4, the susceptible plants were screened in order to map the Xa4 gene genetically and physically. Out of 24 insert ends isolated from the BACs in the contig, three revealed polymorphisms between IR24 and IRBB4. Two insert ends, 56M22F and 26D24R, flanked Xa4 on each side. Based on the overlap of the BACs, six overlapping clones were considered to include the Xa4 allele, one of which, 106P13, was chosen for further investigation.     

Regulation of phospholipase C-gamma(1) by the actin-regulatory protein villin

The actin-regulatory protein villin is tyrosinephosphorylated and associates with phospholipase C-₁(PLC-₁) in the brush border of intestinalepithelial cells. To study the mechanism of villin-associatedPLC-₁ activation, we reconstituted in vitro the tyrosinephosphorylation of villin and its association with PLC-₁. Recombinant villin was phosphorylated in vitro bythe nonreceptor tyrosine kinase c-src or by expression in the TKX1 competent cells that carry an inducible tyrosine kinase gene. Using invitro binding assays, we demonstrated that tyrosine-phosphorylated villin associates with the COOH-terminal Src homology 2 (SH2) domain ofPLC-₁. The catalytic activity of PLC-₁was inhibited by villin in a dose-dependent manner with half-maximalinhibition at a concentration of 12.4 µM. Villin inhibitedPLC-₁ activity by sequestering the substratephosphatidylinositol 4,5-bisphosphate (PIP₂), sinceincreasing concentrations of PIP₂ reversed the inhibitory effects of villin on PLC activity. The inhibition ofPLC-₁ activity by villin was reversed by the tyrosinephosphorylation of villin. Further, we demonstrated that tyrosinephosphorylation of villin abolished villin's ability to associate withPIP₂. In conclusion, tyrosine-phosphorylated villinassociates with the COOH-terminal SH2 domain of PLC-₁and activates PLC-₁ catalytic activity. Villin regulatesPLC-₁ activity by modifying its own ability to bindPIP₂. This study provides biochemical proof of thefunctional relevance of tyrosine phosphorylation of villin andidentifies the molecular mechanisms involved in the activation ofPLC-₁ by villin.

     

A new method for protein coexpression in Escherichia coli using two incompatible plasmids

It is commonly believed that incompatible plasmids carrying the same replicon cannot coexist stably in one Escherichia coli cell. However, we found that two incompatible plasmids carrying different antibiotic resistance genes, if under the selection pressure of the two antibiotics, can coexist in E. coli for at least 14 h, which is adequate for routine culture and protein expression. Based on this discovery, we developed a new method to coexpress foreign proteins in E. coli using two incompatible plasmids. The coding regions of the two subunits (DFF45 and DFF40) of the human DNA fragmentation factor (DFF) were cloned into two incompatible bacterial expression vectors-pET-21a with ampicillin resistance and pET-28a with kanamycin resistance, respectively. The two resulting plasmids were used to cotransform E. coli BL21(DE3) cells. After selection by ampicillin and kanamycin simultaneously, cotransformants that contain both recombinant plasmids were obtained. Induced by isopropyl beta-d-thiogalactoside, DFF45, and DFF40 were coexpressed efficiently in the presence of the two antibiotics. The coexpression product contained adequate soluble portions for both DFF45 and DFF40, while all DFF40 was insoluble if expressed alone. The coexpression product also exhibited the same caspase-activated DNase activity as its natural counterparts, which cannot be obtained if its two subunits are expressed separately.