Evolution and control of imprinted FWA genes in the genus Arabidopsis

A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.     

Synthesis of 1-arylpiperazyl-2-phenylcyclopropanes designed as antidopaminergic agents: cyclopropane-based conformationally restricted analogs of haloperidol

A series of the cyclopropane-based conformationally restricted analogs of haloperidol were designed as potential antidopaminergic agents and were effectively synthesized using highly stereoselective Grignard reaction with tert-butanesulfinyl imines as the key step. Pharmacological evaluation of the compounds showed that the conformational restriction method can effectively work for improving the pharmacological selectivity of a parent compound and also for investigating the bioactive conformation.     

Molecular characterization of organelle-type Nudix hydrolases in Arabidopsis

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its K(m) value was approximately 100-fold lower for ADP-ribose (13.0+/-0.7 microm) than for ADP-glucose (1,235+/-65 microm). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7+/-3.4 microm) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3+/-5.4 microm) and NADPH (36.9+/-3.5 microm). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1+/-0.9 microm). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n=4-5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes.     

Novel Toxin-Antitoxin System Composed of Serine Protease and AAA-ATPase Homologues Determines the High Level of Stability and Incompatibility of the Tumor-Inducing Plasmid pTiC58

Stability of plant tumor-inducing (Ti) plasmids differs among strains. A high level of stability prevents basic and applied studies including the development of useful strains. The nopaline type Ti plasmid pTiC58 significantly reduces the transconjugant efficiency for incoming incompatible plasmids relative to the other type, such as octopine-type plasmids. In this study we identified a region that increases the incompatibility and stability of the plasmid. This region was located on a 4.3-kbp segment about 38 kbp downstream of the replication locus, repABC. We named two open reading frames in the segment, ietA and ietS, both of which were essential for the high level of incompatibility and stability. Plasmid stabilization by ietAS was accomplished by a toxin-antitoxin (TA) mechanism, where IetS is the toxin and IetA is the antitoxin. A database search revealed that putative IetA and IetS proteins are highly similar to AAA-ATPases and subtilisin-like serine proteases, respectively. Amino acid substitution experiments in each of the highly conserved characteristic residues, in both putative enzymes, suggested that the protease activity is essential and that ATP binding activity is important for the operation of the TA system. The ietAS-containing repABC plasmids expelled Ti plasmids even in strains which were tolerant to conventional Ti-curing treatments.Agrobacterium tumefaciens strains bearing a tumor-inducing (Ti) plasmid are the etiological agents of crown gall disease. Most genes required for pathogenicity are located on the plasmids (17, 33). Ti plasmids are kept stable at a low copy number equivalent to that of the chromosomal DNA in the bacterial cells (32) due to the repABC locus (16, 30, 34). The stability of Ti plasmids differs among strains (11).Many genes for keeping plasmids stable have been reported in eubacteria, and these are divided into three categories based on their mechanism: multimer resolution systems, active partitioning systems, and toxin-antitoxin (TA) systems (15). Multimer resolution systems increase the number of plasmid molecules by resolving a multimer plasmid into monomers, resulting in a higher probability of plasmid distribution to daughter cells during cell division even when plasmid distribution occurs randomly (29). Active partitioning systems deliver plasmid copies to each progeny cell at cell division (21). In the repABC locus, the RepA and RepB proteins and parS site(s) ensure stable plasmid inheritance by the active partitioning system (2). TA systems contribute to plasmid maintenance in cell populations by initiating growth inhibition or death of plasmid-free cells and are widely distributed among eubacterial and archaeal plasmids as well as their chromosomes (9). Generally, the TA module consists of two genes which encode toxin and antitoxin. The antitoxin neutralizes the action of a cognate toxin by interaction with the toxin or its target molecules. When a plasmid harboring the TA module is lost from a host cell, the antitoxin molecules decrease to an ineffective level because the antitoxin is degraded quickly or diluted by cell division (15). Thereafter, the toxin exerts its toxicity and inhibits the host cell growth. RNA antitoxins can suppress toxin expression by binding to the toxin mRNA as an antisense RNA or repress toxicity effects by an unknown mechanism (6, 4). In pTi-SAKURA, the Ti plasmid in the A. tumefaciens strain MAFF301001, it was shown that the tiorf24 and tiorf25 module increased plasmid stability by the TA mechanism (40; also S. Yamamoto, unpublished data).Differences in Ti plasmid stability are critical for plasmid engineering and evolution (33). However, little is known about the stability factors of Ti plasmids other than the repABC locus. In our previous study (40), tiorf24 and tiorf25 were shown to increase the segregational stability and incompatibility of Ti plasmids and reduce the efficiency of transconjugants by the introduction of incompatible plasmids into host cells. The two genes are located 2.5-kbp downstream of repABC (8). Generally, incompatibility has been defined as a situation where two plasmids contain a related replication and/or partitioning system and are unable to exist in a cell simultaneously without external selection (1). A. tumefaciens strain C58, which contains a Ti plasmid pTiC58, allows entry of an incompatible repABC plasmid into the cell 60-fold less efficiently than a derivative of C58. The derivative of C58 harbors a small repABC vector instead of pTiC58 (40). This suggests the presence of incompatibility-enhancing genes on pTiC58.In this study, we located the responsible genes in pTiC58 and found that the novel genes ietA and ietS enhance the incompatibility and stability of the plasmid by the TA mechanism.