Mechanism of double-stranded supercoiled DNA cleavage induced by RNA N-glycosidase

Plant RNA N-glycosidase specifically hydrolyzes the N-C glycosidic bond of a conserved adenosine in the sarcin/ricin domain of the largest RNA in ribosome, releasing an adenine base and thus inhibiting protein synthesis. This substrate specificity was challenged later by discovery that various RNA derivatives and DNAs, especially the double-stranded supercoiled DNA could be used as substrate by RNA N-glycosidase. Thus, it was argued whether the DNA-cleaving activity was an intrinsic feature of RNA N-glycosidase or it was contaminated by DNase. In this article, several lines of evidence are presented to show that RNA N-glycosidase can really release the adenine base from the double-stranded supercoi/ed DNA. It was proposed that the cleavage mechanism of supercoiled DNA was the phosphodiester bonds in enzymatically deadenylated regions of the supercoiled DNA would become fragile and liable to produce nicked or linear form owing to the existence of tension in the supercoiled DNA molecule, not direct result of enzymatic action on the phosphodiester bond.

The Arabidopsis LFR Gene Is Required for the Formation of Anther Cell Layers and Normal Expression of Key Regulatory Genes

The anther is the male reproductive organ in flowering plants. Although some genes were reported to be involved in anther development, the molecular mechanisms underlying the transcriptional regulation of these genes is unclear. Ifr-2 （leaf and flower related-2）, the null allele of Arabidopsis thaliana LFR （LEAF AND FLOWER RELATED）, was male-sterile. The anthers of Ifr-2 plants were defective in sporogenous cell formation, tapetum development, and pollen development. In agreement with these phenotypes, expression studies showed that LFR was expressed in all cell layers of the anther, and that expression was particularly strong in the tapetal cells and pollen grains, Quantitative RT-PCR analysis revealed that LFR is required for the normal transcription of some anther development-related genes, such as AMS, CALS5, and DYT1, MS1 and MS2, and ROXY2. Genetic analysis showed that SPL was epistatic to LFR while LFR was epistatic to DYT1. We propose that LFR may be a crucial component in the regulation of a genetic network that modulates anther development.

Extracellular ATP Promotes Stomatal Opening of Arabidopsis thaliana through Heterotrimeric G Protein α Subunit and Reactive Oxygen Species

In recent years, adenosine tri-phosphate （ATP） has been reported to exist in apoplasts of plant cells as a signal molecule. Extracellular ATP （eATP） plays important roles in plant growth, development, and stress tolerance. Here, extra- cellular ATP was found to promote stomatal opening of Arabidopsis thaliana in light and darkness. ADP, GTP, and weakly hydrolyzable ATP analogs （ATPγS, Bz-ATP, and 2meATP） showed similar effects, whereas AMP and adenosine did not affect stomatal movement. Apyrase inhibited stomatal opening. ATP-promoted stomatal opening was blocked by an NADPH oxidase inhibitor （diphenylene iodonium） or deoxidizer （dithiothreitol）, and was impaired in null mutant of NADPH ox- idase （atrbohD/F）. Added ATP triggered ROS generation in guard cells via NADPH oxidase. ATP also induced Ca^2＋ influx and H ＋ efflux in guard cells. In atrbohD/F, ATP-induced ion flux was strongly suppressed. In null mutants of the heterotrimeric G protein α subunit, ATP-promoted stomatal opening, cytoplasmic ROS generation, Ca^2＋ influx, and ^H＋ efflux were all sup- pressed. These results indicated that eATP-promoted stomatal opening possibly involves the heterotrimeric G protein, ROS, cytosolic Ca^2＋, and plasma membrane H＋-ATPase.

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A membrane-bound protein was purified from rat liver mitochondria.After being di-gested with V8 protease,two peptides containing identical 14 amino acid residue sequences were obtained.Using the 14 amino acid peptide derived DNA sequence as gene specific primer,the cDNA of correspondent gene 5′-terminal and 3′-terminal were obtained by RACE technique.The full-length cDNA that encoded a protein of 616 amino acids was thus cloned,which included the above mentioned peptide sequence.The full length cDNA was highly homologous to that of human ETF-QO,indicating that it may be the cDNA of rat ETF-QO.ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center:FAD and[4Fe-4S]center.After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria,it was found that the N terminal 32 amino acid residues did not exist in the mature protein,indicating that the cDNA was that of ETF-QOp.When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors,the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity(NBT re-ductase activity)of ETF-QO.Results demonstrated that the 32 amino acid peptide was a mito-chondrial targeting peptide,and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO.ETF-QO had a high level expression in rat heart,liver and kidney.The fu-sion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.

Conservation and divergence of the histone H2B monoubiquitination pathway from yeast to humans and plants

Histone ubiquitination plays a critical role in the regulation of transcription,and histone H2B monoubiquitination(H2Bub1)is mainly associated with transcriptional activation.Recent studies in yeast,humans,and Arabidopsis have revealed the conservation of chromatin modification via H2Bub1 during evolution.Rad6-Bre1 and their homologs are responsible for H2B monoubiquitination in diverse eukaryotic organisms,and the PAF complex is required for H2Bub1 to proceed.H2Bub1 is involved in many developmental processes in yeast,humans,and Arabidopsis,and it activates gene transcription by regulating the H3K4 methylation state.Notably,the level of H3K4 methylation is entirely dependent on H2Bub1 in yeast and humans,whereas the H3K4 methylation level of only a small number of genes in Arabidopsis is dependent on H2Bub1.In this review,we summarize the enzymes involved in H2B monoubiquitination and deubiquitination,and discuss the biologic functions of H2Bub1 in different organisms.In addition,we focus on recent advances in our understanding of the molecular mechanisms that enable H2Bub1 to perform its function.