Molecular cloning and characterization of a putative proline dehydrogenase gene in the Colorado potato beetle,Leptinotarsa decemlineata

Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate ofL. decemlineata during flight and proline dehydrogenase （ProDH） catalyzes the first step in proline catabolism. Here we identified a putative LdProDHgene; it had three cDNA isoforms which shared the same 5＇UTR and coding region, but differed in the lengths of 3＇UTRs （515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively）. LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDHwas expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH- dsRNA by adults significantly decreased its messenger RNA （mRNA） level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals （AATAAA） were tandemly located in the 3＇UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3＇UTR to control proline catabolism for the season-dependent activity ofL. decemlineata adults.     

Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.     

Cloning and Characterization of miRNAs and Their Targets,Including a Novel miRNA-Targeted NBS-LRR Protein Class Gene in Apple （Golden Delicious）

MicroRNA （miRNA） has emerged as an important regulator of gene expression in plants. 146 miRNAs were identified from apple （Malus domestica cv. Golden Delicious） by bioinformatic analysis and RNA library sequencing. From these, 135 were conserved and 11 were novel miRNAs. Target analysis predicted one of the novel miRNAs, Md-miRLn11 （Malus domestica microRNA Ln11）, targeted an apple nucleotide-binding site （NBS）-Ieucine-rich repeat （LRR） class protein coding gene （Md-NBS）. 5/ RACE assay confirmed the ability of Md-miRLn11 to cleave Md-NBS at the 11-12-nt position. Analysis of the expression of Md-miRLn11 and Md-NBS during the optimum invasion period in 40 apple varieties showed that the expression of Md-NBS gene in resistant varieties is higher than in susceptible varieties, with an inverse pattern for Md-miRLn11. Seedlings from the resistant apple variety ＇JiGuan＇ were used to carry out an Agrobacterium infiltration assay, and then inoculated with the apple leaf spot disease. The result showed a clear decline of disease resistance in JiGuan apples. In contrast, the susceptible variety ＇FuJi＇ infiltrated with the Md-NBS gene showed a significant increase in disease resistance. Based on the above results, we propose that Md-miRLn11 regulates Md-NBS gene expression in particular under the condition of pathogen infection, and that the Md-miRLn11 targeting P-loop site may regulate many NBS-LRR protein class genes in woody plants.     

Interferon-γ mRNA attenuates its own translation by activating PKR： A molecular basis for the therapeutic effect of interferon-β in multiple sclerosis

PKR, the interferon （IFN）-inducible protein kinase activated by double-stranded RNA, inhibits translation by phosphorylating the initiation factor eIF2α chain. Uniquely, human IFN-γ mRNA uses local activation of PKR in the cell to control its own translation yield. IFN-γ mRNA activates PKR through a structure in its 5＇- region harboring a pseudoknot which is critical for PKR activation. Mutations that impair pseudoknot stability reduce the ability of IFN-γ mRNA to activate PKR and strongly increase its translation efficiency. The cis-acting RNA element in IFN-γ mRNA functions as a biological sensor of intracellular PKR levels. During an immune response, as IFN-γ and other inflammatory cytokines build up in the cell＇s microenvironment, they act to induce higher levels of PKR in the cell, resulting in a more extensive activation of PKR by IFN-γ mRNA. With the resulting phosphorylation of eIF2α, a negative feedback loop is created and the production of IFN-γ is progressively attenuated. We propose that the therapeutic effect of IFN-β in multiple sclerosis may rest, at least in part, on its exquisite ability to induce high levels of PKR in the cell and thereby to limit IFN-γ mRNA translation through this negative feedback loop, blocking the excessive IFN-γ gene expression that precedes clinical attacks.     

Sequences near both termini of the C/EBPβ mRNA 3＇ untranslated region are important for its tumor suppression activity

The 3＇ untranslated region （3＇ UTR） of eukaryotic mRNA is an important regulation element that affects not only mRNA translation, but also cell growth. We had found that the 3＇ UTR of CCAAT-enhancerbinding protein β （C/EBPβ） mRNA had tumor suppression activity. Herein, we reported that deletion of two short sequences at both termini of the C/EBPβ 3t UTR reduced the tumor suppression activity of this 3＇ UTR, as demonstrated by reduced cell growth, colony formation ability, and tumorigenicity in nude mice. It is noteworthy that the only deletion of a single such sequence was enough for the reduction of tumor sup- pression effect, and the reducing effect of deletion of the sequence near 3r terminus was stronger. Therefore, specific short sequences in the C/EBPβ 3＇ UTR are crucial for the tumor suppression activity of C/EBPβ.     

Mitochondrial dysfunction in Parkinson＇s disease： a possible target for neuroprotection

Mitochondria are dynamic organelles which are required for maintaining cellular homeostasis. Thus, it is not surprising that irregularities in mitochondrial function result in cellular damage and are linked with neurodegenerative diseases, such as Parkinson＇s disease. Evidence that mitochondrial dysfunction is key to the pathogenesis of Parkinson＇s disease is founded in studies in post-mortem tissue from patients with Parkinson＇s disease, and also from genetic studies stemming from patients with familial Parkinson＇s disease. Whether triggered by environmental or genetic factors, mitochondrial dysfunction occurs early in the pathogenic process, and is central to Parkinson＇s disease pathology. As such, targeting the mitochondria to slow or halt disease progression is an attractive strategy for disease-modifying agents in Parkinson＇s disease. Indeed, several therapies which target the mitochondria have been investigated as neuroproteetive treatments for Parkinson＇s disease. This review will discuss the evidence supporting mitochondrial dysfunction in Parkinson＇s disease pathology as well as treatment strategies that target the mitochondria.     

Methylation Modifications in Eukaryotic Messenger RNA

RNA methylation modifications have been found for decades of years, which occur at different RNA types of numerous species, and their distribution is species-specific. However, people rarely know their biological functions. There are several identified methylation modifications in eukaryotic messenger RNA （mRNA）, such as NT-methylguanosine （mVG） at the cap, Nr-methyl-2＇-O-methyladenosine （m6Am）, 2＇-O-methylation （Nm） within the cap and the internal positions, and internal N6-methyladenosine （m6A） and 5-methylcytosine （mSC）. Among them, mTG cap was studied more clearly and found to have vital roles in several important mRNA processes like mRNA translation, stability and nuclear export, m6A as the most abundant modification in mRNA was found in the 1970s and has been proposed to function in mRNA splicing, translation, stability, transport and so on. mrA has been discovered as the first RNA reversible modification which is demethylated directly by human fat mass and obesity associated protein （FRO） and its homolog protein, alkylation repair ho- molog 5 （ALKBH5）. b-TO has a special demethylation mechanism that demethylases m6A to A through two over-oxidative intermediate states： N6-hydroxymethyladenosine （hm6A） and Nr-formyladenosine （frA）. The two newly discovered m6A demethylases, bTO and ALKBH5, significantly control energy homeostasis and spermatogenesis, respectively, indicating that the dynamic and reversible mrA, analogous to DNA and histone modifications, plays broad roles in biological kingdoms and brings us an emerging field ＂RNA Epige- netics＂. 5-methylcytosine （5mC） as an epigenetic mark in DNA has been studied widely, but mSC in mRNA is seldom explored. The bisulfide sequencing showed mSC is another abundant modification in mRNA, suggesting that it might be another RNA epigenetic mark. This review focuses on the main methylation modifications in mRNA to describe their formation, distribution, function and demethylation from the current knowledge and to provide future 19erspectives on functional studies.     

Parental RNA is Significantly Degraded During Arabidopsis Seed Germination

Germination is the first and maybe the foremost growth stage in the life cycle of a plant. Herein, we report that initiation of germination in the Arabidopsis Columbia ecotype was accompanied by a sharp decrease in the amount of extractable total RNA. At the beginning of our germination experiment, we were usually able to obtain 35-40 IJg total RNA from 100 mg dry seeds. However, after 3 d of cold stratification, we could only obtain less than 5 μg total RNA from the same amount of starting material. Young seedlings contained approximately 100 μg total RNA per 100 mg fresh tissue. Further studies showed that inhibition of de novo RNA synthesis by actinomycin D prevented the degradation of parental RNA and, in the meantime, significantly delayed the germination process. Several ribonuclease-like genes that were highly expressed in dry seeds, and especially during the cold stratification period, were discovered. We propose that these enzymes are involved in the regulation of parental RNA degradation. These results indicate that parental RNA metabolism may be an important process for Arabidopsis seed germination.     

Study on substrate specificity at subsites for severe acute respiratory syndrome coronavirus 3CL protease

Autocleavage assay and peptide-based cleavage assay were used to study the substrate specificity of 3CL protease from the severe acute respiratory syndrome coronavirus. It was found that the recognition between the enzyme and its substrates involved many positions in the substrate, at least including residues from P4 to P2＇. The deletion of either P4 or P2＇ residue in the substrate would decrease its cleavage efficiency dramatically. In contrast to the previous suggestion that only small residues in substrate could be accommodated to the S 1＇ subsite, we have found that bulky residues such as Tyr and Trp were also acceptable. In addition, based on both peptide-based assay and autocleavage assay, Ile at the PI＇ position could not be hydrolyzed, but the mutant L27A could hydrolyze the Ile peptide fragment. It suggested that there was a stereo hindrance between the S 1＇ subsite and the side chain of Ile in the substrate. All 20 amino acids except Pro could be the residue at the P2＇ position in the substrate, but the cleavage efficiencies were clearly different. The specificity information of the enzyme is helpful for potent anti-virus inhibitor design and useful for other coronavirus studies.     

Effects of 2＇-O-methyl nucleotide on ligation capability of T4 DNA ligase

To further understand the ligation mechanism, effects of 2＇-O-methyl nucleotide （2＇-OMeN） on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5＇- and 3＇-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ±4.0%, respectively. Substitutions at both 5＇- and 3＇-ends decreased the ligation efficiency by 76.6%±1.3%. Corresponding kinetic para- meters, Vmax, Kin, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2＇-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5＇- and 3＇-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3＇-end impairs the ligation efficiency more profoundly than that at 5＇-end. Different concentrations of Mg2＋, Ca2＋, K＋, Na＋, and ATP were also demonstrated to affect the T4 DNA ligase activ- ity. These results enriched our knowledge about the effects of 2＇-OMEN substitutions on the T4 DNA ligase.     

Iron homeostasis in plants： when transcription affects translocation

Despite the fact that iron is one of the most abundant elements of the earth＇s crust, iron deficiencies are serious problems both in human nutrition [ 1 ] and in agriculture [2]. Six to eight percent of the world＇s population is potentially affected by iron deficiency induced anemia, a leading cause of maternal death in African and Asian countries where people rely mostly on plants for their daily intake of iron. Iron can also be a limiting factor in the growth of economically important crop plants because of inadequate soil chemistry, and such deficiencies cannot easily be corrected by amending the soil. Improving the plant＇s ability to absorb iron in adverse conditions and to increase their overall content could offer solutions to these dramatic problems. Therefore understanding the molecular mechanisms regulating iron uptake and homeostasis in plants has potentially important practical applications both in agriculture and human health [3].     

Advances in chloroplast engineering

The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world＇s food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus： high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.     

NS5B与HCV负链RNA 3＇末端特异性结合的分析

NS5B是RNA依赖性RNA聚合酶,在病毒RNA合成过程中起到中心催化酶的作用.在大肠杆菌中表达和提纯了GST-NS5B融合蛋白,应用紫外交联试验(UVcross-linking)检测NS5B与丙型肝炎病毒(HCV)负链RNA3＇末端的结合,确定NS5B是否参与HCV负链RNA3＇末端复制体的形成.NS5B可与HCV负链RNA3＇末端发生结合,这种结合存在量效关系,比与正链RNA3＇UTRX区的结合强约10倍,超大量的非同源性RNA和蛋白质不能竞争抑制NS5B与负链RNA3＇末端的结合,证明这种结合存在特异性.结果提示NS5B是HCV负链RNA3＇末端复制体的成分之一.     

Editorial

Plants--of the botanical kind--are not often mentioned in ＇The State of the Union＇ addresses by US Presidents, but when they are, it can have global impacts on plant biology research. In the 1990s, President Clinton ridiculed government support of research on ＇plant stress＇ in one of his addresses, confusing psychological stress with the devastating effects of drought, salinity, and other environmental stresses on agricultural productivity. A decade later, President Bush mentioned the S word （switchgrass） in connection with alternatives to petroleum, thereby encouraging the US Department of Energy to invest heavily in research to understand plant cell walls and to develop efficient processes to harness the sun＇s energy, which is stored on a massive scale in plant cell walls.     

Effects of cations on small fragment of DNA polymerase I using a novel FRET assay

DNA polymerase I （Poll） digested by protease produces a small fragment （SF） containing 5~--~3~ exonuclease activity. The 5~-~3＇ exonuclease activity of poll cleaves the down- stream RNA primer strands during DNA replication in vivo. Previous in vitro studies suggested its capability of cleaving duplex from 5＇ terminal and a flap-structure-spe- cific endonuclease activity. From the crystal structures of other nucleases and biochemical data, a two-metal-ion mechanism has been proposed but has not been deter- mined. In this study, we cloned, expressed, and purified the SF protein, and established a novel fluorescence resonance energy transfer （FRET） assay to analyze the catalytic activ- ity of the SF protein. The effects of several metal ions on its catalytic capability were analyzed using this FRET assay. Results showed that Mg2＋, Mn2＋, and Zn2＋ were able to activate the cleavage of SF, while Ca2＋, Ni2＋, and Co2＋ were not suitable for SF catalysis. The effects of K＋, Na＋, and dNTP were also determined.