Plant ubiquitin-proteasome pathway and its role in gibberellin signaling

The ubiquitin-proteasome system (UPS) in plants, like in other eukaryotes, targets numerous intracellular regulators and thus modulates almost every aspect of growth and development. The well-known and best-characterized outcome of ubiquitination is mediating target protein degradation via the 26S proteasome, which represents the major selective protein degradation pathway conserved among eukaryotes. In this review, we will discuss the molecular composition, regulation and function of plant UPS, with a major focus on how DELLA protein degradation acts as a key in gibberellin signal transduction and its implication in the regulation of plant growth.     

Maturation and germination of walnut somatic embryos

Walnut somatic embryos were multiplied by repetitive embryogenesis on a solid basal DKW medium at 25°C in the dark. When the embryos were isolated at early cotyledonary stage (1–2 mm long) from the primary embryos and cultured on the medium for 3 weeks, they developed into mature embryos showing white, enlarged cotyledons and shoot and root apex. After transfer to light on solid germination medium, however, few mature embryos (0–5%) germinated. Germination percentage increased to about 10% when the mature embryos were pretreated by a storage at 4°C in the dark for 2 months, or by desiccation at 25°C in the dark for 3 or 5 days under an air-humidity conditioned by saturated salt solutions (Mg(NO₃)₂.6H₂O, or ZnSO₄.7H₂O). Similar results were obtained by the addition of gibberellic acid (GA₃) to the germination medium. When mature embryos were desiccated and then placed on medical cotton compresses in liquid germination medium, 45% of the embryos germinated into complete plantlets. These plantlets continued their growth after transplanting to a mixture of peat and vermiculite in pots.Abbreviations GA₃ gibberellic acid - DKW medium Driver & Kuniyuki Walnut medium     

Biphasic transitions of a hairpin hexanucleotide triplex DNA

The conformational transitions (helix-coil transitions) of three hairpin triple helices, models 5'-(A-G)(3) + 5'-(T-C)(3)-T(4)-((br)C-T)(3) [CY], 5'-(A-G)(3) + 5'-(T-(br)C)(3)-T(4)-(C-T)(3) [YC] and 5'-(A-G)(3) + 5'-(T-(br)C)(3)-T(4)-((br)C-T)(3) [YY], are characterized in this work by UV spectroscopy. Melting of these triplexes is biphasic, and the profiles are used to obtain the thermodynamic parameters. The thermodynamic properties of the hairpin triplex are T(m) = 19.45 degrees C and DeltaH(vH) = 293.12 kJ mol(-1) for CY, T(m) = 22.85 degrees C and DeltaH(vH) = 256.63 kJ mol(-1) for YC and T(m) = 28.47 degrees C and DeltaH(vH) = 234.68 kJ mol(-1) for YY at pH 4.4. Those of the duplex are T(m) = 30.50 degrees C and DeltaH(vH) = 427.09 kJ mol(-1) for CY, T(m) = 32.96 degrees C and DeltaH(vH) = 374.47 kJ mol(-1) for YC and T(m) = 33.24 degrees C and DeltaH(vH) = 329.67 kJ mol(-1) for YY at pH 4.4. The distinct transitions of triplex to duplex and duplex to single strands are analyzed using the nearest-neighbor Ising model. Electrostatic effects on each conformation are also analyzed.     

克隆鸭乙型肝炎病毒DNA双体体内转染的研究

用一种含头尾相连DHBVDNA双体的质粒体内转染2日龄芙蓉鸭,大多数鸭（86％）产生了短暂病毒血症。血清DHBs／preSAg和DHBVDNA于转染后第9天出现,第12～14天达峰值,第28天时多数转阴;少数鸭的病毒血症可持续50天以上。转染鸭肝组织中也检测到复制中间型DHBVDNA的存在。用转染鸭病毒血症期的血清作磷钨酸负染电镜观察,找到了完整的DHBV病毒颗粒,并且用此血清腹腔注射1日龄鸭,60％的鸭被感染成功,证明体内转染后有生物活性的DHBV病毒颗粒的产生。该研究方法的建立．对于研究DHBV变异株．DHBV基因结构与功能的关系等,均有一定理论意义及应用价值。     

Paradoxical effects of a stress signal on pro- and anti-apoptotic machinery in HTLV-1 tax expressing cells

Adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) are associated with Human T-cell lymphotropic virus type 1 (HTLV-1) infection. The viral transactivator, Tax is able to mediate the cell cycle progression by targeting key regulators of the cell cycle such as p21/waf1, p16/ink4a, p53, cyclins D1-3/cdk complexes, and the mitotic spindle checkpoint MAD apparatus, thereby deregulating cellular DNA damage and checkpoint control. Genome expression profiling of infected cells exemplified by the development of DNA microarrays represents a major advance in genome-wide functional analysis. Utilizing cDNA microarray analysis, we have observed an apparent opposing and paradoxical regulatory network of host cell gene expression upon the introduction of DNA damage stress signal. We find the apparent induction of cell cycle inhibitors, and pro- as well as anti-apoptotic gene expression is directly linked to whether cells are at either G1, S, or G2/M phases of the cell cycle. Specifically, a G1/S block is induced by p21/waf1 and p16/ink4a, while pro-apoptotic expression at S, and G2/M is associated with caspase activation, and anti-apoptotic gene expression is associated with up regulation of Bcl-2 family member, namely bfl-1 gene. Therefore, the microarray results indicating expression of both pro- and anti-apoptotic genes could easily be explained by the particular stage of the cell cycle. Mechanism and the functional outcome of induction for both pathways are discussed.     

High level soluble expression and one-step purification of IBDV VP2 protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To improve the expression of soluble IBDV VP2 protein by using different tagged vectors in Escherichia coli.

Results

Fusion tags, Grifin, MBP, SUMO, thioredoxin, γ-crystallin, ArsC and PpiB, enhanced the expression and solubility of VP2 protein. The fusion proteins were purified by Ni–NTA chromatography, MBP-VP2 showed the highest purity about 90 %. After removing the MBP tag, VP2 self-assembled into virus-like particles, ~25 nm diam. Results from AGP suggested the recombinant IBDV VP2 protein identified by reference serum like IBDV.

Conclusion

All the seven tags enhanced the expression and solubility of IBDV VP2 protein. The recombinant protein self-assembly into virus like particles and possess antigenicity as reference IBDV.

     

Detection and correction of assembly errors of rice Nipponbare reference sequence

A complete and high‐quality genome reference sequence of an organism provides a solid foundation for a wide research community and determines the outcomes of relevant genomic, genetic, molecular and evolutionary research. Rice is an important food crop and a model plant for grasses, and therefore was the first chosen crop plant for whole genome sequencing. The genome of the japonica representative rice variety, Nipponbare, was sequenced using a gold standard, map‐based clone‐by‐clone strategy. However, although the Nipponbare reference sequence (RefSeq) has the best quality for existing crop genome sequences, it still contains many assembly errors and gaps. To improve the Nipponbare RefSeq, first a robust method is required to detect the hidden assembly errors. Through alignments between BAC‐end sequences (BESs) embedded in the Nipponbare bacterial artificial chromosome (BAC) physical map and the Nipponbare RefSeq, we detected locations on the Nipponbare RefSeq that were inversely matched with BESs and could therefore be candidates for spurious inversions of assembly. We performed further analysis of five potential locations and confirmed assembly errors at those locations; four of them, two on chr4 and two on chr11 of the Nipponbare RefSeq (IRGSP build 5), were found to be caused by reverse repetitive sequences flanking the locations. Our approach is effective in detecting spurious inversions in the Nipponbare RefSeq and can be applied for improving the sequence qualities of other genomes as well.