LPA2蛋白调控PSII组装的分子机理研究

光系统II（PSII）是叶绿体类囊体膜上电子传递链中第一个色素蛋白复合体，由20多个蛋白亚基组成。它催化光驱动的水的裂解和醌的氧化。由于其结构的复杂性，PSII的生物发生和组装是核基因与叶绿体基因编码的蛋白以一定次序多步骤合成、组装的复杂过程，并需要大量的核基因编码的调节组装因子的参与。分离、鉴定拟南芥中这些核基因编码的叶绿体蛋白并研究它们的作用机制有助于我们认识高等植物PSII复合物组装和功能调控的分子机理。因此，我们从T-DNA插入的拟南芥突变体库中筛选到PSII突变体lpa2（low photosystemII accumulation），对LPA2蛋白调控光系统II复合物组装的功能进行了研究，并进一步探讨了LPA2和其他调节因子协同作用参与PSII组装的模式。 突变体lpa2具有高叶绿素荧光表型，与野生型相比生长量、色素含量均显著降低。蛋白免疫印记发现在lpa2突变体中光系统II复合物的累积量明显降低，仅有野生型的30%左右，而其他复合物的含量变化不大。核酸杂交和与多聚核糖体结合的检测表明光系统II亚基在转录及翻译启始水平没有受到影响。拟南芥叶片蛋白标记实验证明在突变体中CP43的合成量明显降低而其他光系统II主要蛋白CP47, D1 和 D2的合成正常，但相对于野生型这些蛋白的周转速率加快。在突变体中，新合成的蛋白亚基可以组装进入光系统II复合物，但新合成的CP43蛋白组装效率降低。以上的结果表明LPA2对光系统II的正常组装起着重要的作用，LPA2的缺失导致CP43不能有效组装进入光系统II，从而引起其他核心蛋白周转加快，光系统 II复合物累积量降低，最终植株光合效率降低。 基因克隆和蛋白定位分析表明LPA2基因编码一个内在的类囊体膜蛋白，但并不是光系统II的亚基组分。进一步采用酵母双杂分析证实了LPA2蛋白与光系统II核心蛋白CP43有相互作用，而与中心蛋白D1和D2没有相互作用。此外实验还表明LPA2蛋白与参与类囊体膜生物发生有关的Alb3蛋白有相互作用。因此LPA2可能是与Alb3形成复合物来协助CP43有效的整合进入光系统II。 另外，我们实验室已鉴定，LPA3，LPA4也是分别特异地参与CP43和D1组装的光系统II分子伴侣。LPA2，LPA3基因共同缺失会使幼苗不能光合自养而致死，因而LPA2和LPA3共同相互作用促进CP43的组装。体内和体外实验证明LPA2，LPA3和LPA4都和Alb3相互作用，而参与D1组装的分子伴侣LPA1不和Alb3以及上述这些伴侣因子作用。因此，Alb3 很有可能与LPA2、LPA3和LPA4形成多蛋白复合物在D1蛋白合成之后的组装过程中起作用。这些结果表明光系统II多亚基复合物组装是多步骤的，并通过一个精确复杂的调控网络确保复合物的有效组装以及功能行使。

蓝藻谷氨酰胺酶的酶学特征及生理功能研究

谷氨酰胺酶是催化谷氨酰胺分解为谷氨酸的氨基酸水解酶。它广泛存在于真核生物和原核生物中，在许多微生物和哺乳动物的氮代谢过程中起重要作用。但蓝藻中谷氨酰胺酶的酶学特征及生理功能尚不清楚，仅在一些蓝藻的基因组中发现有假定谷氨酰胺酶基因，这些基因编码的未知功能蛋白中有谷氨酰胺酶功能结构域，如集胞藻6803基因组中的slr2079基因。因此，本研究以模式蓝藻集胞藻6803 为研究对象，研究蓝藻谷氨酰胺酶的酶学特征及其生理功能。 为研究蓝藻谷氨酰胺酶的酶学特征，本研究克隆了集胞藻6803 slr2079基因，并在大肠杆菌中融合表达，经Ni-NTA亲合柱纯化后，通过对重组蛋白进行酶活测定及动力学分析，发现Slr2079蛋白是以谷氨酰胺为唯一催化底物的谷氨酰胺酶。 重组酶Slr2079的最适反应pH为9;最适反应温度为37C - 42C。该酶和绝大多数微生物源性的谷氨酰胺酶一样均为非磷酸依赖型。有趣的是该酶活性受Na+调节，而这种调节是通过提高对底物的亲和力来实现的。 为研究蓝藻谷氨酰胺酶在细胞内的生理功能，本研究通过基因插入失活，构建了缺失slr2079基因的集胞藻6803突变体，并对其进行生理、生化研究。在正常生长条件下，突变体和野生型蓝藻的生长未见差异，表明该基因不是集胞藻6803生长所必需的基因。但在700 mM NaCl胁迫条件下，突变体的生长速率比野生型快1.25倍。半定量RT-PCR结果显示，几个盐胁迫相关基因在突变体与野生型中的表达有所不同：与耐受盐胁迫的相关基因slr1608 (gdhB) 和slr1751 (prc)在突变体中表达提高，而盐敏感的基因sll0262 (desD) 和 slr0213 (guaA)在突变体中表达下降。由于重组的Slr2079具有谷氨酰胺酶活性，因此我们试图通过检测在蓝藻中参与氨同化作用的关键酶谷氨酸合成酶和谷氨酰胺合成酶在集胞藻6803中的表达情况来揭示Slr2079在集胞藻6803谷氨酰胺代谢中的生理功能。半定量RT-PCR结果显示，仅谷氨酸合成酶在突变体中表达提高，而谷氨酰胺合成酶表达未见明显变化。这些研究结果表明，在集胞藻6803中，Slr2079可能是通过调节与盐胁迫相关基因的表达来参与应对盐胁迫，而在氮代谢中起次要作用。

Maximin 9, a novel free thiol containing antimicrobial peptide with antimycoplasma activity from frog Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides, like maximins and maximin Hs from frog Bombina maxima. Novel cDNA clones encoding a precursor protein, which comprises a novel maximin peptide (maximin 9) and reported maximin H3, were isolated from two constructed skin cDNA libraries of B. maxima. The predicted primary structure of maximin 9 is GIGRKFLGGVKTTFRCGVKDFASKHLY-NH2. A surprising substitution is at position 16, with a free cysteine in maximin 9 rather than usual conserved glycine in other reported maximins. Maximin 9, the homodimer form and its Cys(16) to Gly(16) mutant were synthesized and their antimicrobial activities were evaluated. Unlike previously reported maximin 3, the tested bacterial and fungal strains were resistant to maximin 9, its homodimer and the Cys(16) to Gly(16) mutant (with MICs > 100 mu M). On the other hand, interestingly, while eight clinical Mollicutes strains were generally resistant to maximin 9 homodimer and its Cys(16) to Gly(16) mutant, most of them are sensitive to maximin 9 at a peptide concentration of 30 mu M, especially in the presence of dithiothreitol. These results indicate that the presence of a reactive Cys residue in maximin 9 is important for its antimycoplasma activity. The diversity of antimicrobial peptide cDNA structures encountered in B. maxima skin cDNA libraries and the antimicrobial specificity differences of the peptides may reflect well the species' adaptation to the unique microbial environments. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs.

Role of RNA polymerase IV in plant small RNA metabolism.

In addition to the three RNA polymerases (RNAP I-III) shared by all eukaryotic organisms, plant genomes encode a fourth RNAP (RNAP IV) that appears to be specialized in the production of siRNAs. Available data support a model in which dsRNAs are generated by RNAP IV and RNA-dependent RNAP 2 (RDR2) and processed by DICER (DCL) enzymes into 21- to 24-nt siRNAs, which are associated with different ARGONAUTE (AGO) proteins for transcriptional or posttranscriptional gene silencing. However, it is not yet clear what fraction of genomic siRNA production is RNAP IV-dependent, and to what extent these siRNAs are preferentially processed by certain DCL(s) or associated with specific AGOs for distinct downstream functions. To address these questions on a genome-wide scale, we sequenced approximately 335,000 siRNAs from wild-type and RNAP IV mutant Arabidopsis plants by using 454 technology. The results show that RNAP IV is required for the production of >90% of all siRNAs, which are faithfully produced from a discrete set of genomic loci. Comparisons of these siRNAs with those accumulated in rdr2 and dcl2 dcl3 dcl4 and those associated with AGO1 and AGO4 provide important information regarding the processing, channeling, and functions of plant siRNAs. We also describe a class of RNAP IV-independent siRNAs produced from endogenous single-stranded hairpin RNA precursors.

Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana.

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent--disruption of one class does not affect the other--suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.

RNAi, DRD1, and histone methylation actively target developmentally important Non-CG DNA methylation in Arabidopsis

Cytosine DNA methylation protects eukaryotic genomes by silencing transposons and harmful DNAs, but also regulates gene expression during normal development. Loss of CG methylation in the Arabidopsis thaliana met1 and ddm1 mutants causes varied and stochastic developmental defects that are often inherited independently of the original met1 or ddm1 mutation. Loss of non-CG methylation in plants with combined mutations in the DRM and CMT3 genes also causes a suite of developmental defects. We show here that the pleiotropic developmental defects of drm1 drm2 cmt3 triple mutant plants are fully recessive, and unlike phenotypes caused by met1 and ddm1, are not inherited independently of the drm and cmt3 mutations. Developmental phenotypes are also reversed when drm1 drm2 cmt3 plants are transformed with DRM2 or CMT3, implying that non-CG DNA methylation is efficiently re-established by sequence-specific signals. We provide evidence that these signals include RNA silencing though the 24-nucleotide short interfering RNA (siRNA) pathway as well as histone H3K9 methylation, both of which converge on the putative chromatin-remodeling protein DRD1. These signals act in at least three partially intersecting pathways that control the locus-specific patterning of non-CG methylation by the DRM2 and CMT3 methyltransferases. Our results suggest that non-CG DNA methylation that is inherited via a network of persistent targeting signals has been co-opted to regulate developmentally important genes. © 2006 Chan et al.

Molecular genetic basis for the rhino mouse from Chinese KM subcolony

Both the rhino mouse and hairless mouse resulted from hairless gene mutation, but they show different phenotypes of skin physiology. The rhino mouse has more similar histological characters to human papular alopecia. Therefore rhino mouse is a good experimental animal model for human papular alopecia. This study reports a hairless mouse named rhino KIZ, arose from KM colony in Kunming Institue of Zoology, by systematic studies on morphology, skin histopathology, gene sequence, pedigree and protein domain analysis. The results demonstrate that a C-to-T transition in exon 11 of hr gene (The mutant gene has been applied for a Chinese patent (patent No. 03135280)) results in the rhino KIZ. The rhino KIZ with clear genetic mechanism will be a useful animal model.

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCSM((120-123)), TCSE160A/E189A, with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCSR122G, which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The bacterial cytoskeleton modulates motility, type 3 secretion, and colonization in Salmonella.

Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC.

EARLY FLOWERING4 recruitment of EARLY FLOWERING3 in the nucleus sustains the Arabidopsis circadian clock

The plant circadian clock is proposed to be a network of several interconnected feedback loops, and loss of any component leads to changes in oscillator speed. We previously reported that Arabidopsis thaliana EARLY FLOWERING4 (ELF4) is required to sustain this oscillator and that the elf4 mutant is arrhythmic. This phenotype is shared with both elf3 and lux. Here, we show that overexpression of either ELF3 or LUX ARRHYTHMO (LUX) complements the elf4 mutant phenotype. Furthermore, ELF4 causes ELF3 to form foci in the nucleus. We used expression data to direct a mathematical position of ELF3 in the clock network. This revealed direct effects on the morning clock gene PRR9, and we determined association of ELF3 to a conserved region of the PRR9 promoter. A cis-element in this region was suggestive of ELF3 recruitment by the transcription factor LUX, consistent with both ELF3 and LUX acting genetically downstream of ELF4. Taken together, using integrated approaches, we identified ELF4/ELF3 together with LUX to be pivotal for sustenance of plant circadian rhythms. © 2012 American Society of Plant Biologists.

The application of STEM and in situ controlled dehydration to bacterial systems using ESEM.

Transmission imaging with an environmental scanning electron microscope (ESEM) (Wet STEM) is a recent development in the field of electron microscopy, combining the simple preparation inherent to ESEM work with an alternate form of contrast available through a STEM detector. Because the technique is relatively new, there is little information available on how best to apply this technique and which samples it is best suited for. This work is a description of the sample preparation and microscopy employed by the authors for imaging bacteria with Wet STEM (scanning transmission electron microscopy). Three different bacterial samples will be presented in this study: first, used as a model system, is Escherichia coli for which the contrast mechanisms of STEM are demonstrated along with the visual effects of a dehydration-induced collapse. This collapse, although clearly in some sense artifactual, is thought to lead to structurally meaningful morphological information. Second, Wet STEM is applied to two distinct bacterial systems to demonstrate the novel types of information accessible by this approach: the plastic-producing Cupriavidus necator along with wild-type and ΔmreC knockout mutants of Salmonella enterica serovar Typhimurium. Cupriavidus necator is shown to exhibit clear internal differences between bacteria with and without plastic granules, while the ΔmreC mutant of S. Typhimurium has an internal morphology distinct from that of the wild type.

The application of STEM and in situ controlled dehydration to bacterial systems using ESEM

Transmission imaging with an environmental scanning electron microscope (ESEM) (Wet STEM) is a recent development in the field of electron microscopy, combining the simple preparation inherent to ESEM work with an alternate form of contrast available through a STEM detector. Because the technique is relatively new, there is little information available on how best to apply this technique and which samples it is best suited for. This work is a description of the sample preparation and microscopy employed by the authors for imaging bacteria with Wet STEM (scanning transmission electron microscopy). Three different bacterial samples will be presented in this study: first, used as a model system, is Escherichia coli for which the contrast mechanisms of STEM are demonstrated along with the visual effects of a dehydration-induced collapse. This collapse, although clearly in some sense artifactual, is thought to lead to structurally meaningful morphological information. Second, Wet STEM is applied to two distinct bacterial systems to demonstrate the novel types of information accessible by this approach: the plastic-producing Cupriavidus necator along with wild-type and δmreC knockout mutants of Salmonella enterica serovar Typhimurium. Cupriavidus necator is shown to exhibit clear internal differences between bacteria with and without plastic granules, while the δmreC mutant of S. Typhimurium has an internal morphology distinct from that of the wild type. © 2012 Wiley Periodicals, Inc.

Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities.

Cyanobacteria perform photosynthesis and respiration in the thylakoid membrane, suggesting that the two processes are interlinked. However, the role of the respiratory electron transfer chain under natural environmental conditions has not been established. Through targeted gene disruption, mutants of Synechocystis sp. PCC 6803 were generated that lacked combinations of the three terminal oxidases: the thylakoid membrane-localized cytochrome c oxidase (COX) and quinol oxidase (Cyd) and the cytoplasmic membrane-localized alternative respiratory terminal oxidase. All strains demonstrated similar growth under continuous moderate or high light or 12-h moderate-light/dark square-wave cycles. However, under 12-h high-light/dark square-wave cycles, the COX/Cyd mutant displayed impaired growth and was completely photobleached after approximately 2 d. In contrast, use of sinusoidal light/dark cycles to simulate natural diurnal conditions resulted in little photobleaching, although growth was slower. Under high-light/dark square-wave cycles, the COX/Cyd mutant suffered a significant loss of photosynthetic efficiency during dark periods, a greater level of oxidative stress, and reduced glycogen degradation compared with the wild type. The mutant was susceptible to photoinhibition under pulsing but not constant light. These findings confirm a role for thylakoid-localized terminal oxidases in efficient dark respiration, reduction of oxidative stress, and accommodation of sudden light changes, demonstrating the strong selective pressure to maintain linked photosynthetic and respiratory electron chains within the thylakoid membrane. To our knowledge, this study is the first to report a phenotypic difference in growth between terminal oxidase mutants and wild-type cells and highlights the need to examine mutant phenotypes under a range of conditions.

Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2.

Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.