水稻花粉[肌动蛋白]抑制蛋白基因的克隆和表达分析

[肌动蛋白]抑制蛋白（profilin）是一种低分子量、与肌动蛋白结构的蛋白质。通过筛选水稻成熟花粉的cDNA文库,获得了两个全长cDNA片段,序列分析结果表明,两个cDNA片段长度分别为821bp和805bp;共同拥有一个由131个氨基酸组成的开放密码框、5′末端翻译区和一个带有poly(A)的3′区域。[肌动蛋白]抑制蛋白与玉米、C.dactylon、H.brasiliensis、P.pratense中的该蛋白质的同源性分别为89％、87％、83％、89％。Southern杂交分析显示,在基因组至少有两个基因存在。Northern杂交和RT-PCR结果显示它在花粉和花粉中特异表达。     

前纤维蛋白影响花粉肌动蛋白体外聚合分析

利用超速离心沉淀及紫外分光光度测定等技术 ,研究了不同比例的玉米 (ZeamaysL .)花粉内源前纤维蛋白对玉米 (ZeamaysL .)花粉肌动蛋白 (前纤维蛋白与肌动蛋白摩尔数比分别为 2∶1,1.5∶1,1∶1,0 .5∶1,0 .1∶1)聚合与解聚的影响。初步实验结果显示 ,前纤维蛋白在各种比例下均可与Mg_ATP_肌动蛋白结合并抑制肌动蛋白的聚合作用。这种抑制作用随着前纤维蛋白比例的增加而增大。其解离常数值 (Kd)为 (1.30± 0 .33) μmol/L。在本实验条件下尚未见到有前纤维蛋白促进植物肌动蛋白聚合的作用 ,表明玉米花粉前纤维蛋白具有螯合G_肌动蛋白的作用。     

纤维蛋白影响花粉肌动蛋白体外聚合分析

The effects of different ratio of native profilin on maize (Zea mays L.) pollen actin polymerization in vitro were analyzed by using ultracentrifuging sedimentation and ultraviolet absorption spectrum measurement (the molar ratio of profilin to actin was 2∶1, 1.5∶1, 1∶1, 0.5∶1, 0.1∶1 respectively). Preliminary results showed that profilin bound to G-actin and inhibited its polymerization. The inhibition of actin polymerization by profilin increased with the increasing ratio of profilin to pollen actin. The dissociation constant （Kd） value of profilin for binding to actin was (1.30±0.33) μmol/L. No stimulation effect of profilin on actin polymerization was observed, suggesting that pollen profilin may affect actin organization by sequestering the G-actin.     

RNA原位杂交技术的一些应用技巧

目的:检测基因在动物组织或细胞中的时空表达模式。方法:转录反义RNA探针;利用RNA原位杂交技术检测人和小鼠牙原基中若干基因的表达。结果与结论:通过优化条件,转录出完整的反义RNA探针,并成功地利用RNA原位杂交技术在组织中检测到基因的表达;分析了一些在RNA原位杂交的过程中可能碰到的问题及其解决方法。     

Structural basis for the recruitment of profilin-actin complexes during filament elongation by Ena/VASP

Cells sustain high rates of actin filament elongation by maintaining a large pool of actin monomers above the critical concentration for polymerization. Profilin-actin complexes constitute the largest fraction of polymerization-competent actin monomers. Filament elongation factors such as Ena/VASP and formin catalyze the transition of profilin-actin from the cellular pool onto the barbed end of growing filaments. The molecular bases of this process are poorly understood. Here we present structural and energetic evidence for two consecutive steps of the elongation mechanism: the recruitment of profilin-actin by the last poly-Pro segment of vasodilator-stimulated phosphoprotein (VASP) and the binding of profilin-actin simultaneously to this poly-Pro and to the G-actin-binding (GAB) domain of VASP. The actin monomer bound at the GAB domain is proposed to be in position to join the barbed end of the growing filament concurrently with the release of profilin.     

Arabidopsis group Ie formins localize to specific cell membrane domains, interact with actin-binding proteins and cause defects in cell expansion upon aberrant expression

The closely related proteins AtFH4 and AtFH8 represent the group Ie clade of Arabidopsis formin homologues. The subcellular localization of these proteins and their ability to affect the actin cytoskeleton were examined. AtFH4 protein activity was identified using fluorimetric techniques. Interactions between Arabidopsis profilin isoforms and AtFH4 were assayed in vitro and in vivo using pull-down assays and yeast-2-hybrid. The subcellular localization of group Ie formins was observed with indirect immunofluorescence (AtFH4) and an ethanol-inducible green fluorescent protein (GFP) fusion construct (AtFH8). AtFH4 protein affected actin dynamics in vitro, and yeast-2-hybrid assays suggested isoform-specific interactions with the actin-binding protein profilin in vivo. Indirect immunofluorescence showed that AtFH4 localized specifically to the cell membrane at borders between adjoining cells. Expression of an AtFH8 fusion protein resulted in GFP localization to cell membrane zones, similar to AtFH4. Furthermore, aberrant expression of AtFH8 resulted in the inhibition of root hair elongation. Taken together, these data suggest that the group Ie formins act with profilin to regulate actin polymerization at specific sites associated with the cell membrane.     

Differential expression of mRNAs for sialyltransferase isoenzymes induced in the hippocampus of mouse following kindled seizures

Sialic acids play important roles in various biological functions. In the brain, evidence suggests that sialylation of glycoproteins and glycolipids affects neural plasticity. While the 18 sialyltransferase isoenzymes (STs) identified to date synthesize individual sialyl-oligosaccharide structures, they each exhibit activity toward more than one substrate and can overlap in their specificity. Therefore, the distribution of STs is a secondary factor in the study of specific sialylation. Here, seven STs; ST3Gal I-IV, ST8Sia IV, ST6Gal I and ST6GalNAc II, the expressions of which were identified in the adult hippocampus by RT-PCR, showed diverse localization patterns in the hippocampus on in situ hybridization, suggesting that the individual cells expressed relevant STS: Furthermore, to assay activity-related changes in ST expression, we used amygdaloid-kindling among models of neural plasticity. Differential expression of the STs participating in the kindling, notably, up-regulation of ST3Gal IV and ST6GalNAc II mRNAs, and down-regulation of ST3Gal I and ST8Sia IV mRNAs, were observed in the hippocampus following kindled seizures. These results indicate that ST expressions are regulated by physiological activity and may play a role in neural plasticity.     

ATP和钾离子对肌动蛋白与前纤维蛋白结合的影响及其在植物肌动蛋白纯化中的应用

根据ATP和钾离子影响肌动蛋白聚合及肌动蛋白及肌动蛋白与前纤维蛋白结合的实验结果,通过对以往报道的利用多聚脯氨酸-琼脂糖亲和柱层析法纯化植物肌动蛋白过程中ATP及钾离子浓度的改变,不需要加源前纤维蛋白。可得到较大量高纯度具活性的花粉肌动蛋白。SDS凝胶电泳、免疫印迹、电镜负染及紫外检测表明,所得肌动蛋白在纯度、聚合特性等方面均与原方法所得结果相同,肌动蛋白产率为原方法的73.5%,而整个肌动蛋白提纯过程所需时间缩短了1/3,节省了纯化重组前纤维蛋白所需的费用,消除了外源前纤维蛋白的使用对一些实验室的限制,而且同时得到了大量纯化的花粉前纤维蛋白。     

Two proteins from Saccharomyces cerevisiae: Pfy1 and Pkc1, play a dual role in activating actin polymerization and in increasing cell viability in the adaptive response to oxidative stress

In this work, we show that the proteins Pkc1 and Pfy1 play a role in the repolarization of the actin cytoskeleton and in cell survival in response to oxidative stress. We have also developed an assay to determine the actin polymerization capacity of total protein extracts using fluorescence recovery after photobleaching techniques and actin purified from rabbit muscle. This assay allowed us to demonstrate that Pfy1 promotes actin polymerization under conditions of oxidative stress, while Pkc1 induces actin polymerization and cell survival under all the conditions tested. Our assay also points to a relationship between Pkc1 and Pfy1 in the actin cytoskeleton polymerization that is required to adapt to oxidative stress.     

WH2 and proline‐rich domains of WASP‐family proteins collaborate to accelerate actin filament elongation

WASP‐family proteins are known to promote assembly of branched actin networks by stimulating the filament‐nucleating activity of the Arp2/3 complex. Here, we show that WASP‐family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP‐family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline‐rich sequence that binds profilin–actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline‐rich sequences are required to support polymerase activity by (i) bringing polymerization‐competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin–actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP‐family proteins that create it. Collaboration between WH2 and proline‐rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP‐family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation.     

人DDX36和小鼠Ddx36基因在成年睾丸组织中的表达研究

果蝇是结构基因组学和功能基因组学研究的最为理想的一种模式生物,采用同源克隆的策略,应用生物信息学分析和实验技术相结合的方法分别从人和小鼠中克隆了同源于果蝇MLE蛋白的新基因DDX36和Ddx36。为进一步研究DDX36和Ddx36基因与精子发生的关系,再应用Northrn blotting,RT-PCR和组织原位杂交技术探讨了DDX36和Ddx36基因的表达情况,结果发现人DDX36和小鼠Ddx36基因在成年睾丸组织中高表达。初步证明DDX36和Ddx36基因在精子发生中亦可能发挥重要作用。     

Region-specific and epileptogenic-dependent expression of six subtypes of alpha2,3-sialyltransferase in the adult mouse brain

Sialylated glycoconjugates play important roles in various biological functions. The structures are also observed in brains and it has been proposed that sialylation may affect neural plasticity. To clarify the effects of sialylation in the brain, particular neurons that exhibit sialylation should first be determined. Using in situ hybridization, we performed systematic surveys of the localization of mRNAs encoding the six alpha2,3-sialyltransferases (ST3Gal I-VI) in the adult mouse brain with or without physiological stimulation. First, striking region-specific patterns of expression were observed: While ST3Gal II, III, and V mRNAs were in neuronal cells throughout the brain, ST3Gal I, IV, and VI mRNAs were in restricted brain regions. Next, to assess whether the expression of the six mRNAs can be regulated, we examined the effect of kindling epileptogenesis on the six mRNA levels. Of the six subtypes, upregulation in the ST3Gal IV level in the thalamus was most pronounced; the number of ST3Gal IV-expressing neurons in the anterior thalamic nuclei increased from 2% to 21% in a time-dependent manner during epileptogenesis. Western blot analysis evaluated the increase of the end-products in the thalamus. These findings provide a molecular basis to clarify when and where sialylated glycoconjugates function accompanied by neural plasticity.     

High resolution distribution of mRNA within the cytoskeleton

It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this research was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems involved. This work required the development of novel in situ hybridization methods for use with electron microscopy.