Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturation

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA coding for tobacco profilin, which shared an average sequence identity of 75% with other plant profilins, was isolated from a tobacco pollen cDNA library by antibody screening. Tobacco profilin was expressed in Escherichia coli and purified by affinity to poly-(L-proline) Sepharose. A rabbit antiserum was raised against recombinant tobacco profilin and used to estimate the amount of profilin expressed in different tobacco tissues. Profilin can be detected in different somatic tissues, but the expression is 50–100 fold higher in mature pollen. Immunofluorescence and confocal laser scanning microscopy showed a homogeneous distribution of profilin in the cytoplasm of in vitro cultured pollen grains and pollen tubes of tobacco whereas some growing pollen tubes were stained more intensively a their tip. A possible role of pollen profilin as a developmentally upregulated microfilament precursor in mature pollen is discussed.     

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。     

Arabidopsis ACT11 modifies actin turnover to promote pollen germination and maintain the normal rate of tube growth

Actin is an ancient conserved protein that is encoded by multiple isovariants in multicellular organisms. There are eight functional actin genes in the Arabidopsis genome, and the precise function and mechanism of action of each isovariant remain poorly understood. Here, we report the characterization of ACT11, a reproductive actin isovariant. Our studies reveal that loss of function of ACT11 causes a delay in pollen germination, but enhances pollen tube growth. Cytological analysis revealed that the amount of filamentous actin decreased, and the rate of actin turnover increased in act11 pollen. Convergence of actin filaments upon the germination aperture was impaired in act11 pollen, consistent with the observed delay of germination. Reduction of actin dynamics with jasplakinolide suppressed the germination and tube growth phenotypes in act11 pollen, suggesting that the underlying mechanisms involve an increase in actin dynamics. Thus, we demonstrate that ACT11 is required to maintain the rate of actin turnover in order to promote pollen germination and maintain the normal rate of pollen tube growth.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.     

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.     

How depolymerization can promote polymerization: the case of actin and profilin

Rapid polymerization and depolymerization of actin filaments in response to extracellular stimuli is required for normal cell motility and development. Profilin is one of the most important actin‐binding proteins; it regulates actin polymerization and interacts with many cytoskeletal proteins that link actin to extracellular membrane. The molecular mechanism of profilin has been extensively considered and debated in the literature for over two decades. Here we discuss several accepted hypotheses regarding the mechanism of profilin function as well as new recently emerged possibilities. Thermal noise is routine in molecular world and unsurprisingly, nature has found a way to utilize it. An increasing amount of theoretical and experimental research suggests that fluctuation‐based processes play important roles in many cell events. Here we show how a fluctuation‐based process of exchange diffusion is involved in the regulation of actin polymerization.     

南方鲇Vasa基因两种亚型cDNA的克隆及其表达

采用RT-PCR和RACE相结合的方法,从南方鲇分离到Vasa基因的两个亚型scVasa和scVaga-s。它们是同一基因在5′端经选择性剪接的产物,其cDNA全长分别为2525bp和2438bp,编码662和641个氨基酸。两者均具有DEAD-box家族成员特有的8个保守基序和Vasa的典型特征。南方鲇Vasa与银鲫相似性最高（73．3％）。两个亚型均特异地表达于雌雄性腺中。原位杂交结果表明：scVasa主要在卵巢Ⅰ、Ⅱ时相的卵母细胞和精巢的精原细胞和初级精母细胞中表达。半定量PCR结果显示,在生殖周期中,两种亚型在以Ⅱ时相卵母细胞为主体的卵巢恢复期表达均高于以Ⅲ-Ⅳ时相卵母细胞为主体的卵黄生成期[动物学报54（6）：1051—1060,2008]。     

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。     

Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate-lysine-2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the ultrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside-induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes. Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0.1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes. The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.     

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.     

人类第五染色体5q13.3带区一新基因的分子克隆初报

人类第五染色体５ｑ１３．３带区是毛发样白血病、ｒｅｆａｃｔｏｒｙ白血病、骨髓增生不良综合征、卵巢癌、肺癌等恶性肿瘤的共同畸变区域,表明该区域可能存在一个或多个同恶性肿瘤发生有关的基因（癌基因或抑癌基因）．克隆这些有关基因并进一步鉴定,对于阐明恶性肿瘤的发病机理,进而应用于基因治疗具有重要的理论和实践意义．本文报道位于该区域的一个新基因的ｃＤＮＡ克隆及在人体不同组织表达的初步结果．     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.