鸡脂肪酸结合蛋白基因的克隆和测序分析

根据哺乳动物脂肪酸结合蛋白基因序列设计一对引物对鸡基因组进行PCR扩增,将163bp扩增片段进行克隆和测序,并与猪的脂肪酸结合蛋白（fatty acid binding protein,FABP）基因序列进行同源性比较。该基因因片段与猪的心脏脂肪酸结合蛋白（heart fatty acid binding protein,H-FABP）基因有68%的同源性,与猪的脂肪型脂肪酸结合蛋白（adipocyte fatty acid binding protein,A-FABP）基因有75%的同源性,演绎成氨基酸之后与猪的脂肪型脂肪酸结合蛋白相应的氨基酸有75%的同源性。Northern结果表明该基因只在脂肪组织中表达。     

Reg基因蛋白家族的研究进展

Reg基因蛋白（regenerating gene protein）属于钙依赖的植物血凝素超家族,其功能类似应激蛋白、抗凋亡因子或生长因子。Reg基因蛋白的促进胰岛β细胞分裂和诱导再生作用最早是在糖尿病研究中被发现。Reg基因蛋白在人体多种组织中均表达,与细胞增殖、炎症创伤、感染和神经系统发育关系密切。随着研究的深入,Reg基因蛋白在胰腺损伤修复、神经系统损伤、消化系统肿瘤、脓毒血症及其他疾病中的作用逐渐引起人们重视。     

胆固醇酯转运蛋白的基因多态性

胆固醇酯转运蛋白（cholesteryl ester transfer protein,CETP）通过介导胆固醇酯在高密度脂蛋白和富含载脂蛋白B的脂蛋白之间的交换,在胆固醇逆向转运过程中起着关键的作用。流行病学研究资料已经阐明CETP基因多态同血浆CETP浓度和脂蛋白水平相关联,因而CETP基因被认为是冠心病（coronary heart disease,CHD）的易感基因之一。本文重点阐述CETP基因单核苷酸多态与脂蛋白代谢及临床疾病易感性关系的最新研究进展。     

PCR一步法构建融合蛋白基因fpg

采用一种不需要限制核酸酶和连接酶的新方法——“PCR一步法”将芳香烃化合物降解的关键基因pheB和绿色荧光蛋白编码基因gfp融合,构建得到融合蛋白基因fpg。该方法在一个PCR反应体系中通过三个引物、两个模板扩增得到一个含有中间柔性肽段-Gly4Ser-的融合基因fpg。本文研究结果表明,PCR一步法是一种快速方便的构建融合基因的方法。Abstract: TP-PCR,a method developed for fusion gene construction without the use of endonuclease and ligase, was performed to construct a fused fpg gene. The TP-PCR reaction system contained three primers and two templates and resulting PCR product, fused fpg gene, consisted of three sections: pheB gene, which was responsible for catechol 2,3-dioxygenase, gfp gene for GFP protein and the intermediate ligation segment which was designed for the correct expression of the fusion gene. The result in this paper showed that the TP-PCR method is one of rapid and convenient methods for fused gene construction.     

小G蛋白Ran的研究进展

真核细胞中含量最丰富的小G蛋白Ran(Ras-related nuclear protein),具有与其所属的Ras家族其它成员不同的生化特点,其活性主要受到Ran的鸟苷酸交换因子（RanGEF)RCC1（regulator of chromosome condensation-1),GTP酶激活蛋白（GTPase-activating protein,GAP）以及Ran结合蛋白（Ran binding protein,RanBP)的调节,Ran主要参与了物质的核浆转运,此外在RNA出核,RNA合成,加工及细胞周期调控中也起到一定作用,并且是LPS活化基因的产物。     

长春花黄化植原体核糖体蛋白基因片段序列分析*

对自然表现典型黄化症的长春花植株总RNA进行植原体核糖体蛋白基因（ribosomal protein gene,rp gene）PCR扩增,得到约1.3kb的特异片段。将此特异片段与pGEM-T Easy载体连接并转化到大肠杆菌JM109感受态细胞中,通过PCR鉴定、限制性内切酶(EcoRI)酶切分析、核苷酸序列测定及分析,结果表明该株系核糖体蛋白基因片段长1,44bp,包含rp122、rps3基因,分别编码129和252个氨基酸,且这两个基因为重叠基因。该植原体核糖体蛋白基因特性与其它植原体相似。     

小麦抗叶锈病近等基因系TcLr41 SSH文库构建与分析

以小麦抗叶锈病近等基因系TcLr41和感病亲本Thatcher的叶片cDNA分别作为试验方和驱动方,利用抑制差减杂交技术,构建了一个包含2544个克隆的差减文库。随机提取阳性克隆质粒DNA后经PCR检测,插入片段大部分集中在200～1000bp之间,证明所构建的文库符合要求。在功能已知的基因中,推测过氧化氢酶（catalasc）基因、抗秆锈病基因（rust resistance gene）、铜蓝结合蛋白（blue copper—binding protein）基因、锌指蛋白（ring zinc finger protein）基因、胁迫反应蛋白（stress responsive protein）基因等可能是TcLr41中抗病相关差异表达基因。     

Dlmo基因编码一个32kDa的蛋白

为了获取研究Dlmo（果蝇LMO基因的简称）的功能信息,使用耦联网织红细胞体外翻译系统将Dlmo基因进行体外转录翻译得到32kDa的蛋白产物。该蛋白产物与抗人类LMO2蛋白的多抗抗体发生免疫沉淀,并得到了32kDa的阳性带。为了证实Dlmo基因在体外和体内翻译能得到同一大小蛋白,从2～4小时果蝇胚盘中分别抽提核和胞浆提取物进行Western印迹分析,免疫血清可以识别胞浆提取物中的32kDa蛋白,而在核提取物中未曾见到,证实体外和体内翻译产物相同。免疫组织化学的分析在0～4小时胚盘外周胞浆中有Dlmo基因的阳性染色信号,结果证实,Dlmo与人类LMO不同,其表达产物是一个胞浆蛋白。 Abstract：In order to gain some insight into a possible function of Dlmo gene,we used the TNT coupled reticulocyte lysate systems as an in vitro translation system to detect the Drosoplila protein. We found a 32kDa protein product.To demonstrate that the DLMO protein has the same size in vivo as in vitro we studied nuclear and cytoplasmic extracts from 2-4h embryos in Western blots. The immuno serum recognizes a 32kDa protein in the cytoplasm that is not present in the nucleus.The products were immune precipitated with the polyclonal anti-LMO2 antibody raised against the human protein and seen a positive band of 32kDa.Using immuno histochemical analysis was seen a positive staining in the basal cytoplasm of blastoderm embryos at 4 hours. This result confirms that the DLMO is a cytoplasmprotein, not like human LMO protein.     

单纯疱疹病毒1型单链结合蛋白ICP8研究进展

在单纯疱疹病毒1型（Herpes simplex virus 1,HSV-1）中，感染细胞蛋白8(Infected cell protein 8,ICP8)是由UL29基因编码的一种单链DNA结合蛋白（Single strand DNA-binding protein,SSBP），该蛋白在病毒复制中必不可缺，具有维持单链DNA的稳定性，并与其他病毒蛋白相互结合，促进病毒复制室的形成，介导晚期基因的表达。除此之外，ICP8还具有促进UL9编码的起源结合蛋白（Origin binding protein,OBP）和UL5/UL8/UL52蛋白的酶活性，与UL12蛋白共同介导链交换等功能。本文就上述目前国内外对HSV-1 ICP8的研究进展作一综述，以期为后续研究ICP8的作用机制提供参考。     

The survival motor neuron protein of Schizosacharomyces pombe. Conservation of survival motor neuron interaction domains in divergent organisms

Spinal muscular atrophy is a common often lethal neurodegenerative disease resulting from deletions or mutations in the survival motor neuron gene (SMN). SMN is ubiquitously expressed in metazoan cells and plays a role in small nuclear ribonucleoprotein assembly and pre-mRNA splicing. Here we characterize the Schizosacharomyces pombe orthologue of SMN (yeast SMN (ySMN)). We report that the ySMN protein is essential for viability and localizes in both the cytoplasm and the nucleus. Like human SMN, we show that ySMN can oligomerize. Remarkably, ySMN interacts directly with human SMN and Sm proteins. The highly conserved carboxyl-terminal domain of ySMN is necessary for the evolutionarily conserved interactions of SMN and required for cell viability. We also demonstrate that the conserved amino-terminal region of ySMN is not required for SMN and Sm binding but is critical for the housekeeping function of SMN.     

SMN tudor domain structure and its interaction with the Sm proteins

Spinal muscular atrophy (SMA) is a common motor neuron disease that results from mutations in the Survival of Motor Neuron (SMN) gene. The SMN protein plays a crucial role in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes via binding to the spliceosomal Sm core proteins. SMN contains a central Tudor domain that facilitates the SMN-Sm protein interaction. A SMA-causing point mutation (E134K) within the SMN Tudor domain prevents Sm binding. Here, we have determined the three-dimensional structure of the Tudor domain of human SMN. The structure exhibits a conserved negatively charged surface that is shown to interact with the C-terminal Arg and Gly-rich tails of Sm proteins. The E134K mutation does not disrupt the Tudor structure but affects the charge distribution within this binding site. An intriguing structural similarity between the Tudor domain and the Sm proteins suggests the presence of an additional binding interface that resembles that in hetero-oligomeric complexes of Sm proteins. Our data provide a structural basis for a molecular defect underlying SMA.     

Purification of native survival of motor neurons complexes and identification of Gemin6 as a novel component.

The survival of motor neurons (SMN) protein, the product of the gene responsible for the motor neuron degenerative disease spinal muscular atrophy (SMA), is part of a large macromolecular complex. The SMN complex is localized in both the cytoplasm and the nucleus and contains SMN, Gemin2, Gemin3, Gemin4, Gemin5, and a few not yet identified proteins. The SMN complex plays a key role in the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and other ribonucleoprotein particles. As a step toward the complete characterization of the components of the SMN complex, we generated stable cell lines that express FLAG-tagged SMN or Gemin2 under the control of a tetracycline-inducible promoter. Native SMN complexes of identical protein composition to those isolated by immunoprecipitation with anti-SMN antibodies were purified by affinity chromatography from extracts of both cell lines. Here we report the identification by mass spectrometry of a novel protein component of the SMN complex termed Gemin6. Co-immunoprecipitation, immunolocalization, and in vitro binding experiments demonstrate that Gemin6 is a component of the SMN complex that localizes to gems and interacts with several Sm proteins of the spliceosomal snRNPs.     

The Methylosome, a 20S Complex Containing JBP1 and pICln, Produces Dimethylarginine-Modified Sm Proteins

snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.     

Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly

The survival motor neuron (SMN) protein is the product of the spinal muscular atrophy disease gene. SMN and Gemin2-7 proteins form a large macromolecular complex that localizes in the cytoplasm as well as in the nucleoplasm and in nuclear Gems. The SMN complex interacts with several additional proteins and likely functions in multiple cellular pathways. In the cytoplasm, a subset of SMN complexes containing unrip and Sm proteins mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, by mass spectrometry analysis of SMN complexes purified from HeLa cells, we identified a novel protein that is evolutionarily conserved in metazoans, and we named it Gemin8. Co-immunoprecipitation and immunolocalization experiments demonstrated that Gemin8 is associated with the SMN complex and is localized in the cytoplasm and in the nucleus, where it is highly concentrated in Gems. Gemin8 interacts directly with the Gemin6-Gemin7 heterodimer and, together with unrip, these proteins form a heteromeric subunit of the SMN complex. Gemin8 is also associated with Sm proteins, and Gemin8-containing SMN complexes are competent to carry out snRNP assembly. Importantly, RNA interference experiments indicate that Gemin8 knock-down impairs snRNP assembly, and Gemin8 expression is down-regulated in cells with low levels of SMN. These results demonstrate that Gemin8 is a novel integral component of the SMN complex and extend the repertoire of cellular proteins involved in the pathway of snRNP biogenesis.     

Gemin3: A novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of gems

The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.     

Spinal Muscular Atrophy and the Antiapoptotic Role of Survival of Motor Neuron (SMN) Protein

Spinal muscular atrophy (SMA) is a devastating and often fatal neurodegenerative disease that affects spinal motor neurons and leads to progressive muscle wasting and paralysis. The survival of motor neuron (SMN) gene is mutated or deleted in most forms of SMA, which results in a critical reduction in SMN protein. Motor neurons appear particularly vulnerable to reduced SMN protein levels. Therefore, understanding the functional role of SMN in protecting motor neurons from degeneration is an essential prerequisite for the design of effective therapies for SMA. To this end, there is increasing evidence indicating a key regulatory antiapoptotic role for the SMN protein that is important in motor neuron survival. The aim of this review is to highlight key findings that support an antiapoptotic role for SMN in modulating cell survival and raise possibilities for new therapeutic approaches.     

The Gemin6-Gemin7 heterodimer from the survival of motor neurons complex has an Sm protein-like structure

The survival of motor neurons (SMN) protein, product of the disease gene of the common neurodegenerative disease spinal muscular atrophy, is part of the large multiprotein "SMN complex." The SMN complex functions as an assembly machine for small nuclear ribonucleoproteins (snRNPs)-the major components of the spliceosome. Here, we report the crystal structure of two components of the human SMN complex, Gemin6 and Gemin7. Although Gemin6 and Gemin7 have no significant sequence similarity with Sm proteins, both adopt canonical Sm folds. Moreover, Gemin6 and Gemin7 exist as a heterodimer, and interact with each other via an interface similar to that which mediates interactions among the Sm proteins. Together with binding experiments that show that the Gemin6/Gemin7 complex binds to Sm proteins, these findings provide a framework for considering how the SMN complex, with Gemin6 and Gemin7 as tools, might organize Sm proteins for formation of Sm rings on snRNA targets.     

A comprehensive interaction map of the human survival of motor neuron (SMN) complex

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs.