小鼠富亮氨酸重复结构蛋白Lrig2基因、蛋白结构及组织定位分析

目的：分析小鼠富亮氨酸重复结构蛋白家族成员Lrig2的基因与蛋白的结构,明确其组织分布和定位,并对其功能进行初步预测。方法：利用生物信息学分析技术对小鼠Ln萨基因的染色体定位、蛋白结构进行分析预测;通过RT-PCR、mRNA原位杂交技术检测Lrig2基因在小鼠不同组织中的表达定位;通过系统进化树分析“薛与其他Lrrs蛋白家族成员的同源性。结果：生物信息学分析显示,H薛是一种跨膜蛋白受体,是Lrrs蛋白超家族成员之一,胞外区含有15个m模序、3个免疫球蛋白样结构域,存在单一的跨膜结构域;Lrig2在小鼠的多个组织中表达,其中在胸腺、脾脏等组织中表达较强;系统树分析显示,Lrigs蛋白是sLrPs超家族成员,是一种跨膜蛋白受体。结论：Lrig2在免疫组织中表达较强,推测其可能在肿瘤免疫应答进程中发挥重要的效能。     

小鼠脂联素受体2基因编码区的克隆及序列分析

构建小鼠脂联素受体2(mAdipoR2)基因cDNA克隆,并进行序列及基因结构分析,为进一步研究小鼠AdipoR2的表达和生物学活性奠定基础。用RT—PCR方法扩增mAdipoR2基因cDNA,获得的片段连接至pGEM-T载体,转化JM109大肠杆菌,经酶切鉴定后,进行序列测定。利用因特网生物信息学资源分析mAdipoR2基因序列。结果成功地构建了mAdipoR2基因cDNA克隆,其序列与GenBank登录序列一致。通过与小鼠基因组序列比对,分析了mAdipoR2基因结构,其编码序列由7个外显子组成,编码1个含7个跨膜区的膜蛋白,但该受体不属于G蛋白偶联受体家族(GPCRs)。mAdipoR2与人及大鼠蛋白的同源性分别为91％和95％。     

烟草C2H2锌指蛋白转录因子家族成员的鉴定与表达分析

C2H2锌指蛋白转录因子家族在真核生物中具有重要的生物学功能,广泛参与植物叶的发生、花器官的调控、侧枝的形成及逆境胁迫等生命过程。植物C2H2锌指蛋白不仅结合DNA和RNA,而且与蛋白质之间相互作用。本研究利用普通烟草(Nicotiana tabacum)基因组数据库,运用Blastp比对,结合Pfam和SMART分析,鉴定了118条普通烟草C2H2锌指蛋白家族成员;对烟草C2H2锌指蛋白家族进行了进化树分析、结构域分析、物理化学性质分析、染色体定位、基因结构分析、三维结构分析及组织表达分析等。结果表明：不同成员的氨基酸长度差异较大;系统进化及结构域分析显示,所有C2H2家族成员可以被分为5个亚家族,同一亚家族成员之间在结构域和理化性质上呈现较高一致性;每个成员都含有C2H2结构域,在数量上存在较大差异;将所有基因家族成员定位在22条染色体上;组织表达分析表明,每个C2H2亚家族都有成员在不同组织中表达,在叶及根中有些基因的表达量较高。     

家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

基于生物信息学方法预测人线粒体转录终止因子3蛋白的结构与功能

目的:基于生物信息学预测人线粒体转录终止因子3(hMTERF3)蛋白的结构与功能。方法:利用GenBank、Uniprot、ExPASy、SWISS-PROT数据库资源和不同的生物信息学软件对hMTERF3蛋白进行系统研究,包括hMTERF3的理化性质、跨膜区和信号肽、二级结构功能域、亚细胞定位、蛋白质的功能分类预测、同源蛋白质多重序列比对、系统发育树构建、三级结构同源建模。结果:软件预测hMTERF3蛋白的相对分子质量为47.97×103,等电点为8.60,不具信号肽和跨膜区;二级结构分析显示主要为螺旋和无规则卷曲,包含6个MTERF基序,三级结构预测结果与二级结构预测结果相符;亚细胞定位分析结果显示该蛋白定位于人线粒体;功能分类预测其为转运和结合蛋白,参与基因转录调控;同源蛋白质多重序列比对和进化分析显示,hMTERF3蛋白与大鼠、小鼠等哺乳动物的MTERF3蛋白具有高度同源性,在系统发育树上聚为一类。结论:hMTERF3蛋白的生物信息学分析为进一步开展对该蛋白的结构和功能的实验研究提供了理论依据。     

人p17.3基因及其推导蛋白的结构和功能分析

用生物信息学技术研究人类神经元蛋白p17.3的基因结构、染色体定位、组织表达特征及其推导蛋白的理化特性、空间结构和功能等。以高通量基因组序列(HTGS)数据库及SAGE文库为基础对p17.3基因进行染色体定位及组织表达谱分析。通过DAS和TMpred等程序预测其蛋白质结构及功能。利用PDB程序模拟其三维空间结构。研究表明p17.3基因定位于人类染色体Xp11.2-3,在多种组织中广谱表达,但在前列腺表达量最高。其编码蛋白含有一个PEST区域、一个线粒体靶向序列和一个潜在“跨膜螺旋”结构。p17.3基因可能是一个参与神经元发育调控的基因。     

人Transgelin蛋白家族生物信息学分析

本研究通过对人Transgelin蛋白家族(Transgelins)3位成员(Transgelin,-2,-3)的生物信息学预测分析,显示Transgelin和-2等电点分别为8.87和8.41,Transgelin-3等电点为6.84,3位成员均为非分泌型蛋白,且极有可能是跨膜型蛋白.蛋白质稳定性分析结果显示,除Tr...     

家鸡G蛋白偶联受体78基因的克隆与组织表达图谱分析

G蛋白偶联受体(GPCR)是一大类膜受体超家族,参与了机体几乎所有的生理过程,是一类重要信号分子受体。GPR78作为GPCR超家族的成员之一,属于孤儿型受体。目前,在脊椎动物中,针对该基因结构与功能的研究极少。本研究以家鸡为动物模型,通过RT-PCR方法克隆了GPR78基因的编码区序列,并探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR78基因编码区全长为1020 bp,含3个外显子,可编码1个长为339个氨基酸的7次跨膜受体;该基因在家鸡脑各功能区及垂体中高表达,而在其他外周组织中均不表达。本研究为后续探究GPR78基因在家鸡中的生理功能奠定基础。     

雪里蕻非特异脂质转运蛋白基因的克隆与分析

非特异脂质转运蛋白是植物生命活动中一类重要的活性蛋白,这类蛋白在植物的抗性和防御中行使着重要的功能。近年来研究发现这类蛋白还与植物的有性生殖密切相关。通过已得到的普通白菜的脂质转运蛋白基因BcMF15的核苷酸序列,在其基因全长两侧设计引物,从雪里蕻中克隆得到该类活性蛋白基因,命名为BjLTP (登陆号: EU082009)。该基因全长650bp,不含内含子。不同组织的表达特征分析发现,BjLTP在雪里蕻的花蕾、开放的花中特异表达,推测BjLTP可能与花粉的发育有关。蛋白质特征预测及蛋白序列结构分析发现BjLTP是一个跨膜蛋白,具有显著的疏水区。序列同源比对表明该基因与白花芥蓝、拟南芥等的脂质转运蛋白基因有很高的相似性,证明BjLTP是LTP家族的成员之一。     

杨树基因组AMT转运蛋白的生物信息学特性

本研究中,通过隐马尔科夫模型(HMM)和杨树蛋白质库搜索,共找到17个杨树铵转运体蛋白(PtAMTs).利用生物信息学方法,我们对杨树家族17条AMT蛋白序列的系统发生和AMT基因组定位进行分析,然后对其氨基酸组成成分、理化性质以及二级结构进行预测和分析,同时还分析了杨树与拟南芥、水稻、番茄、百脉根和欧洲油菜的AMT基因家族之间的联系.二级结构预测结果发现不同成员间氨基酸数目、氨基酸序列间的疏水性存在一定的差异;α-螺旋和无规则卷曲为主要二级结构组成部分.同源性比对分析表明,PtAMT基因家族主要分为2个亚家族,AMT1 (11个成员)和AMT2 (6个成员),基因结果分析表明AMT2亚家族成员不含内含子.杨树AMT蛋白的亚细胞定位分析表明PtAMT主要定位于膜结构上.电子表达图谱分析结果表明:只有XP_002309151和 XP_002334025基因有对应的EST序列,并有相应的电子表达谱,并主要在花蕾表达.     

Functional domains required for the Saccharomyces cerevisiae Mus81-Mms4 endonuclease complex formation and nuclear localization

The Saccharomyces cerevisiae Mms4 and Mus81 proteins form a specific complex, which functions as an endonuclease specific for branched DNA molecules and protects cells from killing by DNA alkylation damage, but not damage induced by ionizing radiations. In an effort to further understand the structure and functions of the Mus81-Mms4 complex, we attempted to define domains required for complex formation and nuclear localization through deletion and mutagenesis analyses. Combined yeast two-hybrid and co-immunoprecipitation experiments indicate that the C-terminal 100 amino acids of both Mus81 and Mms4 are required and sufficient for heterodimer formation. However, a single amino acid substitution in Mms4 in the N-terminal region is able to abolish the interaction, which suggests that the three-dimensional structure is also important for Mms4 to interact with Mus81. By fusion to green fluorescent protein and in vivo subcellular localization studies, we demonstrate that Mms4 and Mus81 are nuclear proteins and can be localized to the nucleus independently. Deletion analyses indicate that one of two putative nuclear localization signals (residues 244-263) in Mms4 is required for localization, whereas the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.     

Leucine-rich Repeat and Immunoglobulin Domain-containing Protein-1 (Lrig1) Negative Regulatory Action toward ErbB Receptor Tyrosine Kinases Is Opposed by Leucine-rich Repeat and Immunoglobulin Domain-containing Protein 3 (Lrig3)

Lrig1 is the founding member of the Lrig family of transmembrane leucine-rich repeat proteins, which also includes Lrig2 and Lrig3. Lrig1 is a negative regulator of oncogenic receptor tyrosine kinases, including ErbB and Met receptors, and promotes receptor degradation. Lrig1 has recently emerged as both a tumor suppressor and a key regulator of epidermal and epithelial stem cell quiescence. Despite this, little is known of the mechanisms by which Lrig1 is regulated. Lrig3 was recently reported to increase ErbB receptor expression suggesting that it may function in a manner opposite to Lrig1. In this study, we explore the interaction between Lrig1 and Lrig3 and demonstrate that Lrig1 and Lrig3 functionally oppose one another. Lrig3 opposes Lrig1 negative regulatory activity and stabilizes ErbB receptors. Conversely, Lrig1 destabilizes Lrig3, limiting Lrig3''s positive effects on receptors and identifying Lrig3 as a new target of Lrig1. These studies provide new insight into the regulation of Lrig1 and uncover a complex cross-talk between Lrig1 and Lrig3.     

Lrig1 is an estrogen-regulated growth suppressor and correlates with longer relapse-free survival in ERα-positive breast cancer

Lrig1 is the founding member of the Lrig family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth-inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor α (ERα)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ERα-positive disease than in ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by showing that Lrig1 is a target of ERα. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen-regulated growth suppressor. In addition, we find that Lrig1 expression correlates with prolonged relapse-free survival in ERα-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we show that ErbB2 activation antagonizes ERα-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth-inhibitory role for Lrig1 in breast cancer.     

In Vivo Analysis of Lrig Genes Reveals Redundant and Independent Functions in the Inner Ear

Lrig proteins are conserved transmembrane proteins that modulate a variety of signaling pathways from worm to humans. In mammals, there are three family members – Lrig1, Lrig2, and Lrig3 – that are defined by closely related extracellular domains with a similar arrangement of leucine rich repeats and immunoglobulin domains. However, the intracellular domains show little homology. Lrig1 inhibits EGF signaling through internalization and degradation of ErbB receptors. Although Lrig3 can also bind ErbB receptors in vitro, it is unclear whether Lrig2 and Lrig3 exhibit similar functions to Lrig1. To gain insights into Lrig gene functions in vivo, we compared the expression and function of the Lrigs in the inner ear, which offers a sensitive system for detecting effects on morphogenesis and function. We find that all three family members are expressed in the inner ear throughout development, with Lrig1 and Lrig3 restricted to subsets of cells and Lrig2 expressed more broadly. Lrig1 and Lrig3 overlap prominently in the developing vestibular apparatus and simultaneous removal of both genes disrupts inner ear morphogenesis. This suggests that these two family members act redundantly in the otic epithelium. In contrast, although Lrig1 and Lrig2 are frequently co-expressed, Lrig1^−/−;Lrig2^−/− double mutant ears show no enhanced structural abnormalities. At later stages, Lrig1 expression is sustained in non-sensory tissues, whereas Lrig2 levels are enhanced in neurons and sensory epithelia. Consistent with these distinct expression patterns, Lrig1 and Lrig2 mutant mice exhibit different forms of impaired auditory responsiveness. Notably, Lrig1^−/−;Lrig2^−/− double mutant mice display vestibular deficits and suffer from a more severe auditory defect that is accompanied by a cochlear innervation phenotype not present in single mutants. Thus, Lrig genes appear to act both redundantly and independently, with Lrig2 emerging as the most functionally distinct family member.     

Lrig1 is a cell‐intrinsic modulator of hippocampal dendrite complexity and BDNF signaling

Even though many extracellular factors have been identified as promoters of general dendritic growth and branching, little is known about the cell‐intrinsic modulators that allow neurons to sculpt distinctive patterns of dendrite arborization. Here, we identify Lrig1, a nervous system‐enriched LRR protein, as a key physiological regulator of dendrite complexity of hippocampal pyramidal neurons. Lrig1‐deficient mice display morphological changes in proximal dendrite arborization and defects in social interaction. Specifically, knockdown of Lrig1 enhances both primary dendrite formation and proximal dendritic branching of hippocampal neurons, two phenotypes that resemble the effect of BDNF on these neurons. In addition, we show that Lrig1 physically interacts with TrkB and attenuates BDNF signaling. Gain and loss of function assays indicate that Lrig1 restricts BDNF‐induced dendrite morphology. Together, our findings reveal a novel and essential role of Lrig1 in regulating morphogenic events that shape the hippocampal circuits and establish that the assembly of TrkB with Lrig1 represents a key mechanism for understanding how specific neuronal populations expand the repertoire of responses to BDNF during brain development.     

HIV-1 CAP2NC蛋白的表达及体外自组装

构建并表达HIV-1 CAP2NC蛋白,探索其体外自组装条件。通过PCR技术扩增HIV-1(NL4-3毒株)CAP2NC基因片段,并将其连接到原核表达载体pTO-T7,获得重组质粒pTO-T7-CAP2NC,然后转化至大肠杆菌BL21(DE3)菌株,经疏水层析纯化后获得重组蛋白CAP2NC。SDS-PAGE结果表明,重组蛋白CAP2NC可在大肠杆菌可溶高效表达,经纯化后纯度约为95%。ELISA检测表明重组蛋白CAP2NC可被HIV-1衣壳蛋白特异性单克隆抗体识别,具有较好反应活性。重组蛋白透析后在非原性SDS-PAGE中呈现为多种聚体形式。分子筛排阻层析分析CAP2NC蛋白透析后可进行组装,负染电镜进一步观察显示CAP2NC蛋白在RNA存在条件下,可形成空心管状颗粒,其形态结构与HIV-1病毒衣壳体外自组装形成的类似。上述结果表明HIV-1 CAP2NC蛋白具有体外自组装的性质,为进一步在体外研究非成熟病毒样颗粒结构奠定基础。     

Better prediction of sub-cellular localization by combining evolutionary and structural information

The native sub-cellular compartment of a protein is one aspect of its function. Thus, predicting localization is an important step toward predicting function. Short zip code-like sequence fragments regulate some of the shuttling between compartments. Cataloguing and predicting such motifs is the most accurate means of determining localization in silico. However, only few motifs are currently known, and not all the trafficking appears regulated in this way. The amino acid composition of a protein correlates with its localization. All general prediction methods employed this observation. Here, we explored the evolutionary information contained in multiple alignments and aspects of protein structure to predict localization in absence of homology and targeting motifs. Our final system combined statistical rules and a variety of neural networks to achieve an overall four-state accuracy above 65%, a significant improvement over systems using only composition. The system was at its best for extra-cellular and nuclear proteins; it was significantly less accurate than TargetP for mitochondrial proteins. Interestingly, all methods that were developed on SWISS-PROT sequences failed grossly when fed with sequences from proteins of known structures taken from PDB. We therefore developed two separate systems: one for proteins of known structure and one for proteins of unknown structure. Finally, we applied the PDB-based system along with homology-based inferences and automatic text analysis to annotate all eukaryotic proteins in the PDB (http://cubic.bioc.columbia.edu/db/LOC3D). We imagine that this pilot method-certainly in combination with similar tools-may be valuable target selection in structural genomics.     

SARS-CoV推测N蛋白功能结构的生物信息学研究

目的:利用生物信息学方法理论分析不同地区来源的SARS冠状病毒(SARSCoV)推断N蛋白的基因组与氨基酸序列的差异及分子生物学特征以及基因突变对蛋白结构功能的影响。方法:针对GenBank上发布的来自不同国家地区的15条SARSCoV基因组序列,采用生物信息学软件分析其推测N蛋白的CDS和氨基酸序列,分别找出突变位点并预测其等电点及功能结构域。结果:SARSCoV推测N蛋白基因组序列存在5个变异位点导致蛋白序列有4个位点发生突变。在该蛋白上发现四个有意义的低成分复杂性区域;未发现卷曲螺旋、跨膜螺旋和信号肽序列。基因突变造成4条序列在功能位点数量上减少,但未影响抗原决定簇。预测发现两个保守的Domain和一个丝氨酸富集区。结论:不同地区来源的15条推测N蛋白序列的变异很少。基因突变导致部分序列功能位点数量发生改变,但未影响抗原决定簇的数量。     

Actin in Mucor rouxii

Rhodamine-conjugated phalloidin was used to analyze the actin distribution during hyphal formation in Mucor rouxii. The occurrence of actin patches in the cortical region of the cells was seen in the initial stages of growth. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-sepharose. This peptide was identified on immunoblots when polyclonal antibodies against rabbit muscle actin were used as a probe. These results indicate: (1) that changes in actin localization accompany the hyphal development and (2) the fungal 43 kDa protein shares properties that are common to muscle actin.