斑马鱼神经介素S直系同源基因的克隆、序列分析及组织表达

旨在更深入和广泛了解脊椎动物NMS前体基因的结构、进化和生理功能,利用生物信息学和分子生物学技术从斑马鱼脑组织中克隆得到了NMS前体基因序列,并对其基因和蛋白结构以及组织表达等方面进行了分析。结果表明,斑马鱼NMS前体基因cDNA序列编码110个氨基酸组成的多肽,其中包含22个氨基酸的信号肽。脊椎动物NMS前体蛋白序列比对结果显示,与哺乳动物不同,硬骨鱼类NMS前体蛋白在C端缺失很大一部分序列,不能形成类似哺乳动物的NMS成熟肽,经蛋白酶切割可能形成同源性较高的34个氨基酸的多肽。进化树和基因组分析显示,斑马鱼和青NMS前体蛋白聚类在一起,位于NMS这一分支上。同时,NMS前体基因在斑马鱼、青和人基因组中具有同线性关系。半定量RT-PCR结果显示,NMS前体基因在所检测的斑马鱼各组织中均有表达,其中以脑中表达量为最高。     

蛋白质将成为生物学研究重点

在科学家对人类基因组进行初步研究后发现 ,人类的基因数量远少于原来的预想 ,科学家由此认为 ,生命的特性并不是全部由基因决定的。人类基因只有 3万至 4万个 ,而蛋白质却有 2 5万种 ,因此对蛋白质研究将成为今后生物学研究的一个新学科前沿。科学家研究发现 ,人类基因是果蝇的两倍 ,比老鼠多 30 0个 ,远少于人类有 6万至 10万个基因的原来预测。负责人类基因组研究科学家认为 ,这表明也许基因并不能包含生命的全部信息 ;同时也表明科学家原来的观点 ,认为一种基因只负责合成一种蛋白质可能是错误的 ,一种基因可能要负责合成几种蛋白质。美…     

基于组学数据构建基因调控网络

在生命体内,基因以及其它分子间相互作用形成复杂调控网络,生命过程都是以调控网络的形式存在,如从代谢通路网络到转录调控网络,从信号转导网络到蛋白质相互作用网络等等。因此,网络现象是生命现象的复杂本质和主要特征。本文系统地介绍了基于表达谱数据构建基因调控网络的布尔网络模型,线性模型,微分方程模型和贝叶斯网络模型,并对各种网络构建模型进行了深入的分析和总结。同时,文章从基因组序列信息、蛋白质相互作用信息和生物医学文献信息等方面讨论了基因调控网络方面构建的研究,这对从系统生物学水平揭示生命复杂机制具有重要的参考价值。     

斑马鱼一个IFIT 家族基因的鉴定及启动子分析

IFIT 家族由一类受干扰素诱导表达并具有TPR 结构域的蛋白组成, 但是在鱼类关于IFIT 基因的研究还很少。研究利用哺乳类IFIT 家族基因IFI56 的序列搜索斑马鱼基因组数据库鉴定出一个未知基因, 该基因具有哺乳类IFIT 家族保守的基因组结构, 编码蛋白具有保守的TPR 结构域, 暂命名为IFIT-A。RT-PCR 分析表明, Poly I:C 能够诱导IFIT-A 基因转录水平上调。与哺乳类IFIT 家族基因相似, 斑马鱼IFIT-A 启动子存在ISG 基因特有的典型ISRE 结构域。荧光素酶活性实验揭示Poly I:C 和重组IFN 蛋白能激活斑马鱼IFIT-A 启动子活性。此外, 过量表达IFN 调控因子IRF3 和IRF7 能诱导斑马鱼IFIT-A 启动子活性。实验结果证明IFIT-A基因是斑马鱼IFIT 家族成员, IRF3 和IRF7 在其诱导表中具有重要调控作用。       

用于通路(Gateway)克隆技术的植物表达载体研究进展

通路(Gateway)克隆技术是根据λ噬菌体基因组和大肠杆菌基因组之间的位点专一性重组分子机制开发的一套分子克隆新技术.利用该技术LR反应构建目的基因的表达载体时不需要经过酶切和连接等繁琐而又费时的过程,因此,可以节省很多时间.为了扩大Gateway技术在植物基因工程领域的应用,最近有很多研究机构和研究小组开发了能用于组成型或诱导型表达目的基因、基因沉默、启动子分析、蛋白质亚细胞定位、蛋白质/蛋白质相互作用、多个DNA片段的模块化组装和DNA组片段功能验证等研究用的植物表达载体.该文对这些技术的研究进展进行了综述.     

斑点叉尾鮰Cbx基因家族的全基因组鉴定与进化分析

利用Cbx家族基因高度保守的Chromo结构域氨基酸序列作为种子序列,检索斑点叉尾鮰(Ictalurus punetaus)基因组注释的蛋白数据库,共鉴定分离出14个Cbx基因。并在全基因组水平对斑点叉尾鮰Cbx基因家族进行了保守结构域序列、进化、基因结构、染色体定位的系统分析。本研究中鉴定出的斑点叉尾鮰Cbx家族基因蛋白序列与已知的人类、小鼠、鸡、斑马鱼等物种Cbx蛋白序列构建系统进化树。根据系统进化树分析结果,14个斑点叉尾鮰Cbx基因分成两个亚族,5个隶属于Hp1s亚族,9个隶属于PcGs亚族。Cbx基因主要分布于斑点叉尾鮰7条染色体中,且呈不均匀分布。本研究对于后续斑点叉尾鮰Cbx基因家族深入的功能验证及分子作用机制研究具有重要的意义,也为日益丰富的水产基因组资源的挖掘利用提供了参考。     

快速碱化因子基因BcRALF在菜心中的克隆及序列分析

快速碱化因子是近年来新发现的一种植物多肽类信号分子,广泛存在于高等植物中。通过已得到的普通白菜的快速碱化因子基因BcMF14（GenBank序列登录号EF523516）的核苷酸序列,在其编码框两侧设计引物,从菜心中克隆出该类信号分子基因,命名为BcRALF（登陆号：GU086228）。序列同源比对表明该基因与花椰菜、拟南芥等的快速碱化因子基因有很高的相似性,证明BcRALF属于快速碱化因子家族。蛋白质特征预测以及蛋白序列结构分析发现BcRALF蛋白包含有多个生物活性位点,符合其作为多肽类信号分子类蛋白的特征。     

氧化葡萄糖酸杆菌中一种潜在的双组分系统蛋白的功能研究

氧化葡萄糖酸杆菌(Gluconobacteroxydans)基因组编码的蛋白质中,有相当数量的传感器激酶和反应调控蛋白组成了细菌的多个双组分信号转导系统(two-componentsignaltransduction systems, TCSs),这些系统能够介导细菌对外界环境变化做出反应。但目前对G. oxydans中潜在的双组分系统成员蛋白质结构和功能缺少必要的研究【。目的】研究菌株G. oxydans 621H中GOX0645基因序列所编码蛋白质的自磷酸化活性,探究其与细菌趋化性运动的关联,揭示其是否作为一种双组分系统成员蛋白在细胞内发挥作用。【方法】以菌株G. oxydans 621H基因组中一段可能编码双组分系统蛋白质的基因GOX0645为基础,通过生物信息学分析其保守结构域;采用体外化学发光实验证明其编码蛋白的自磷酸化活性;利用基因定点突变筛选出与自磷酸化活性相关的氨基酸位点;通过差速离心法寻找双组分蛋白的亚细胞定位;最后运用体内双分子荧光互补和体外生物大分子相互作用实验印证其与下游鞭毛马达蛋白之间的相互作用。【结果】生物信息学分析发现GOX0645编码蛋白同时具有组氨酸激...     

双杂交和质谱技术在蛋白质-蛋白质相互作用研究中的应用

基因的功能是由蛋白质来执行的,而蛋白质要通过与其他生物分子相互作用来完成其各种生物功能。因此,如果能够快速做出蛋白质在不同时间、空间和不同环境中的相互作用图谱,就会帮助我们了解这些蛋白质的功能,进而了解许多生命活动的机制。目前,用于大规模研究蛋白质间相互作用的方法主要有酵母双杂交系统及其衍生系统、亲和纯化与质谱分析联用技术,前者用于研究蛋白分子间的两两相互作用,后者用于研究蛋白质复合物间的相互作用。本文主要阐述了酵母双杂交、细菌双杂交、哺乳动物细胞双杂交、亲和纯化与质谱联用技术在大规模蛋白质相互作用研究中的应用。     

斑马鱼模型在孤独症谱系障碍研究中应用进展

斑马鱼(Danio rerio,zebra fish)是一种新型脊椎动物模型,由于具有与人类同源性高、价格低廉、生长周期短、透明度高等优点,已应用于人类疾病研究。该文分析对比了斑马鱼12个已通过测量目标mRNA或蛋白质水平证明功能丧失的ASD风险基因与人类基因的同源性百分比,从基因组靶向诱变方面介绍了9个斑马鱼功能敲低或敲除且有显著ASD行为的ASD模型,从药物环境诱导方面介绍了丙戊酸钠、戊四唑等环境因素诱导的斑马鱼ASD模型,以及对应的ASD行为表现。斑马鱼对于孤独症谱系障碍(autism spectrum disorder, ASD)的机理研究、药物治疗与筛选等研究具有独特的优势,在人类疾病的研究中将会带来极大的研究前景和应用价值。     

Disease-Aging Network Reveals Significant Roles of Aging Genes in Connecting Genetic Diseases

One of the challenging problems in biology and medicine is exploring the underlying mechanisms of genetic diseases. Recent studies suggest that the relationship between genetic diseases and the aging process is important in understanding the molecular mechanisms of complex diseases. Although some intricate associations have been investigated for a long time, the studies are still in their early stages. In this paper, we construct a human disease-aging network to study the relationship among aging genes and genetic disease genes. Specifically, we integrate human protein-protein interactions (PPIs), disease-gene associations, aging-gene associations, and physiological system–based genetic disease classification information in a single graph-theoretic framework and find that (1) human disease genes are much closer to aging genes than expected by chance; and (2) diseases can be categorized into two types according to their relationships with aging. Type I diseases have their genes significantly close to aging genes, while type II diseases do not. Furthermore, we examine the topological characters of the disease-aging network from a systems perspective. Theoretical results reveal that the genes of type I diseases are in a central position of a PPI network while type II are not; (3) more importantly, we define an asymmetric closeness based on the PPI network to describe relationships between diseases, and find that aging genes make a significant contribution to associations among diseases, especially among type I diseases. In conclusion, the network-based study provides not only evidence for the intricate relationship between the aging process and genetic diseases, but also biological implications for prying into the nature of human diseases.     

A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance

The completion of the genome sequences of both rice and Magnaporthe oryzae has strengthened the position of rice blast disease as a model to study plant-pathogen interactions in monocotyledons. Genetic studies of blast resistance in rice were established in Japan as early as 1917. Despite such long-term study, examples of cultivars with durable resistance are rare, partly due to our limited knowledge of resistance mechanisms. A rising number of blast resistance genes and quantitative trait loci (QTL) have been genetically described, and some have been characterized during the last 20 years. Using the rice genome sequence, can we now go a step further toward a better understanding of the genetics of blast resistance by combining all these results? Is such knowledge appropriate and sufficient to improve breeding for durable resistance? A review of bibliographic references identified 85 blast resistance genes and approximately 350 QTL, which we mapped on the rice genome. These data provide a useful update on blast resistance genes as well as new insights to help formulate hypotheses about the molecular function of blast QTL, with special emphasis on QTL for partial resistance. All these data are available from the OrygenesDB database.     

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.     

Studying synthetic lethal interactions in the zebrafish system: insight into disease genes and mechanisms

The post-genomic era is marked by a pressing need to functionally characterize genes through understanding gene-gene interactions, as well as interactions between biological pathways. Exploiting a phenomenon known as synthetic lethality, in which simultaneous loss of two interacting genes leads to loss of viability, aids in the investigation of these interactions. Although synthetic lethal screening is a powerful technique that has been used with great success in many model organisms, including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans, this approach has not yet been applied in the zebrafish, Danio rerio. Recently, the zebrafish has emerged as a valuable system to model many human disease conditions; thus, the ability to conduct synthetic lethal screening using zebrafish should help to uncover many unknown disease-gene interactions. In this article, we discuss the concept of synthetic lethality and provide examples of its use in other model systems. We further discuss experimental approaches by which the concept of synthetic lethality can be applied to the zebrafish to understand the functions of specific genes.     

Molecular and chemical genetic approaches to developmental origins of aging and disease in zebrafish

The incidence of diseases increases rapidly with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but mostly inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states in response to different environmental or genetic perturbations. On the one hand, we hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds of adaptive plasticity by chemical genetic approaches, we have been investigating whether any relationship exists between the regulatory mechanisms that function in early development and in senescence using the zebrafish (Danio rerio), a small freshwater fish and a useful model animal for genetic studies. We have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during embryogenesis (“embryonic senescence”), subsequently showing shortened lifespan in adulthoods. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and regulation. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes and small molecular compounds that can be linked to the senescence phenotype, and thereby facilitates searching for the evolutionary and developmental origins of aging in vertebrates. This article is part of a Special Issue entitled: Animal Models of Disease.     

Systems biology of pathogen-host interaction: Networks of protein-protein interaction within pathogens and pathogen-human interactions in the post-genomic era

Infectious diseases comprise some of the leading causes of death and disability worldwide. Interactions between pathogen and host proteins underlie the process of infection. Improved understanding of pathogen-host molecular interactions will increase our knowledge of the mechanisms involved in infection, and allow novel therapeutic solutions to be devised. Complete genome sequences for a number of pathogenic microorganisms, as well as the human host, has led to the revelation of their protein-protein interaction (PPI) networks. In this post-genomic era, pathogen-host interactions (PHIs) operating during infection can also be mapped. Detailed systematic analyses of PPI and PHI data together are required for a complete understanding of pathogenesis of infections. Here we review the striking results recently obtained during the construction and investigation of these networks. Emphasis is placed on studies producing large-scale interaction data by high-throughput experimental techniques.     

From fish bowl to bedside: The power of zebrafish to unravel melanoma pathogenesis and discover new therapeutics

Melanoma is the most aggressive and deadliest form of skin cancer. A detailed knowledge of the cellular, molecular, and genetic events underlying melanoma progression is highly relevant to diagnosis, prognosis and risk stratification, and the development of new therapies. In the last decade, zebrafish have emerged as a valuable model system for the study of melanoma. Pathway conservation, coupled with the availability of robust genetic, transgenic, and chemical tools, has made the zebrafish a powerful model for identifying novel disease genes, visualizing cancer initiation, interrogating tumor–microenvironment interactions, and discovering new therapeutics that regulate melanocyte and melanoma development. In this review, we will give an overview of these studies, and highlight recent advancements that will help unravel melanoma pathogenesis and impact human disease.     

Predicting Abdominal Aortic Aneurysm Target Genes by Level-2 Protein-Protein Interaction

Abdominal aortic aneurysm (AAA) is frequently lethal and has no effective pharmaceutical treatment, posing a great threat to human health. Previous bioinformatics studies of the mechanisms underlying AAA relied largely on the detection of direct protein-protein interactions (level-1 PPI) between the products of reported AAA-related genes. Thus, some proteins not suspected to be directly linked to previously reported genes of pivotal importance to AAA might have been missed. In this study, we constructed an indirect protein-protein interaction (level-2 PPI) network based on common interacting proteins encoded by known AAA-related genes and successfully predicted previously unreported AAA-related genes using this network. We used four methods to test and verify the performance of this level-2 PPI network: cross validation, human AAA mRNA chip array comparison, literature mining, and verification in a mouse CaPO₄ AAA model. We confirmed that the new level-2 PPI network is superior to the original level-1 PPI network and proved that the top 100 candidate genes predicted by the level-2 PPI network shared similar GO functions and KEGG pathways compared with positive genes.     

Identification and Expression of the Family of Classical Protein-Tyrosine Phosphatases in Zebrafish

Protein-tyrosine phosphatases (PTPs) have an important role in cell survival, differentiation, proliferation, migration and other cellular processes in conjunction with protein-tyrosine kinases. Still relatively little is known about the function of PTPs in vivo. We set out to systematically identify all classical PTPs in the zebrafish genome and characterize their expression patterns during zebrafish development. We identified 48 PTP genes in the zebrafish genome by BLASTing of human PTP sequences. We verified all in silico hits by sequencing and established the spatio-temporal expression patterns of all PTPs by in situ hybridization of zebrafish embryos at six distinct developmental stages. The zebrafish genome encodes 48 PTP genes. 14 human orthologs are duplicated in the zebrafish genome and 3 human orthologs were not identified. Based on sequence conservation, most zebrafish orthologues of human PTP genes were readily assigned. Interestingly, the duplicated form of ptpn23, a catalytically inactive PTP, has lost its PTP domain, indicating that PTP activity is not required for its function, or that ptpn23b has lost its PTP domain in the course of evolution. All 48 PTPs are expressed in zebrafish embryos. Most PTPs are maternally provided and are broadly expressed early on. PTP expression becomes progressively restricted during development. Interestingly, some duplicated genes retained their expression pattern, whereas expression of other duplicated genes was distinct or even mutually exclusive, suggesting that the function of the latter PTPs has diverged. In conclusion, we have identified all members of the family of classical PTPs in the zebrafish genome and established their expression patterns. This is the first time the expression patterns of all members of the large family of PTP genes have been established in a vertebrate. Our results provide the first step towards elucidation of the function of the family of classical PTPs.     

Zebrafish disease models in hematology: Highlights on biological and translational impact

Zebrafish (Danio rerio) has proven to be a versatile and reliable in vivo experimental model to study human hematopoiesis and hematological malignancies. As vertebrates, zebrafish has significant anatomical and biological similarities to humans, including the hematopoietic system. The powerful genome editing and genome-wide forward genetic screening tools have generated models that recapitulate human malignant hematopoietic pathologies in zebrafish and unravel cellular mechanisms involved in these diseases. Moreover, the use of zebrafish models in large-scale chemical screens has allowed the identification of new molecular targets and the design of alternative therapies. In this review we summarize the recent achievements in hematological research that highlight the power of the zebrafish model for discovery of new therapeutic molecules. We believe that the model is ready to give an immediate translational impact into the clinic.