Phylogenetic relationships among Hystrix species and related species based on expressed sequence tag-polymerase chain reaction

Twelve species, including three Hystrix species, five Leymus species, Hordeum bogdanii, Pseudoroegneria spicata, Psathyrostachys huashanica, and Roegneria ciliaris, were used for expressed sequence tag-polymerase chain reaction (EST-PCR) assay. A total of 125 products were amplified by 72 sets of EST-PCR markers developed in barley, among which 106 (84.8%) products were found to be polymorphic. Each EST-PCR marker produced 0–6 polymorphic bands, with an average of 1.47. The relationship between H. duthiei s...     

两种鲤鱼胰凝乳蛋白酶原基因的电子克隆及分析

为研究胰凝乳蛋白酶原在鱼类中的生理功能和作用机制,利用生物信息学的方法,成功获得了鲤鱼两种胰凝乳蛋白酶原的cDNA序列(ccCHTR1和ccCHTR2)并对其进行序列分析。结果显示,ccCHTR1 cDNA含有792 bp的开放阅读框,编码263个氨基酸;ccCHTR2 cDNA含有798 bp的开放阅读框,编码265个氨基酸。二者氨基端均含有18个氨基酸组成的信号肽,同时,在成熟肽的第15和16个氨基酸(R-I)之间存在一个切割位点。氨基酸比对结果显示,ccCHTR1和ccCHTR2具备胰凝乳蛋白酶原的保守结构特征,同时二者有72.8%的同源性,且都与斑马鱼有最高的同源性,分别是93.3%和73.5%。进化分析显示,二者分别与斑马鱼和鳕鱼亲缘关系最近,与哺乳动物的亲缘关系较远。     

Utilization of fluorescently-labeled tetracysteine-tagged proteins to study virus entry by live cell microscopy

Viruses exploit cellular machinery to gain entry and initiate their replication cycle within host cells. The development of methods to visualize virus entry in live cells has provided new insights to the cellular processes involved in virus entry and the intracellular locations where viral payloads are deposited. The use of fluorescently labeled virus and high-resolution microscopy is currently the method of choice to study virus entry in live cells. While fluorescent protein fusions (e.g. viral proteins fused to GFP) have been used, the labeling of viral proteins that contain a small tetracysteine (tc) tag with biarsenical fluorescent compounds (e.g. FlAsH, ReAsH, Lumio-x) offers several advantages over conventional xFP-fusion constructs. This article describes methods for generating fluorescently labeled viruses encoding tc-tagged proteins that are suitable for the study of virus entry in live cells by fluorescence microscopy. Critical parameters required to quantify fluorescence signals from the labeled, tc-tagged proteins in individual virus particles during the entry process and the subsequent fate of the labeled viral proteins after virus uncoating are also described.     

A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

Expression and purification of aggregation‐prone and disulfide‐containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high‐value target proteins. Here, we present a novel gene‐fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag‐Acidic‐Target Tag) results in near‐complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one‐step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization.     

Optimized E. coli expression strain LOBSTR eliminates common contaminants from His‐tag purification

His‐tag affinity purification is one of the most commonly used methods to purify recombinant proteins expressed in E. coli. One drawback of using the His‐tag is the co‐purification of contaminating histidine‐rich E. coli proteins. We engineered a new E. coli expression strain, LOBSTR (lo w b ackground str ain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging low‐expressing protein targets by reducing background contamination with no additional purification steps, materials, or costs, and thus pushes the limits of standard His‐tag purifications. Proteins 2013; 81:1857–1861. © 2013 Wiley Periodicals, Inc.     

无标记microRNA芯片分析方法的建立与优化

MicroRNAs(miRNAs)是生物体内源的一类非编码小分子RNA,它与癌症的发生息息相关,是一个有潜质的生物标志物,细胞的发育、分化、增殖、凋亡都与miRNAs调控有关．miRNAs研究中关键环节是其表达谱的分析,用芯片分析miRNAs表达谱,通常要在杂交前对样品进行分离、标记、纯化,这是整个检测过程中最耗时耗力、费用昂贵的一个步骤,且在此过程中由于酶的使用及步骤的增加还可能改变样品中目标序列的初始比例,影响实验结果的可靠性．为解决这些问题,作者建立了一种新型的“无标记microRNA芯片分析” 方法,该技术基于堆积杂交(stacking hybridization)原理,引入一段预先标记荧光的通用标签序列(universal tag,UT),因而被命名为SHUT检测方法．本文主要对SHUT assay的整个实验过程进行了系统优化,并对其灵敏度和特异性等相关指标进行了评价．实验结果表明：该新型芯片技术检测灵敏度可达2 fmol/L,不但可以区分只有1个碱基差异的miRNAs家族成员,还可以有效排除非活性pri-miRNA与pre-miRNA前体的交叉杂交信号,而且100 ng的总RNA即可用于检测分析;该新型芯片技术对于miRNAs及其他短链核酸分子的检测分析是一个理想快速的检测平台．     

PRODUCTION OF SMALL ANTIBACTERIAL PEPTIDES USING SILKWORM–BACULOVIRUS PROTEIN EXPRESSION SYSTEM

The recombinant proteins with strong antimicrobial activity are known to be very difficult to express using bacterial expression system. Here, human β-defensin (DEFB) 1, DEFB2, and DEFB3 were successfully produced using a silkworm–baculovirus protein expression system. We have generated four baculoviruses for each DEFB protein to compare the effect of different peptide tags in secretion into silkworm larval hemolymph. Interestingly, the best performing peptide tags for the secretion were different among DEFBs: C-terminal GST-H8 tag for DEFB1, N-terminal H8 tag for DEFB2, and C-terminal H8 tag for DEFB3, respectively. In addition, the colony count assay demonstrated that the recombinant DEFB2 s showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus thiaminolyticus.     

Effects of geolocation archival tags on reproduction and adult body mass of sooty shearwaters (Puffinus griseus)

Abstract

We attached 11 g (1.4% body‐mass equivalent) global location sensing (GLS) archival tag packages to tarsi of 25 breeding sooty shearwaters (Puffinus griseus, titi) on Whenua Hou (Codfish Island), New Zealand during the chick‐rearing period in 2005. Compared with chicks reared by non‐handled adults that did not carry tags, deployment of tags on one or both adult parents ultimately resulted in 35% reduction in chick body mass and significantly reduced chick skeletal size preceding fledging (19 April). However, body mass between chick groups was not significantly different after controlling for skeletal size. Effects on chicks were more pronounced in six pairs where both parents carried tags. Chick mass was negatively related to the duration that adults carried tags. In this study, none of the chicks reared by pairs where both parents were tagged, 54% of chicks reared by pairs where one parent was tagged, and 83% of chicks reared by non‐handled and non‐tagged parents achieved a previously determined pre‐fiedging mass threshold (564 g; Sagar & Horning 1998). Body mass of adults carrying tags and returning from trans‐equatorial migration the following year were 4% lighter on average than non‐tagged birds, but this difference was not statistically significant. Reduced mass among chicks reared by adults carrying tags during the chick‐provisioning period indicated that adults altered “normal” provisioning behaviours to maintain their own body condition at the expense of their chicks. Population‐level information derived from telemetry studies can reveal important habitat‐linked behaviours, unique aspects of sea‐bird foraging behaviours, and migration ecology. Information for some species (e.g., overlap with fisheries) can aid conservation and marine ecosystem management. We advise caution, however, when interpreting certain data related to adult provisioning behaviours (e.g., time spent foraging, provisioning rates, etc.). If effects on individuals are of concern, we suggest shorter‐term deployments, smaller and lighter tags, and alternative attachment techniques, especially when investigating threatened or endangered species.