The position of the epistatic S locus in the halothane linkage group in pigs

The linkage of the Phi, Pgd, Po2, S, H and halothane sensitivity loci was followed in a Belgian Landrace family, heterozygous for these systems over 6 generations. Recombination next to the S locus occurred mainly in pigs belonging to this particular family. From this investigation the position of the S locus is proved to be outwith the Phi-Pgd region, next to Phi . Therefore the gene sequence S - Phi - Hal -H- Po2 -Pgd is proposed. Higher recombination rates were observed in the female parental line of the multiheterozygous family when compared to the male parental line. Additional data from animals, unrelated to this strain, confirm the evidence of close linkage of the S system to the nearest marker loci.     

ΦC31位点特异性整合酶DNA结合功能域的鉴定

DNA结合功能域的确定是阐明位点特异性重组酶整合机制的关键,而对酶DNA结合功能域进行突变研究是提高酶整合效率和整合特异性的重要方法.为了鉴定ΦC31位点特异性整合酶的DNA结合功能域,依据对ΦC31整合酶序列的生物信息学分析结果,利用PCR和克隆技术在pET22b原核表达载体上构建ΦC31整合酶重组截短突变体表达质粒,将获得的表达质粒转化入大肠杆菌BL21(DE3)菌株扩大培养并用IPTG诱导融合蛋白的表达,经镍柱纯化获得了纯度达90%以上的重组蛋白,分子量也与预期大小一致,Western印迹确定了重组蛋白的特异性.凝胶迁移滞后实验显示野生型以及截短突变体蛋白ΦC311-528、ΦC311-472、ΦC311-413能与细菌附着位点DNAattB和噬菌体附着位点DNAattP结合的条带,而截短突变体ΦC311-353、ΦC311-279、ΦC311-120观察不到相应的结合条带.6个截短突变体质粒在体内重组活性蓝白斑实验中均表现为蓝斑,显示出皆丧失体内重组活性.研究证实,ΦC31整合酶半胱氨酸富集域(第353～413位氨基酸)具有DNA结合的功能,而C末端缬氨酸富集区(第528～613位氨基酸)也与其重组活性相关.这为进一步了解ΦC31整合酶的结构与功能,最终引导其结构进化,提高其特异性和整合效率奠定了基础.     

A high-throughput genome-walking method and its use for cloning unknown flanking sequences

We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5′ ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5′ end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5′ flanking regions/promoters of selected plant genes.     

Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymerase

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.     

Investigating the Effect of Chain Connectivity on the Folding of a Beta-Sheet Protein On and Off the Ribosome

Determining the relationship between protein folding pathways on and off the ribosome remains an important area of investigation in biology. Studies on isolated domains have shown that alteration of the separation of residues in a polypeptide chain, while maintaining their spatial contacts, may affect protein stability and folding pathway. Due to the vectorial emergence of the polypeptide chain from the ribosome, chain connectivity may have an important influence upon cotranslational folding. Using MATH, an all β-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome. From Φ-value analysis, we show that the most structured region of the transition state for folding in MATH includes the N and C terminal strands, which are located adjacent to each other in the structure. However, arrest peptide force-profile assays show that wild-type MATH is able to fold cotranslationally, while some C-terminal residues remain sequestered in the ribosome, even when destabilized by 2–3?kcal?mol^?1. We show that, while this pattern of Φ-values is retained in two circular permutants in our studies of the isolated domains, one of these permutants can fold only when fully emerged from the ribosome. We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both β-sheets, rather than by the location of the terminal strands in the ribosome tunnel.     

ΦC31整合酶介导猪基因组定点修饰的探讨

高效与特异的基因组定点修饰是基因工程动物研究的前沿与难点.链霉菌噬菌体ΦC31整合酶能介导含attB位点的外源基因定点整合于多种真核生物基因组的假attP位点,可维持外源基因的正常结构及高效表达.本文探讨ΦC31整合酶介导外源基因在猪基因组内定点整合的分子基础.构建含attB位点的报告载体pEGFP-N1-attB,与ΦC31整合酶表达载体pCMV-INT共转染猪肾PK15细胞,G418筛选获得单克隆细胞系.实时荧光定量PCR筛选出单拷贝整合的转基因细胞系.TAIL-PCR鉴定出1个猪基因组假attP位点,位于猪1号染色体,watson链,坐标114220087-114220126,命名为pig-attP-1.测序结果显示,pEGFP-N1-attB在attB位点处断开插入到pig-attP-1.用荧光计测定细胞培养基上清EGFP含量发现,该转基因细胞系EGFP的表达水平是本底的50倍(13500 AU vs.280 AU),表明pig-attP-1是利于外源基因高效表达的"友好位点".该研究不仅为实现外源基因在猪基因组内的定点整合提供了新策略,也为创制基因工程猪、建立动物生物反应器等研究注入了新思路.     

滚环DNA扩增的原理、应用和展望

滚环DNA扩增 (rollingcircleDNAamplification ,RCA)是一种等温信号扩增方法 ,其线性扩增倍数为 1 0 ⁵,指数化扩增能力大于 10⁹,产生的扩增产物连接在固相支持物 (如玻片、微孔板等 )表面的DNA引物或抗体上。RCA是一种适合在芯片上 (on chip)进行信号扩增的新技术 ,它既能提供研究分析的敏感性和特异性 ,又能保持立体分析的多元性。RCA亦是一种痕量的分子检测方法 ,可用于极其微量的生物大分子和生物标志的检测与研究     

Bacteria and archaea as the sources of traits for enhanced plant phenotypes

Rising global demand for food and population increases are driving the need for improved crop productivity over the next 30 years. Plants have inherent metabolic limitations on productivity such as inefficiencies in carbon fixation and sensitivity to environmental conditions. Bacteria and archaea inhabit some of the most inhospitable environments on the planet and possess unique metabolic pathways and genes to cope with these conditions. Microbial genes involved in carbon fixation, abiotic stress tolerance, and nutrient acquisition have been utilized in plants to enhance plant phenotypes by increasing yield, photosynthesis, and abiotic stress tolerance. Transgenic plants expressing bacterial and archaeal genes will be discussed along with emerging strategies and tools to increase plant growth and yield.     

A novel method to perform genomic walks using a combination of single strand DNA circularization and rolling circle amplification

Characterization of regions flanking a known sequence within a genome, known as genome walking, is a cornerstone technique in modern genetic analysis. In the present work we have developed a new PCR-dependent, directional genome walking protocol based on the unique circularization property of a novel DNA ligase, CircLigase. In the first step, PCR based primer extension is performed using a phosphorylated primer, designed to extend from the boundary of the known sequence, into the flanking region. This linear amplification results in the generation of single-stranded (ss) DNA, which is then circularized using CircLigase. Using the hyperbranching activity of Phi29 DNA polymerase, the circular ssDNA is then linearized by rolling circle amplification, resulting in copious amounts of double stranded concatameric DNA. Nested primers are used to amplify the flanking sequence using inverse PCR. The products are resolved on an agarose gel and the bands whose mobility change due to the nested location of the primer combination used are identified, extracted, and cloned into a plasmid vector for sequencing. Empirical proof for this concept was generated on two antimicrobial biosynthetic genes in Pseudomonas sp. LBUM300. Using the hcnB and phlD genes as starting points, ca 1 kb of flanking sequences were successfully isolated. The use of locus specific primers ensured both directionality and specificity of the walks, alleviating the generation of spurious amplicons, typically observed in randomly primed walking protocols. The presented genome walking protocol could be applied to any microbial genome and requires only 100-150 bp of prior sequence information. The proposed methodology does not entail laborious testing of restriction enzymes or adaptor ligation. This is the first report of a successful application of the novel ligase enzyme, CircLigase for genomic walking purposes.