Phage integrases: biology and applications

Phage integrases are enzymes that mediate unidirectional site-specific recombination between two DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB. Integrases may be grouped into two major families, the tyrosine recombinases and the serine recombinases, based on their mode of catalysis. Tyrosine family integrases, such as lambda integrase, utilize a catalytic tyrosine to mediate strand cleavage, tend to recognize longer attP sequences, and require other proteins encoded by the phage or the host bacteria. Phage integrases from the serine family are larger, use a catalytic serine for strand cleavage, recognize shorter attP sequences, and do not require host cofactors. Phage integrases mediate efficient site-specific recombination between two different sequences that are relatively short, yet long enough to be specific on a genomic scale. These properties give phage integrases growing importance for the genetic manipulation of living eukaryotic cells, especially those with large genomes such as mammals and most plants, for which there are few tools for precise manipulation of the genome. Integrases of the serine family have been shown to work efficiently in mammalian cells, mediating efficient integration at introduced att sites or native sequences that have partial identity to att sites. This reaction has applications in areas such as gene therapy, construction of transgenic organisms, and manipulation of cell lines. Directed evolution can be used to increase further the affinity of an integrase for a particular native sequence, opening up additional applications for genomic modification.     

Strategies for transgenic manipulation of monoterpene biosynthesis in plants

Monoterpenes, the C(10) isoprenoids, are a large family of natural products that are best known as constituents of the essential oils and defensive oleoresins of aromatic plants. In addition to ecological roles in pollinator attraction, allelopathy and plant defense, monoterpenes are used extensively in the food, cosmetic and pharmaceutical industries. The importance of these plant products has prompted the definition of many monoterpene biosynthetic pathways, the cloning of the relevant genes and the development of genetic transformation techniques for agronomically significant monoterpene-producing plants. Metabolic engineering of monoterpene biosynthesis in the model plant peppermint has resulted in yield increase and compositional improvement of the essential oil, and also provided strategies for manipulating flavor and fragrance production, and plant defense.     

Integration of chromosome set manipulation and transgenic technologies for fishes.

Chromosome set manipulation techniques have significant implications for research of transgenic fish. Gynogenesis can be used to generate isogenic lines, to map genes in relation to their centromeres, and to produce fish carrying extra chromosome fragments of foreign origin. Androgenesis can be used to produce isogenic lines and to recover strains from cryopreserved sperm. Triploidy can be induced in fish with heat or pressure treatment of fertilized eggs and by crossing tetraploid individuals with normal diploids. Triploid fish are typically effectively sterile. Triploid interspecific hybrids are usually more viable than the corresponding diploid hybrids. Given concerns about potential reproduction of transgenic fish in the wild, induced triploidy could facilitate application of transgenic technologies in some situations.     

Achievements and problems in cloning and making transgenic mammals

The current use of nuclear transfer for cloning of mammals providing its application in agriculture, for biomedical aims and restoration of biodiversity was analyzed. Main problems associated with the method complexity and its low efficiency as well as ethic problems were discussed. Future application prospects were evaluated.     

Genetic manipulation of gibberellin metabolism in transgenic rice

The 'green revolution' was fueled by the introduction of the semi-dwarf trait into cereal crop cultivars. The semi-dwarf cultivars--which respond abnormally to the plant growth hormone gibberellin (GA)--are more resistant to wind and rain damage and thus yield more grain when fertilized. To generate dwarf rice plants using a biotechnological approach, we modified the level of GA by overproduction of a GA catabolic enzyme, GA 2-oxidase. When the gene encoding GA 2-oxidase, OsGA2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism but failed to set grain because GA is involved in both shoot elongation and reproductive development. In contrast, OsGA2ox1 ectopic expression at the site of bioactive GA synthesis in shoots under the control of the promoter of a GA biosynthesis gene, OsGA3ox2 (D18), resulted in a semi-dwarf phenotype that is normal in flowering and grain development. The stability and inheritance of these traits shows the feasibility of genetic improvement of cereal crops by modulation of GA catabolism and bioactive GA content.     

A system for the three-dimensional construction,manipulation and display of microbiological models

A system is described for building up serial sections into a three dimensional structure, incorporating density, that can be displayed and then further manipulated by rotation about three orthogonal axes. The initial application was to produce a computer model of a protein structure and to compare the diverse images obtained from rotation with the two dimensional images observed in related electron micrographs. To obtain sufficient contrast in the electron microscope images of protein structures, the specimens need to be stained and since this can cause some deformation of the observed images, it is also necessary to simulate the possible effects of stain on the protein model. Because of the need to compare numerous orientations of the combined model, techniques are available either for speeding up the comparison or for obtaining better accuracy. The methods have been applied to the interpretation of electron micrograph images of microbiological specimens, where the three dimensional structure of the specimen is an important aid in understanding its biological function, but the techniques are also applicable to more general serial reconstruction requirements.     

hGFAP‐cre transgenic mice for manipulation of glial and neuronal function in vivo

With the goal of performing astrocyte‐specific modification of genes in the mouse, we have generated a transgenic line expressing Cre recombinase under the control of the human glial fibrillary acidic protein (hGFAP) promoter. Activity was monitored by crossing the hGFAP‐cre transgenics with either of two reporter lines carrying a lacZ gene whose expression requires excision of loxP‐flanked stop sequences. We found that lacZ expression was primarily limited to the central nervous system, but therein was widespread in neurons and ependyma. Cell types within the brain that notably failed to activate lacZ expression included Purkinje neurons of the cerebellum and choroid plexus epithelium. Onset of Cre expression began in the forebrain by e13.5, suggesting that the hGFAP promoter is active in a multi‐potential neural stem cell. genesis 31:85–94, 2001. © 2001 Wiley‐Liss, Inc.     

Pleiotropic effect of flavonoid biosynthesis manipulation in transgenic potato plants

Three approaches were successfully used to manipulate content of flavonoids in transgenic plants. Overexpressing either the adaptor 14-3-3 protein or genes coding the key enzymes of the flavonoid biosynthesis pathway resulted in a significant increase in the compound content in potato tuber epidermis. The opposite effect was observed in transgenic plants in which these proteins were repressed; this strongly supports the view that the gene construct determines transgenic plant features. The most effective construct was, however, the one containing single dihydroflavonol reductase (DFR) gene in sense orientation. In all cases the increase in flavonoid content resulted in the expected enhancement of the antioxidant capacity of tuber extract. At the biochemical level a decrease in the starch content in transgenic plant overexpressing proteins regulating flavonoid biosynthesis was detected. In the case of glucosyl transferase (GT) gene overexpression, the content of phenolic compounds remained at the control level, however, the antioxidant capacity of tuber extracts significantly decreased. The GT plants grew faster and were more resistant to pathogen attacks, the tuber yield was significantly higher than that of nontransformants. Thus it is speculated that it is the chemical structure and degree of glucosylation of flavonoids rather than their quantity which determines transgenic plant features.     

A transgenic Tbx6;CreERT2 line for inducible gene manipulation in the presomitic mesoderm

The rhythmic segmentation process of the presomitic mesoderm (PSM) orchestrates the formation of somites, the fundamental units for the vertebrate axial body plan. To aid the investigation of molecular components governing the conversion from PSM into somites, we generated a transgenic mouse line that expresses a tamoxifen (tmx) inducible CreER(T2) under the control of a 2.5 kb enhancer element of Tbx6, a gene essential for PSM formation and somite patterning. Combined with Cre reporters, this Tbx6;CreER(T2) line displays robust tmx-inducible Cre activity in the PSM at various embryonic stages. This tool should be useful for studying gene function during somitogenesis by either conditional inactivation or mis-expression, and potentially coupled with cell marking.     

The design, construction and use of a radio-tracking system for some British mammals

Radio-tracking equipment designed by the late G. E. Ashwell is described. Detailed instructions are given for building transmitters suitable for foxes and rats so that the circuits can be made by people with only limited electronic knowledge. Constructional details are also provided for receiver aerials. The address of a firm that can supply receivers is given. The function and performance of each part of the equipment is described and details provided of the methods used by the authors to track mammals. The handling of mammals and attachment of transmitters is covered briefly. Some comments are included on the limitations of radio-tracking as a means of studying wild mammals.     

Nucleophile selection for the endonuclease activities of human, ovine, and avian retroviral integrases

Retroviral integrases catalyze four endonuclease reactions (processing, joining, disintegration, and nonspecific alcoholysis) that differ in specificity for the attacking nucleophile and target DNA sites. To assess how the two substrates of this enzyme affect each other, we performed quantitative analyses, in three retroviral systems, of the two reactions that use a variety of nucleophiles. The integrase proteins of human immuno- deficiency virus type 1, visna virus, and Rous sarcoma virus exhibited distinct preferences for water or other nucleophiles during site-specific processing of viral DNA and during nonspecific alcoholysis of nonviral DNA. Although exogenous alcohols competed with water as the nucleophile for processing, the alcohols stimulated nicking of nonviral DNA. Moreover, different nucleophiles were preferred when the various integrases acted on different DNA targets. In contrast, the nicking patterns were independent of whether integrase was catalyzing hydrolysis or alcoholysis and were not influenced by the particular exogenous alcohol. Thus, although the target DNA influenced the choice of nucleophile, the nucleophile did not affect the choice of target sites. These results indicate that interaction with target DNA is the critical step before catalysis and suggest that integrase does not reach an active conformation until target DNA has bound to the enzyme.     

Genetic manipulation of Aspergillus nidulans: meiotic progeny for genetic analysis and strain construction

The multicellular microbial eukaryote Aspergillus nidulans is an excellent model for the study of a wide array of biological processes. Studies in this system contribute significantly to understanding fundamental biological principles and are relevant for biotechnology and industrial applications, as well as human, animal and plant fungal pathogenesis. A. nidulans is easily manipulated using classical and molecular genetics. Here, we describe the storage and handling of A. nidulans and procedures for genetic crossing, progeny analysis and growth testing. These procedures are used for Mendelian analysis of segregation of alleles to show whether a mutant phenotype segregates as a single gene and independent assortment of genes to determine the linkage relationship between genes. Meiotic crossing is used for construction of multiple mutant strains for genetic analysis. Genetic crossing and analysis of progeny can be undertaken in 2-3 weeks and growth testing takes 2-3 days.     

人源抗狂犬病毒单克隆抗体载体的构建及烟草转基因研究(四)

为了构建高效表达人源抗狂犬病毒单克隆抗体载体,首先对人源抗狂犬病毒抗体(SO57)重链和轻链编码基因的密码子进行偏好性改造,添加增强外源基因表达的控制元件,然后分别与花椰菜花叶病毒35s启动子和木薯叶脉花叶病毒启动子融合,连接至植物表达载体pBI121上,然后将构建好的载体转入农杆菌LB4404,采用叶盘法转化烟草叶片.用分子生物学技术,对6株转基因烟草进行检测,电泳检测结果均为阳性.用酶联接免疫吸附剂法,检测6株烟草叶片中人源抗狂犬病毒单克隆抗体的表达.结果表明,6株烟草均成功表达人源抗狂犬病毒抗体.     

几种新型植物基因表达载体的构建方法

利用基因工程技术手段研究基因功能过程中,构建基因表达载体处于转基因植物的主导地位,采用合适的构建方法会使实验效果事半功倍。植物基因表达载体的构建方法除了传统构建法、Gateway技术、三段T-DNA法、一步克隆法等,还有近年来出现的几种新型的载体构建方法:基于竞争性连接原理快速构建小片段基因表达载体;MicroRNA前体PCR置换法适用于构建小分子RNA表达载体;重组融合PCR法特别适用于插入片段中含有较多限制性酶切位点的载体构建;利用In-Fusion试剂盒可以将任何目的片段插入一个线性化载体的某个区域;构建多片段复杂载体可采用不依赖序列和连接的克隆方法(Sequence and ligation-independent cloning,SLIC)法;Gibson等温拼接法;Golden Gate拼接法。本文将在总结分析前人工作的基础上,结合自己工作的体会和经验分析这7种新方法的特点,期望通过这几种新的方法给植物基因工程表达载体的构建提供新的思路。     

Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse.

Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene segment of >7 kb with the neo gene inserted for positive selection of the targeting event. More complex targeting vectors carry additional genetic elements (e.g. lacZ, loxP, point mutations). Here we use homologous recombination in yeast to construct targeting vectors for the incorporation of genetic elements (GEs) into mouse genes. The precise insertion of GEs into any position of a mouse gene segment cloned in an Escherichia coli/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking the GEs. In this way, complex targeting vectors can be engineered with considerable ease and speed, obviating extensive gene mapping in search for suitable restriction sites.     

Attachment site recognition and regulation of directionality by the serine integrases

Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between ‘attachment sites’ in the phage (attP) and the host (attB). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase bound to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP × attB synaptic complexes to initiate recombination, and how attL × attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR. The results provide a structural framework for understanding, testing and engineering serine integrase function.     

Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants

Excessive softening is the main factor limiting fruit shelf life and storage. Transgenic plants modified in the expression of cell wall modifying proteins have been used to investigate the role of particular activities in fruit softening during ripening, and in the manufacture of processed fruit products. Transgenic experiments show that polygalacturonase (PG) activity is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG -subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of -subunit protein accumulation increases softening. All these pectin-modifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of -galactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. Suppression or overexpression of endo-(1\to4)-d-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansin protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably -galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process.     

辅助噬菌体在噬菌体展示中的应用及研究进展

噬菌体展示技术是一种将外源肽或蛋白质与特定噬菌体衣壳蛋白相融合,展示于噬菌体表面来构建蛋白质或多肽文库,并从中筛选目的蛋白、多肽或抗体的基因工程高新技术。噬菌粒/辅助噬菌体系统是最常用的噬菌体展示系统,此系统中辅助噬菌体对噬菌粒的复制和组装发挥着至关重要的作用。本文结合当今该领域的最新研究动态,概述了噬菌粒和辅助噬菌体双基因组系统,着重介绍了不同辅助噬菌体的特点及其突变机制,并对其应用前景进行了展望,以期为该技术的进一步完善提供一定的借鉴作用。