The authors have nothing to disclose.

molecular evolution of the tre recombinase

Burada yönettiği, moleküler evrim yoluyla Tre recombinase nesil raporu. Tre recombinase enfekte insan hücreleri provirus eksizyon ve eradikasyon, HIV-1 provirus LTR dizileri içinde önceden tanımlanmış bir hedef sırası tanır. Biz Cre loxP olarak bilinen 34 bp çift iplikli DNA dizisi tanıyan bir 38-kDa recombinase ile başladı. Cre etkili genomik dizileri ortadan kaldırmak, çünkü biz terzi dizisi arasında entegre bir HIV-1 provirus 5&#39;-LTR ve 3&#39;-LTR kaldırmak bir recombinase yola. Bir ilk adım olarak biz loxP için benzer ve rekombinasyon aktivite için test LTR siteleri içinde dizileri belirledi. Başlangıçta Cre ve mutagenized Cre kütüphaneler, HIV-1 provirus seçilen loxLTR siteleri rekombinasyona başarısız oldu. Herhangi bir yönlendirilmiş moleküler evrim sürecinin başlamasından en az rezidüel aktivitesi gerektirdiği gibi, orijinal asimetrik loxLTR dizileri alt kümelerini içine bölünmüş ve rekombinasyon aktivite için tekrar test edildi. Ara ürün olarak hareket ederek, rekombinasyon faaliyet alt grupları ile gösterilmiştir. Sonra, recombinase kütüphaneler reiterative evrim döngüleri ile zenginleştirildi. Daha sonra, zenginleştirilmiş kütüphaneler karıştırılır ve recombined edildi. Farklı mutasyonlar kombinasyonu sinerjik kanıtladı ve recombinases loxLTR1 ve loxLTR2 rekombinasyona başardık oluşturuldu. Bu ara yoluyla evrimsel bir strateji başarılı olabilir dair kanıt oldu. Toplam 126 tekamül döngüsünde sonra bireysel recombinases, işlevsel ve yapısal analiz edildi. En aktif recombinase - Tre - Cre ile karşılaştırıldığında 19 amino asit değişiklikleri vardı. Tre recombinase tüketim genom HIV-1 ile enfekte HeLa hücreleri (bkz. &quot;Evolved Recombinase kullanarak HIV-1 Proviral DNA eksizyonu&quot; Hauber J. Deneysel Viroloji ve İmmünoloji Heinrich-Pette Enstitüsü, HIV-1 provirus başardı Hamburg, Almanya). Hala emekleme döneminde olsa da, yönlendirilmiş moleküler evrim &quot;moleküler cerrahi&quot; ve moleküler tıp araçları olarak hizmet verecek özel enzimlerin yaratılmasına izin verecek.

Watch this Scientific Journal Video about Molecular Evolution of the Tre Recombinase at JoVE.com

Molecular Evolution of the Tre Recombinase

Burada yönettiği, moleküler evrim yoluyla Tre recombinase nesil raporu. Tre recombinase enfekte insan hücreleri provirus eksizyon ve eradikasyon, HIV-1 provirus LTR dizileri içinde önceden tanımlanmış bir hedef sırası tanır. Hala emekleme döneminde olsa da, yönlendirilmiş moleküler evrim moleküler cerrahi ve moleküler tıp araçları olarak hizmet verecek özel enzimlerin yaratılmasına izin verecek.

Tre Recombinase Moleküler Evrim

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

molecular-evolution-of-the-tre-recombinase

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9.7K Görüntüleme.  Max Plank Institute for Molecular Cell Biology and Genetics, Dresden.  Burada yönettiği, moleküler evrim yoluyla Tre recombinase nesil raporu. Tre recombinase enfekte insan hücreleri provirus eksizyon ve eradikasyon, HIV-1 provirus LTR dizileri içinde önceden tanımlanmış bir hedef sırası tanır. Hala emekleme döneminde olsa da, yönlendirilmiş moleküler evrim moleküler cerrahi ve moleküler tıp araçları olarak hizmet verecek özel enzimlerin yaratılmasına izin verecek. 

Video: Molecular Evolution of the Tre Recombinase

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Site Özel Recombinase (SSR) Teknoloji İlkeleri

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Biyoloji

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.

Röportaj: Evolved Recombinase kullanarak HIV-1 Proviral DNA eksizyonu

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

Predasyon Risk, Herbivore Fizyolojik Stres ve Bitki Çöp mikrobiyal bozunma Bağlama

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

Genetik olarak kodlanmış Molecular Probes G protein-bağlanmış alıcılar Çalışması için

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Hayatta kalma Süre deneyinde<em&gt; Brachionus</em&gt; Rotiferler: Maternal Koşullarının Senkronizasyon

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Genom kapsamındaki bir tanımlama ve ifade Meta-Analizi ATL E3 Ubikuitin ligaz gen ailesinin asma için kapsamlı iş akışı

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Mezenkimal endotel geçiş büyüme faktörü β sinyal dönüşüm tarafından indüklenen moleküler Analizi

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

Diseksiyon ve DiceCT gizli ortaya başka veri evrim primat yüz ve ters

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

Otomatik Görüntüleme Sistemi kullanarak iPSC'lerin Birleşme Noktasının Ölçülmesi.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

RNA-seq Kullanarak Moleküler Evrimi ve Gen ekspresyonını Araştırmak için Biyoinformatik Boru Hattı

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus ...