Recombinase-mediated cassette exchange (RMCE) — A rapidly-expanding toolbox for targeted genomic modifications

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.     

Quantifying soil organic carbon in complex landscapes: an example of grassland undergoing encroachment of woody plants

The invasion of woody plants into grass‐dominated ecosystems has occurred worldwide during the past century with potentially significant impacts on soil organic carbon (SOC) storage, ecosystem carbon sequestration and global climate warming. To date, most studies of tree and shrub encroachment impacts on SOC have been conducted at small scales and results are equivocal. To quantify the effects of woody plant proliferation on SOC at broad spatial scales and to potentially resolve inconsistencies reported from studies conducted at fine spatial scales, information regarding spatial variability and uncertainty of SOC is essential. We used sequential indicator simulation (SIS) to quantify spatial uncertainty of SOC in a grassland undergoing shrub encroachment in the Southern Great Plains, USA. Results showed that both SOC pool size and its spatial uncertainty increased with the development of woody communities in grasslands. Higher uncertainty of SOC in new shrub‐dominated communities may be the result of their relatively recent development, their more complex above‐ and belowground architecture, stronger within‐community gradients, and a greater degree of faunal disturbance. Simulations of alternative sampling designs demonstrated the effects of spatial uncertainty on the accuracy of SOC estimates and enabled us to evaluate the efficiency of sampling strategies aimed at quantifying landscape‐scale SOC pools. An approach combining stratified random sampling with unequal point densities and transect sampling of landscape elements exhibiting strong internal gradients yielded the best estimates. Complete random sampling was less effective and required much higher sampling densities. Results provide novel insights into spatial uncertainty of SOC and its effects on estimates of carbon sequestration in terrestrial ecosystem and suggest effective protocol for the estimating of soil attributes in landscapes with complex vegetation patterns.     

Birds adjust acoustic directionality to beam their antipredator calls to predators and conspecifics

Animals in many vertebrate species vocalize in response to predators, but it is often unclear whether these antipredator calls function to communicate with predators, conspecifics or both. We evaluated the function of antipredator calls in 10 species of passerines by measuring the acoustic directionality of these calls in response to experimental presentations of a model predator. Acoustic directionality quantifies the radiation pattern of vocalizations and may provide evidence about the receiver of these calls. We predicted that antipredator calls would have a lower directionality if they function to communicate with surrounding conspecifics, and a higher directionality and aimed at the receiver if they function to communicate with the predator. Our results support both of these functions. Overall, the birds produce antipredator calls that have a relatively low directionality, suggesting that the calls radiate in many directions to alert conspecifics. However, birds in some species increase the directionality of their calls when facing the predator. They can even direct their calls towards the predator when facing lateral to it—effectively vocalizing sideways towards the predator. These results suggest that antipredator calls in some species are used to communicate both to conspecifics and to predators, and that birds adjust the directionality of their calls with remarkable sophistication according to the context in which they are used.     

Efficient reversal of phiC31 integrase recombination in mammalian cells

Over the past decade, the integrase enzyme from phage phiC31 has proven to be a useful genome engineering tool in a wide variety of species, including mammalian cells. The enzyme efficiently mediates recombination between two distinct sequences, attP and attB, producing recombinant product sites, attL and attR. The reaction proceeds exclusively in a unidirectional manner, because integrase is unable to synapse attL and attR. To date, use of phiC31 integrase has been limited to attP × attB recombination. The factor needed for the reverse reaction – the excisionase or recombination directionality factor (RDF) – was identified recently and shown to function in vitro and in bacterial cells. To determine whether the phiC31 RDF could also function in mammalian cells, we cloned and tested several vectors that permit assessment of phiC31 RDF activity in mammalian environments. In the human and mouse cell lines tested (HeLa, HEK293, and NIH3T3), we observed robust RDF activity, using plasmid and/or genomic assays. This work is the first to demonstrate attL–attR serine integrase activity in mammalian cells and validates phiC31 RDF as a new tool that will enable future studies to take advantage of phiC31 integrase recombination in the forward or reverse direction.     

The post-rigor structure of myosin VI and implications for the recovery stroke

Myosin VI has an unexpectedly large swing of its lever arm (powerstroke) that optimizes its unique reverse direction movement. The basis for this is an unprecedented rearrangement of the subdomain to which the lever arm is attached, referred to as the converter. It is unclear at what point(s) in the myosin VI ATPase cycle rearrangements in the converter occur, and how this would effect lever arm position. We solved the structure of myosin VI with an ATP analogue (ADP.BeF3) bound in its nucleotide-binding pocket. The structure reveals that no rearrangement in the converter occur upon ATP binding. Based on previously solved myosin structures, our structure suggests that no reversal of the powerstroke occurs during detachment of myosin VI from actin. The structure also reveals novel features of the myosin VI motor that may be important in maintaining the converter conformation during detachment from actin, and other features that may promote rapid rearrangements in the structure following actin detachment that enable hydrolysis of ATP.     

Fine-Resolution Mapping of TF Binding and Chromatin Interactions

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Serine Integrase attP Binding and Specificity

Serine integrases catalyze the site-specific insertion of viral DNA into a host's genome. The minimal requirements and irreversible nature of this integration reaction have led to the use of serine integrases in applications ranging from bacterial memory storage devices to gene therapy. Our understanding of how the integrase proteins recognize the viral (attP) and host (attB) attachment sites is limited, with structural data available for only a Listeria integrase C-terminal domain (CTD) bound to an attP half-site. Here we report quantitative binding and saturation mutagenesis analyses for the Listeria innocua prophage attP site and a new 2.8-Å crystal structure of the CTD?attP half site. We find that Int binds with high affinity to attP (6.9?nM), but the Int CTD binds to attP half-sites with only 7- to 10-fold lower affinity, supporting the idea that free energy is expended to open an Int dimer for attP binding. Despite the 50-bp Int–attP interaction surface, only 20 residues are sensitive to mutagenesis, and of these, only 6 require a specific residue for efficient Int binding and integration activity. One of the integrase DNA-binding domains, the recombinase domain, appears to be primarily non-specific. Several substitutions result in an improved attP site, indicating that higher-efficiency attachment sites can be obtained through site engineering. These findings advance our understanding of serine integrase function and provide important data for efforts towards engineering this family of enzymes for a variety of biotechnology applications.     

杨树光肩星天牛种群空间格局的地统计学研究

根据在宁夏青铜峡市４块不同类型样地上收集到的光肩星天牛卵、幼虫和成虫的数量和位置资料,应用地统计学方法对其处群的空间格局进行了研究,结果表明,在各种林型中天牛卵和幼虫的数量具有明显的空间依赖性,其变程分别为２０－４０ｍ和２８－１７０ｍ,局部空间连续性强度分别为０．２６－０．４７和０．１４－０．５５５;而成虫的数量没有表现出空间依赖性,在空间呈随机分布。对不同样方大小的变异曲线图进行比较得知;在一定