Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology

We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two ∼750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5–10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5′ overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5′ overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.     

The role of microtubules in rapid hyphal tip growth of Aspergillus nidulans

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-alpha-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew approximately 5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10x reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.     

Tied hands: synchronism between beak development and feeding-related morphological changes in ommastrephid squid paralarvae

Ommastrephid squid produce some of the smallest cephalopod hatchlings, whose feeding behavior has not been observed. The present study aimed at indirectly filling this knowledge gap by describing ontogenetic changes in beak morphology and morphometry and integrating these results with published datasets on Illex argentinus arm crown morphology and gut contents. Individuals [0.7–15 mm mantle length (ML)] were measured, weighed, and had their buccal mass extracted. Jaw measurements were correlated with ML to determine whether jaw development occurred linearly with ML. For a 10 mm increment in ML, weight increased 430-fold. The jaws of hatchlings were rudimentary, but in larger paralarvae the rostrum protrudes and the jaw features (teeth, slit, groove) disappear. Increases in ML were predicted by beak robustness indices and rostrum protrusion, with growth discontinuities pointing to faster growth in individuals ≤ 2 mm ML. Morphological changes in the beak and arm crown are in synchrony with a transitional event in the feeding ecology of paralarvae: the onset of active predation on crustaceans and masticating their exoskeletons for ingestion. Integration of the results with published data has led to the proposal of a hypothesis of four size-differentiated developmental stages in the feeding ecology of I. argentinus rhynchoteuthions.

     

Under pressure: Short- and long-term response to predation varies in two populations of a live-bearing fish

The non-lethal effects of predation can significantly influence animal behavior and population composition. Research has often centered around prey response to predator exposure in the short term, but fewer studies have highlighted the effects of long-term predator exposures. In addition, studies of responses to predation risk are not always calibrated against the ecological history of predation risk in specific populations. We address these gaps by examining the effects of both long- and short-term predator exposure on the behavior of individuals from populations that have different ecological histories of predation risk. We exposed individuals from high-predation and low-predation populations of the live-bearing freshwater poeciliid, Heterandria formosa, to predators to assess changes in male reproductive behavior toward females. We also assessed longer-term reproductive responses by exposing male and female H. formosa to predators at a random time of day, every day, for 30 days. In the presence of a predator, in the short term, males changed the frequency of their behaviors and females varied in their concentration of cortisol, demonstrating immediate responses to the perceived risk. The magnitude of these changes was larger in the population without a long history of predator exposure. However, we found that males and females did not change their reproductive output when exposed to predators over longer periods of time, suggesting that individuals acclimatize to the level of predation risk they experience. These results also suggest that short-term variation in behavior or stress hormone responses should not be used as proxies for long-term responses or fitness effects. Future work should assess both short-term behavior and long-term responses while simultaneously considering the ecological history of populations.     

HRG1 Is Essential for Heme Transport from the Phagolysosome of Macrophages during Erythrophagocytosis

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Highlights? Humans have about 25 trillion RBCs, each containing over a billion heme moieties ? Five million senescent RBCs are recycled per second by erythrophagocytosis in macrophages to make new RBCs ? HRG1, the mammalian homolog of C. elegans heme transporter, recycles heme-iron from the macrophage phagolysosome ? Missense polymorphisms in human HRG1 may be genetic modifiers of iron metabolism     

Alpine forest genomics network (AForGeN): a report of the first annual meeting

The Alpine Forest Genomics Network was formed in 2011 and held its first annual meeting on June 24–26, 2012, in the Natural Park Adamello Brenta in Trentino Region, Italy. The meeting was attended by 30 researchers from the alpine region of Europe and had two primary goals: (1) for researchers to introduce each other to current and planned research activities in forest landscape genomics and (2) to develop a strategic vision for the network. A steering committee was elected and will develop a white paper over the next year. The next annual meeting will be held in Austria in June 2013.