Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement

To more fully characterize the internal structure of transgene loci and to gain further understanding of mechanisms of transgene locus formation, we sequenced more than 160 kb of complex transgene loci in two unrelated transgenic oat (Avena sativa L.) lines transformed using microprojectile bombardment. The transgene locus sequences from both lines exhibited extreme scrambling of non-contiguous transgene and genomic fragments recombined via illegitimate recombination. A perfect direct repeat of the delivered DNA, and inverted and imperfect direct repeats were detected in the same transgene locus indicating that homologous recombination and synthesis-dependent mechanism(s), respectively, were also involved in transgene locus rearrangement. The most unexpected result was the small size of the fragments of delivered and genomic DNA incorporated into the transgene loci via illegitimate recombination; 50 of the 82 delivered DNA fragments were shorter than 200 bp. Eleven transgene and genomic fragments were shorter than the DNA lengths required for Ku-mediated non-homologous end joining. Detection of these small fragments provided evidence that illegitimate recombination was most likely mediated by a synthesis-dependent strand-annealing mechanism that resulted in transgene scrambling. Taken together, these results indicate that transgene locus formation involves the concerted action of several DNA break-repair mechanisms.     

Arg tyrosine kinase is involved in homologous recombinational DNA repair

c-Abl plays important roles in cellular response to DNA damage. However, possible roles for Arg (Abl-related gene) in DNA damage response are unknown. Here, we show that ionizing radiation (IR)-induced Rad51 focus formation is reduced in Arg-deficient cells generated from a chicken B cell line by targeted disruption. This is consistent with the findings that Arg-deficient cells display hypersensitivity to IR, elevated frequencies of IR-induced chromosomal aberrations, and reduced targeted integration frequencies. All of these abnormalities in DNA damage repair are also observed in ATM-deficient cells but not in c-Abl-deficient cells. Finally, we show that Arg interacts with and phosphorylates Rad51 in 293T cells. These results suggest that Arg plays a role in homologous recombinational (HR) DNA repair by phosphorylating Rad51.     

RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms

Several aspects of the mechanism of homologous double-strand break repair remain unclear. Although intensive efforts have focused on how recombination reactions initiate, far less is known about the molecular events that follow. Based upon biochemical studies, current models propose that RecBCD processes double-strand ends and loads RecA to initiate recombinational repair. However, recent studies have shown that RecBCD plays a critical role in completing replication events on the chromosome through a mechanism that does not involve RecA or recombination. Here, we examine several studies, both early and recent, that suggest RecBCD also operates late in the recombination process – after initiation, strand invasion, and crossover resolution have occurred. Similar to its role in completing replication, we propose a model in which RecBCD is required to resect and resolve the DNA synthesis associated with homologous recombination at the point where the missing sequences on the broken molecule have been restored. We explain how the impaired ability to complete chromosome replication in recBC and recD mutants is likely to account for the loss of viability and genome instability in these mutants, and conclude that spontaneous double-strand breaks and replication fork collapse occur far less frequently than previously speculated.     

Kinematics of primate midfoot flexibility

This study describes a unique assessment of primate intrinsic foot joint kinematics based upon bone pin rigid cluster tracking. It challenges the assumption that human evolution resulted in a reduction of midfoot flexibility, which has been identified in other primates as the “midtarsal break.” Rigid cluster pins were inserted into the foot bones of human, chimpanzee, baboon, and macaque cadavers. The positions of these bone pins were monitored during a plantarflexion‐dorsiflexion movement cycle. Analysis resolved flexion‐extension movement patterns and the associated orientation of rotational axes for the talonavicular, calcaneocuboid, and lateral cubometatarsal joints. Results show that midfoot flexibility occurs primarily at the talonavicular and cubometatarsal joints. The rotational magnitudes are roughly similar between humans and chimps. There is also a similarity among evaluated primates in the observed rotations of the lateral cubometatarsal joint, but there was much greater rotation observed for the talonavicular joint, which may serve to differentiate monkeys from the hominines. It appears that the capability for a midtarsal break is present within the human foot. A consideration of the joint axes shows that the medial and lateral joints have opposing orientations, which has been associated with a rigid locking mechanism in the human foot. However, the potential for this same mechanism also appears in the chimpanzee foot. These findings demonstrate a functional similarity within the midfoot of the hominines. Therefore, the kinematic capabilities and restrictions for the skeletal linkages of the human foot may not be as unique as has been previously suggested. Am J Phys Anthropol 155:610–620, 2014. © 2014 Wiley Periodicals, Inc.     

TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.     

Pol32 is required for Pol zeta-dependent translesion synthesis and prevents double-strand breaks at the replication fork

POL32 encodes a non-essential subunit of Polδ and plays a role in Polδ processivity and DNA repair. In order to understand how Pol32 is involved in these processes, we performed extensive genetic analysis and demonstrated that POL32 is required for Polζ-mediated translesion synthesis, but not for Polη-mediated activity. Unlike Polζ, inactivation of Pol32 does not result in decreased spontaneous mutagenesis, nor does it limit genome instability in the absence of the error-free postreplication repair pathway. In contrast, inactivation of Pol32 results in an increased rate of replication slippage and recombination. A genome-wide synthetic lethal screen revealed that in the absence of Pol32, homologous recombination repair and cell cycle checkpoints play crucial roles in maintaining cell survival and growth. These results are consistent with a model in which Pol32 functions as a coupling factor to facilitate a switch from replication to translesion synthesis when Polδ encounters replication-blocking lesions. When Pol32 is absent, the S-phase checkpoint complex Mrc1–Tof1 becomes crucial to stabilize the stalled replication fork and recruit Top3 and Sgs1. Lack of any of the above activities will cause double strand breaks at or near the replication fork that require recombination as well as Rad9 for cell survival.     

Dynamic change of histone H2AX phosphorylation independent of ATM and DNA-PK in mouse skin in situ

Histone H2AX undergoes phosphorylation on Ser 139 (γ-H2AX) rapidly in response to DNA double-strand breaks induced by exogenous stimuli, such as ionizing radiation. However, the endogenous phosphorylation pattern and modifier of H2AX remain unclear. Here we show that H2AX is regulated physically at the level of phosphorylation at Ser139 during a hair cycle in the mouse skin. In anagen hair follicles, γ-H2AX-positive cells were observed in the outer root sheath (ORS) and hair bulb in a cycling inferior region but not in a permanent superficial region. In telogen hair follicles, γ-H2AX-positive cells were only detected around the germ cell cap. In contrast, following X-irradiation, γ-H2AX was observed in various cell types including the ORS cells in the permanent superficial region. Furthermore, γ-H2AX-positive cells were detected in the skin of mice lacking either ATM or DNA-PK, suggesting that these kinases are not essential for phosphorylation in vivo.     

Recurrent gene amplification and soft selective sweeps during evolution of multidrug resistance in malaria parasites

When selection is strong and beneficial alleles have a single origin, local reductions in genetic diversity are expected. However, when beneficial alleles have multiple origins or were segregating in the population prior to a change in selection regime, the impact on genetic diversity may be less clear. We describe an example of such a "soft" selective sweep in the malaria parasite Plasmodium falciparum that involves adaptive genome rearrangements. Amplification in copy number of genome regions containing the pfmdr1 gene on chromosome 5 confer resistance to mefloquine and spread rapidly in the 1990s. Using flanking microsatellite data and real-time polymerase chain reaction determination of copy number, we show that 5-15 independent amplification events have occurred in parasites on the Thailand/Burma border. The amplified genome regions (amplicons) range in size from 14.7 to 49 kb and contain 2-11 genes, with 2-4 copies arranged in tandem. To examine the impact of drug selection on flanking variation, we genotyped 48 microsatellites on chromosome 5 in 326 parasites from a single Thai location. Diversity was reduced in a 170- to 250-kb (10-15 cM) region of chromosomes containing multiple copies of pfmdr1, consistent with hitchhiking resulting from the rapid recent spread of selected chromosomes. However, diversity immediately flanking pfmdr1 is reduced by only 42% on chromosomes bearing multiple amplicons relative to chromosomes carrying a single copy. We highlight 2 features of these results: 1) All amplicon break points occur in monomeric A/T tracts (9-45 bp). Given the abundance of these tracts in P. falciparum, we expect that duplications will occur frequently at multiple genomic locations and have been underestimated as drivers of phenotypic evolution in this pathogen. 2) The signature left by the spread of amplified genome segments is broad, but results in only limited reduction in diversity. If such "soft" sweeps are common in nature, statistical methods based on diversity reduction may be inefficient at detecting evidence for selection in genome-wide marker screens. This may be particularly likely when mutation rate is high, as appears to be the case for gene duplications, and in pathogen populations where effective population sizes are typically very large.     

Direct Delivery of Inoculum to Shoot Tissue Interferes with Genotypic Resistance to Ralstonia solanacearum in Tomato Seedlings

Employing known susceptible and resistant genotypes and pure bacterial inoculum (0.1 OD; 10⁸ CFU/ml^?1), five different inoculation methods were tried to assess the response of tomato genotypes to Ralstonia solanacearum. This included seed‐soaking inoculation, seed‐sowing followed by inoculum drenching, or at 2‐week stage through petiole‐excision inoculation, soaking of planting medium with inoculum either directly or after imparting seedling root‐injury. Seed‐based inoculations or mere inoculum drenching at 2 weeks did not induce much disease in seedlings. Petiole inoculation induced 90–100% mortality in susceptible checks but also 50–60% mortality in normally resistant genotypes within 7–10 days. Root‐injury inoculation at 2‐week seedling stage appeared the best for early and clearer distinction between resistant and susceptible lines. The observations suggest a role played by the root system in governing genotypic resistance to the pathogen. Direct shoot inoculation is to be adopted only for selecting highly resistant lines or to thin down segregating populations during resistance breeding.