Capture of linear fragments at a double-strand break in yeast

Double-strand breaks (DSBs) are dangerous chromosomal lesions that must be efficiently repaired in order to avoid loss of genetic information or cell death. In all organisms studied to date, two different mechanisms are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Previous studies have shown that during DSB repair, non-homologous exogenous DNA (also termed ‘filler DNA’) can be incorporated at the site of a DSB. We have created a genetic system in the yeast Saccharomyces cerevisiae to study the mechanism of fragment capture. Our yeast strains carry recognition sites for the HO endonuclease at a unique chromosomal site, and plasmids in which a LEU2 gene is flanked by HO cut sites. Upon induction of the HO endonuclease, a linear extrachromosomal fragment is generated in each cell and its incorporation at the chromosomal DSB site can be genetically monitored. Our results show that linear fragments are captured at the repaired DSB site at frequencies of 10−6 to 10−4 per plated cell depending on strain background and specific end sequences. The mechanism of fragment capture depends on the NHEJ machinery, but only partially on the homologous recombination proteins. More than one fragment can be used during repair, by a mechanism that relies on the annealing of small complementary sequences. We present a model to explain the basis for fragment capture

The "Black Box" Behind Prison-Based Vocational Training Programs

Despite the great importance of prison vocational programs, studies have pointed to a wide variety of barriers that inhibit the released prisoner's chances to integrate into the labor market. The present qualitative investigation was designed to crack the "black box" behind six vocational programs implemented in the Israel Prison Service (IPS). Our findings based on the interviews with all the supervisors in the programs emphasized several factors that seem necessary for the success of the different vocational programs. The interviews show that training in a correctional environment poses a number of major difficulties for the respondents. Furthermore, a significant part of the prisoners' motivation to participate in the training programs is not necessarily related to the desire to find work after release. It was also found that a relatively long training, which makes it possible to find work in the field even during the period of incarceration, holistically addresses the various needs of the prisoner, and corresponds to the job market requirements, increases the chances of its participants to find employment also after their release from prison. When the characteristics of the various programs were examined, it was found that in addition to the characteristics of prisoners, one must also consider structural characteristics such: to what extent do the programs comply with the requirements of the Israeli labor market, confer a formal diploma at the end of the training period, or allow integration into the work force after release

The FOXO Transcription Factor DAF-16 Bypasses ire-1 Requirement to Promote Endoplasmic Reticulum Homeostasis

SummaryThe unfolded protein response (UPR) allows cells to adjust the capacity of the endoplasmic reticulum (ER) to the load of ER-associated tasks. We show that activation of the Caenorhabditis elegans transcription factor DAF-16 and its human homolog FOXO3 restore secretory protein metabolism when the UPR is dysfunctional. We show that DAF-16 establishes alternative ER-associated degradation systems that degrade misfolded proteins independently of the ER stress sensor ire-1 and the ER-associated E3 ubiquitin ligase complex sel-11/sel-1. This is achieved by enabling autophagy-mediated degradation and by increasing the levels of skr-5, a component of an ER-associated ubiquitin ligase complex. These degradation systems can act together with the conserved UPR to improve ER homeostasis and ER stress resistance, beyond wild-type levels. Because there is no sensor in the ER that activates DAF-16 in response to intrinsic ER stress, natural or artificial interventions that activate DAF-16 may be useful therapeutic approaches to maintain ER homeostasis

Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7.

In C. elegans, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named irg-7, as a novel mediator of longevity in germlineless animals. We consider irg-7 to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, irg-7 activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks

Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7

<div><p>In <i>C</i>. <i>elegans</i>, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named <i>irg-7</i>, as a novel mediator of longevity in germlineless animals. We consider <i>irg-7</i> to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, <i>irg-7</i> activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks.</p></div

The transcription factor DAF-12 is activated in <i>irg-7(zc6)</i> mutants.

<p>(A-B) The <i>irg-7(zc6)</i> mutation is sufficient to increase the expression of the <i>Pcdr-6</i>::<i>gfp</i> reporter (Student's t-test, p<0.0001) in a <i>daf-12(+)</i> background but not in a <i>daf-12(-)</i> background (Student's t-test, p = 0.075). Asterisks mark Student's t-test values of P<0.001 compared to the fluorescence in wild-type animals. 30–35 animals analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) The <i>irg-7(zc6)</i> mutation is not sufficient to increase the expression of the DAF-16 reporter <i>Psod-3</i>::<i>gfp</i> (Student's t-test, P = 0.067). (D) The <i>irg-7(zc6)</i> mutation is not sufficient for the accumulation of DAF-16::GFP translational reporter in the intestine.</p

<i>irg-7(zc6)</i> is a gain of function mutation in F40F4.6.

<p>(A) <i>F40F4</i>.<i>6</i> RNAi significantly shortened the lifespan of <i>irg-7(zc6)</i> mutants (Mantel-Cox, P<0.0001) but did not affect the lifespan of otherwise wild-type animals (Mantel-Cox,P = 0.9). Mean lifespans are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s004" target="_blank">S2 Table</a></b> for more lifespan data. (B) Representative fluorescence micrographs (100-fold magnification) of day-1 adults harboring an integrated <i>Phsp-4</i>::<i>gfp</i> transgene. <i>F40F4</i>.<i>6</i> RNAi significantly reduced the expression of the <i>Phsp-4</i>::<i>gfp</i> in <i>zc6</i> mutants. Asterisks mark Student's T-test values of P<0.001 compared to fluorescence on control RNAi. 40 animals were analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) Expression of fosmid WRM0619aB08 (which includes F40F4.6) significantly extended the lifespan of wild-type animals (Mantel-Cox, P<0.001), whereas expression of the partially overlapping fosmid WRM0635bF05 (which does not include F40F4.6) did not extend the lifespan of wild-type animals (Mantel-Cox, P = 0.4). Mean lifespans are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s004" target="_blank">S2 Table</a></b> for more lifespan data. (D) Expression of an NLS-RFP reporter fused to the putative promoter upstream of the F40F4.6 gene drives expression in the posterior cells of the intestine. Fluorescence of this reporter increased upon exposure of L4 animals to Photorhabdus luminescens subsp Hb bacteria (HB) or Enterococcus faecalis bacteria (EF). Asterisks mark Student's T-test values of P<0.001 compared to wild-type fluorescence on OP50 bacteria. (E) Schematic representation of the major domains in the F40F4.6 protein. The region deleted by the <i>zc6</i> mutation is indicated by a dashed line. This region includes an EGF domain. Note that the deletion preserves the ORF of the original gene. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s002" target="_blank">S2 Fig</a> for sequence data.</p

<i>irg-7(zc6)</i> mutation extends lifespan by increasing pathogen resistance.

<p>(A) Feeding animals with dead OP50 bacteria extended the lifespan of wild-type animals (Mantel-Cox, P<0.001), but did not extend the lifespan of <i>irg-7(zc6)</i> mutants (Mantel-Cox, P = 0.53). (B) The <i>irg-7(zc6) gof</i> mutation improved the survival of animals fed with pathogenic HB bacteria (Mantel-Cox, P<0.0001). (C-D) <i>irg-7</i> inactivation by pre-treatment with F40F4.6 RNAi from eggs to early adulthood hindered survival of animals fed henceforth with pathogenic HB bacteria (C) (Mantel-Cox, P<0.0001), but did not affect the survival of animals fed henceforth with OP50 bacteria (D) (Mantel-Cox, P = 0.7). (E) <i>irg-7</i> inactivation by pre-treatment with F40F4.6 RNAi from eggs to early adulthood did not affect the survival of <i>daf-16(mu86)</i> mutants fed henceforth with HB bacteria (Mantel-Cox, P = 0.43). Mean lifespan are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s005" target="_blank">S3 Table</a></b> for additional lifespan data.</p

Sensitivity of germline homeostasis to pathogenic HB bacteria improves the animals' survival.

<p>(A) Treatment with pathogenic HB bacteria induced germline apoptosis in an <i>egl-1</i> dependent manner. Asterisks mark Mann-Whitney P values of P<0.001 of HB-treated animals (gray) compared to OP50 treated animals (black) of the same genotype. 45–50 animals were analyzed per genotype. Error bars represent SD. (B) Amount of mitotic germ cells was determined in wild-type animals and in animals with activated <i>irg-7</i> upon exposure to OP50 (black) or HB bacteria (gray). Asterisks mark Mann-Whitney values of P<0.001 of HB-treated animals (gray) compared to OP50 treated animals (black) of the same genotype. Additional Mann-Whitney P values are indicated in the graph. Note that treatment with pathogenic HB bacteria reduced the amount of mitotic germ cells in the gonad of wild-type animals but not in animals with activated <i>irg-7</i>. Also note that activation of <i>irg-7</i> did not reduce, and even increased, the amount of mitotic germ cells in the animals. 25–30 DAPI-stained gonads were analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) In the absence of pathogen-induced germline apoptosis, animals are more sensitive to the pathogenic bacteria. Mean survival and Mantel-Cox P-values are indicated within the graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s009" target="_blank">S7 Table</a></b> for additional survival data.</p