Category Archives: Genome

In the largest genetic study of bipolar disorder to date, researchers have identified 64 regions of the genome containing DNA variations that increase risk of this condition more than double the number previously identified. Bipolar disorder is known to be one of the most heritable mental illnesses, but unravelling the genetics of this condition has been challenging.

The research team also found overlap in the genetic bases of bipolar disorder and other psychiatric disorders. Furthermore, the study supports a role of sleep habits, alcohol, and substance usage in the development of bipolar disorder. Their study results were published today in Nature Genetics. http://dx.doi.org/10.1038/s41588-021-00857-4

It is well-established that bipolar disorder has a substantial genetic basis and identifying DNA variations that increase risk can yield insights into the conditions underlying biology, says Niamh Mullins, Ph.D., Assistant Professor of Psychiatric Genomics at the Icahn School of Medicine at Mount Sinai and lead author of the paper. Our study found DNA variations involved in brain cell communication and calcium signaling that increase risk of bipolar disorder.

Bipolar disorder is a complex psychiatric disorder characterized by recurrent episodes of severely high and low mood, and affects an estimated 40 to 50 million people worldwide. It typically begins in young adulthood, often takes a chronic course, and carries an increased risk of suicide, making it a major public health concern and cause of global disability. It is associated with substance abuse, as well as other psychiatric disorders such as anxiety, eating disorders, and chronic affective symptoms. Its estimated that in the US alone, this condition leads to $31 billion in direct costs and $120 billion in indirect costs.

More than 1,300 clinical trials are currently ongoing for treatments of bipolar disorder.

To help elucidate the underlying biology of this condition, an international team of scientists from within the Psychiatric Genomics Consortium conducted a genome-wide association study. This involved scanning more than 7.5 million common variations in the DNA sequence of nearly 415,000 people, more than 40,000 of whom had bipolar disorder. The study identified 64 regions of the genome containing DNA variations that increase risk of bipolar disorder.

These findings suggest that drugs, such as calcium channel blockers, already used for the treatment of high blood pressure and other conditions of the circulatory system, could be potential treatments for bipolar disorder.

The study also found overlap in the genetic basis of bipolar disorder and that of other psychiatric disorders and confirmed the existence of partially genetically distinct subtypes of the disorder. Specifically, they found that bipolar I disorder shows a strong genetic similarity with schizophrenia and bipolar II disorder is more genetically similar to major depression.

This research would not have been possible without the collaborative efforts of scientists worldwide that enabled the study of hundreds of thousands of DNA sequences, said Ole Andreassen, MD, PhD, Professor of Psychiatry, Institute of Clinical Medicine and Oslo University Hospital and senior author of the paper. Through this work, we prioritized some specific genes and DNA variations which can now be followed up in laboratory experiments to better understand the biological mechanisms through which they act to increase risk of bipolar disorder.

The researchers say that further genetic studies in larger and more diverse populations are needed to pinpoint the genes relevant to risk of bipolar disorder in other areas of the genome.

The Psychiatric Genomics Consortium (PGC) is an international consortium of scientists dedicated to studying the genetic basis of psychiatric disorders and includes over 800 researchers, from more than 150 institutions from over 40 countries.

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Bipolar Disorder Linked to Variants in 64 Regions of the Genome - Clinical OMICs News

Study shows erosion of ant genome tied to loss of functional, behavioral and social traits in 3 inquiline species.

Ants are renowned in the insect world for their complex social structure and behaviors. Workers and foragers support the queen, faithfully carrying out their social roles for the overall health of the colony. This complex superorganism as scientists have dubbed it has become a prime model to explore the genetic and behavioral roots of social organisms.

Remarkably, there are also rare instances of ants not playing well with others and shrugging off their societal duties to become free-loading parasites amongst their free-living relatives.

Now, in a new study published inNature Communications, an international collaboration of researchers from Europe (the Universities of Mnster and Copenhagen), South America (University of the Republic in Montevideo, Uruguay), and the U.S., (led by Arizona State University), teamed up to discover and collect these rare ant social parasites. Together, they have obtained and analyzed the full DNA genome sequences of three rare social parasite leaf-cutting ant species (called Acromyrmex inquilines) to better understand the differences between them and their respective host species.

A new study, led by ASU SOLS professor Christian Rabeling, has provided detailed insights into the molecular evolution of social parasitism in ants. Credit: Martin Bollazzi

Its the first time several species of socially parasitic ants could have their genomes sequenced.

Our findings advance our understanding of the genomic consequences of transitioning to a novel, highly specialized life history and provide detailed insights into the molecular evolution of social parasitism in ants, said Christian Rabeling, an associate professor in ASUs School of Life Sciences and a corresponding author of the study.

The unusual social parasite transition is important to understand because the genomes of ants have evolved for more than 100 million years. A single major transition occurred to introduce the novel superorganism level of social organizational structure with queen-worker caste segregation and unconditional altruism. This superorganism was so successful, it produced a biodiversity of 17 subfamilies, 338 genera and more than 13,900 living species.

It is therefore no surprise that parallel shifts to a highly specialized socially parasitic behavior and lifestyle abandoning this fundamental ancestral condition, usually based on outbreeding and larger effective populations, leave significant genomic footprints, said Rabeling. The results of our analyses of just three of these species confirm that ant social parasites offer important study systems for identifying hallmarks of cooperative social colony life.

And in doing so, their analyses have confirmed that over a time span of about a million and a half years, these ant species have each found independent, separate ways to evolve and become social parasites. The signatures of genome-wide and trait-specific genetic erosion were found to be most extreme in social parasite ants.

Divergence estimates for Acromyrmex host and inquiline parasite species. ime-calibrated phylogeny of the fungus-growing ants for which genomes have been sequenced, including the three inquiline social parasite species and their two host species. The two origins of social parasitism in Acromyrmex (orange dots and boxes) occurred ca. 0.96 Ma ago for A. insinuator (1) and ca. 2.50 Ma ago when the ancestor of A. heyeri diverged from the stem group representative of Pseudoatta argentina (2) and A. charruanus (3). Credit: Arizona State University

Think of how it would start. A group of queen ants wants to just live in a colony without doing the work. And not work on the nest anymore. Next, the queen ants focus on solely producing new queens and males, and this small population size of social parasites would start frequent inbreeding to survive. This immediately reduces their genomic diversity over time. Then, over a blink in evolutionary time, due to natural selection and an increase in the prevalence of genetic drift, it would enhance the rates by which ancestral traits were lost while also slowing down the rates by which new, more adaptive traits could emerge.

Its almost like a snooze and lose it phenomena occurred within the parasitic ant DNA to trigger the genome erosion.

To prove this effect within the ant genome, the research team investigated the overall genomic structure and the individual genes that may be affected by this genomic decay. First, they found widespread evidence of genomic rearrangements and inversions that are hallmarks of instability and decay. Then, within gene networks, they identified 233 genes that showed evidence of relaxed selection in at least one of the social parasite branches and signatures of intensified selection in 102 genes. Our analysis showed that gene family evolution at three of the four social parasite nodes is indeed largely characterized by gene losses, said Rabeling.

The genome losses and reductions most affected were in the social parasite ants sense of smell and to a lesser degree taste.

Not only did some of the genes responsible for ant smell become lost over time, but as a result, the ants also showed a reduced size in the olfactory lobes in their brains when microCT scans were performed.

This is no surprise because ants predominantly communicate via chemical cues and have once been described as chemical factories, explains Rabeling. So, the loss of olfactory genes is correlated with an extreme transition of extensive morphological and behavioral changes.

This includes the reduction or complete loss of the worker caste system, simplified mouthparts, antennae and integuments, loss of certain hormonal glands, and a nervous system of reduced complexity likely associated with a drastically narrowed behavioral repertoire.

Micro CT scans show the relative olfactory lobe (OL) size of the hosts and inquilines. The phylogram is an ancestral state reconstruction of OL volumes relative to total brain volumes across the social parasites (A. insinuator, A. charruanus and P. argentina) and their hosts (A. echinatior, and A. heyeri). Barplots show ratios of OL volume to total brain volume in inquiline parasites (in orange) relative to their hosts (in blue). Circles inserted at the tips of bars are proportional to the measured total brain volumes, while the smaller contained circles represent the measured volumes of the right and left OLs. On average, Panamanian species have larger brains than Uruguayan species (2-sample t-test, pt-test = 0.005, df = 2.97, t = ?7.74, n = 5). Relative OL volumes became reduced (pt-test = 0.059, df =2, t = ?2.65, n = 5) as inquiline social parasites evolved their different degrees of specialization along the gradient of inquiline adaptations known as the inquiline syndrome27. Shown below are 3D surface reconstructions of the brains (with the OLs highlighted in yellow) and of the head capsules of A. heyeri, A. charruanus, and P. argentina (from top to bottom). Credit: Arizona State University

From their comparative analysis, they could also put these changes into the larger perspective of evolutionary time. They were also able to date the origins of social parasitism within the leaf-cutting ant family tree.

Two independent origins of social parasitism occurred in the ant genus Acromyrmex. Within this genus, A. heyeri, a social ant, is the host species of both A. charruanus and P. argentina parasitic species.

First, a South American lineage of social ants (A. heyeri) separated from the last common (thought to be socially parasitic) ancestor of A. charruanus and P. argentina before the two social parasites diverged. Second, a Central American speciation event occurred when A. insinuator diverged from its host A. echinatior.

Both origins of social parasitism are evolutionarily recent, estimated to be about 2.5 million years ago for the divergence between A. heyeri and the last common ancestor of A. charruanus and P. argentina, and about 1 million years ago for the divergence between A. insinuator and A. echinatior.

We infer that relaxed natural selection accelerated general genome erosion in social parasites and alleviated evolutionary constraints, which facilitated rapid adaptive evolution of specific traits associated with a socially parasitic lifestyle, said Rabeling.

Why did it take so long to do the genome analysis? It turns out that the easiest part of the study may have been the comparative genome analysis. Finding the ants in the first place proved to be the greatest major hurdle. Why?

Populations of ant social parasites are almost invariably small and patchily distributed. How patchy?

Well, the last time that one of the species, P. argentina was seen in the wild was 1924, a time well before the discovery of DNA as the hereditary chemical unit of life.

Rabeling remembers prior trips to South America that were in vain because they could not find P. argentina. Then, about a decade ago, a phone call from colleague Martin Bollazzi and study co-author changed his life.

Martin Bollazzi said his wife Leticia just re-discovered P. argentina!!!

Rabeling hopped on a plane as fast as he could. When he saw P. argentina up close, it was a moment of discovery hell never forget.

Leticias rediscovery of P. argentina was the find of a lifetime. What I especially love is to connect the ant field work and natural history observations with the new technologies like whole genome sequencing, and to have the opportunity to do so was such a joy.

Now, they could make their research dreams a reality by collecting P. argentina and put their field work-based hypotheses to the test by doing the first modern whole genome sequencing of social parasitic ants.

Their results are not only important to understanding ants, but offer insights into the role of these genomic loss-of-function study systems in other parasites and for identifying hallmarks of cooperative social colony life at both the phenotypic and the genomic levels.

Social parasites came to exploit the foraging efforts, nursing behavior and colony infrastructure of their hosts, said Rabeling.

Rabeling also points to other species, such as the Mexican blind cave-dwelling fish or other parasites such as tapeworms as examples of organisms that lost important traits over time. In each case, they have developed and exploited novel ecological niches. for their species survival.

From these first 3 social parasite ant species, they have learned a lot. Next, they plan on future genomics studies of these ant social parasites to generate exciting further insights, particularly with long-read sequencing technologies allowing analyses in even greater detail.

But Rabeling and his colleagues are now involved in another race against time as every year, more and more natural ant habitats are lost to deforestation and development. Now, our understanding of ant evolution depends on people to cooperate to save biodiversity while we still can.

We hope such future studies can expand our knowledge on the signatures of the evolution of social behavior in ants, for which few other model systems can offer such species-level sample sizes of several dozens.

Reference: Relaxed selection underlies genome erosion in socially parasitic ant species by Lukas Schrader, Hailin Pan, Martin Bollazzi, Morten Schitt, Fredrick J. Larabee, Xupeng Bi, Yuan Deng, Guojie Zhang, Jacobus J. Boomsma and Christian Rabeling, 18 May 2021, Nature Communications.DOI: 10.1038/s41467-021-23178-w

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How to Become "Ant-I-Social" Erosion of Ant Genome Tied to Loss of Functional, Behavioral and Social Traits - SciTechDaily

Express News Service

DEHRADUN: Genome sequencing of samples in Uttarakhand has revealed that there are at least 16 variants of the Covid-19 virus active in the hilly state.

Health department officials said that reports of 531 samples are still pending to decide which variant is dominating the scene. "The genome sequencing has revealed that 16 variants are active as we speak. However, a majority of samples are still pending to determine which dominant variant is prevailing," an official said on the condition of anonymity.

Officials from the state health department said that total 851 simples were sent to a Delhi lab out of which reports of 285 samples have arrived.

Out of these 285 samples, reports of 208 samples were detected with Sara Cov 2 variant (original variant) while UK variant B117 was detected in 32 samples, one sample has B16171 variant, two have B16172 variant and 42 were detected with various other kind of variants of the virus.

Other strains include UK variant B16171, UK variant B16172, P681R, P681H, P681RQ1071H, Q677H, N440K, N501Y, L452R, E484K, L452RP681R, N501YP681H, DN50N and E484QLRP681RQ1071H.

Last month with the National Centre for Disease Control (NCDC) confirming detection of a total of six cases of various strains, according to doctors in Dehraduns Viral Research and Diagnostic Laboratory (VRDL), it was confirmed that highly contagious double mutant strain of SARS-CoV-2 had registered its presence before the Mahakumbh 2021 started on April 1.

The experts confirmed that three of the six are of the double mutant strain, two are of the UK strain and one is yet to be identified.

The samples which confirmed the double mutant strain was sent to the NCDC in March end.

The mega religious event was attended by millions of people from across the country with 35 lakh on April 12 Shahi Snan and 13.51 lakh on April 14 Shahi Snan.

At present Uttarakhand has 78802 active cases of Covid 19. The number of active cases came down from 80,000 in comparison with Friday as 5034 people recovered from Covid 19 on Sunday while 4496 new cases were added.

Sunday also marked lowest number of new cases surfacing in one day since last 21 days. The number of recoveries were also higher than new cases.

Last time this happened on March 30 with 147 recoveries and 128 new cases in one day.

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16 variants of Covid active in Uttarakhand, reveals genome sequencing - The New Indian Express

Virologist Shahid Jameel, one of the most prominent scientific voices of the pandemic, has resigned as the head of the Indian SARS-COV-2 Genomics Consortia (INSACOG), the scientific advisory group coordinating the countrys genome sequencing work.

INSACOG had come into being in January this year as a scientific body to promote and expedite genome sequencing of the SARS-CoV2 virus and its multiple variants. The consortium had established a network of ten leading laboratories to carry out gene sequencing of virus samples from across the country. The consortium was initially given a tenure of six months, but later got an extension. The genome sequencing work, which had been progressing at a very slow pace, gathered momentum only after the constitution of INSACOG.

Jameel, a widely respected scientist, has been speaking and writing frequently on the pandemic, including in The Indian Express. Only last week, he was the guest at the Explained Live event organized by this newspaper. Known to speak his mind on scientific matters, Jameel had been critical of the governments efforts to contain the spread of the virus, particularly during the second wave.

During the Explained Live event, he had said that government authorities had erred in prematurely believing that the pandemic was over in January, and by folding up many temporary facilities that had been set up in previous months.

Recently, he also wrote a piece in the New York Times in which he stressed on the need to increase testing and isolation, ramp up hospital beds by creating more temporary facilities, rope in retired doctors and nurses, and strengthen the supply chain for critical medicines and oxygen.

All these measures have wide support among my fellow scientists in India. But they are facing stubborn resistance to evidence-based policymaking, he wrote.

Decision-making based on data is yet another casualty, as the pandemic in India has spun out of control. The human cost we are enduring will leave a permanent scar, he wrote.

But Jameel had also criticised the Supreme Courts recent decision to appoint a task force to manage oxygen supplies.

This is really unfortunate. We are short on doctors and we have taken some of our best doctors and told them you play oxygen-oxygen. You decide who will get oxygen. It is really a sad day for us. These good doctors know about medicine, but what do they know about oxygen supply chain and logistics, he had said at the Explained event.

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Virologist Shahid Jameel quits govts genome mapping group - The Indian Express

RESULTS AND DISCUSSION

To address the fate of DNA downstream of physiologically relevant RFBs and to assess the consequences for genetic integrity, we monitored mutagenesis at endogenous G-quadruplex (G4) motifs. It was previously demonstrated in Caenorhabditis elegans that a single G4 structure under physiological conditions can impose a persistent impediment to DNA replication, requiring the helicase FANCJ/DOG-1 for resolution (35). Failure to unwind the impediment results in a DNA double-strand break (DSB) that requires polymerase (POLQ)mediated end-joining (TMEJ) for its repair (4). A distinct mutation profile results in small deletions, typically 70 to 200 base pairs (bp) in size, which have one junction mapping to the stem of the G4 and the other junction ~70 to 200 bp downstream of it. While the nascent strand blocked at the RFB likely defines the proximal deletion junction, it is currently unknown what biology is dictating the location of the junction distal to the RFB, and thus which enzymes suppress excessive DNA loss as the result of underreplication or because of endo- or exonucleic attack on incomplete replication intermediates.

We sought to test the idea that DNA synthesis downstream of an RFB limits the size of a vulnerable single-stranded (ssDNA) gap by that act producing an ssDNA/double-stranded DNA (dsDNA) transition point that could define the RFB-distal deletion junctionthe size distribution of deletions thus reflecting the ssDNA gap width (Fig. 1A). Previous work indicated that such ssDNA gaps can be converted to DSBs in the next round of replication after the premutagenic lesion has been passed on to daughter cells (6). Genetic testing of the logical candidate to initiate DNA synthesis, the DNA polymerase (pol )primase complex, by knockout is impossible given its essential function in genome duplication (7, 8). Instead, we made use of an established biochemical property of this enzyme. RNA primer initiation by the primase subunit is, in fact, not random: All tested eukaryotic pol -primase complexes use purine as a cofactor to kickstart RNA synthesis and thus require a mandatory pyrimidine template (811). We exploited this necessity by genetically engineering DNA stretches consisting exclusively of purines, which we termed primase deserts (PDs) into the C. elegans genome downstream of a replication-blocking G4 motif (Fig. 1, A and B). To monitor mutagenic events, we placed a G4 motif flanked by a stop codon in the reading frame of the unc22 gene, hence generating UNC-22 loss-of-function animals, which move uncoordinatedly (twitching; Fig. 1B). G4-induced deletion events that remove the stop codon can lead to restoration of the deletion-tolerant UNC-22 open reading frame (ORF) and reversion to wild-type moving animals, which can easily be isolated from populations of twitching animals. To increase the rate of mutagenesis at replication-blocking G4s, we used animals that lack DOG-1/FANCJ helicase (fig. S1). The deletion spectrum of >50 independently isolated revertant animals is consistent with earlier work: Deletions have one junction flanking the G4 motif immediately upstream and have the other junction 70 to 200 bp downstream of the G4. In addition, these deletions rely on TMEJ (Fig. 1C and fig. S1) (4). Next, we inserted PDs (which by themselves do not have predicted G4 folding capability) of different lengths (56, 100, 140, and 160 bp), ~50 bp downstream of the G4 motif (see Fig. 1B and fig. S2 for a schematic illustration) (12). None of these deserts affected the reversion rate as compared to the allele without such PD (fig. S1). However, we observed a profound influence on the position of the deletion junction distal to the G4 (Fig. 1C): The vast majority of deletion junctions were found outside the deserts, and the median deletion size shifted proportionally with the PD size. As expected, the position of the proximal junction, likely reflecting the position of the stalled nascent strand, was unaffected (fig. S1). The altered deletion spectrum induced by PDs was completely dependent on the orientation of the desert: Insertion of an inverted sequence at the same location such that a track results exclusively consisting of pyrimidines downstream of the RFB had no effect on the position of the distal deletion junction (Fig. 1C and fig. S2). This outcome suggests that primase activity downstream of an RFB suppresses extensive DNA loss by reducing the ssDNA gap, thereby defining the position where DNA eventually becomes susceptible to end-joining (EJ) activity. Later steps in the biology of G4-induced mutagenesis, i.e., processing of DSBs by TMEJ, appear unaffected as deletions taking out the PDs have microhomology at the junction, occasionally have template insertions, and are completely dependent on functional POLQ (fig. S1).

(A) A schematic representation of a model explaining G4-induced deletion mutagenesis, where one deletion junction (proximal to the RFB) is defined by the blocked nascent strand, the other (distal to the RFB) by the ssDNA/dsDNA transition of downstream Okazaki fragments. Stretches of pyrimidines, which we termed primase deserts (PDs), are expected to be devoid of primer initiation because primase requires a template pyrimidine to initiate synthesis of the RNA primer. G4e is an endogenous G4 motif inserted in unc-22. (B) The C. elegans genome was engineered to include a G4 sequence at the endogenous unc-22 gene, which rendered the UNC-22 ORF out-of-frame. The unc-22(G4) gene was modified to contain PDs of different length at distinct positions from the G4. These alleles disrupt UNC-22 functionality, yet deletion mutagenesis induced by the G4 can restore the downstream ORF resulting in wild-type moving animals (see fig. S2 for details). WT, wild type. (C to E) Deletion spectra of dog-1deficient animals with different PDs and a control PD (stretch of pyrimidines) positioned downstream of the G4 motif in unc-22. PDs are positioned 47 bp downstream of G4 motif, unless the label contains @ sign, e.g., 125PD@25, means a 125-bp PD at 25 bp from the G4 motif. Dots represent independently derived deletion alleles and indicate the position of the distal junctions (in base pairs) relative to the G4 motif set at 0. Blue rectangles indicate the position and size of the PDs, and gray rectangle indicates the position and size of the control PD. Red lines indicate the median. ns, nonsignificant; *P < 0.05, ***P < 0.001, and ****P < 0.0001 by Dunnetts multiple comparisons test.

While junctions are greatly underrepresented in PDs, some were found (Fig. 1C). Although promiscuity of RNA primases, potentially templating on purines, cannot be excluded, these outcomes could also result from priming upstream of the deserts: There are nine pyrimidine bases between the G4 and the deserts, providing templates for primer initiation. Replacement of eight of these nine pyrimidines by purines (the ninth is part of the stop codon downstream of the G4 motif and is essential to the assay) indeed further reduced the number of deletions with a junction within the desert (from 17 to 5%; Fig. 1D). This outcome also suggests (i) that deletion junctions can be located numerous bases away from where a primer starts and (ii) that primase can initiate much closer to an RFB than was suspected on the basis of deletion junctions being >70 bp downstream of the RFB. Priming in close proximity to the stalled replisome may, however, be rare, as analysis of the distributions presented in Fig. 1D points to restricted template availability: The distribution at normal sites (e.g., Fig. 1D, top) resembles that at desert-containing sites if one envisions an occlusion zone from 0 to ~50 bp downstream of the G4. To test this idea directly, we engineered a 46-bp desert immediately adjacent to the G4 motif and found this desert to have no effect on the deletion distribution, strengthening the notion that priming rarely occurs in very close proximity of an RFB, possibly due to steric hindrance by either the RFB or the blocked replisome (Fig. 1D, bottom). Last, we tested how PDs affected the deletion landscape when positioned at 80 and 100 bp downstream of the RFB, where most of the deletion junctions map under unperturbed circumstances. In such a scenario, there is an ample opportunity for primase initiation between the replication stall and the desert. Figure 1E shows a profound and highly indicative disturbance: The deserts split the distributions in two; deletion junctions now predominantly map to either sides of the desert. However, the deserts are not devoid of junctions; for the desert 80 bp from the G4, we found many junctions within the desert, close to its 5 border. This outcome may be best explained by abundant primase initiation in the region between G4 and desert, while other subsequent biology contributes to the loss of bits of DNA before repair by TMEJ. Later in the manuscript, we will describe one of these contributing activities.

We next aimed to identify genes that contribute to genome stability at sites of stalled replication. Motivated by the suggestion that DNA priming downstream of an RFB affects the degree of DNA loss in a polarized manner, we generated an in vivo reporter system that not only visualizes G4-induced deletion formation but is also able to discriminate between categorically different deletion sizes. We inserted enhanced green fluorescent protein (eGFP) and wrmScarlet (13) separated by a 2A sequence (to ensure physical separation of the fluorescent markers upon expression), C-terminal to a small sequence directly downstream of the ATG start codon. This sequence contains a G4 motif and a stop codon to prevent expression of eGFP and wrmScarlet (Fig. 2A). Deletions that take out the stop codon can bring the downstream ORF in-frame with the upstream ATG, resulting in reporter expression. The chosen length of the N-terminal sequence dictates that deletions smaller than 160 bp can lead to expression of eGFP and wrmScarlet, whereas in-frame deletions of 160 to 1000 bp exclusively activate wrmScarlet as GFP-encoding sequence is lost. The reporter functionality and G4 specificity were confirmed by detecting elevated levels of activation in DOG-1/FANCJ-deficient animals (Fig. 2, B and C): 15% of animals have stochastic patches of somatic cells expressing either eGFP and wrmScarlet (50%) or exclusively wrmScarlet (50%). This 50:50 ratio is in good agreement with deletion size distributions at endogenous G4 motifs. In line with the directionality and asymmetry of deletions, no worms were observed that only expressed eGFP.

(A) Fluorescent-based reporter able to discriminate G4-induced deletion mutagenesis based on size: Deletions that are <160 bp and bring the downstream ORF in frame with the upstream ATG result in mEGFP and wrmScarlet expression. In-frame deletions that are 160 to 1000 bp will express wrmScarlet exclusively. (B) Quantification of reporter activation for ~250 synchronized animals of the indicated genotype. Experiments are performed in triplicate. Error bars denote SD. TL, transmitted light. (C) Representative images of fluorescent animals. Long dashes indicate an eGFP- and wrmScarlet-positive animal; short dashes indicate a wrmScarlet-positive animal. (D) Ratio of fluorescent animals expressing wrmScarlet exclusively upon RNAi-mediated knockdown of genome stability genes (see table S4). RNAi (L4440) in red. (E) Validation of POLA2/DIV-1 by RNAi [Ahringer (50) and Vidal (51) library clones] in triplicate; and by genetics: dog-1 div-1(ts) animals versus dog-1. Green indicates animals expressing both mEGFP and wrmScarlet, and red indicates animals exclusively expressing wrmScarlet. Error bars denote SD; ***P < 0.001 and ****P < 0.0001 by t test. (F) Deletion spectra from the unc-22 G4 assay, with or without a PD (in blue), for the indicated genotypes. Dots represent independently derived deletion alleles and indicate the position of the distal junctions relative to the G4 motif set at 0. Red lines indicate the median; ****P < 0.0001 by Mann-Whitney test. (G) Size representation of deletions at endogenous G4 loci that were found in animals of the indicated genotype. Each dot represents the distal junction relative to the G4 sequence set at 0. ****P < 0.0001 by Mann-Whitney test.

We next used these reporter animals to perform a candidate-based RNA interference (RNAi) screen, targeting enzymes that are involved in DNA repair, DNA damage signaling, and DNA replication (table S4). While none of the RNAi clones led to a complete loss of reporter activation, we found two clones that selectively reduced eGFP activation (Fig. 2D): In these knockdowns, 90 to 95% of events exclusively expressed wrmScarlet, indicative of larger deletions. Both RNAi clones target DIV-1/POLA2, which encodes the DNA pol -subunit B that is part of the DNA pol -primase complex. We validated our screen by retesting the top 20 RNAi hits in triple and found that only DIV-1 RNAi displayed a consistent increase in wrmScarlet expression (fig. S3). Targeting other members of the DNA pol -primase complex by RNAi induced embryonic lethality, precluding an assessment of their involvement. Fortuitously, previous genetic studies in C. elegans have led to the isolation of a temperature-sensitive (ts) allele of div-1/POLA2, which contains a leucine residue instead of an evolutionary highly conserved proline at amino acid position 329 (14). We tested animals that are homozygous for this allele at a growth-permissive temperature of 20C using our reporter and confirmed the RNAi results (Fig. 2E). To obtain a more detailed deletion spectrum, we performed the unc-22 assay described above and observed a profound effect: The median deletion size of 125 bp in DIV-1 wild-type animals shifted to 262 bp in DIV-1(P329L) animals grown at 20C (Fig. 2F). When assayed at even lower culturing temperatures (15C), this shift was less pronounced yet still clearly present (fig. S4), arguing that the P329L mutation affects DIV-1 functionality in a temperature-dependent manner and also in conditions where population growth is seemingly unaffected. In agreement with a proposed role for pol -primase acting downstream of the RFB, the increase in deletion size can, in its entirety, be explained by nucleotide loss at the RFB distal site (fig. S5). A similar increase was observed in animals where the unc-22 allele contains a 100-bp PD: Here, the median deletion size shifted from 199 to 362 bp when DIV-1(P329L) animals were assayed (Fig. 2F). To further substantiate the involvement of DNA pol -primase activity in suppressing DNA loss at RFBs, we assayed G4-induced deletion formation throughout the C. elegans genome in an unselected manner: We clonally grew separate populations of dog-1 div-1(ts) animals in parallel to dog-1 controls for 50 generations, after which we sequenced their genomes. We found similar rates for deletion formation at genomic G4 sites in these genetic backgrounds, demonstrating that DIV-1(P329L) expression does not cause elevated fragility or number of G4s (fig. S6). However, and in perfect agreement with the reporter and unc-22 data, we found a profoundly altered size distribution at G4 loci, with the median deletion size shifting from 125 to 270 bp as a result of altered DIV-1 functionality (Fig. 2G).

We thus found that disruption of the DNA pol -primase complex via RNAi or genetic mutation leads to a very similar outcome for RFB-induced mutagenesis as the local insertion of sequences that inhibit DNA pol -primase activity in cis. Together, this provides strong support for the hypothesis that primase activity directly downstream of an RFB protects the genome locally from genetic deterioration. At least two plausible scenarios can be envisaged for recruitment and positioning of the DNA pol -primase complex at sites of stalled replication: (i) For RFBs located in the lagging strand, Okazaki fragment production by the progressing replisome provides a mechanism for placing RNA primers close to the RFB, and (ii) for RFBs in the leading strand, we favor a prominent role for a converging replication fork, which, upon its approach, will start Okazaki fragment synthesis in close proximity to the RFB. A potential explanation for the observed increased deletion size in DIV-1compromised C. elegans may be a reduced incidence of primer deposition, leading to Okazaki fragments being initiated further away from the RFB. In line with this idea is the recent observation that reduced primase expression in yeast leads to increased Okazaki fragment size (15).

At present, it is unclear whether Okazaki fragments that are located downstream of an RFB are subjected to exonucleic attack by, e.g., 5 to 3 resection enzymes. The presence of deletion junctions within the PDs, in some cases >100 bp away from the nearest primase template, hints toward this DNA processing. One candidate for this activity is EXO1 because of its demonstrated 5 to 3 exonuclease activity toward both DNA and RNA in vitro (16). We thus generated exo-1 dog-1 animals and measured G4-induced deletion formation in unc-22(G4). Figure 3A shows that the deletion junctions in EXO1-deficient animals are indeed, on average, positioned closer to the G4 motif than in EXO1-proficient animals, the median deletion size being 94 bp instead of 125 bp. A similar reduction in deletion size is observed in alleles carrying a 100-bp PD in addition to the G4 motif: 164 bp versus 199 bp for exo-1 mutant versus wild type, respectively, arguing that EXO1 activity, on average, removes 30 nucleotides of the 5 end of newly synthesized DNA at this RFB. To address the generality of this activity, we also determined the sizes of G4-induced deletions that accumulate throughout the genome in exo-1 dog-1 animals upon prolonged culturing. Compared to EXO1-proficient worms, genomic deletions mapping to G4 loci were, on average, ~40 bp smaller in worms that lost EXO1 activity (Fig. 3B and fig. S6). Our data combined suggest that Okazaki fragment production prevents excessive loss of DNA at RFBs yet are subject to EXO1-dependent degradation.

(A) Deletion spectra from the unc-22 G4 assay, with or without a PD (in blue), for the indicated genotypes. Dots represent independently derived deletion alleles and indicate the position of the distal junctions (in base pairs) relative to the G4 motif set at 0. Red lines indicate the median; ****P < 0.0001 by Mann-Whitney test. (B) Size representation of deletions at endogenous G4 loci that were obtained after prolonged culturing of animals of the indicated genotype. Each dot represents the distal junction relative to the G4 sequence set at 0. ****P < 0.0001 by Mann-Whitney test. (C) Deletion spectra from the unc-22 G4 assay for the indicated genotypes. Dots represent independently derived deletion alleles and indicate the position of the distal junctions (in base pairs) relative to the G4 motif set at 0. Red lines indicate the median; ****P < 0.0001 by Dunnetts multiple comparisons test. (D) Quantification of reporter activation (as described in Fig. 2A) for 200 to 300 synchronized L4 animals of the indicated genotype. Green indicates the ratio of animals expressing both mEGFP and wrmScarlet over the total of animals that express one or both fluorochromes, and red indicates the ratio of animals exclusively expressing wrmScarlet. Experiments were performed in triplicate. Error bars denote SD. ****P < 0.0001 by t test. (E) Size representation of deletions at endogenous G4 loci that were obtained after prolonged culturing of animals of the indicated genotype. Each dot represents the distal junction relative to the G4 sequence set at 0. ****P < 0.0001 by Mann-Whitney test. (F) Deletion spectra from the unc-22 G4 assay for the indicated genotypes. Dots represent independently derived deletion alleles and indicate the position of the distal junctions (in base pairs) relative to the G4 motif set at 0. Red lines indicate the median; ***P < 0.001 and ****P < 0.0001 by Dunnetts multiple comparisons test.

Since EXO1 is known to perform long-range resection (17), we were surprised to observe such a modest loss of only ~30 to 40 nucleotides, which may point to inhibiting factors that we next sought to identify. We focused our attention on the 9-1-1 (RAD9a/HUS1/RAD1) heterotrimeric complex, because it is recruited to sites of stalled replication forks, where it fulfills an essential function in DNA damage-induced checkpoint activation; hence, this complex is also called the checkpoint clamp (18, 19). The 9-1-1 complex structurally resembles PCNA, and an interesting concept here emerges of similar ring-like protein structures providing physical boundaries and functional scaffolds on both ends of an RFB. Consequences of 9-1-1 loss include genomic instability, telomere shortening, and cell death (20, 21). In contrast to mammalian cells, nematodes tolerate a complete loss of 9-1-1, at least for some generations, up to the point that telomeres become critically short, leading to growth arrest, telomere fusions, and animal sterility (2224). These delayed detrimental phenotypes provide a sufficient window of opportunity to test the involvement of 9-1-1 complex members in protecting Okazaki fragment deterioration at sites of stalled replication. To this end, we established G4-induced deletion profiles in animals carrying null mutations in HPR-9/RAD9a, in HUS-1/HUS1, and in MRT-2/RAD1. The absence of any single member of the 9-1-1 clamp is known to destabilize the complex, and we therefore expect different null mutations to behave similarly (2224). We found that the loss of 9-1-1 had a profound effect on the size of the deletions induced at G4s: While the proximal junction was unaffected, we observed a marked increase in distance and spread of the distal junction, with the median increasing three to four times to 435 to 551 bp and the 10 to 90 percentile ranging from 140 to 1591 bp (82 to 288 bp in 9-1-1proficient animals) (Fig. 3C and fig. S6). We found an identical outcome by knocking out the clamp loader RAD17 (Fig. 3C) (25), arguing that for 9-1-1 to suppress DNA loss at sites of stalled replication, it needs to be physically loaded onto DNA. While the increased loss of DNA is nonsymmetric with respect to the RFB, which argues for a role for 9-1-1 specifically in protecting DNA at the RFB downstream site, we wished to formally exclude the possibility that disturbed repair of consequential DSBs explains our observation: We found that CRISPR-induced DSB repair by TMEJ is not affected by 9-1-1 deficiency (fig. S5). Next, we verified more excessive loss at G4s by 9-1-1 dysfunction in vivo by demonstrating a greatly increased wrmScarlet over eGFP ratio in animals carrying the size-discriminatory G4-deletion reporter (Fig. 3D). Last, we performed whole-genome sequencing of hus-1 dog-1deficient animals after prolonged clonal growth and found the protective effect of 9-1-1 on sites of RFBs acting throughout the genome (Fig. 3E).

We next tested whether 9-1-1 protects the 5 dsDNA end from EXO1-dependent resection by removing EXO1 from 9-1-1deficient animals. We observed a significant reduction in the median deletion size in these animals as compared to EXO1-proficient 9-1-1mutant animals: 196 bp versus 441 bp, respectively (Fig. 3F). While most of the deletions are smaller in an exo-1 mutant background, large deletions also remain, pointing to previously reported redundancy in processing 5 DNA ends (26).

Our data, for which we used G4s as a model substrate, establish a new role for the 9-1-1 complex in limiting loss of genetic information downstream of RFBs. To address the generality of this protective function at RFBs, we extended our investigation to psoralen adducts and spontaneous damage that are dependent on replicative bypass by translesion synthesis (TLS) polymerases (2729). Exposing C. elegans to trioxsalen (TMP) followed by ultraviolet A (UVA) irradiation leads to replication-blocking psoralen cross-links, which in wild-type animals give rise to deletions randomly spread throughout the genome in the same size range as those accumulating at G4s in dog-1 animals (27). We monitored mutagenesis in wild-type and 9-1-1(hus-1)mutant animals using the wild-type 40-kb-sized unc-22 gene as a mutational target, isolating UNC-22deficient worms out of the progeny of exposed hermaphrodites (Fig. 4A). Approximately 50% of the mutants had a deletion disrupting the unc-22 ORF, and subsequent molecular characterization revealed that these were larger in hus-1deficient animals than in wild-type animals (Fig. 4B). For a third RFB category, we focused on spontaneously occurring DNA lesions that require TLS polymerases (polh-1) and (polk-1) to be bypassed: Previous work has shown that 50- to 200-bp deletions spontaneously accumulate in the genomes of animals that have impaired TLS activity (28). We here performed whole-genome sequencing of animals in which such a TLS defect (polh-1 polk-1) is combined with a 9-1-1 defect (hus-1) and found that also for this biological context, a 9-1-1 deficiency results in extensive loss of DNA: The median deletion size increased from 104 bp in HUS-1proficient to 466 bp in HUS-1deficient animals (Fig. 4C and fig. S6). Together, these data illustrate that 9-1-1 function suppresses extensive loss of DNA downstream of RFBs by counteracting EXO1-dependent nucleolytic degradation of a newly formed 5 dsDNA segment initiated by the DNA pol -primase complex. While a mechanism of physical inhibition at the site of the RFB is appealing, it could also be that disturbed 9-1-1mediated checkpoint activation is causing altered mutagenesis, e.g., by disrupted ATR (ataxia telangiectasia and Rad3-related protein) signaling (30).

(A) Mutation induction by UV/TMP treatment using the endogenous unc-22 locus as a mutational target. Wild-type (N2) and hus-1 mutant animals were either mock-treated or exposed to UV/TMP. Error bar denotes SD. (B) Size spectrum of UV/TMP-induced deletion mutations captured at the unc-22 locus. **P < 0.01 by Mann-Whitney test. (C) Size representation of genomic deletions that accumulated in the genomes of the indicated genotype upon prolonged culturing. Red lines indicate median deletion size. ****P < 0.0001 by Mann-Whitney test. (D) Tentative model for preserving lagging strand integrity at sites of stalled replication. Upon replication fork stalling at an RFB, the 9-1-1 complex protects newly made Okazaki fragments against nucleolytic degradation by EXO1. In the absence of RFB bypass, the ssDNA gaps will give rise to DSBs that are subject to polymerase mediated end joining (4, 6).

In this study, we used genetic tools to address the fate of DNA downstream of physiologically relevant RFBs and to identify factors that impact on genetic vulnerability resulting from these RFBs. To study potential deleterious consequences at nucleotide resolution, we made use of two sequence motifs that affect the replication machinery in different ways: While a G4 motif has the ability to fold into a secondary structure that can block the replication fork, hence defining the location of a stalled nascent strand, the newly introduced PD motif prevents the initiation of DNA synthesis and can thus be used to modulate Okazaki fragment positioning in vivo. The combined usage of these two motifs creates an opportunity to temporarily capture an RFB at a fixed genomic position. From the data obtained using this well-defined genetic context, we conclude that (i) Okazaki fragment deposition, either through lagging strand synthesis or brought in by a converging forks, limits the size of vulnerable ssDNA gaps at RFBs; (ii) 5 ends of Okazaki fragments at RFBs are subject to EXO1-dependent degradation; and (iii) the 9-1-1 complex protects Okazaki fragments against endonucleolytic attack, hence preventing excessive loss of genetic information, meanwhile acting as a damage sensor for checkpoint signaling (Fig. 4D) (18, 19, 25, 31, 32).

Replication stress is considered a universal phenomenon in tumorigenesis (33). Arrested forks can evolve into highly toxic and recombinogenic DSBs. It was recently found that mutagenic repair, particularly TMEJ, of replication-associated DSBs results in genomic scars, which are found in disease alleles and in cancer genomes (3441). In-depth knowledge on the processing of stalled forks thus contributes to our understanding of genome alterations during cell and organismal evolution, while some of the molecules acting on RFBs are considered promising targets for anticancer therapy (35, 37, 38, 42).

All strains were cultured according to standard methods (43) and grown at 20C unless otherwise stated. See table S1 for a complete strain list.

To obtain independent reversion events, single animals were put on 9-cm nematode growth media (NGM) plates (100 to 200) and grown until the food was exhausted. From each wild-type moving animalcontaining plate, a single animal was transferred to a new plate to obtain a collection of independently derived deletion alleles. Populations were subsequently lysed in lysis buffer, and DNA was polymease chain reaction (PCR)amplified with primers surrounding the G4 motif to obtain the deletion products, which were analyzed by Sanger sequencing. The reversion frequency was determined by assaying 75 cultures, starting with placing one animal on a 6-cm plate seeded with 25 l of the E. coli strain OP50. When half of the control dog-1 populations contained wild-type moving animals, all genotypes were scored for revertants. The reversion frequency is calculated by assuming a Poisson distribution: Reversion frequency = ln(P0)/2n, where P0 is the fraction of plates that did not yield revertants and n is the number of animals that were screened per plate. Frequencies were determined at least in duplicate and normalized to dog-1 animals (set to 1).

Plasmids were injected using standard C. elegans microinjection procedures. In brief, 1 day before injection, L4 animals of strain XF320 [already containing an inserted G4 sequence (4)] were transferred to OP50-containing 6-cm plates and cultured at 15C. The next day, the gonads of young adults were injected with a solution containing pDD162 [20 ng/l; Peft-3::Cas9, Addgene #47549; (44)], pRS27-29 (20 ng/l; U6 promoter + sgRNA, Addgene #75026; see table S3 for details on sgRNA sequence), ssODN (20 ng/l; see table S3 for details), and pBluescript (40 ng/l). Three to four days after injection, 100 to 200 l of levamisol (20 mM in M9 salt solution) were added to the plates to find animals with altered unc-22 alleles. All levamisole-resistant animals were grown to populations for further inspection. unc-22 mutant progeny animals were analyzed for the presence of a PD.

Plasmids were injected using standard C. elegans microinjection procedures. In brief, 1 day before injection, L4 animals were transferred to OP50-containing 6-cm plates and cultured at 15C. The next day, the gonads of young adults were injected with a solution containing pDD162 [20 ng/l; Peft-3::Cas9, Addgene #47549; (44)], pRS32 (20 ng/l; U6 promoter + sgRNA; see table S3 for details on sgRNA sequence), pBluescript (60 ng/l), pGH8 (10 ng/l), pCFJ90 (2.5 ng/l), and pCFJ104 (5 ng/l). Three to four days after injection, mCherry-positive F1 animals were transferred to 6-cm plates. PCRs were performed on animals from plates with germline dpy-10 mutations in the F2 generation with the following primers: 5-CAACGAACTATTCGCGTCAG-3 and 5-GTGGTGGCTCACGAACTTG-3. PCR products were send for Sanger sequencing to obtain the specific mutation.

pSR02 (Addgene #69149) was digested with Nhe I and subsequently ligated to remove eGFP to create pRS67. A PCR was performed on pSR02 with a G4-containing primer to create G4::eGFP::T2A and Xba I restriction sites. This PCR product was cloned into pCloneJet and subsequently cut out with Xba I and inserted into Xba-Idigested pRS67 to obtain rps-27:G4::eGFP::T2A::mCherry (pRS68). T2A::mCherry-NLS was then replaced by egl-13::F2A::wrmScarlet::egl-13 (ordered as gBlock) through NEBuilder Hifi DNA assembly to create pRS88: rps-27:ATG::G4::eGFP::egl-13::F2A::wrmScarlet::egl-13. The F2A sequence was added to the design, as an earlier version of this reporter only displayed eGFP and wrmScarlet activation but not wrmScarlet activation alone. We attributed this to degradation of misfolded eGFP protein because of deletions into eGFP. The addition of this sequence solved this issue. pRS88 was cut with Avr II and Hind III and cloned into miniMos vector pCFJ1663 (Addgene #51484) cut with Spe I and Hind III, generating pRS89. N2 worms were injected with a mix containing pRS89 (10 ng l1), pCFJ601 (50 ng l1; Addgene #34874), pGH8 (10 ng l1), pCFJ90 (2.5 ng l1), and pCFJ104 (5 ng l1). Five hundred microliters of hygromycin (5 mg/ml) was added to the plates 3 days after injection to select for hygromycin-resistant animals. Plates containing living animals were heat-shocked 7 days after injection for 2 hours at 34C to counterselect for animals containing extrachromosomal arrays. Animals were then inspected for the presence of both eGFP and wrmScarlet signal. An eGFP- and wrmScarlet-positive strain was obtained, and this strain was subsequently targeted by CRISPR-HDR to switch off the reporter by introducing stops in every frame directly downstream of the G4 motif and to increase the distance between the base of the G4 and eGFP to 160 bp.

RNAi feeding was performed as previously described (45). In brief, we grew RNAi clones against different targets (see table S4) in 2 ml of LB supplemented with ampicillin. The following day, isopropyl--D-thiogalactopyranoside (IPTG) was added to the RNAi bacteria to induce dsRNA expression for 2 hours. Six-centimeter NGM plates supplemented with ampicillin and IPTG was seeded with 100 l of RNAi bacteria and was kept at room temperature overnight. Five L4 animals were transferred to each RNAi-containing plate. After 3 to 4 days, the animals were rinsed off the plate with M9 and inspected for eGFP and wrmScarlet expression.

Animals of the indicated genotypes were synchronized by hypochlorite treatment, and surviving eggs were hatched in M9 overnight. L1 animals were plated out, and 48 hours later, the animals were rinsed off the plate with M9, sedated with 40 mM NaN3, and inspected for eGFP and wrmScarlet expression. To this end, animals were mounted on microscope slides containing dried 2% agarose pads and inspected for eGFP and wrmScarlet expression using a Zeiss Axio imager D2.

Mutation accumulation (MA) lines were generated by cloning out F1 animals from one hermaphrodite. All experiments were performed at 20C. Each generation, three worms were transferred to new plates. MA lines were maintained for 50 generations (dog-1 exo-1, dog-1, and div-1) or 10 generations (polh-1 polk-1 hus-1, dog-1, and hus-1). After 10 or 50 generations, single animals were cloned out and allowed to generate a full population that was used for DNA isolation. To remove bacteria from the sample and from the animals intestine, rinsed-off worms were washed three times with M9 and subsequently incubated for 2 hours at room temperature while shaking. After allowing the sample to set down, the supernatant was removed, and the QIAGEN Blood and Tissue Kit was used to extract DNA according to the manufacturers protocol with some minor adjustments: 200 l of ATL buffer and 20 l of ProtK were added and incubated for 1.5 hours at 60C in a shaker incubator at 1400 rpm. The samples were spun down for 30 s at 2000 rpm, and the supernatant was transferred to new tubes to prevent blocking of the columns by cellular debris. Then, 5 l of ribonuclease A (100 mg/ml) was added and samples were incubated for 5 to 10 min, after which 200 l of AL buffer was first added (mixed thoroughly) followed by 200 l of EtOH (mixed thoroughly). Spin columns were used and washed with the appropriate buffers, AW1 and AW2, and DNA was eluted in 100 to 150 l of H2O. DNA samples were subsequently prepared according to Illuminas protocol and sequenced on either HiSeq4000 or NovaSeq.

Mapping of paired-end next-generation sequencing (NGS) reads was performed by BWA-MEM (Burrows-Wheeler Aligner). For each MA line, at least three independently grown samples were analyzed (table S2). For strains that were crossed before growing as MA line, we also sequenced generation 0 to filter out background single-nucleotide variants (SNVs) and copy number variations (CNVs) unrelated to the MA experiment, which may segregate differently in subpopulations. For CNV detection, we made use of Pindel, GRIDSS, GATK, and Manta (4649). Only unique events that were supported by at least two callers or called with high confidence (5 unique reads supporting the CNV) by a single caller were included in the analysis. Metadata such as homology, topology, and templated insertions were analyzed and categorized using a custom Java program (data file S2). SNV calling was performed by GATK (data file S2).

To annotate the unc-22 alleles obtained in the unc-22 G4 RFB assay and UV/TMP assay, a custom Java program was written to extract high-confidence sequences from Sanger sequence files; high-confidence sequence defined as a sequence of >30 nt where all nucleotides have an error probability of <0.05. This sequence is then mapped to a reference FASTA file containing the appropriate unc-22 allele using k-mer mapping. Differences between the Sanger sequences and the reference are further classified into wild type, SNV, insertion, deletion, or deletion-insertion (delins). Additional informationsuch as location, homology, and likelihood of templated insertionwas added, leading to output in a TSV format (data file S1).

Animals were synchronized by alkaline hypochlorite treatment (0.5 M NaOH and 2% hypochlorite), and eggs were allowed to hatch overnight. L1 worms were placed on 9-cm NGM agar plates seeded with Escherichia coli (OP50) and grown at 20C. After 48 hours, L4 worms were washed off the plates and treated for 1 hour with TMP (10 g/ml; Sigma-Aldrich, T6137, stock: 100 mg dissolved in 40 ml of acetone) in M9. Animals were then distributed on nonseeded NGM plates and exposed to UVA irradiation (366 nm; CAMAG 29200 Universal UV LAMP) at a dose rate of 160 W/cm2 (Blak-Ray UV meter, model no. J221). Thereafter, animals were transferred to standard 9-cm OP50/NGM plates (10 P0 animals per plate; 75 plates with treated animals and 50 plates with mock-treated animals). Animals of the F2 generation were washed off the plates with 2 mM levamisole and transferred to six-well plates to facilitate scoring of unc-22 mutants that are insensitive to the hypercontracting effects of the drug levamisole. For each well, we searched for a levamisole-resistant animal for 120 s. If found, a single levamisole-resistant animal was picked from the well, and homozygous mutants were grown to a full 9-cm plate. Genomic DNA was isolated and analyzed by PCR and Sanger sequencing. The mutation frequency was calculated assuming a Poisson distribution: MF = ln(P0)/2n, where P0 is the fraction of plates without reverted animals and n is the number of animals that were screened per plate.

Acknowledgments: We thank E. Klaassen and R. Profijt for experimental support. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Funding: This work was funded by an ALW OPEN grant (OP.393) from the Netherlands Organization for Scientific Research for Earth and Life Sciences to M.T. Author contributions: R.v.S: Conceptualization, software, data analysis, visualization, validation, and writing. R.R.: Validation. H.B.: Validation. M.T.: Conceptualization, funding acquisition, and writing. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Sequence data can be found in the NCBI SRA repository under project number: PRJNA639450. Some sequences have been previously published and can be found in the NCBI SRA repository under project numbers PRJNA196232 and PRJNA260487. Additional data related to this paper may be requested from the authors.

See the article here:
Preservation of lagging strand integrity at sites of stalled replication by Pol -primase and 9-1-1 complex - Science Advances

Study Take-Aways:

FORT LAUDERDALE/DAVIE, Fla. They are some of the most beautiful, and elusive, animals on the planet. Leopards.

These majestic animals are the only great cat species (Genus Panthera) to roam about both Africa and Asia today. Leopards are considered as highly vulnerable, classified by the Convention on International Trade of Endangered Species of Wild Fauna and Flora (CITES). Because of their elusive nature, and their adaptation to multiple landscapes (rain forest, savannah, deserts and mountain sides) an accurate estimation of their global census has not been possible.

In a major scientific step to reconstruct the evolutionary history plus their conservation status, the whole genome DNA sequence of 23 individual leopards sampled from eight geographically separated subspecies locales were interpreted using the latest technologies of population ecology and molecular evolution. Ancient DNA sequences for 18 archival specimens along with 5 living leopards were combined to refine our understanding of the leopards movements, population reductions, divergence and isolation, and over the past half million years.

The new study was published today in Current Biology

An international team involving scientists from Nova Southeastern University (NSU), Nottingham Trent University, the University of Cambridge, University of Leicester in U.K. and the University of Potsdam in Germany carried out genetic analysis of modern and historical specimens stored in natural history museums as part of the study.

This study changes everything about genetic contributions to conservation management of the worlds leopards, particularly the highly threatened Amur leopard, said Stephen J. OBrien, Ph.D., a professor and research scientist in NSUs Halmos College of Arts and Sciences, who is a collaborating author and also led the genetic analyses the Florida panther restoration two decades ago.

OBrien, is also the Chief Scientific Officer at the Theodosius Dobzhansky Center for Genome Bioinformatics, St. Petersburg State University, Russia, and is a member of theNational Academy of Sciences.

Genetic distinctiveness of nine previously suggested subspecies was re-affirmed with increased precision. Different African populations were genetically interrelated suggesting abundant gene flow across Africa such that all African populations should be considered together as a single subspecies. By contrast Asian leopard populations were geographically distinct along ten prior recognized subspecies boundaries.

There appeared a striking genomic distance between leopards living in Asia vs. leopards in Africa. Asian leopards are more genetically separated from African leopards than brown bear species are from polar bear species, the researchers found. The two leopard groups actually diverged around the same time as Neanderthals split apart from modern humans. The genetic differences between African and Asian leopards have been maintained since 500,000 to 600,000 years ago. Asian leopards retain markedly less overall genetic variation than is seen in African leopards.

While they both have spots, the African and Asian leopards are quite different when looking at their DNA, said Johanna Paijmans, Ph.D., lead author, Research Fellow at the University of Cambridge and Honorary Fellow at the University of Leicester. Given their mobility, it is surprising that they have remained so distinct and do not carry more shared genetic material with one another. Our study demonstrates that many more exciting genetic discoveries could be hidden among the shelves of natural history museums around the world.

The simplest explanation for both low Asian diversity and this large genetic difference may involve an origin for all leopards in Africa, with a single founding out of Africa migration to Asia occurring 500,000 600,000 years ago. Evidence of subsequent additional northern migrations were not detected, perhaps because the Asian habitats were protected by behavioural reinforcement, a term used to describe the blocking of immigrants across a narrow migration corridor. For example, a similar pattern had occurred with puma/cougar population migrations to North America from South American isthmus 1-12,000 years ago.

The depletion of genetic diversity observed in Asian leopard subspecies extends to the extreme in the critically endangered Amur leopard ( Panthera pardi orientalis ). Amur leopards comprise a tiny relict subspecies living on the edge of extinction in the Primorsky Krai region of the Russian Far East-RFE, along the North Chinese border. The Amur leopard population has dropped below 60 individuals and is now showing congenital traits that derive from close inbreeding. Conservation organizations in the RFE are proposing a genetic restoration of re-introduction of Amur leopards to Ussurijsky and Lazovsky Nature Reserves in the RFE (similar to what was successful for the Florida Panther restoration 15 years ago to augment the struggling wild population). The new Amur leopard genomic diversity data shall form a baseline for monitor of Amur leopard re-introduction consequences.

Despite the fact that they the most widely distributed big cats, the evolutionary development and population history of leopards across Africa and Asia has not been studied with genomic tools before now, said Axel Barlow, Ph.D., an expert in palaeogenomics and molecular bioscience in Nottingham Trent Universitys School of Science and Technology. Our results have highlighted marked genetic differences between African and Asian leopards, that Asian leopards originated from a single out-of-Africa dispersal event, and that the distinctiveness of leopards on the two continents has been maintained.

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NSU Researcher Part of Team to Sequence the Genome of One of the Worlds Most Elusive Big Cats the Leopard - Newswise

The Union Health Ministry on Monday announced that CoWIN portal, through which slots are to be booked for Covid vaccination, will be made available in Hindi and 14 regional languages by next week. It also said that 17 more laboratories will be added to the INSACOG network to conduct genome surveillance and monitor the variants of Covid-19.

The two key decisions were announced at the 26th meeting of the High-level Group of Ministers (GoM) on COVID-19, chaired by Health Minister Harsh Vardhan.

This came on a day Prime Minister Narendra Modi held an interaction with a group of doctors from across the country, via video conferencing, to discuss the Covid situation. During the meeting, the Prime Minister urged the doctors to include oxygen audits in their daily efforts.

Noting that a large number of patients are undergoing treatment in home isolation, he requested the doctors to ensure that the home-based care of every patient is SOP driven. He said telemedicine has played a big role for patients in home isolation, and this service needs to be expanded in rural areas as well, the PMO said in a statement.

During the meeting, the PMO said, the Prime Minister appealed to doctors across the states to form teams, train final year MBBS students and MBBS interns, and work towards ensuring that all tehsils and districts of the country have telemedicine service.

He further underscored the importance of psychological care, along with the importance of physical care. He said continuously fighting this long battle against the virus must be mentally challenging for the medical fraternity, but the power of faith of citizens stands with them in this fight, the PMO said in a statement.

During the GoM meeting, Vardhan, informed the ministers that 17 new labs are going to be added to the INSACOG (Indian SARS-CoV-2 Consortium on Genomics) network to increase the number of samples screened and allow for more spatial analysis. The network is currently served by 10 laboratories located in different locations of the country.

Indias Covid-19 new cases have dropped to less than 3 lakh for the first time after 26 days. Also, a net decline of 1,01,461 cases have been recorded in the active caseload in the last 24 hours, Vardhan said during the meeting.

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CoWIN in Hindi, vernacular soon; 17 more labs to test genome - The Indian Express

Though common, Triple Black is a staple amongst sneakers. And whenever in doubt, its easy for brands to fall back on the colorless palette. Nike seems to have found themselves in that corner, as they bring the scheme to the recently debuted Air Max Genome.

Straightforward, understated, and inarguably subdued, the colorway is nothing short of simple. It takes the neutral tone and applies to every fixture, including the mesh tongue, laces, and synthetic-filled upper construction. Even the tooling follows suit, pairing a rather lackluster midsole with a tinted Air Unit window. Above, where branding would typical sport pops of color, theres little immediately of note; however, its likely both the side Swoosh and tongue logo are both reflective.

For a closer look, check the official images below. If youd like to add these to the rotation, theyre sure to hit Nike.com and select retailers soon.

In other news, in-hand images of the 2021 Air Jordan 5 Oreo are here.

Where to Buy

Make sure to follow @kicksfinder for live tweets during the release date.

Mens: $170Style Code: CW1648-001

After MarketAvailable Now

Excerpt from:
Triple Black Is Coming To The Nike Air Max Genome - Sneaker News

Use of the genomic prostate score (GPS) on prostate cancer biopsy tissue resulted in a lower rate of selection of active surveillance among African American men with very lowrisk to low intermediaterisk prostate cancer, according to the results of a randomized controlled trial published in the Journal of Clinical Oncology.

GPS can predict adverse outcomes in men with prostate cancer and may be used to select patients for active surveillance rather than active treatment. However, its impact on treatment choice in high-risk populations of African Americans is largely unknown, the authors stated.

The trial (ClinicalTrials.gov Identifier: NCT02668276) randomly assigned 200 patients with very lowrisk to low intermediaterisk prostate cancer to undergo standard counseling or counseling with the 12-gene GPS assay results. The primary endpoint was treatment choice at the second visit after diagnosis.

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At baseline, the median age was 64 years; 70% of the patients were African American, 16.5% were European American, 12.5% were Hispanic or Latino, and 1.0% were Asian. The patients (40%) who had some college were the largest subgroup followed by high school (27.5%), less than high school (17.0%), and a bachelors degree or higher (15.5%). The median health literacy was 8.6, and a score lower than 9 was considered low literacy.

Active surveillance was selected by fewer patients (77%) who received GPS results compared with 88% of the patients who underwent standard counseling, although this result was not statistically significant (P =.067).

Lower health literacy was associated with lower rates of active surveillance selection among the patients who received GPS counseling compared with those who underwent standard counseling (P =.022). However, there was no difference among the men with higher health literacy.

The authors concluded that Population characteristics should be accounted for when predicting the effects of a complex biomarker such as GPS.

Disclosures: Several study authors declared affiliations with the pharmaceutical industry. Please see the original reference for a full list of authors disclosures.

Reference

Murphy AB, Abern MR, Wang H, et al. Impact of a genomic test on treatment decision in a predominantly African American population with favorable-risk prostate cancer: a randomized trial. J Clin Oncol. Published online April 9, 2021. doi:10.1200/JCO.20.02997

This article originally appeared on Cancer Therapy Advisor

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Effect of Genomic Prostate Score on Active Surveillance Selection in African American Population - Renal and Urology News

In the world of microbial warfare, sometimes you have to change the very fabric of who you are.

Viruses that infect bacteria fittingly called bacteriophages - and their prey have been at war for eons, each side evolving more devilish tactics to infect or destroy each other. Eventually, some bacteriophages took this arms race to a new level by changing the way they code their DNA.

At least, that's what we think happened.Once thought to be an outlier, new research published in three separate papers shows that there's a whole army of bacteriophages with non-standard DNA, which researchers call a Z-genome.

"Genomic DNA is composed of four standard nucleotides These nucleobases form the genetic alphabet, ATCG, which is conserved across all domains of life," biologists Michael Grome and Farren Isaacs write in a recent Scienceeditorialaccompanying the new research on bacteriophage genetics.

"However, in 1977, the DNA virus cyanophage S-2L was discovered with all instances of 'A' substituted with 2-aminoadenine (Z) throughout its genome forming the genetic alphabet ZTCG."

The reason appeared to be self-protection. Within the connecting 'rungs' of a DNA double helix, the 'Z' base forms a triple bond to the opposite 'T' base, one more than the two bonds of the regular A:T connection. This makes the viral genome hardier and more difficult for bacteria to prise apart with chemicals called nucleases.

Although scientists were fascinated, no other bacteriophages were found with the Z-genome, and with the difficulty of culturing S-2L in a lab, the Z-genome was set aside as a curiosity.

Now, research documented in three separate studies from researchers in France and China shows that this was not a one-off, whilst also characterizing how the Z-genome works and how it's assembled.

"Scientists have long dreamed of increasing the diversity of bases. Our work shows that nature has already come up with a way to do that," one of the teams, led by first author Yan Zhou from Tianjin University, wrote in their paper.

Zhou's team, along with another group led by Institut Pasteur microbiologist Dona Sleiman, found two major proteins which they called PurZ and PurB; these make up the 'Z' base.

A third group, led by Universit Paris-Saclay synthetic biologist Valerie Pezo, corroborated those findings and analysed an enzyme called DpoZ which is responsible for assembling the whole Z-genome together.

All three searched genetic sequence databases for the sequences relating to their proteins and enzymes, and found a wide variety of bacteriophages with similar genes.

"[The authors] have done an amazingly comprehensive job of showing that this is not one crazy outlier, but there's a whole group of bacteriophages that have this kind of genetic material," Jef Boeke, a molecular biologist at New York University who was not involved in the work, told The Scientist.

There are still plenty of questions to answer about the Z-genome.

For example, is a Z-genome compatible with regular cell machinery such as ours? And could it be used in the same way that artificial DNA is starting to be?

"The Z base has been unambiguously identified in a carbonaceous meteorite and proposed as a nucleobase that could have been available for the origin of life," the team lead by Zhou wrote in their paper.

"Considering that the Z base was discovered in a meteorite, our work may spark interest in interdisciplinary research on the origins of life and astrobiology."

The three papers have been published in Sciencehere, here, and here.

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Some Viruses Have a Completely Different Genome to The Rest of Life on Earth - ScienceAlert