Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

BACKGROUND: The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR) in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. RESULTS: Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR) at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα) but only requires a phage annealing protein (RecT or Redβ). Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. CONCLUSION: Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ

TB176: Agrelation: A Computerized Decision-making Tool for Coloraod Potato Beetle Population Management and Environmental Quality Concerns

Many facets of Maine potato production have been simulated with computer models and expert systems. Given the ongoing improvement of computer technology and validation of past efforts, scientists can now combine several agricultural submodels into one holistic and user-friendly computer application. This bulletin reports on the development of one such application—Agrelation—aimed at modeling a portion of Maine potato production and management.https://digitalcommons.library.umaine.edu/aes_techbulletin/1032/thumbnail.jp

Single-stranded heteroduplex intermediates in λ Red homologous recombination

<p>Abstract</p> <p>Background</p> <p>The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined.</p> <p>Results</p> <p>Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Redα exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule.</p> <p>Conclusions</p> <p>Hence we propose a new model for dsDNA recombination, termed 'beta' recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i) an <it>in situ </it>assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii) the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s) appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins.</p

Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.

Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following de novo metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to basidiomycete transcripts dominated, and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate-active enzymes (CAZymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases, including highly expressed cellulases or hemicellulases. The CAZymes also included 162 predicted redox enzymes classified within auxiliary activity (AA) families. Eighteen of these were manganese peroxidases, which are key components of ligninolytic white rot fungi. The expression of other redox enzymes supported the working of hydroquinone reduction cycles capable of generating reactive hydroxyl radicals. These have been implicated as diffusible oxidants responsible for cellulose depolymerization by brown rot fungi. Thus, enzyme diversity and the coexistence of brown and white rot fungi suggest complex interactions of fungal species and degradative strategies during the decay of lodgepole pine.IMPORTANCE The deconstruction of recalcitrant woody substrates is a central component of carbon cycling and forest health. Laboratory investigations have contributed substantially toward understanding the mechanisms employed by model wood decay fungi, but few studies have examined the physiological processes in natural environments. Herein, we identify the functional genes present in field samples of extensively decayed lodgepole pine (Pinus contorta), a major species distributed throughout the North American Rocky Mountains. The classified transcripts and proteins revealed a diverse array of oxidative and hydrolytic enzymes involved in the degradation of lignocellulose. The evidence also strongly supports simultaneous attack by fungal species employing different enzymatic strategies

Measurement and modelling of the residual stresses in autogenous and narrow gap laser welded AISI grade 316L stainless steel plates

Thick-section austenitic stainless steels have widespread industrial applications, where stress-corrosion cracking is often of major concern. Problems tend to arise in the vicinity of welds, where substantial residual stresses often reside. This paper describes an investigation into the residual stresses in autogenous high power laser welds and narrow gap laser welds (NGLW) in 10 mm thick AISI grade 316L steel plates, using both neutron diffraction and the contour method. The influences of laser power, welding speed and the time interval between weld passes on residual stress were analysed. For the NGLW process, finite element modelling was employed to understand the influence of thermal history on residual stress. The results for the NGLW technique show that the laser power has a significant effect on the peak value of residual stress, while the welding speed has a more significant influence on the width of the region sustaining tensile stresses

Metatranscriptomics reveal differences in in situ energy and nitrogen metabolism among hydrothermal vent snail symbionts

Despite the ubiquity of chemoautotrophic symbioses at hydrothermal vents, our understanding of the influence of environmental chemistry on symbiont metabolism is limited. Transcriptomic analyses are useful for linking physiological poise to environmental conditions, but recovering samples from the deep sea is challenging, as the long recovery times can change expression profiles before preservation. Here, we present a novel, in situ RNA sampling and preservation device, which we used to compare the symbiont metatranscriptomes associated with Alviniconcha, a genus of vent snail, in which specific host–symbiont combinations are predictably distributed across a regional geochemical gradient. Metatranscriptomes of these symbionts reveal key differences in energy and nitrogen metabolism relating to both environmental chemistry (that is, the relative expression of genes) and symbiont phylogeny (that is, the specific pathways employed). Unexpectedly, dramatic differences in expression of transposases and flagellar genes suggest that different symbiont types may also have distinct life histories. These data further our understanding of these symbionts’ metabolic capabilities and their expression in situ, and suggest an important role for symbionts in mediating their hosts’ interaction with regional-scale differences in geochemistry.National Science Foundation (U.S.) (OCE-0732369)National Science Foundation (U.S.) (GRF grant no. DGE-1144152)Gordon and Betty Moore Foundation (Investigator)Agouron Institut

Male Perspectives on Incorporating Men into Antenatal HIV Counseling and Testing

Male partner involvement in antenatal voluntary HIV counseling and testing (VCT) has been shown to increase uptake of interventions to reduce the risk of HIV transmission in resource-limited settings. We aimed to identify methods for increasing male involvement in antenatal VCT and determine male correlates of accepting couple counseling in these settings.We invited women presenting to a Nairobi antenatal clinic to return with their male partners for individual or couples VCT. Male attitudes towards VCT and correlates of accompanying female partners to antenatal clinic and receiving couple counseling were determined. Of 1,993 women who invited their partner, 313 (16%) returned with their partners to ANC. Men attending antenatal clinic were married (>99%), employed (98%), and unlikely to report prior HIV testing (14%). Wanting an HIV test (87%) or health information (11%) were the most commonly cited reasons for attending. Most (95%) men who came to antenatal clinic accepted HIV testing and 39% elected to receive counseling as a couple. Men who received counseling with partners were younger, had fewer children, and were less knowledgeable about prevention of mother-to-child HIV transmission (PMTCT) than those who received counseling individually (p<0.05). Only 27% of men stated they would prefer HIV testing at a site other than the ANC. There was agreement between male and female reports for sociodemographic characteristics; however, men were more likely to report HIV preventive behaviors and health communication within the partnership than their partners (p<0.05).Offering VCT services to men at antenatal clinic with options for couple and individual counseling is an important opportunity and acceptable strategy for increasing male involvement in PMTCT and promoting male HIV testing

Mutation of cancer driver MLL2 results in transcription stress and genome instability

Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly,MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels ofγH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis

Exploring the responses of people who never attend church to the pre-Christmas son et lumiere at Liverpool Cathedral: ‘I am not religious, but I had the best experience ever’

Two conceptual strands of research within the field of cathedral studies have theorised the capacity of Anglican cathedrals to engage more successfully than parish churches with the wider non-churchgoing community: one strand has explored mobilising cathedral metaphors, and the other strand has explored the notion of implicit religion. Both strands illuminate the power of events and installations to soften the boundaries between common ground and sacred space. Drawing on a quantitative survey among 978 people who attended the pre-Christmas son et lumiere at Liverpool Cathedral during December 2022, the present study analyses the qualitative responses of 123 participants who never attend church services. Three categories of themes emerged from these data, concerning the Cathedral itself, the installation, and discordant experience. Contribution: Situated within the science of cathedral studies, this paper draws on original qualitative data to illuminate the experiences of participants who never attend church services when engaging with the pre-Christmas son et lumiere at a major cathedral. Conceptualised within the framework of implicit religion these data confirmed how the son et lumiere succeeded in softening boundaries between the sacred and the secular and provided a deeply moving experience. As one participant said, ‘I am not religious, but I had the best experience ever’