Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Condition‐dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State‐of‐the‐art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double‐mutant strains, does not scale readily to multi‐condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG‐GI), by which double‐mutant strains generated via en masse “party” mating can also be monitored en masse for growth to detect genetic interactions. By using site‐specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG‐GI enables multiplexed quantitative tracking of double mutants via next‐generation sequencing. We applied BFG‐GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4‐nitroquinoline 1‐oxide (4NQO), bleomycin, zeocin, and three other DNA‐damaging environments. BFG‐GI recapitulated known genetic interactions and yielded new condition‐dependent genetic interactions. We validated and further explored a subnetwork of condition‐dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.     

Seasonal patterns in rainforest litterfall: Detecting endogenous and environmental influences from long‐term sampling

Tropical rainforests play an important role in the storage and cycling of global terrestrial carbon. In the carbon cycle, net primary productivity of forests is linked to soil respiration through the production and decomposition of forest litter. Climate seasonality appears to influence the production of litter although there is considerable variability within and across forests that makes accurate estimates challenging. We explored the effects of climate seasonality on litterfall dynamics in a lowland humid rainforest over a 7‐year period from 2007 to 2013, including an El Niño/La Niña cycle in 2010/2011. Litterfall was sampled fortnightly in 24 traps of 0.50 m diameter within a 1‐ha forest plot. Total mean litterfall was 10.48 ± 1.32 (±SD, dry weight) Mg ha^?1 year^?1 and seasonal in distribution. The different components of litterfall were divided into L_Leaf (63.5%), L_Wood (27.7%) and L_{FF[flowers & fruit]} (8.8%), which all demonstrated seasonal dynamics. Peak falls in L_Leaf and L_Wood were highly predictable, coinciding with maximum daily temperatures and 1 and 2 months prior to maximum monthly rainfall. The El Niño/La Niña cycle coincided with elevated local winter temperatures and peak falls of L_Leaf and L_Wood. Importantly, we establish how sampling length and generalized additive models eliminate the requirement for extensive within‐site sampling when the intention is to describe dynamics in litterfall patterns. Further, a greater understanding of seasonal cycles in litterfall allows us to distinguish between endogenous controls and environmental factors, such as El Niño events, which may have significant impacts on biochemical cycles.     

Construction of a Panel of Canine–Rodent Hybrid Cell Lines for Use in Partitioning of the Canine Genome

We have constructed a collection of canine–rodent microcell hybrid cell lines by fusion of canine fibroblast microcell donors with immortalized rodent recipient cells. Characterization of the hybrid cell lines using a combination of fluorescencein situhybridization and PCR analysis of canine microsatellite repeat sequences allowed selection of a panel of hybrids in which most canine chromosomes are represented. Approximately 90% of genetic markers and genes that were tested could be assigned to 1 of 31 anonymous canine chromosome groups, based on common patterns of retention in the hybrid set. Many of these putative chromosome groups have now been validated by linkage analysis. This panel of cell lines provides a tool for development of genetic, physical, and comparative maps of the canine genome.     

The impact of bird herbivory on macrophytes and the resilience of the clear-water state in shallow lakes: a model study

Land-use change associated with human development can alter aquatic habitat and imperil aquatic species. Fish are challenged when urban streams are altered, for example for stormwater conveyance, but little is known about how such activities influence the space use of individual fish. Electronic tagging and experimental displacement of fish can be used to explore site fidelity and homing behaviour of fish and can therefore be useful for testing hypotheses about space use and habitat selection. In this study, we used experimental displacement to determine how longnose dace (LND, Rhinichthys cataractae) utilize reaches within a watershed that have varying degrees of degradation. LND were tagged using passive integrated transponders (PIT tags), transported upstream, and released either into the natural stream reach, impaired stormwater drain reach, or at their confluence. Fixed PIT antennas were used to monitor movement of the PIT-tagged fish among the three reaches for a period of 3 weeks. LND exhibited dramatic and rapid selection against the stormwater drain. No LND moved into the drain and 97% of fish transported to the drain left within 24 h. LND were actively avoiding the stormwater drain, emphasizing the need for enhancement work to improve the biological connectivity of the system.     

Carry‐over effects provide linkages across the annual cycle of a Neotropical migratory bird,the Louisiana Waterthrush Parkesia motacilla

Population limitation models of migratory birds have sought to include impacts from events across the full annual cycle. Previous work has shown that events occurring in winter result in some individuals transitioning to the breeding grounds earlier or in better physical condition than others, thereby affecting reproductive success (carry‐over effects). However, evidence for carry‐over effects from breeding to wintering grounds has been shown less often. We used feather corticosterone (CORT_f) levels of the migratory Louisiana Waterthrush Parkesia motacilla as a measure of the physiological state of birds at the time of moult on the breeding territory to investigate whether carry‐over effects provide linkages across the annual cycle of this stream‐obligate bird. We show that birds arriving on wintering grounds with lower CORT_f scores, indicating reduced energetic challenges or stressors at the time of moult, occupied higher quality territories, and that these birds then achieved a better body condition during the overwinter period. Body condition, in turn, was important in determining whether adult birds returned the following winter, with birds in better condition returning at higher rates. Together these data suggest a carry‐over effect from the breeding grounds to the wintering grounds that is further extended with respect to annual return rates. Very few other studies have linked conditions during the previous breeding season with latent effects during the subsequent overwintering period or with annual survival. This study shows that the effects of variation in energetic challenges or stressors can potentially carry over from the natal stream and accumulate over more than one life‐history period before being manifested in reduced survival. This is of particular relevance to models of population limitation in migratory birds.     

Crusticorallina gen. nov., a nongeniculate genus in the subfamily Corallinoideae (Corallinales,Rhodophyta)

Molecular phylogenetic analyses of 18S rDNA (SSU) gene sequences confirm the placement of Crusticorallina gen. nov. in Corallinoideae, the first nongeniculate genus in an otherwise geniculate subfamily. Crusticorallina is distinguished from all other coralline genera by the following suite of morpho‐anatomical characters: (i) sunken, uniporate gametangial and bi/tetrasporangial conceptacles, (ii) cells linked by cell fusions, not secondary pit connections, (iii) an epithallus of 1 or 2 cell layers, (iv) a hypothallus that occupies 50% or more of the total thallus thickness, (v) elongate meristematic cells, and (vi) trichocytes absent. Four species are recognized based on rbcL, psbA and COI‐5P sequences, C. painei sp. nov., the generitype, C. adhaerens sp. nov., C. nootkana sp. nov. and C. muricata comb. nov., previously known as Pseudolithophyllum muricatum. Type material of Lithophyllum muricatum, basionym of C. muricata, in TRH comprises at least two taxa, and therefore we accept the previously designated lectotype specimen in UC that we sequenced to confirm its identity. Crusticorallina species are very difficult to distinguish using morpho‐anatomical and/or habitat characters, although at specific sites, some species may be distinguished by a combination of morpho‐anatomy, habitat and biogeography. The Northeast Pacific now boasts six coralline endemic genera, far more than any other region of the world.     

The mammals of northern Melanesia: speciation,ecology, and biogeography

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PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses,cytosolic survival,and virulence in Listeria monocytogenes

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a ΔprkA mutant to nearly wild-type levels. While a ΔprkA mutant is defective for PG synthesis during cell wall stress, a double ΔreoM ΔprkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a ΔprkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.     

A framework for exhaustively mapping functional missense variants

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here, we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin‐like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.