Genetic Crossovers Are Predicted Accurately by the Computed Human Recombination Map

Hotspots of meiotic recombination can change rapidly over time. This instability and the reported high level of inter-individual variation in meiotic recombination puts in question the accuracy of the calculated hotspot map, which is based on the summation of past genetic crossovers. To estimate the accuracy of the computed recombination rate map, we have mapped genetic crossovers to a median resolution of 70 Kb in 10 CEPH pedigrees. We then compared the positions of crossovers with the hotspots computed from HapMap data and performed extensive computer simulations to compare the observed distributions of crossovers with the distributions expected from the calculated recombination rate maps. Here we show that a population-averaged hotspot map computed from linkage disequilibrium data predicts well present-day genetic crossovers. We find that computed hotspot maps accurately estimate both the strength and the position of meiotic hotspots. An in-depth examination of not-predicted crossovers shows that they are preferentially located in regions where hotspots are found in other populations. In summary, we find that by combining several computed population-specific maps we can capture the variation in individual hotspots to generate a hotspot map that can predict almost all present-day genetic crossovers

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA.

International audienceMeiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method--single-stranded DNA (ssDNA) sequencing (SSDS)--that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired

Long terminal repeats of human endogenous retrovirus K family (HERV-K) specifically bind host cell nuclear proteins

AbstractSolitary long terminal repeats (LTRs) of the human endogenous retroviruses, scattered in several thousand copies throughout the human genome, are potentially capable of affecting the expression of closely located genes. To assess their regulatory potential, the LTR sequences of one of the most abundant HERV families (HERV-K) were screened for the presence of binding sites for the host cell nuclear factors using mobility shift and UV-crosslinking assays. It was shown that the LTR sequences of two subfamilies harbor a specific binding site for a complex consisting of at least three proteins, ERF1, ERF2 and ERF3 of 98, 91 and 88 kDa apparent molecular mass, respectively. This binding site is located in the 5′ region of the LTR U3 element. The preservation of the specific protein binding site in different HERV-K LTR sequences suggests their possible role in regulation of nearby located genes

Hypermutator strains of Pseudomonas aeruginosa reveal novel pathways of resistance to combinations of cephalosporin antibiotics and beta-lactamase inhibitors.

Hypermutation due to DNA mismatch repair (MMR) deficiencies can accelerate the development of antibiotic resistance in Pseudomonas aeruginosa. Whether hypermutators generate resistance through predominantly similar molecular mechanisms to wild-type (WT) strains is not fully understood. Here, we show that MMR-deficient P. aeruginosa can evolve resistance to important broad-spectrum cephalosporin/beta-lactamase inhibitor combination antibiotics through novel mechanisms not commonly observed in WT lineages. Using whole-genome sequencing (WGS) and transcriptional profiling of isolates that underwent in vitro adaptation to ceftazidime/avibactam (CZA), we characterized the detailed sequence of mutational and transcriptional changes underlying the development of resistance. Surprisingly, MMR-deficient lineages rapidly developed high-level resistance (>256 μg/mL) largely without corresponding fixed mutations or transcriptional changes in well-established resistance genes. Further investigation revealed that these isolates had paradoxically generated an early inactivating mutation in the mexB gene of the MexAB-OprM efflux pump, a primary mediator of CZA resistance in P. aeruginosa, potentially driving an evolutionary search for alternative resistance mechanisms. In addition to alterations in a number of genes not known to be associated with resistance, 2 mutations were observed in the operon encoding the RND efflux pump MexVW. These mutations resulted in a 4- to 6-fold increase in resistance to ceftazidime, CZA, cefepime, and ceftolozane-tazobactam when engineered into a WT strain, demonstrating a potentially important and previously unappreciated mechanism of resistance to these antibiotics in P. aeruginosa. Our results suggest that MMR-deficient isolates may rapidly evolve novel resistance mechanisms, sometimes with complex dynamics that reflect gene inactivation that occurs with hypermutation. The apparent ease with which hypermutators may switch to alternative resistance mechanisms for which antibiotics have not been developed may carry important clinical implications

Shared DEGs in MPAO1-<i>mutS</i>^<i>Tn</i> lineages following CZA selection.

Number of DEGs shared by the 3 MPAO1-mutSTn lineages are shown, either up-regulated (left, LFC1, p-adj S2 Data. (EPS)</p

Dissimilar transcriptome profiles highlight lack of involvement of major cephalosporin resistance genes in CZA resistance in MMR-deficient lineages and demonstrate MexVW overexpression.

Three lineages of MPAO1-WT and MPAO1-mutSTn were selected for transcriptomic analysis and 3 biological replicates from 1 isolate per passage underwent RNA sequencing. (A) A clustered heatmap of the top 750 most variable genes across CZA-resistant isolates is shown. Rlog normalized levels of expression of each gene (rows) were scaled within each row. Isolates (columns) are grouped by lineage and arranged by genotype with WT on the left and hypermutators on the right. Within each lineage, isolates are arranged from left to right in order of increasing number of passages under CZA selection, with the corresponding MIC indicated at the bottom. 1X and 2X represent the MPAO1-WT and MPAO1-mutSTn parental strains of the respective lineages. (B) LFC with respect to their corresponding ancestor of the terminal MPAO1-WT isolates (first 3 columns) and CZA-adapted MPAO1-mutSTn isolates (last 9 columns). The rows represent 6 major beta-lactam resistance genes in P. aeruginosa from 4 major resistance pathways (encoding the MexAB-OprM efflux pump, OprD outer membrane porin, PDC beta-lactamase, and penicillin-binding-protein 3 FtsI). Data for 2 differentially expressed RND efflux pumps in the MPAO1-mutSTn lineages (MexVW and MexGHI-OpmD) are additionally shown. (C) Expression levels of RND efflux pumps and outer membrane porins in MPAO1-mutSTn lineages during the course of the CZA adaptation experiment. The vertical axis represents the LFC of expression levels compared to the early isolates, and the horizontal axis shows the passage number. CZA MIC is indicated with color tiles under the axis. Lines are colored and labeled if the respective gene reached a LFC > 1.0 in the terminal isolates of the lineage. The underlying data to generate this figure can be found in S2 Data. CZA, ceftazidime/avibactam; MMR, mismatch repair; WT, wild type.</p

Colonies observed inside the zones of inhibition in disk diffusion susceptibility tests in MPAO1-<i>mutS</i>^Tn isolates.

Representative photos of Kirby–Bauer susceptibility testing with ATM, IPM, MEM, and piperacillin-tazobactam (TZP) in the MPAO1-WT ancestor (1X), MPAO1-mutSTn ancestor (2X), and 2 CZA-resistant MPAO1-mutSTn isolates (lineage 2A passage 12 and lineage 2B passage 7). Pictures display readings at both 16 h, as recommended per CLSI guidelines, as well as at 24 h of incubation at 37°C. Scattered colonies can be observed inside some zones of inhibition at 16 h, particularly in the MPAO1-mutSTn isolates and more markedly in the evolved CZA-resistant isolates. ATM, aztreonam; IPM, imipenem; MEM, meropenem; TZP, piperacillin-tazobactam. (PNG)</p

Consensus sequences and nucleotide and amino acid frequency tables in proximity to PA4373-mexV (4903384 T>C) and MexW E36K variants in 7,493 genomes from the NCBI pathogen database.

Orthologous regions neighboring 2 mutations in the MexVW operon were identified in a set of 7,493 P. aeruginosa genomes from the NCBI Pathogen Detection database. (a) Summary of the nucleotide sequence alignment in proximity to PA4373-mexV +/- 10 bases. (b) Amino acid sequence surrounding MexW E36K with the corresponding residue highlighted in orange background. (DOCX)</p