Frequent Homologous Recombination Research Articles

PARP inhibitor boost the efficacy of photothermal therapy to TNBC through enhanced DNA damage and inhibited homologous recombination repair.

Triple-negative breast cancer (TNBC) could benefit from PARP inhibitors (PARPi) for their frequent defective homologous recombination repair (HR). However, the efficacy of PARPi is limited by their lower bioavailability and high susceptibility to drug resistance, so it often needs to be combined with other treatments. Herein, polydopamine nanoparticles (PDMN) were constructed to load Olaparib (AZD) as two-channel therapeutic nanoplatforms. The PDMN has a homogeneous spherical structure around 100nm and exhibits a good photothermal conversion efficiency of 62.4%. The obtained AZD-loaded nanoplatform (PDMN-AZD) showed enhanced antitumor effects through the combination of photothermal therapy (PTT) and PARPi. By western blot and flow cytometry, we found that PTT and PARPi could exert synergistic antitumor effects by further increasing DNA double-strand damage (DSBs) and enhancing HR defects. The strongest therapeutic effect of PDMN-AZD was observed in a BRCA-deficient mouse tumor model. In conclusion, the PDMN-AZD nanoplatform designed in this study demonstrated the effectiveness of PTT and PARPi for synergistic treatment of TNBC and preliminarily explained the mechanism.

Genome Editing of Plant Mitochondrial and Chloroplast Genomes.

Plastids (including chloroplasts) and mitochondria are remnants of endosymbiotic bacteria, yet they maintain their own genomes, which encode vital components for photosynthesis and respiration, respectively. Organellar genomes have distinctive features, such as being present as multicopies, being mostly inherited maternally, having characteristic genomic structures and undergoing frequent homologous recombination. To date, it has proven to be challenging to modify these genomes. For example, while CRISPR/Cas9 is a widely used system for editing nuclear genes, it has not yet been successfully applied to organellar genomes. Recently, however, precise gene-editing technologies have been successfully applied to organellar genomes. Protein-based enzymes, especially transcription activator-like effector nucleases (TALENs) and artificial enzymes utilizing DNA-binding domains of TALENs (TALEs), have been successfully used to modify these genomes by harnessing organellar-targeting signals. This short review introduces and discusses the use of targeted nucleases and base editors in organellar genomes, their effects and their potential applications in plant science and breeding.

Primary azoospermia factor C duplication associated with spermatogenic impariment: a case-control study based on Y-chromosome haplogrouping in a Han Chinese population.

Azoospermia factor C (AZFc) in the male-specific region of Y-chromosome (MSY) presents wide structure variation mainly due to frequent non-allele homologous recombination, leading to significant copy number variation of the AZFc-linked coding sequences involving in spermatogenesis. A large number of studies had been conducted to investigate the association between AZFc deletions and male infertility in certain Y chromosome genetic backgrounds, however, the influence ofprimary AZFc duplication on spermatogenesis remained controversial and the cause of the discrepant outcomes is unknown. In the present study, a total of 1,102 unrelated Han Chinese males without any detectable AZF deletions were recruited from 2014 to 2019, including 411 controls with normozoospermia and 691 patients with idiopathic spermatogenic failure. Using multiple paralog ratio tests (PRTs), the structure duplications were classified by the copy number of the AZFc-linked amplicons and genes. The Y-chromosome haplogroup (Y-hg) was categorized by genetyping of MSY-linked polymorphism loci. The association of primary AZFc duplication with spermatogenic phenotype was investigated in males with the same Y-hg. Within Y-hg O3* group, the frequency of the gr/gr duplication in patients is significantly higher than that of controls (P=1.29×10-3 , odds ratio (OR) 7.64, 95% confidence interval (CI) 1.79-32.57). Moreover, Y-hg O3* males with the gr/gr duplication presented a significantly lower sperm production compared with non-AZFc duplicated ones (sperm concentration: P=1.46×10-3 ; total sperm count: P=1.82 ×10-3 ). The b2/b3 duplication were identified clustered in Y-hg Cα2*, and the significant difference in the distribution was not observed between patients with spermatogenic failure and controls. The results suggest that, in the Han Chinese population, the gr/gr duplication is a predisposinggenetic factor for spermatogenic impairment in males harboring Y-hg O3* . Meanwhile, the b2/b3 duplication may be fixed ona yet-unidentified subbranch of Y-hg Cα2* without significantly deleterious effect on spermatogenesis. Our findings provide evidence that the difference in the Y-hg composition may cause the discrepancy on the association of AZFc duplication with spermatogenic failure among the studied populations.

Clinical Implications of Chromosome 16 Copy Number Variation

Chromosome 16 is one of the gene-rich chromosomes; however, approximately 10% of the chromosome 16 sequence is composed of segmental copies, which renders this chromosome instable and predisposes it to rearrangements via frequent nonallelic homologous recombination. Microarray technologies have enabled the analysis of copy number variations (CNV), which may be associated with the risk of developing complex diseases. Through comparative genomic hybridisation in 1,298 patients, we detected 18 cases with chromosome 16 CNV. We identified 2recurrent CNV regions, including 1 at 16p13.11 in 4 patients and another at 16p11.2 in 7 patients. We also detected atypical chromosome 16 rearrangements in 7 patients. Furthermore, we noted an increased frequency of co-occurring genomic changes, supporting the two-hit hypothesis to explain the phenotypic variability in the clinical presentation of CNV syndromes. Our findings can contribute to the creation of a chromosome 16 disease map based on regions that may be associated with disease development.

Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis.

Plant mitochondrial genomes undergo frequent homologous recombination (HR). Ectopic HR activity is inhibited by the HR surveillance pathway, but the underlying regulatory mechanism is unclear. Here, we show that the mitochondrial RNase H1 AtRNH1B impairs the formation of RNA:DNA hybrids (R-loops) and participates in the HR surveillance pathway in Arabidopsis thaliana. AtRNH1B suppresses ectopic HR at intermediate-sized repeats (IRs) and thus maintains mitochondrial DNA (mtDNA) replication. The RNase H1 AtRNH1C is restricted to the chloroplast; however, when cells lack AtRNH1B, transport of chloroplast AtRNH1C into the mitochondria secures HR surveillance, thus ensuring the integrity of the mitochondrial genome and allowing embryogenesis to proceed. HR surveillance is further regulated by the single-stranded DNA-binding protein ORGANELLAR SINGLE-STRANDED DNA BINDING PROTEIN1 (OSB1), which decreases the formation of R-loops. This study uncovers a facultative dual targeting mechanism between organelles and sheds light on the roles of RNase H1 in organellar genome maintenance and embryogenesis.

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana.

CRISPR/Cas systems enable gene editing through the induction of site-specific DNA double-strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non-homologous end joining (NHEJ)-mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T-rich regions. Most recently, we tested an improved, temperature-tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants.

A coarse-graining, ultrametric approach to resolve the phylogeny of prokaryotic strains with frequent homologous recombination

BackgroundA frequent event in the evolution of prokaryotic genomes is homologous recombination, where a foreign DNA stretch replaces a genomic region similar in sequence. Recombination can affect the relative position of two genomes in a phylogenetic reconstruction in two different ways: (i) one genome can recombine with a DNA stretch that is similar to the other genome, thereby reducing their pairwise sequence divergence; (ii) one genome can recombine with a DNA stretch from an outgroup genome, increasing the pairwise divergence. While several recombination-aware phylogenetic algorithms exist, many of these cannot account for both types of recombination; some algorithms can, but do so inefficiently. Moreover, many of them reconstruct the ancestral recombination graph (ARG) to help infer the genome tree, and require that a substantial portion of each genome has not been affected by recombination, a sometimes unrealistic assumption.MethodsHere, we propose a Coarse-Graining approach for Phylogenetic reconstruction (CGP), which is recombination-aware but forgoes ARG reconstruction. It accounts for the tendency of a higher effective recombination rate between genomes with a lower phylogenetic distance. It is applicable even if all genomic regions have experienced substantial amounts of recombination, and can be used on both nucleotide and amino acid sequences. CGP considers the local density of substitutions along pairwise genome alignments, fitting a model to the empirical distribution of substitution density to infer the pairwise coalescent time. Given all pairwise coalescent times, CGP reconstructs an ultrametric tree representing vertical inheritance.ResultsBased on simulations, we show that the proposed approach can reconstruct ultrametric trees with accurate topology, branch lengths, and root positioning. Applied to a set of E. coli strains, the reconstructed trees are most consistent with gene distributions when inferred from amino acid sequences, a data type that cannot be utilized by many alternative approaches.ConclusionsThe CGP algorithm is more accurate than alternative recombination-aware methods for ultrametric phylogenetic reconstructions.

Serotype-specific evolutionary patterns of antimicrobial-resistant Salmonella enterica

BackgroundThe emergence of antimicrobial-resistant (AMR) strains of the important human and animal pathogen Salmonella enterica poses a growing threat to public health. Here, we studied the genome-wide evolution of 90 S. enterica AMR isolates, representing one host adapted serotype (S. Dublin) and two broad host range serotypes (S. Newport and S. Typhimurium).ResultsAMR S. Typhimurium had a large effective population size, a large and diverse genome, AMR profiles with high diversity, and frequent positive selection and homologous recombination. AMR S. Newport showed a relatively low level of diversity and a relatively clonal population structure. AMR S. Dublin showed evidence for a recent population bottleneck, and the genomes were characterized by a larger number of genes and gene ontology terms specifically absent from this serotype and a significantly higher number of pseudogenes as compared to other two serotypes. Approximately 50% of accessory genes, including specific AMR and putative prophage genes, were significantly over- or under-represented in a given serotype. Approximately 65% of the core genes showed phylogenetic clustering by serotype, including the AMR gene aac (6′)-Iaa. While cell surface proteins were shown to be the main target of positive selection, some proteins with possible functions in AMR and virulence also showed evidence for positive selection. Homologous recombination mainly acted on prophage-associated proteins.ConclusionsOur data indicates a strong association between genome content of S. enterica and serotype. Evolutionary patterns observed in S. Typhimurium are consistent with multiple emergence events of AMR strains and/or ecological success of this serotype in different hosts or habitats. Evolutionary patterns of S. Newport suggested that antimicrobial resistance emerged in one single lineage, Lineage IIC. A recent population bottleneck and genome decay observed in AMR S. Dublin are congruent with its narrow host range. Finally, our results suggest the potentially important role of positive selection in the evolution of antimicrobial resistance, host adaptation and serotype diversification in S. enterica.

The whole genome landscape of adult metastatic sarcoma.

3137 Background: Metastatic sarcomas represent a heterogeneous, difficult to treat family of cancers with poor median overall survival of 18 months. While global sequencing initiatives have catalogued genomic variation among primary sarcomas, metastatic sarcoma is less well understood. Genome-guided targeted therapy has made promising advances but is difficult to study in sarcomas due to heterogeneity and low prevalence. Whole genome and transcriptome analysis (WGTA) can help elucidate sarcoma oncogenesis, metastasis, and potential therapeutic targets. Methods: Using whole genome (80X) and transcriptome (200M read) sequencing of 43 metastatic sarcomas across 19 subtypes, we analyzed structural variants (SV), copy-number variants (CNV), mutation signatures, gene expression, and the immune microenvironment. All prior treatments were retrieved through chart review. Results: 17 patients (40%) attempted WGTA-informed therapy, of which 8 (47%) were classified as responders. Metastatic sarcomas demonstrated recurrent CNVs, with 17p11-p12 amplification in 42% of cases. Some recurrent expression outliers were associated with potential targets (e.g. MYOCD, PMP22, COPS3) while others (e.g. ADORA2B) have not been previously observed in sarcoma. Discovery of oncogenic fusions refined diagnoses in two cases with atypical histology. Clustering by mutation signatures distinguished histological subtypes, and two signatures were novel in sarcoma: (1) a strong base excision repair signature associated with NTHL1 loss and (2) a cisplatin-associated signature exclusive to platinum-treated cases. Frequent homologous recombination deficiency was observed and was associated with response to ifosfamide in three leiomyosarcomas. Of four immunotherapy-treated cases, the only responder demonstrated outlier CIBERSORT immune infiltration score, which did not correlate with PD-L1 expression. Conclusions: This is the first in-depth WGTA of metastatic sarcoma. We found recurrent and potentially targetable CNVs, expression outliers, mutation signatures, and immune markers. Our results suggest that clinical translation is promising using actionable insights obtained through WGTA.

Frequent Homologous Recombination Deficiency in High-grade Endometrial Carcinomas.

The elevated levels of somatic copy-number alterations (SCNAs) in a subset of high-risk endometrial cancers are suggestive of defects in pathways governing genome integrity. We sought to assess the prevalence of homologous recombination deficiency (HRD) in endometrial cancers and its association with histopathologic and molecular characteristics. Fresh tumor tissue was prospectively collected from 36 endometrial cancers, and functional HRD was examined by the ability of replicating tumor cells to accumulate RAD51 protein at DNA double-strand breaks (RAD51 foci) induced by ionizing radiation. Genomic alterations were determined by next-generation sequencing and array comparative genomic hybridization/SNP array. The prevalence of BRCA-associated genomic scars, a surrogate marker for HRD, was determined in the The Cancer Genome Atlas (TCGA) endometrial cancer cohort. Most endometrial cancers included in the final analysis (n = 25) were of non-endometrioid (52%), grade 3 (60%) histology, and FIGO stage I (72%). HRD was observed in 24% (n = 6) of cases and was restricted to non-endometrioid endometrial cancers (NEEC), with 46% of NEECs being HRD compared with none of the endometrioid endometrial cancers (EEC, P = 0.014). All but 1 of the HRD cases harbored either a pathogenic BRCA1 variant or high somatic copy-number (SCN) losses of HR genes. Analysis of TCGA cases supported these results, with BRCA-associated genomic scars present in up to 48% (63/132) of NEEC versus 12% (37/312) of EEC (P < 0.001). HRD occurs in endometrial cancers and is largely restricted to non-endometrioid, TP53-mutant endometrial cancers. Evaluation of HRD may help select patients that could benefit from treatments targeting this defect, including platinum compounds and PARP inhibitors.

Inherited DNA-Repair Defects in Colorectal Cancer

Colorectal cancer (CRC) heritability has been estimated to be around 30%. However, mutations in the known CRC-susceptibility genes explain CRC risk in fewer than 10% of affected individuals. Germline mutations in DNA-repair genes (DRGs) have recently been reported in CRC, but their contribution to CRC risk is largely unknown. We evaluated the gene-level germline mutation enrichment of 40 DRGs in 680 unselected CRC individuals and 27,728 ancestry-matched cancer-free adults. Significant findings were then examined in independent cohorts of 1,661 unselected CRC individuals and 1,456 individuals with early-onset CRC. Of the 680 individuals in the discovery set, 31 (4.56%) individuals harbored germline pathogenic mutations in known CRC-susceptibility genes, and another 33 (4.85%) individuals had DRG mutations that have not been previously associated with CRC risk. Germline pathogenic mutations in ATM and PALB2 were enriched in both the discovery (OR = 2.81 and p = 0.035 for ATM and OR = 4.91 and p = 0.024 for PALB2) and validation (OR = 2.97 and adjusted p = 0.0013 for ATM and OR = 3.42 and adjusted p = 0.034 for PALB2) sets. Biallelic loss of ATM was evident in all individuals with matched tumor profiling. CRC individuals also had higher rates of actionable mutations in the HR pathway, which can substantially increase the risk of developing cancers other than CRC. Our analysis provides evidence for ATM and PALB2 as CRC-risk genes, underscoring the importance of the homologous recombination pathway in CRC. In addition, we identified frequent complete homologous recombination deficiency in CRC tumors, representing a unique opportunity to explore targeted therapeutic interventions such as poly-ADP ribose polymerase inhibitor (PARPi).

Pold3 is required for genomic stability and telomere integrity in embryonic stem cells and meiosis.

Embryonic stem cells (ESCs) and meiosis are featured by relatively higher frequent homologous recombination associated with DNA double strand breaks (DSB) repair. Here, we show that Pold3 plays important roles in DSB repair, telomere maintenance and genomic stability of both ESCs and spermatocytes in mice. By attempting to generate Pold3 deficient mice using CRISPR/Cas9 or transcription activator-like effector nucleases, we show that complete loss of Pold3 (Pold3−/−) resulted in early embryonic lethality at E6.5. Rapid DNA damage response and massive apoptosis occurred in both outgrowths of Pold3-null (Pold3−/−) blastocysts and Pold3 inducible knockout (iKO) ESCs. While Pold3−/− ESCs were not achievable, Pold3 iKO led to increased DNA damage response, telomere loss and chromosome breaks accompanied by extended S phase. Meanwhile, loss of Pold3 resulted in replicative stress, micronucleation and aneuploidy. Also, DNA repair was impaired in Pold3+/− or Pold3 knockdown ESCs. Moreover, Pold3 mediates DNA replication and repair by regulating 53BP1, RIF1, ATR and ATM pathways. Furthermore, spermatocytes of Pold3 haploinsufficient (Pold3+/−) mice with increasing age displayed impaired DSB repair, telomere shortening and loss, and chromosome breaks, like Pold3 iKO ESCs. These data suggest that Pold3 maintains telomere integrity and genomic stability of both ESCs and meiosis by suppressing replicative stress.

PGAP-X: extension on pan-genome analysis pipeline

BackgroundSince PGAP (pan-genome analysis pipeline) was published in 2012, it has been widely employed in bacterial genomics research. Though PGAP has integrated several modules for pan-genomics analysis, how to properly and effectively interpret and visualize the results data is still a challenge.ResultTo well present bacterial genomic characteristics, a novel cross-platform software was developed, named PGAP-X. Four kinds of data analysis modules were developed and integrated: whole genome sequences alignment, orthologous genes clustering, pan-genome profile analysis, and genetic variants analysis. The results from these analyses can be directly visualized in PGAP-X. The modules for data visualization in PGAP-X include: comparison of genome structure, gene distribution by conservation, pan-genome profile curve and variation on genic and genomic region. Meanwhile, result data produced by other programs with similar function can be imported to be further analyzed and visualized in PGAP-X. To test the performance of PGAP-X, we comprehensively analyzed 14 Streptococcus pneumonia strains and 14 Chlamydia trachomatis. The results show that, S. pneumonia strains have higher diversity on genome structure and gene contents than C. trachomatis strains. In addition, S. pneumonia strains might have suffered many evolutionary events, such genomic rearrangements, frequent horizontal gene transfer, homologous recombination, and other evolutionary process.ConclusionBriefly, PGAP-X directly presents the characteristics of bacterial genomic diversity with different visualization methods, which could help us to intuitively understand dynamics and evolution in bacterial genomes. The source code and the pre-complied executable programs are freely available from http://pgapx.ybzhao.com.

The chromosomal organization of horizontal gene transfer in bacteria

Bacterial adaptation is accelerated by the acquisition of novel traits through horizontal gene transfer, but the integration of these genes affects genome organization. We found that transferred genes are concentrated in only ~1% of the chromosomal regions (hotspots) in 80 bacterial species. This concentration increases with genome size and with the rate of transfer. Hotspots diversify by rapid gene turnover; their chromosomal distribution depends on local contexts (neighboring core genes), and content in mobile genetic elements. Hotspots concentrate most changes in gene repertoires, reduce the trade-off between genome diversification and organization, and should be treasure troves of strain-specific adaptive genes. Most mobile genetic elements and antibiotic resistance genes are in hotspots, but many hotspots lack recognizable mobile genetic elements and exhibit frequent homologous recombination at flanking core genes. Overrepresentation of hotspots with fewer mobile genetic elements in naturally transformable bacteria suggests that homologous recombination and horizontal gene transfer are tightly linked in genome evolution.

Sequential displacement of Type VI Secretion System effector genes leads to evolution of diverse immunity gene arrays in Vibrio cholerae

Type VI secretion systems (T6SS) enable bacteria to engage neighboring cells in contact-dependent competition. In Vibrio cholerae, three chromosomal clusters each encode a pair of effector and immunity genes downstream of those encoding the T6SS structural machinery for effector delivery. Different combinations of effector-immunity proteins lead to competition between strains of V. cholerae, which are thought to be protected only from the toxicity of their own effectors. Screening of all publically available V. cholerae genomes showed that numerous strains possess long arrays of orphan immunity genes encoded in the 3′ region of their T6SS clusters. Phylogenetic analysis reveals that these genes are highly similar to those found in the effector-immunity pairs of other strains, indicating acquisition by horizontal gene transfer. Extensive genomic comparisons also suggest that successive addition of effector-immunity gene pairs replaces ancestral effectors, yet retains the cognate immunity genes. The retention of old immunity genes perhaps provides protection against nearby kin bacteria in which the old effector was not replaced. This mechanism, combined with frequent homologous recombination, is likely responsible for the high diversity of T6SS effector-immunity gene profiles observed for V. cholerae and closely related species.

A New Metazoan Recombination Rate Record and Consistently High Recombination Rates in the Honey Bee Genus Apis Accompanied by Frequent Inversions but Not Translocations.

Western honey bees (Apis mellifera) far exceed the commonly observed 1–2 meiotic recombination events per chromosome and exhibit the highest Metazoan recombination rate (20 cM/Mb) described thus far. However, the reasons for this exceptional rate of recombination are not sufficiently understood. In a comparative study, we report on the newly constructed genomic linkage maps of Apis florea and Apis dorsata that represent the two honey bee lineages without recombination rate estimates so far. Each linkage map was generated de novo, based on SNP genotypes of haploid male offspring of a single female. The A. florea map spans 4,782 cM with 1,279 markers in 16 linkage groups. The A. dorsata map is 5,762 cM long and contains 1,189 markers in 16 linkage groups. Respectively, these map sizes result in average recombination rate estimates of 20.8 and 25.1 cM/Mb. Synteny analyses indicate that frequent intra-chromosomal rearrangements but no translocations among chromosomes accompany the high rates of recombination during the independent evolution of the three major honey bee lineages. Our results imply a common cause for the evolution of very high recombination rates in Apis. Our findings also suggest that frequent homologous recombination during meiosis might increase ectopic recombination and rearrangements within but not between chromosomes. It remains to be investigated whether the resulting inversions may have been important in the evolutionary differentiation between honey bee species.

Widespread interspecies homologous recombination reveals reticulate evolution within the genus Streptomyces

Homologous recombination is increasingly being recognized as a driving force in microbial evolution. However, recombination in streptomycetes, a rich source of diverse secondary metabolites, particularly among different species, remains minimally investigated. In this study, the largest sample of Streptomyces species to date, consisting of 142 type strains spanning the genus, with available sequences of 16S rRNA, atpD, gyrB, recA, rpoB and trpB genes, were collected and subjected to a comprehensive population genetic analysis to generate an overall estimate of the level of Streptomyces interspecies genetic exchange and its effect on the evolution of this genus. The results indicate frequent homologous recombination among Streptomyces species, which occurred three times more frequently and was nearly 14 times more important than point mutation in nucleotide sequence divergence (ρ/θw=3.10, r/m=13.74). As a result, a facilitating effect on the evolutionary process and confusion in phylogenetic relationships were observed, as well as a number of specific transfer events of the six gene fragments. A resultant phylogenetic network depicted extensive horizontal genetic exchange which decays clonality in streptomycetes. Moreover, seven evolutionary lineage groups were identified in the present sample in the Structure analysis, generally consistent with morphological and physiological data, and the contribution of recombination was detected to be varied among them. Our analyses demonstrated a reticulate evolution within Streptomyces due to the high level of interspecies gene exchange, which greatly challenges the traditional tree-shaped phylogeny in this genus and may advance our evolutionary understanding of a genuine Streptomyces species.

Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation.

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating - a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility.

Islands of linkage in an ocean of pervasive recombination reveals two-speed evolution of human cytomegalovirus genomes.

Human cytomegalovirus (HCMV) infects most of the population worldwide, persisting throughout the host's life in a latent state with periodic episodes of reactivation. While typically asymptomatic, HCMV can cause fatal disease among congenitally infected infants and immunocompromised patients. These clinical issues are compounded by the emergence of antiviral resistance and the absence of an effective vaccine, the development of which is likely complicated by the numerous immune evasins encoded by HCMV to counter the host's adaptive immune responses, a feature that facilitates frequent super-infections. Understanding the evolutionary dynamics of HCMV is essential for the development of effective new drugs and vaccines. By comparing viral genomes from uncultivated or low-passaged clinical samples of diverse origins, we observe evidence of frequent homologous recombination events, both recent and ancient, and no structure of HCMV genetic diversity at the whole-genome scale. Analysis of individual gene-scale loci reveals a striking dichotomy: while most of the genome is highly conserved, recombines essentially freely and has evolved under purifying selection, 21 genes display extreme diversity, structured into distinct genotypes that do not recombine with each other. Most of these hyper-variable genes encode glycoproteins involved in cell entry or escape of host immunity. Evidence that half of them have diverged through episodes of intense positive selection suggests that rapid evolution of hyper-variable loci is likely driven by interactions with host immunity. It appears that this process is enabled by recombination unlinking hyper-variable loci from strongly constrained neighboring sites. It is conceivable that viral mechanisms facilitating super-infection have evolved to promote recombination between diverged genotypes, allowing the virus to continuously diversify at key loci to escape immune detection, while maintaining a genome optimally adapted to its asymptomatic infectious lifecycle.

Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice.

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression—not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.