Successive Rearrangements Research Articles

Notch and the pre-TCR coordinate thymocyte proliferation by induction of the SCF subunits Fbxl1 and Fbxl12.

Proliferation is tightly regulated during T cell development and is limited to immature CD4−CD8− thymocytes. The major proliferative event is initiated at the ‘β-selection’ stage following successful rearrangement of Tcrβ and is triggered by and dependent on concurrent signaling by Notch and the pre-TCR; however, it is unclear how these signals cooperate to promote cell proliferation. Here we found that β-selection-associated proliferation required the combined activity of two SCF ubiquitin ligase complexes that included as substrate recognition subunits the F-box proteins Fbxl1 or Fbxl12. Both SCF complexes targeted the cyclin-dependent kinase inhibitor Cdkn1b for ubiquitinylaton and degradation. We found that Notch signals induced the transcription of Fbxl1 whereas pre-TCR signals induced the transcription of Fbxl12. Thus, concurrent Notch and pre-TCR signaling induced the expression of two genes, Fbxl1 and Fbxl12, whose products functioned identically but additively to promote degradation of Cdkn1b, cell cycle progression, and proliferation of β-selected thymocytes.

ISWI ATPase Smarca5 Regulates Differentiation of Thymocytes Undergoing β-Selection.

Development of lymphoid progenitors requires a coordinated regulation of gene expression, DNA replication, and gene rearrangement. Chromatin-remodeling activities directed by SWI/SNF2 superfamily complexes play important roles in these processes. In this study, we used a conditional knockout mouse model to investigate the role of Smarca5, a member of the ISWI subfamily of such complexes, in early lymphocyte development. Smarca5 deficiency results in a developmental block at the DN3 stage of αβ thymocytes and pro-B stage of early B cells at which the rearrangement of Ag receptor loci occurs. It also disturbs the development of committed (CD73+) γδ thymocytes. The αβ thymocyte block is accompanied by massive apoptotic depletion of β-selected double-negative DN3 cells and premitotic arrest of CD4/CD8 double-positive cells. Although Smarca5-deficient αβ T cell precursors that survived apoptosis were able to undergo a successful TCRβ rearrangement, they exhibited a highly abnormal mRNA profile, including the persistent expression of CD44 and CD25 markers characteristic of immature cells. We also observed that the p53 pathway became activated in these cells and that a deficiency of p53 partially rescued the defect in thymus cellularity (in contrast to early B cells) of Smarca5-deficient mice. However, the activation of p53 was not primarily responsible for the thymocyte developmental defects observed in the Smarca5 mutants. Our results indicate that Smarca5 plays a key role in the development of thymocytes undergoing β-selection, γδ thymocytes, and also B cell progenitors by regulating the transcription of early differentiation programs.

The transcription factor Duxbl mediates elimination of pre-T cells that fail β-selection.

T cell development is critically dependent on successful rearrangement of antigen-receptor chains. At the β-selection checkpoint, only cells with a functional rearrangement continue in development. However, how nonselected T cells proceed in their dead-end fate is not clear. We identified low CD27 expression to mark pre-T cells that have failed to rearrange their β-chain. Expression profiling and single-cell transcriptome clustering identified a developmental trajectory through β-selection and revealed specific expression of the transcription factor Duxbl at a stage of high recombination activity before β-selection. Conditional transgenic expression of Duxbl resulted in a developmental block at the DN3-to-DN4 transition due to reduced proliferation and enhanced apoptosis, whereas RNA silencing of Duxbl led to a decrease in apoptosis. Transcriptome analysis linked Duxbl to elevated expression of the apoptosis-inducing Oas/RNaseL pathway. RNaseL deficiency or sustained Bcl2 expression led to a partial rescue of cells in Duxbl transgenic mice. These findings identify Duxbl as a regulator of β-selection by inducing apoptosis in cells with a nonfunctional rearrangement.

Visible-Light Photoredox Catalysis Enables the Biomimetic Synthesis of Nyingchinoids A, B, and D, and Rasumatranin D.

The total synthesis of nyingchinoids A and B has been achieved through successive rearrangements of a 1,2-dioxane intermediate that was assembled using a visible-light photoredox-catalysed aerobic [2+2+2] cycloaddition. Nyingchinoid D was synthesised with a competing [2+2] cycloaddition. Based on NMR data and biosynthetic speculation, we proposed a structure revision of the related natural product rasumatranin D, which was confirmed through total synthesis. Under photoredox conditions, we observed the conversion of a cyclobutane into a 1,2-dioxane through retro-[2+2] cycloaddition followed by aerobic [2+2+2] cycloaddition.

Visible‐Light Photoredox Catalysis Enables the Biomimetic Synthesis of Nyingchinoids A, B, and D, and Rasumatranin D

AbstractThe total synthesis of nyingchinoids A and B has been achieved through successive rearrangements of a 1,2‐dioxane intermediate that was assembled using a visible‐light photoredox‐catalysed aerobic [2+2+2] cycloaddition. Nyingchinoid D was synthesised with a competing [2+2] cycloaddition. Based on NMR data and biosynthetic speculation, we proposed a structure revision of the related natural product rasumatranin D, which was confirmed through total synthesis. Under photoredox conditions, we observed the conversion of a cyclobutane into a 1,2‐dioxane through retro‐[2+2] cycloaddition followed by aerobic [2+2+2] cycloaddition.

Peroxisome Proliferator-Activated Receptor–δ Supports the Metabolic Requirements of Cell Growth in TCRβ-Selected Thymocytes and Peripheral CD4+ T Cells

During T cell development, progenitor thymocytes undergo a large proliferative burst immediately following successful TCRβ rearrangement, and defects in genes that regulate this proliferation have a profound effect on thymus cellularity and output. Although the signaling pathways that initiate cell cycling and nutrient uptake after TCRβ selection are understood, less is known about the transcriptional programs that regulate the metabolic machinery to promote biomass accumulation during this process. In this article, we report that mice with whole body deficiency in the nuclear receptor peroxisome proliferator-activated receptor-δ (PPARδmut) exhibit a reduction in spleen and thymus cellularity, with a decrease in thymocyte cell number starting at the double-negative 4 stage of thymocyte development. Although in vivo DNA synthesis was normal in PPARδmut thymocytes, studies in the OP9-delta-like 4 in vitro system of differentiation revealed that PPARδmut double-negative 3 cells underwent fewer cell divisions. Naive CD4+ T cells from PPARδmut mice also exhibited reduced proliferation upon TCR and CD28 stimulation in vitro. Growth defects in PPAR-δ-deficient thymocytes and peripheral CD4+ T cells correlated with decreases in extracellular acidification rate, mitochondrial reserve, and expression of a host of genes involved in glycolysis, oxidative phosphorylation, and lipogenesis. By contrast, mice with T cell-restricted deficiency of Ppard starting at the double-positive stage of thymocyte development, although exhibiting defective CD4+ T cell growth, possessed a normal T cell compartment, pointing to developmental defects as a cause of peripheral T cell lymphopenia in PPARδmut mice. These findings implicate PPAR-δ as a regulator of the metabolic program during thymocyte and T cell growth.

SOX4 controls invariant NKT cell differentiation by tuning TCR signaling.

Natural killer T (NKT) cells expressing the invariant T cell receptor (iTCR) serve an essential function in clearance of certain pathogens and have been implicated in autoimmune and allergic diseases. Complex effector programs of these iNKT cells are wired in the thymus, and upon thymic egress, they can respond within hours of antigenic challenges, classifying iNKT cells as innate-like. It has been assumed that the successful rearrangement of the invariant iTCRα chain is the central event in the divergence of immature thymocytes to the NKT cell lineage, but molecular properties that render the iTCR signaling distinct to permit the T cell lineage diversification remain obscure. Here we show that the High Mobility Group (HMG) transcription factor (TF) SOX4 controls the production of iNKT cells by inducing MicroRNA-181 (Mir181) to enhance TCR signaling and Ca2+ fluxes in precursors. These results suggest the existence of tailored, permissive gene circuits in iNKT precursors for innate-like T cell development.

Successive Domain Rearrangements Underlie the Evolution of a Regulatory Module Controlled by a Small Interfering Peptide.

The establishment of new interactions between transcriptional regulators increases the regulatory diversity that drives phenotypic novelty. To understand how such interactions evolve, we have studied a regulatory module (DDR) composed by three MYB-like proteins: DIVARICATA (DIV), RADIALIS (RAD), and DIV-and-RAD-Interacting Factor (DRIF). The DIV and DRIF proteins form a transcriptional complex that is disrupted in the presence of RAD, a small interfering peptide, due to the formation of RAD–DRIF dimers. This dynamic interaction result in a molecular switch mechanism responsible for the control of distinct developmental processes in plants. Here, we have determined how the DDR regulatory module was established by analyzing the origin and evolution of the DIV, DRIF, and RAD protein families and the evolutionary history of their interactions. We show that duplications of a pre-existing MYB domain originated the DIV and DRIF protein families in the ancestral lineage of green algae, and, later, the RAD family in seed plants. Intraspecies interactions between the MYB domains of DIV and DRIF proteins are detected in green algae, whereas the earliest evidence of an interaction between DRIF and RAD proteins occurs in the gymnosperms, coincident with the establishment of the RAD family. Therefore, the DDR module evolved in a stepwise progression with the DIV–DRIF transcription complex evolving prior to the antagonistic RAD–DRIF interaction that established the molecular switch mechanism. Our results suggest that the successive rearrangement and divergence of a single protein domain can be an effective evolutionary mechanism driving new protein interactions and the establishment of novel regulatory modules.

Rebuilding C60: Chlorination-Promoted Transformations of the Buckminsterfullerene into Pentagon-Fused C60 Derivatives.

In recent years, many higher fullerenes that obey the isolated pentagon rule (IPR) were found capable of rearranging into molecules with adjacent pentagons and even with heptagons via chlorination-promoted skeletal transformations. However, the key fullerene, buckminsterfullerene I h-C60, long seemed insusceptible to such rearrangements. Now we demonstrate that buckminsterfullerene yet can be transformed by chlorination with SbCl5 at 420-440 °C and report X-ray structures for the thus-obtained library of non-IPR derivatives. The most remarkable of them are non-IPR C60Cl24 and C60Cl20 with fundamentally rearranged carbon skeletons featuring, respectively, four and five fused pentagon pairs (FPPs). Further high-temperature trifluoromethylation of the chlorinated mixture afforded additional non-IPR derivatives C60(CF3)10 and C60(CF3)14, both with two FPPs, and a nonclassical C60(CF3)15F with a heptagon, two FPPs, and a fully fused pentagon triple. We discuss the general features of the addition patterns in the new non-IPR compounds and probable pathways of their formation via successive Stone-Wales rearrangements.

Impaired serial ordering in nondemented patients with mild Parkinson's disease.

The ability to arrange thoughts and actions in an appropriate serial order (the problem of serial order) is essential to complex behaviors such as language, reasoning and cognitive planning. Patients with Parkinson’s disease (PD) perform poorly in tasks that rely on the successful rearrangement of working memory representations. We hypothesized that serial ordering is impaired in nondemented patients with mild PD. We recruited 49 patients with mild idiopathic PD (Hoehn and Yahr Scale 1–2.5) and 51 matched healthy adults. Nineteen patients had normal global cognition (PD-NC, Montreal Cognitive Assessment, MoCA≥26/30) and thirty patients had mild cognitive impairment (PD-MCI, 21≤MoCA≤25). All participants underwent three working memory assessments: two experimental tests that require reordering random digits following a particular rule (adaptive digit ordering test and digit span backward test) and a control test that requires maintaining but no reordering (digit span forward test). PD-NC and PD-MCI patients performed significantly worse (with lower test scores and larger ordering costs) than healthy controls in both digit ordering and backward tests, although they performed normally in the forward test. The ordering cost increased as a function of age across groups, indicating an aging-related decline in the ability of serial ordering. However, individual patients’ task performances were not correlated with their severity or duration of motor symptoms, or daily exposure to dopaminergic drugs. These results suggested that serial ordering deficits exist in early stages of PD, prior to subtle changes in global cognition and in parallel with motor symptoms.

Genomic properties of chromosomal bands are linked to evolutionary rearrangements and new centromere formation in primates.

Chromosomal rearrangements in humans are largely related to pathological conditions, and phenotypic effects are also linked to alterations in the expression profile following nuclear relocation of genes between functionally different compartments, generally occupying the periphery or the inner part of the cell nuclei. On the other hand, during evolution, chromosomal rearrangements may occur apparently without damaging phenotypic effects and are visible in currently phylogenetically related species. To increase our insight into chromosomal reorganisation in the cell nucleus, we analysed 18 chromosomal regions endowed with different genomic properties in cell lines derived from eight primate species covering the entire evolutionary tree. We show that homologous loci, in spite of their evolutionary relocation along the chromosomes, generally remain localised to the same functional compartment of the cell nuclei. We conclude that evolutionarily successful chromosomal rearrangements are those that leave the nuclear position of the regions involved unchanged. On the contrary, in pathological situations, the effect typically observed is on gene structure alteration or gene nuclear reposition. Moreover, our data indicate that new centromere formation could potentially occur everywhere in the chromosomes, but only those emerging in very GC-poor/gene-poor regions, generally located in the nuclear periphery, have a high probability of being retained through evolution. This suggests that, in the cell nucleus of related species, evolutionary chromosomal reshufflings or new centromere formation does not alter the functionality of the regions involved or the interactions between different loci, thus preserving the expression pattern of orthologous genes.

Dual Mechanism of Rag Gene Repression by c-Myb during Pre-B Cell Proliferation.

Developing B lymphocytes undergo clonal expansion following successful immunoglobulin heavy chain gene rearrangement. During this proliferative burst, expression of the Rag genes is transiently repressed to prevent the generation of double-stranded DNA (dsDNA) breaks in cycling large pre-B cells. The Rag genes are then reexpressed in small, resting pre-B cells for immunoglobulin light chain gene rearrangement. We previously identified c-Myb as a repressor of Rag transcription during clonal expansion using Abelson murine leukemia virus-transformed B cells. Nevertheless, the molecular mechanisms by which c-Myb achieved precise spatiotemporal repression of Rag expression remained obscure. Here, we identify two mechanisms by which c-Myb represses Rag transcription. First, c-Myb negatively regulates the expression of the Rag activator Foxo1, an activity dependent on M303 in c-Myb's transactivation domain, and likely the recruitment of corepressors to the Foxo1 locus by c-Myb. Second, c-Myb represses Rag transcription directly by occupying the Erag enhancer and antagonizing Foxo1 binding to a consensus forkhead site in this cis-regulatory element that we show is crucial for Rag expression in Abelson pre-B cell lines. This work provides important mechanistic insight into how spatiotemporal expression of the Rag genes is tightly controlled during B lymphocyte development to prevent mistimed dsDNA breaks and their deleterious consequences.

Archival Transformations in Early Modern European History

This special issue addresses a double transformation. The first is the historical process that saw a dramatic increase in the production of documents and a substantial improvement in their management and preservation throughout Europe between the fifteenth and the eighteenth centuries. The early modern period, broadly conceived, is often described as the age of print, but it was also the great time of archives, understood as both the physical repositories and organized offices established by institutions or collectivities to store handwritten documents produced in the course of continuous functions with a view to long-term use. For many European historians, the process of centralization, expansion and (more or less successful) rearrangement of archives is symbolized by the establishment of the great Simancas and Vatican archives in 1540 and 1612 respectively. But, as the articles collected here demonstrate, smaller states also enacted reforms in record-keeping, and these changes were more concerned with archives than with central institutions. The second transformation is interpretive and methodological. Archives have long been at the centre of historians’ research, but over the last ten to fifteen years, an ‘archival turn’ in disciplines ranging from history, literature, anthropology and the social sciences has transformed archives from sites of research into objects of enquiry in their own right. These works study the evolving processes of selection, ordering and usage that produced archives not as neutral repositories of sources but as historically constructed tools of power relations, deeply embedded in changing social and cultural contexts. The history of archives has been long practised by archivists, who are professionally aware that documents were neither produced nor arranged as

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint.

Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation.

Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins.

Disruptive ARID1A Mutations in Follicular Lymphoma Impair DNA Repair Efficiency and Are Associated with Favorable Outcome in Patients Receiving First-Line Immunochemotherapy

Follicular lymphoma (FL) is among the most common lymphomas worldwide and considered incurable for the majority of patients who present with advanced disease. FL is a clinically and molecularly heterogeneous disease. We have recently established a clinicogenetic risk model (m7-FLIPI) that integrates the mutation status of 7 genes, including ARID1A, into a predictive algorithm for improved risk stratification for failure-free survival in patients receiving first-line immunochemotherapy (Pastore et al., Lancet Oncology 2015). When adjusted for FLIPI and ECOG performance status, the presence of ARID1A mutations correlated with longer failure-free survival (HR 2.85, 95% confidence interval (CI) 1.12-7.27; p=0.049) in patients receiving first-line R-CHOP. The underlying molecular mechanism for better treatment outcome of patients with ARID1A mutated FL is unclear.ARID1A is a member of SWI/SNF nucleosome remodeling complex. Evolving evidence indicates that the SWI/SNF complex functions as a tumor suppressor and is implicated in DNA damage repair. ARID1Ais the most commonly mutated SWI/SNF complex member in FL: in our series, we identified 44 ARID1A mutations in 304 FL cases (14%). These mutations were primarily heterozygous and disruptive (66%), another 6 cases harbored splice site mutations. Primary patient samples and lymphoma cell lines that harbored ARID1A mutations (e.g., SU-DHL5, WSU-FSCCL, Namalwa) were had lower ARID1A protein expression as compared to ARID1A wild type (wt) t(14;18)-positive lymphoma cell lines (OCI-Ly1, -Ly8, DB).Non-homologous end joining (NHEJ) has been reported to be the predominant pathway of DNA double strand break (DSB) repair in FL (Koues et al., Immunity 2015) and is indispensible for successful VDJ rearrangement during early B-cell maturation.To analyze the impact of Arid1a disruption on B-cell maturation in vivo, we used a conditional Arid1a knock-out mouse model (Arid1af/f; Gao, PNAS 2008). 8-10 week old Mx1-Cre+Arid1af/f mice and control (Mx1-Cre-Arid1af/f) mice were treated with poly(IC). Bone marrow (BM) were harvested from these animals 14 days later. Conditionally deletion of Arid1a (n=3) resulted in severely impaired B-cell maturation in BM cells affecting Hardy fractions B/C though F. E.g., flow cytometric analysis showed that the frequency of fraction C (B220+ CD43+ HSA+ BP-1+) was greatly reduced in BM from Mx1-Cre+Arid1af/f vs in the control group (0.1 % vs 1.2 %, n=3), consistent with the inability to successfully complete VDJ recombination.As a proof-of-principle experiment we assayed the impact of Arid1a loss on DSB repair efficiency using mouse embryo fibroblasts (MEFs) from either wt or conditional Arid1a knock-out mice. MEFs were irradiated with 2 Gy 36 hrs after retroviral transduction with Cre and stained for γH2A.X and 53BP1 to assay for NHEJ. In three independent and blinded experiments, evaluation of >100 cells per condition demonstrated significantly more double-positive cells (>6 foci/cell) in Arid1a-/- MEFs (43+/-14%) compared to both Arid1af/f MEFs (9+/-1%) and Cre-exposed wt MEFs (15+/-5%) at 16 hrs post irradiation, whereas there was no significant difference for Arid1af/f or wt MEFs with or without Cre at baseline (all <10%), and 1 hr post irradiation (all >50%).ARID1A haplodeficient lymphoma cell lines (SU-DHL5, WSU-FSCCL, Namalwa) were 10-100 fold more sensitive to the DSB inducing doxorubicin treatment compared to ARID1A wt lymphoma cell lines (OCI-Ly1, -Ly8, DB). Tet-induced shRNA knock-down (via pTRIPZ vectors) of endogenous ARID1A in two t(14;18)-positive lymphoma cell lines with wt ARID1A (OCI-Ly1 and OCI-Ly8) doubled the number of NHEJ foci by 53-BP1 staining in two independent experiments. Furthermore, shRNA knock-down of ARID1A resulted in increased sensitivity to doxorubicine (IC50 <10 nM) in OCI-Ly8 cell lines as compared to 25 nM for scrambled shRNA.We conclude that ARID1A is recurrently and significantly mutated in FL. Most mutations are disruptive and result in functionally relevant ARID1A protein loss. ARID1A disruption slows the repair kinetics of NHEJ and delays the clearance of DSB both in a genetically clean MEF model and in t(14;18)-positive lymphoma cell lines. As a result, ARID1A loss might underlie the genomic instability of FL, but -at the same time- sensitize tumors to DNA damaging agents and irradiation. DisclosuresNo relevant conflicts of interest to declare.

Stereoselective Synthesis of β-Lactam-triflones under Catalyst-Free Conditions.

The first example of the synthesis of β-lactam-triflones is described. Treatment of 2-diazo-1-aryl-2-(trifluoromethylsulfonyl)ethanones 1c-f with imines 2 under catalyst-free heating conditions provides pharmaceutically attractive multisubstituted β-lactam-triflones 3 in good to high yields with regio- and diastereoselectivities. A successive Wolff rearrangement and Staudinger [2 + 2] cycloaddition reaction are key elements for the success of this transformation.

Antidepressants are selective serotonin neuronal reuptake inhibitors: 40-year history

The paper presents historical prerequisites for designing antidepressants from a group of selective serotonin neuronal reuptake inhibitors (SSRIs): to determine a lower serotonin concentration in the different tissues of depressed patients; to establish a higher serotonin concentration in the treatment of depressed patients with tricyclic antidepressants, and to formulate the serotonergic theory of depression. It also provides a consecutive account of the history of clinical introduction of individual SSRI representatives, such as fluoxetine, zimelidine, fluvoxamine, indalpine, citalopram, sertraline, paroxetine, and escitalopram. There are data from the history of studying the mechanism of SSRI action: from the theory of the importance of an increase in the concentration of serotonin in the synaptic cleft to the current understanding of complex successive intracellular rearrangements at the level of the postsynaptic neuron. The history of studying the efficacy of SSRIs in treating depression is considered in detail. Emphasis is laid on the reasons for a paradoxical difference in the evaluations of the efficiency of therapy with SSRIs versus other groups of antidepressants at different developmental stages of psychopharmacology. The role of marketing technologies in disseminating the data on the efficacy of this or that group of antidepressants is described. The practical significance of differences in individual SSRI representatives (the potency of serotonin uptake inhibition; the degree of selectivity and activity against the serotonergic system; the likelihood of an unfavorable pharmacokinetic interaction with other drugs; the half-life of elimination; the quickness of achieving a therapeutic dose) is analyzed. Whether it is possible and reasonable to differentially choose different SSRI representatives in the treatment of depressions at the present stage is discussed. The authors state their belief that researches should be continued to specify the place of SSRIs among other groups of current antidepressants for the treatment of depressions.

Independent recruitment of Igh alleles in V(D)J recombination.

How the vast majority of B cells express only one of the two alleles at their immunoglobulin loci remains a biological puzzle. Here, in mice reconstituted with a single haematopoietic stem cell, we demonstrate that each of the two immunoglobulin heavy chain (Igh) alleles has a similar probability to be the first to undergo VH to DJH rearrangement. We also observe this similar probability in clones from multipotent and common lymphoid precursors. The extreme biases in the expression of the alleles that we find in more differentiated subsets are mostly due to constraints imposed by early rearrangements. Our data demonstrate that each of the two Igh alleles in a B cell behaves independently of the other, up to the moment when a successful rearrangement in one allele triggers a feedback mechanism that prevents further recombination.

Large chromosomal rearrangements during a long-term evolution experiment with Escherichia coli.

ABSTRACTLarge-scale rearrangements may be important in evolution because they can alter chromosome organization and gene expression in ways not possible through point mutations. In a long-term evolution experiment, twelve Escherichia coli populations have been propagated in a glucose-limited environment for over 25 years. We used whole-genome mapping (optical mapping) combined with genome sequencing and PCR analysis to identify the large-scale chromosomal rearrangements in clones from each population after 40,000 generations. A total of 110 rearrangement events were detected, including 82 deletions, 19 inversions, and 9 duplications, with lineages having between 5 and 20 events. In three populations, successive rearrangements impacted particular regions. In five populations, rearrangements affected over a third of the chromosome. Most rearrangements involved recombination between insertion sequence (IS) elements, illustrating their importance in mediating genome plasticity. Two lines of evidence suggest that at least some of these rearrangements conferred higher fitness. First, parallel changes were observed across the independent populations, with ~65% of the rearrangements affecting the same loci in at least two populations. For example, the ribose-utilization operon and the manB-cpsG region were deleted in 12 and 10 populations, respectively, suggesting positive selection, and this inference was previously confirmed for the former case. Second, optical maps from clones sampled over time from one population showed that most rearrangements occurred early in the experiment, when fitness was increasing most rapidly. However, some rearrangements likely occur at high frequency and may have simply hitchhiked to fixation. In any case, large-scale rearrangements clearly influenced genomic evolution in these populations.