RESUMEN
Evolution is often an obstacle to the engineering of stable biological systems due to the selection of mutations inactivating costly gene circuits. Gene overlaps induce important constraints on sequences and their evolution. We show that these constraints can be harnessed to increase the stability of costly genes by purging loss-of-function mutations. We combine computational and synthetic biology approaches to rationally design an overlapping reading frame expressing an essential gene within an existing gene to protect. Our algorithm succeeded in creating overlapping reading frames in 80% of E. coli genes. Experimentally, scoring mutations in both genes of such overlapping construct, we found that a significant fraction of mutations impacting the gene to protect have a deleterious effect on the essential gene. Such an overlap thus protects a costly gene from removal by natural selection by associating the benefit of this removal with a larger or even lethal cost. In our synthetic constructs, the overlap converts many of the possible mutants into evolutionary dead-ends, reducing the evolutionary potential of the system and thus increasing its stability over time.
Asunto(s)
Genes Esenciales/genética , Ingeniería Genética/métodos , Mutación/genética , Biología Sintética/métodos , Algoritmos , Escherichia coli/genética , Evolución Molecular , Genómica , Sistemas de Lectura/genética , Análisis de Secuencia de ADNRESUMEN
Natural selection is thought to shape the evolution of aging patterns, although how life-history trajectories orchestrate the inherently stochastic processes associated with aging is unclear. Tracking clonal growth-arrested Escherichia coli cohorts in an homogeneous environment at single-cell resolution, we demonstrate that the Gompertz law of exponential mortality characterizes bacterial lifespan distributions. By disentangling the rate of aging from age-independent components of longevity, we find that increasing cellular maintenance through the general stress pathway reduces the aging rate and rescales the lifespan distribution at the expense of growth. This trade-off between aging and growth underpins the evolutionary tuning of the general stress response pathway in adaptation to the organism's feast-or-famine lifestyle. It is thus necessary to involve both natural selection and stochastic physiology to explain aging patterns.
Asunto(s)
Adaptación Biológica/fisiología , Escherichia coli/fisiología , Evolución Biológica , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Análisis de la Célula Individual/métodos , Factores de TiempoRESUMEN
Bacterial genes that confer crucial phenotypes, such as antibiotic resistance, can spread horizontally by residing on mobile genetic elements (MGEs). Although many mobile genes provide strong benefits to their hosts, the fitness consequences of the process of transfer itself are less clear. In previous studies, transfer has been interpreted as a parasitic trait of the MGEs because of its costs to the host but also as a trait benefiting host populations through the sharing of a common gene pool. Here, we show that costly donation is an altruistic act when it spreads beneficial MGEs favoured when it increases the inclusive fitness of donor ability alleles. We show mathematically that donor ability can be selected when relatedness at the locus modulating transfer is sufficiently high between donor and recipients, ensuring high frequency of transfer between cells sharing donor alleles. We further experimentally demonstrate that either population structure or discrimination in transfer can increase relatedness to a level selecting for chromosomal transfer alleles. Both mechanisms are likely to occur in natural environments. The simple process of strong dilution can create sufficient population structure to select for donor ability. Another mechanism observed in natural isolates, discrimination in transfer, can emerge through coselection of transfer and discrimination alleles. Our work shows that horizontal gene transfer in bacteria can be promoted by bacterial hosts themselves and not only by MGEs. In the longer term, the success of cells bearing beneficial MGEs combined with biased transfer leads to an association between high donor ability, discrimination, and mobile beneficial genes. However, in conditions that do not select for altruism, host bacteria promoting transfer are outcompeted by hosts with lower transfer rate, an aspect that could be relevant in the fight against the spread of antibiotic resistance.
Asunto(s)
Bacterias/genética , Farmacorresistencia Bacteriana/genética , Transferencia de Gen Horizontal , Genes Bacterianos/genética , Algoritmos , Conjugación Genética , Escherichia coli/genética , Evolución Molecular , Aptitud Genética , Genética de Población , Secuencias Repetitivas Esparcidas/genética , Modelos Genéticos , Plásmidos/genética , Selección GenéticaRESUMEN
Many bacterial species are social, producing costly secreted "public good" molecules that enhance the growth of neighboring cells. The genes coding for these cooperative traits are often propagated via mobile genetic elements and can be virulence factors from a biomedical perspective. Here, we present an experimental framework that links genetic information exchange and the selection of cooperative traits. Using simulations and experiments based on a synthetic bacterial system to control public good secretion and plasmid conjugation, we demonstrate that horizontal gene transfer can favor cooperation. In a well-mixed environment, horizontal transfer brings a direct infectious advantage to any gene, regardless of its cooperation properties. However, in a structured population transfer selects specifically for cooperation by increasing the assortment among cooperative alleles. Conjugation allows cooperative alleles to overcome rarity thresholds and invade bacterial populations structured purely by stochastic dilution effects. Our results provide an explanation for the prevalence of cooperative genes on mobile elements, and suggest a previously unidentified benefit of horizontal gene transfer for bacteria.
Asunto(s)
Bacterias/genética , Transferencia de Gen Horizontal/fisiología , Genes Bacterianos/fisiología , Plásmidos/genética , Bacterias/patogenicidadRESUMEN
CONTEXT: Twin and family studies indicate a significant heritability of pubertal timing and more specifically of age at menarche (AAM). OBJECTIVE: Test the association of AAM with common variants of three candidate genes suspected to have a prominent role in reproductive physiology: leptin (LEP), neuropeptide Y receptor 1 (NPY1R) and GPR54. DESIGN AND METHODS: We selected the -2459 LEP, the rs7687423 NPY1R and thers350132 GPR54 variants as the more common coding or regulatory variants (minor allelic frequency >0.10) in these gene regions. To avoid stratification problems that can impair association studies, we used the Q-TDT method based on allele transmission to evaluate the relationship of these variants with AAM in 245 healthy women from 107 families of European ancestry. RESULTS: We found no association of AAM with any of the studied variants. CONCLUSIONS: Keeping in mind that common variants do not recapitulate the whole genetic variation in a given gene region, this study indicates that the studied LEP, NPY1R and GPR54 variants do not have a major influence upon pubertal timing in Caucasian women. Effects of these genetic loci on age at menarche can definitively be excluded only through determination of extended haplotypes in a larger cohort.
Asunto(s)
Variación Genética , Leptina/genética , Menarquia/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropéptido Y/genética , Adolescente , Niño , Femenino , Frecuencia de los Genes , Humanos , Receptores de Kisspeptina-1 , Población Blanca/genéticaRESUMEN
CONTEXT: Because IGF-I is the main mediator of GH action on osteogenic cells, individual differences in IGF-I sensitivity are expected to contribute to the variations of GH effects on growth. In GH-treated children, the variable responses in growth rates at a specific IGF-I target level indicate heterogeneity of responses to serum IGF-I exposures. OBJECTIVES: This study tested a cell-based assay as an index of individual IGF-I sensitivity that could help dissect GH pharmacogenetics. DESIGN: Akt phosphorylation (P-Akt) was quantified in response to IGF-I in fresh lymphocytes from 50 short children (25 with idiopathic short stature and 25 born short for gestational age) whose growth parameters were being prospectively monitored during the first year of GH therapy (86 +/- 20 mug/kg.d). RESULTS: Intra-individual triplicate measurements of IGF-I-stimulated P-Akt were reasonably consistent (0.11 < or = sd; mean < or = 0.23). Among the 50 children, the distribution of P-Akt in lymphocytes stimulated by 125 ng/ml IGF-I was closely associated with the growth response to GH administration (univariate P = 0.001). Both GH dosage (P = 0.006) and the fold increase in IGF-I levels (P = 0.04) in response to GH (P = 0.04) were also correlated with the growth response. CONCLUSION: Lymphocytes are the only IGF-I target cells that can be easily studied in clinical research. IGF-I-stimulated P-Akt in these cells was found to be a predictor of GH efficacy, supporting a significant role of the first steps of IGF-I signaling in the individual variability of GH effects on growth.
Asunto(s)
Crecimiento/efectos de los fármacos , Hormona de Crecimiento Humana/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Linfocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Femenino , Hormona de Crecimiento Humana/uso terapéutico , Humanos , Factor I del Crecimiento Similar a la Insulina/análisis , Linfocitos/metabolismo , Masculino , FosforilaciónRESUMEN
OBJECTIVE: In search of functional polymorphisms associated with the genetics of insulin resistance, we studied a variant in the promoter of PIK3CB, the gene coding for the catalytic p110beta subunit of phosphatidylinositol (PI) 3-kinase, a major effector of insulin action. RESEARCH DESIGN AND METHODS: The rs361072 C/T variant was selected among single nucleotide polymorphisms of the PIK3CB region because we suspected that its common C allele (allelic frequency approximately 50% in Europeans) could create a GATA-binding motif and was genotyped in five cohorts of obese (n = 1,876) and two cohorts of nonobese (n = 1,490) European children. To estimate insulin resistance in these children, the homeostasis model assessment for insulin resistance (HOMA-IR) index was measured in strict nutritional conditions. GATA-binding and functional effects of rs361072 were explored in transfected cell lines and in lymphocytes from obese children. RESULTS: The rs361072 C/T variant was associated with HOMA-IR in the obese children cohorts (1.7 x 10(-12) < P < 2 x 10(-4) for C/C vs. T/T using regression analysis). HOMA-IR averaged 3.3 +/- 0.1 in C/C and 4.5 +/- 0.2 in T/T obese children (P = 4.5 x 10(-6) by ANOVA). C/T patients had intermediate values. As shown by the interaction between BMI and genotype (P = 2.1 x 10(-9)), the association of rs361072 with HOMA-IR depended on BMI and was only marginal in nonobese children (P = 0.04). At the molecular level, the C allele of rs361072 was found to create a GATA-binding site able to increase transcription of PIK3CB. CONCLUSIONS: We postulate that the C allele of rs361072 is a causal variant capable of attenuating insulin resistance in obese children through increased expression of p110beta.
Asunto(s)
Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Variación Genética , Resistencia a la Insulina/genética , Obesidad/genética , Fosfatidilinositol 3-Quinasas/genética , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Sitios de Unión , Glucemia/análisis , Índice de Masa Corporal , Niño , Fosfatidilinositol 3-Quinasas Clase II , Estudios de Cohortes , Predisposición Genética a la Enfermedad , Genotipo , Homeostasis , Humanos , Insulina/sangre , Regiones Promotoras Genéticas , ARN Mensajero/genéticaRESUMEN
Proinsulin is a key Ag in type 1 diabetes, but the mechanisms regulating proinsulin immune tolerance are unknown. We have shown that preproinsulin-2 gene-deficient mice (proins-2(-/-)) are intolerant to proinsulin-2. In this study, we analyzed the mechanisms underlying T cell-mediated tolerance to proinsulin-2 in 129/Sv nonautoimmune mice. The expression of one proinsulin-2 allele, whatever its parental origin, was sufficient to maintain tolerance. The site of proinsulin-2 expression relevant to tolerance was evaluated in thymus and bone marrow chimeras. CD4+ T cell reactivity to proinsulin-2 was independent of proinsulin-2 expression in radiation-sensitive bone marrow-derived cells. A wt thymus restored tolerance in proins-2(-/-) mice. Conversely, the absence of the preproinsulin-2 gene in radioresistant thymic cells was sufficient to break tolerance. Although chimeric animals had proinsulin-2-reactive CD4+ T cells in their peripheral repertoire, they displayed no insulitis or insulin Abs, suggesting additional protective mechanisms. In a model involving transfer to immunodeficient (CD3epsilon(-/-)) mice, naive and proinsulin-2-primed CD4+ T cells were not activated, but could be activated by immunization regardless of whether the recipient mice expressed proinsulin-2. Furthermore, we could not identify a role for putative specific T cells regulating proinsulin-2-reactive CD4+ T in transfer experiments. Thus, proinsulin-2 gene expression by radioresistant thymic epithelial cells is involved in the induction of self-tolerance, and additional factors are required to induce islet abnormalities.
Asunto(s)
Proinsulina/inmunología , Autotolerancia , Timo/inmunología , Timo/efectos de la radiación , Traslado Adoptivo , Animales , Trasplante de Médula Ósea/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/efectos de la radiación , Linfocitos T CD4-Positivos/trasplante , Proliferación Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Femenino , Interferón gamma/biosíntesis , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/patología , Islotes Pancreáticos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proinsulina/biosíntesis , Proinsulina/deficiencia , Proinsulina/genética , Quimera por Radiación , Autotolerancia/efectos de la radiación , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/efectos de la radiación , Timo/citología , Timo/trasplanteRESUMEN
Deciphering mechanisms involved in failure of self tolerance to preproinsulin-2 is a key issue in type 1 diabetes. We used nonautoimmune 129SV/Pas mice lacking preproinsulin-2 to study the immune response to preproinsulin-2. In these mice, a T cell response was detected after immunization with several preproinsulin-2 peptides and confirmed by generating hybridomas. Activation of some of these hybridomas by wild-type (wt) islet cells or recombinant murine proinsulin-2 demonstrated that two epitopes can be generated from the naturally expressed protein. Although T cells from wt mice responded to preproinsulin-2 peptides, we could not detect a response to the naturally processed epitopes in these mice. Moreover, after immunization with recombinant whole proinsulin-2, a T cell response was detected in preproinsulin-2-deficient but not in wt mice. This suggests that islet preproinsulin-2-autoreactive T cells are functionally eliminated in wt mice. We used a transplantation model to evaluate the relevance of reactivity to preproinsulin-2 in vivo. Wild-type preproinsulin-2-expressing islets transplanted in preproinsulin-2-deficient mice elicited a mononuclear cell infiltration and insulin Abs. Graft infiltration was further increased by immunization with preproinsulin-2 peptides. Preproinsulin-2 expression thus shapes the immune response and prevents self reactivity to the islet. Moreover, islet preproinsulin-2 primes an immune response to preproinsulin-2 in deficient mice.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Proinsulina/genética , Proinsulina/inmunología , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/genética , Hibridomas , Insulina , Interleucina-2/metabolismo , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Trasplante de Islotes Pancreáticos/inmunología , Trasplante de Islotes Pancreáticos/patología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Proinsulina/administración & dosificación , Proinsulina/deficiencia , Isoformas de Proteínas/administración & dosificación , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Precursores de Proteínas/administración & dosificación , Precursores de Proteínas/deficiencia , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Tolerancia al Trasplante/genética , VacunaciónRESUMEN
Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that results from the destruction of insulin secreting beta cells by diabetogenic T cells. The time and location of the encounter of autoantigen(s) by naive autoreactive T cells in normal NOD mice are still elusive. To address these issues, we analyzed diabetes development in mice whose spleen or pancreatic lymph nodes (panLNs) had been removed. Excision of panLNs (panLNx) at 3 wk protected mice against insulin autoantibodies (IAAs), insulitis, and diabetes development almost completely, but had no effect when performed at 10 wk. The protection afforded by panLNx at weaning was not due to modifications of the immune system, the absence of autoreactive T cells, or the increase in the potency of regulatory T cells. That panLNs are dispensable during adult life was confirmed by the capacity of 10-wk-old panLNx irradiated recipients to develop diabetes upon transfer of diabetogenic T cells. In contrast, splenectomy had no effect at any age. Partial excision of mesenteric LN at 3 wk did not prevent accelerated diabetes by cyclophosphamide as panLNx did. Thus, in normal NOD mice, autoreactive T cell initial priming occurs in LNs draining the target organ of the disease from 3 wk of age.