RESUMEN
Triacylglycerols (TAGs) are the main source of stored energy in the body, providing an important substrate pool for mitochondrial beta-oxidation. Imbalances in the amount of TAGs are associated with obesity, cardiac disease and various other pathologies1,2. In humans, TAGs are synthesized from excess, coenzyme A-conjugated fatty acids by diacylglycerol O-acyltransferases (DGAT1 and DGAT2)3. In other organisms, this activity is complemented by additional enzymes4, but whether such alternative pathways exist in humans remains unknown. Here we disrupt the DGAT pathway in haploid human cells and use iterative genetics to reveal an unrelated TAG-synthesizing system composed of a protein we called DIESL (also known as TMEM68, an acyltransferase of previously unknown function) and its regulator TMX1. Mechanistically, TMX1 binds to and controls DIESL at the endoplasmic reticulum, and loss of TMX1 leads to the unconstrained formation of DIESL-dependent lipid droplets. DIESL is an autonomous TAG synthase, and expression of human DIESL in Escherichia coli endows this organism with the ability to synthesize TAG. Although both DIESL and the DGATs function as diacylglycerol acyltransferases, they contribute to the cellular TAG pool under specific conditions. Functionally, DIESL synthesizes TAG at the expense of membrane phospholipids and maintains mitochondrial function during periods of extracellular lipid starvation. In mice, DIESL deficiency impedes rapid postnatal growth and affects energy homeostasis during changes in nutrient availability. We have therefore identified an alternative TAG biosynthetic pathway driven by DIESL under potent control by TMX1.
Asunto(s)
Aciltransferasas , Triglicéridos , Animales , Humanos , Ratones , Aciltransferasas/metabolismo , Coenzima A/metabolismo , Diacilglicerol O-Acetiltransferasa/metabolismo , Escherichia coli/metabolismo , Homeostasis , Triglicéridos/biosíntesis , Metabolismo Energético , Nutrientes/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismoRESUMEN
The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to cancer. Here, we study the homozygous mutation V63E in MSH2 that was found in the germline of a patient with suspected constitutional mismatch repair deficiency syndrome who developed colorectal cancer before the age of 30. Characterization of the mutant in mouse models, as well as slippage and repair assays, shows a mildly pathogenic phenotype. Using cryogenic electron microscopy and surface plasmon resonance, we explored the mechanistic effect of this mutation on MutSα function. We discovered that V63E disrupts a previously unappreciated interface between the mismatch binding domains (MBDs) of MSH2 and MSH6 and leads to reduced DNA binding. Our research identifies this interface as a 'safety lock' that ensures high-affinity DNA binding to increase replication fidelity. Our mechanistic model explains the hypomorphic phenotype of the V63E patient mutation and other variants in the MBD interface.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Reparación del ADN , Proteína 2 Homóloga a MutS , Animales , Ratones , ADN/química , Mutación , Proteína 2 Homóloga a MutS/metabolismoRESUMEN
Through transfection of short single-stranded oligodeoxyribonucleotides (ssODNs) small genomic alterations can be introduced into mammalian cells with high precision. ssODNs integrate into the genome during DNA replication, but the resulting heteroduplex is prone to detection by DNA mismatch repair (MMR), which prevents effective gene modification. We have previously demonstrated that the suppressive action of MMR can be avoided when the mismatching nucleotide in the ssODN is a locked nucleic acid (LNA). Here, we reveal that LNA-modified ssODNs (LMOs) are not integrated as intact entities in mammalian cells, but are severely truncated before and after target hybridization. We found that single additional (non-LNA-modified) mutations in the 5'-arm of LMOs influenced targeting efficiencies negatively and activated the MMR pathway. In contrast, additional mutations in the 3'-arm did not affect targeting efficiencies and were not subject to MMR. Even more strikingly, homology in the 3'-arm was largely dispensable for effective targeting, suggestive for extensive 3'-end trimming. We propose a refined model for LMO-directed gene modification in mammalian cells that includes LMO degradation.
Asunto(s)
Replicación del ADN/genética , ADN de Cadena Simple/genética , Oligonucleótidos/genética , Animales , Línea Celular , Reparación de la Incompatibilidad de ADN/genética , Edición Génica , Marcación de Gen , Humanos , Mamíferos/genética , Mutación/genética , TransfecciónRESUMEN
BACKGROUND: Inactivating mutations in the MLH1 DNA mismatch repair (MMR) gene underlie 42% of Lynch syndrome (LS) cases. LS is a cancer predisposition causing early onset colorectal and endometrial cancer. Nonsense and frameshift alterations unambiguously cause LS. The phenotype of missense mutations that only alter a single amino acid is often unclear. These variants of uncertain significance (VUS) hinder LS diagnosis and family screening and therefore functional tests are urgently needed. We developed a functional test for MLH1 VUS termed 'oligonucleotide-directed mutation screening' (ODMS). METHODS: The MLH1 variant was introduced by oligonucleotide-directed gene modification in mouse embryonic stem cells that were subsequently exposed to the guanine analogue 6-thioguanine to determine whether the variant abrogated MMR. RESUTS: In a proof-of-principle analysis, we demonstrate that ODMS can distinguish pathogenic and non-pathogenic MLH1 variants with a sensitivity of >95% and a specificity of >91%. We subsequently applied the screen to 51 MLH1 VUS and identified 31 pathogenic variants. CONCLUSION: ODMS is a reliable tool to identify pathogenic MLH1 variants. Implementation in clinical diagnostics will improve clinical care of patients with suspected LS and their relatives.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Homólogo 1 de la Proteína MutL/genética , Animales , Codón sin Sentido/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Modelos Animales de Enfermedad , Mutación del Sistema de Lectura/genética , Variación Genética/genética , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Mutagénesis Sitio-Dirigida , Mutación Missense/genéticaRESUMEN
Lynch syndrome (LS) is a hereditary cancer predisposition caused by inactivating mutations in DNA mismatch repair (MMR) genes. Mutations in the MSH6 DNA MMR gene account for approximately 18% of LS cases. Many LS-associated sequence variants are nonsense and frameshift mutations that clearly abrogate MMR activity. However, missense mutations whose functional implications are unclear are also frequently seen in suspected-LS patients. To conclusively diagnose LS and enroll patients in appropriate surveillance programs to reduce morbidity as well as mortality, the functional consequences of these variants of uncertain clinical significance (VUS) must be defined. We present an oligonucleotide-directed mutagenesis screen for the identification of pathogenic MSH6 VUS. In the screen, the MSH6 variant of interest is introduced into mouse embryonic stem cells by site-directed mutagenesis. Subsequent selection for MMR-deficient cells using the DNA damaging agent 6-thioguanine (6TG) allows the identification of MMR abrogating VUS because solely MMR-deficient cells survive 6TG exposure. We demonstrate the efficacy of the genetic screen, investigate the phenotype of 26 MSH6 VUS and compare our screening results to clinical data from suspected-LS patients carrying these variant alleles.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas de Unión al ADN/genética , Pruebas Genéticas/métodos , Mutación Missense , Fenotipo , Animales , Células Cultivadas , Células Madre Embrionarias/metabolismo , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Tioguanina/toxicidadRESUMEN
BACKGROUND: One way to achieve universal health coverage (UHC) in low- and middle-income countries (LMIC) is the implementation of health insurance schemes. A robust and up to date overview of empirical evidence assessing and substantiating health equity impact of health insurance schemes among specific vulnerable populations in LMICs beyond the more common parameters, such as income level, is lacking. We fill this gap by conducting a systematic review of how social inclusion affects access to equitable health financing arrangements in LMIC. METHODS: We searched 11 databases to identify peer-reviewed studies published in English between January 1995 and January 2018 that addressed the enrolment and impact of health insurance in LMIC for the following vulnerable groups: female-headed households, children with special needs, older adults, youth, ethnic minorities, migrants, and those with a disability or chronic illness. We assessed health insurance enrolment patterns of these population groups and its impact on health care utilization, financial protection, health outcomes and quality of care. RESULTS: The comprehensive database search resulted in 44 studies, in which chronically ill were mostly reported (67%), followed by older adults (33%). Scarce and inconsistent evidence is available for individuals with disabilities, female-headed households, ethnic minorities and displaced populations, and no studies were yielded reporting on youth or children with special needs. Enrolment rates seemed higher among chronically ill and mixed or insufficient results are observed for the other groups. Most studies reporting on health care utilization found an increase in health care utilization for insured individuals with a disability or chronic illness and older adults. In general, health insurance schemes seemed to prevent catastrophic health expenditures to a certain extent. However, reimbursements rates were very low and vulnerable individuals had increased out of pocket payments. CONCLUSION: Despite a sizeable literature published on health insurance, there is a dearth of good quality evidence, especially on equity and the inclusion of specific vulnerable groups in LMIC. Evidence should be strengthened within health care reform to achieve UHC, by redefining and assessing vulnerability as a multidimensional process and the investigation of mechanisms that are more context specific.
Asunto(s)
Países en Desarrollo , Reforma de la Atención de Salud , Equidad en Salud , Gastos en Salud , Seguro de Salud , Cobertura Universal del Seguro de Salud , Poblaciones Vulnerables , Humanos , Renta , PobrezaRESUMEN
Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that "oligo targeting" can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Reparación de la Incompatibilidad de ADN , Proteína 2 Homóloga a MutS/genética , Mutagénesis , Oligonucleótidos/genética , HumanosRESUMEN
Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.
Asunto(s)
ADN de Cadena Simple , Escherichia coli/genética , Animales , Disparidad de Par Base , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación de la Incompatibilidad de ADN , Reparación del ADN , Oligonucleótidos/genéticaRESUMEN
BACKGROUND & AIMS: Lynch syndrome, a nonpolyposis form of hereditary colorectal cancer, is caused by inherited defects in DNA mismatch repair (MMR) genes. Most patients carry a germline mutation in 1 allele of the MMR genes MSH2 or MLH1. With spontaneous loss of the wild-type allele, cells with defects in MMR exist among MMR-proficient cells, as observed in healthy intestinal tissues from patients with Lynch syndrome. We aimed to create a mouse model of this situation to aid in identification of environmental factors that affect MMR-defective cells and their propensity for oncogenic transformation. METHODS: We created mice in which the MMR gene Msh2 can be inactivated in a defined fraction of crypt base columnar stem cells to generate MSH2-deficient intestinal crypts among an excess of wild-type crypts (Lgr5-CreERT2;Msh2(flox/-) mice). Intestinal tissues were collected; immunohistochemical analyses were performed for MSH2, along with allele-specific PCR assays. We traced the fate of MSH2-deficient crypts under the influence of different external factors. RESULTS: Lgr5-CreERT2;Msh2(flox/-) mice developed more adenomas and adenocarcinomas than control mice; all tumors were MSH2 deficient. Exposure of Lgr5-CreERT2;Msh2(flox/-) mice to the methylating agent temozolomide caused MSH2-deficient intestinal stem cells to proliferate more rapidly than wild-type stem cells. The MSH2-deficient intestinal stem cells were able to colonize the intestinal epithelium and many underwent oncogenic transformation, forming intestinal neoplasias. CONCLUSIONS: We developed a mouse model of Lynch syndrome (Lgr5-CreERT2;Msh2(flox/-) mice) and found that environmental factors can modify the number and mutability of the MMR-deficient stem cells. These findings provide evidence that environmental factors can promote development of neoplasias and tumors in patients with Lynch syndrome.
Asunto(s)
Adenocarcinoma/inducido químicamente , Adenocarcinoma/genética , Adenoma/inducido químicamente , Adenoma/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/inducido químicamente , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Reparación de la Incompatibilidad de ADN/efectos de los fármacos , Dacarbazina/análogos & derivados , Intestinos/efectos de los fármacos , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenoma/metabolismo , Adenoma/patología , Animales , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Neoplasias Colorrectales Hereditarias sin Poliposis/metabolismo , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Dacarbazina/toxicidad , Modelos Animales de Enfermedad , Femenino , Mucosa Intestinal/metabolismo , Intestinos/patología , Masculino , Ratones Noqueados , Proteína 2 Homóloga a MutS/deficiencia , Proteína 2 Homóloga a MutS/genética , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Factores de Riesgo , Temozolomida , Factores de TiempoRESUMEN
Replication-coupled gene editing using locked nucleic acid-modified single-stranded DNA oligonucleotides (LMOs) can genetically engineer mammalian cells with high precision at single nucleotide resolution. Based on this method, oligonucleotide-directed mutation screening (ODMS) was developed to determine whether variants of uncertain clinical significance of DNA mismatch repair (MMR) genes can cause Lynch syndrome. In ODMS, the appearance of 6-thioguanine-resistant colonies upon introduction of the variant is indicative for defective MMR and hence pathogenicity. Whereas mouse embryonic stem cells (mESCs) hemizygous for MMR genes were used previously, we now show that ODMS can also be applied in wild-type mESCs carrying two functional alleles of each MMR gene. 6-Thioguanine resistance can result from two possible events: first, the mutation is present in only one allele, which is indicative for dominant-negative activity of the variant; and second, both alleles contain the planned modification, which is indicative for a regular loss-of-function variant. Thus, ODMS in wild-type mESCs can discriminate fully disruptive and dominant-negative MMR variants. The feasibility of biallelic targeting suggests that the efficiency of LMO-mediated gene targeting at a nonselectable locus may be enriched in cells that had undergone a simultaneous selectable LMO targeting event. This turned out to be the case and provided a protocol to improve recovery of LMO-mediated gene modification events.
Asunto(s)
Alelos , Reparación de la Incompatibilidad de ADN , Edición Génica , Reparación de la Incompatibilidad de ADN/genética , Edición Génica/métodos , Ratones , Animales , Células Madre Embrionarias de Ratones/metabolismo , Humanos , Proteína 2 Homóloga a MutS/genética , Mutación con Pérdida de Función , Replicación del ADN/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteína 3 Homóloga de MutS/genética , Mutación , Genes Dominantes , Proteínas de Unión al ADNRESUMEN
While there is increasing academic analysis and policy concern regarding growing inequality and the need for more inclusive development trajectories, it is equally important to advance our understanding of the pathways to attain more inclusive development in practice. This paper serves as the introduction to a special issue examining the empirical outcomes and processes of inclusive development policies in selected countries in Africa. The paper presents a policy implementation and assessment framework as a lens that connects the different case studies. The framework links general inclusive development strategies in employment, social protection and governance, to the participation and representation of the various stakeholders as well as the monetary and non-monetary transaction costs in accessing and/or implementing these programmes on the ground in different national and sub-national contexts. Based on the findings of the 9 case studies, the paper also advances policy directions and operational frameworks to attain more inclusive development in practice.
Bien qu'il existe une analyse académique croissante, et une préoccupation politique envers la montée des inégalités et la nécessite de trajectoires de développement plus inclusives, il est tout aussi important d'avancer notre compréhension des chemins qui permettentdans la pratiqued'atteindre un développement plus inclusif. Cet article sert d'introduction à un numéro spécial examinant les résultats empiriques et les processus de développement inclusifs dans certains pays en Afrique. L'article présente un cadre d'implémentation et évaluation des politiques, tel que un prisme qui connecte les différents études de cas. Ce cadre relie des stratégies de développement générales (dans l'emploi, la protections sociale, et la gouvernance) à la participation et la représentation de différents partis intéressés, aussi qu'aux aux couts de transaction (monétaires et autres) dans l'accès à ces programmes et à leur implémentation sur le terrain, dans des contextes nationales et sous-nationales. Nous basant sur les résultats de ces 10 études de cas, l'article avance des directions de politique et des cadres opérationnels, ayant comme but la réalisation pratique d'un développement plus inclusif.
RESUMEN
Short synthetic single-stranded oligodeoxyribonucleotides (ssODNs) can be used to introduce subtle modifications into the genome of mouse embryonic stem cells (ESCs). We have previously shown that effective application of ssODN-mediated gene targeting in ESC requires (transient) suppression of DNA mismatch repair (MMR). However, whereas transient down-regulation of the mismatch recognition protein MSH2 allowed substitution of 3 or 4 nucleotides, 1 or 2 nucleotide substitutions were still suppressed. We now demonstrate that single- or dinucleotide substitution can effectively be achieved by transient down-regulation of the downstream MMR protein MLH1. By exploiting highly specific real-time PCR, we demonstrate the feasibility of substituting a single basepair in a non-selectable gene. However, disabling the MMR machinery may lead to inadvertent mutations. To obtain insight into the mutation rate associated with transient MMR suppression, we have compared the impact of transient and constitutive MMR deficiency on the repair of frameshift intermediates at mono- and dinucleotide repeats. Repair at these repeats relied on the substrate specificity and functional redundancy of the MSH2/MSH6 and MSH2/MSH3 MMR complexes. MLH1 knockdown increased the level of spontaneous mutagenesis, but modified ESCs remained germ line competent. Thus, transient MLH1 suppression provides a valuable extension of the MSH2 knockdown strategy, allowing rapid generation of mice carrying single basepair alterations in their genome.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Reparación de la Incompatibilidad de ADN , ADN de Cadena Simple , Proteínas Nucleares/metabolismo , Oligonucleótidos , Animales , Línea Celular , Repeticiones de Dinucleótido , Regulación hacia Abajo , Células Madre Embrionarias , Mutación del Sistema de Lectura , Técnicas de Silenciamiento del Gen , Marcación de Gen , Ratones , Homólogo 1 de la Proteína MutL , MutagénesisRESUMEN
We use three years of household panel data to analyze the effects of ill-health on household economic outcomes in rural Ethiopia. We examine the immediate effects of various ill-health measures on health expenditure and labor supply, the subsequent coping responses, and finally the effect on income and consumption. We find evidence of substantial economic risk in terms of increased health expenditure and reduced agricultural productivity. Households are able to smooth consumption by resorting to intra-household labor substitution, borrowing and depleting assets. However, maintaining current consumption through borrowing and depletion of assets is unlikely to be sustainable and displays the need for health financing reforms and safety nets that reduce the financial consequences of ill-health.
Asunto(s)
Gastos en Salud , Pobreza , Etiopía , Composición Familiar , Humanos , Población RuralRESUMEN
Gene inactivation in mouse embryonic stem (ES) cells usually affects a single allele that is subsequently transmitted to the mouse germline. Upon breeding to homozygosity the consequences of complete gene ablation can be studied in the context of the complete organism. In many cases, it can be useful to study the consequences of gene ablation already in ES cells, for example, when a cellular phenotype is expected. This requires both alleles of a gene to be disrupted. Besides consecutive targeting by using different selectable marker genes, homozygosity for gene disruption can also be obtained by selecting cells for duplication of (part of) the chromosome carrying the targeted allele with concomitant loss of the wild-type allele.
Asunto(s)
Células Madre Embrionarias/fisiología , Marcación de Gen/métodos , Animales , Ratones , Ratones NoqueadosRESUMEN
Oligonucleotide-mediated gene targeting is an attractive alternative to current procedures to subtly modify the genome of mouse embryonic stem (ES) cells. However, oligonucleotide-directed substitution, insertion or deletion of a single or a few nucleotides was hampered by DNA mismatch repair (MMR). We have developed strategies to circumvent this problem based on findings that the central MMR protein MSH2 acts in two different mismatch recognition complexes: MSH2/MSH6, which mainly recognizes base substitutions; and MSH2/MSH3, which has more affinity for larger loops. We found that oligonucleotide-mediated base substitution could effectively be obtained upon transient suppression of MSH2 protein level, while base insertions were effective in ES cells deficient for MSH3. This method allows substitution of any codon of interest in the genome.
Asunto(s)
ADN de Cadena Simple/genética , Células Madre Embrionarias/fisiología , Marcación de Gen/métodos , Oligonucleótidos/genética , Proteínas/fisiología , Animales , Secuencia de Bases , Reparación de la Incompatibilidad de ADN , Ratones , Datos de Secuencia Molecular , Proteína 3 Homóloga de MutS , Homología de Secuencia de Ácido NucleicoRESUMEN
Loss of activity of the retinoblastoma pathway is a common event in human cancer. Mouse models have revealed that tumorigenesis by loss of Rb was accelerated by concomitant loss of the cell cycle inhibitor p27KIP1. This has been attributed to reduced apoptosis and weakening of the G1 checkpoint. However, the role of p27KIP1 in a recently identified G2 restriction point may offer an alternative explanation for this synergy. Here, we have investigated the significance of the G2 restriction point in Rb-deficient pituitaries. We show that Rb loss in the pituitary gland activated the G2 restriction point, as evidenced by the appearance of cyclin B1-p27KIP1 complexes. Somewhat unexpectedly, these complexes remained present in Rb-deficient tumors. These results indicate that the G2 restriction point does operate in vivo. However, in the pituitary gland, this mechanism seems to retard rather than to prevent tumor growth.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/deficiencia , Fase G2/genética , Neoplasias Hipofisarias/patología , Proteína de Retinoblastoma/deficiencia , Animales , Transformación Celular Neoplásica/genética , Ciclina B/metabolismo , Ciclina B1 , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ratones , Hipófisis/citología , Hipófisis/metabolismo , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismoRESUMEN
Oligonucleotide-mediated gene targeting is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. However, its efficacy is strongly suppressed by DNA mismatch repair (MMR). Here we report a simple and rapid procedure for the generation of mouse mutants using transient down regulation of the central MMR protein MSH2 by RNA interference. We demonstrate that under this condition, unmodified single-stranded DNA oligonucleotides can be used to substitute single or several nucleotides. In particular, simultaneous substitution of four adjacent nucleotides was highly efficient, providing the opportunity to substitute virtually any given codon. We have used this method to create a codon substitution (N750F) in the Rb gene of mouse ES cells and show that the oligonucleotide-modified Rb allele can be transmitted through the germ line of mice.
Asunto(s)
Células Madre Embrionarias/metabolismo , Marcación de Gen/métodos , Ratones Mutantes , Sustitución de Aminoácidos , Animales , Células Cultivadas , Codón , Reparación de la Incompatibilidad de ADN , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Genes de Retinoblastoma , Ratones , Ratones Noqueados , Proteína 2 Homóloga a MutS/antagonistas & inhibidores , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga de MutS , Mutagénesis , Oligonucleótidos , Mutación Puntual , Proteínas/genética , Interferencia de ARNRESUMEN
In cancer cells, loss of G1/S control is often accompanied by p53 pathway inactivation, the latter usually rationalized as a necessity for suppressing cell cycle arrest and apoptosis. However, we found an unanticipated effect of p53 loss in mouse and human G1-checkpoint-deficient cells: reduction of DNA damage. We show that abrogation of the G1/S-checkpoint allowed cells to enter S-phase under growth-restricting conditions at the expense of severe replication stress manifesting as decelerated DNA replication, reduced origin firing and accumulation of DNA double-strand breaks. In this system, loss of p53 allowed mitogen-independent proliferation, not by suppressing apoptosis, but rather by restoring origin firing and reducing DNA breakage. Loss of G1/S control also caused DNA damage and activation of p53 in an in vivo retinoblastoma model. Moreover, in a teratoma model, loss of p53 reduced DNA breakage. Thus, loss of p53 may promote growth of incipient cancer cells by reducing replication-stress-induced DNA damage.
Asunto(s)
Daño del ADN/genética , Replicación del ADN/genética , Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Humanos , Ratones , Neoplasias/patología , Fase S/genética , Teratoma/genética , Teratoma/patologíaRESUMEN
Gene targeting through homologous recombination in murine embryonic stem (ES) cells is already strongly suppressed by DNA mismatch-repair (MMR)-dependent anti-recombination when targeting construct and target locus differ at <1% of the nucleotide positions. We demonstrate that MMR activity also raises a strong impediment to gene modification mediated by small synthetic DNA oligonucleotide sequences. In the absence of the DNA MMR gene MSH2, synthetic single-stranded deoxyribo-oligonucleotides can be used to site-specifically modify the ES cell genome. We show that PCR-based procedures can be used to identify and clone modified cells. By this method we have substituted a single codon in the retinoblastoma gene.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN , Embrión de Mamíferos/metabolismo , Marcación de Gen/métodos , Oligonucleótidos/genética , Células Madre/metabolismo , Sustitución de Aminoácidos , Animales , Secuencia de Bases , ADN de Cadena Simple/genética , Embrión de Mamíferos/citología , Mutación del Sistema de Lectura/genética , Vectores Genéticos/genética , Ratones , Proteína 2 Homóloga a MutS , Mutación , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteína de Retinoblastoma/genética , Eliminación de SecuenciaRESUMEN
The primary role of the mismatch repair (MMR) system is the avoidance of mutations caused by replication and recombination errors. Furthermore, the lethality of methylating agents has been attributed to the processing of O(6)-methylguanine lesions in DNA by MMR. Loss of the MSH2 protein completely abolishes repair function and results in reduced cell killing by methylating agents and accelerated accumulation of methylation-damage-induced mutations. This has raised the question as to whether MMR is also involved in the cellular response to other genotoxic insults. Here we describe that in mice deficient for Msh2, lymphomagenesis was strongly accelerated by an ethylating agent, N-ethyl-N-nitrosourea (ENU), given at a dose that did not induce lymphomas in wild-type mice. This suggests that MMR deficiency and ENU-induced mutagenesis synergistically collaborate in inducing tumorigenesis. To study the interaction between MMR and ENU-induced DNA damage, we compared the lethality and mutagenicity of ENU in MSH2-proficient and -deficient mouse embryonic stem cells. Although MSH2-deficiency only slightly reduced the lethality of ENU, it strongly enhanced the mutagenicity of ENU. Mutation analysis of ENU-induced Hprt mutants revealed that base substitutions occurred predominantly at A-T base-pairs. These results suggest that MMR modulates the processing of ethylation damage at AT base-pairs.