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1.
Genetics ; 2021 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-34850872

RESUMEN

Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant Caenorhabditis elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mouse mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, Nphp4;Bbs5 double mutant mice were not viable and there were fewer mice than expected carrying three mutant alleles. In addition, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with an Nphp4 allele. As cilia are still formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes.

2.
Cell Death Dis ; 12(7): 659, 2021 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-34193827

RESUMEN

Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Daño del ADN , Estrés del Retículo Endoplásmico , Estrés Oxidativo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteínas Reguladoras de la Apoptosis/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica , Macrólidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/farmacología , Factores de Tiempo , Transactivadores/genética , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Rayos X , Pez Cebra/genética , Proteínas de Pez Cebra/genética
3.
Dis Model Mech ; 8(8): 941-55, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-26044958

RESUMEN

Mutations in ESCO2, one of two establishment of cohesion factors necessary for proper sister chromatid cohesion (SCC), cause a spectrum of developmental defects in the autosomal-recessive disorder Roberts syndrome (RBS), warranting in vivo analysis of the consequence of cohesion dysfunction. Through a genetic screen in zebrafish targeting embryonic-lethal mutants that have increased genomic instability, we have identified an esco2 mutant zebrafish. Utilizing the natural transparency of zebrafish embryos, we have developed a novel technique to observe chromosome dynamics within a single cell during mitosis in a live vertebrate embryo. Within esco2 mutant embryos, we observed premature chromatid separation, a unique chromosome scattering, prolonged mitotic delay, and genomic instability in the form of anaphase bridges and micronuclei formation. Cytogenetic studies indicated complete chromatid separation and high levels of aneuploidy within mutant embryos. Amongst aneuploid spreads, we predominantly observed decreases in chromosome number, suggesting that either cells with micronuclei or micronuclei themselves are eliminated. We also demonstrated that the genomic instability leads to p53-dependent neural tube apoptosis. Surprisingly, although many cells required Esco2 to establish cohesion, 10-20% of cells had only weakened cohesion in the absence of Esco2, suggesting that compensatory cohesion mechanisms exist in these cells that undergo a normal mitotic division. These studies provide a unique in vivo vertebrate view of the mitotic defects and consequences of cohesion establishment loss, and they provide a compensation-based model to explain the RBS phenotypes.


Asunto(s)
Acetiltransferasas/genética , Cromátides/metabolismo , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/patología , Ectromelia/genética , Ectromelia/patología , Hipertelorismo/genética , Hipertelorismo/patología , Mutación/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Acetiltransferasas/deficiencia , Acetiltransferasas/metabolismo , Animales , Apoptosis , Segregación Cromosómica , Cromosomas/metabolismo , Pérdida del Embrión/metabolismo , Pérdida del Embrión/patología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Inestabilidad Genómica , Índice Mitótico , Modelos Biológicos , Mutagénesis Insercional/genética , Tubo Neural/metabolismo , Tubo Neural/patología , Fenotipo , Retroviridae/genética , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/metabolismo
4.
Drug Metab Dispos ; 43(7): 1037-44, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25934576

RESUMEN

Since its identification in 2000, sulfotransferase (SULT) 4A1 has presented an enigma to the field of cytosolic SULT biology. SULT4A1 is exclusively expressed in neural tissue, is highly conserved, and has been identified in every vertebrate studied to date. Despite this singular level of conservation, no substrate or function for SULT4A1 has been identified. Previous studies demonstrated that SULT4A1 does not bind the obligate sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, yet SULT4A1 is classified as a SULT superfamily member based on sequence and structural similarities to the other SULTs. In this study, transcription activator-like effector nucleases were used to generate heritable mutations in the SULT4A1 gene of zebrafish. The mutation (SULT4A1(Δ8)) consists of an 8-nucleotide deletion within the second exon of the gene, resulting in a frameshift mutation and premature stop codon after 132 AA. During early adulthood, casual observations were made that mutant zebrafish were exhibiting excessively sedentary behavior during the day. These observations were inconsistent with published reports on activity in zebrafish that are largely diurnal organisms and are highly active during the day. Thus, a decrease in activity during the day represents an abnormal behavior and warranted further systematic analysis. EthoVision video tracking software was used to monitor activity levels in wild-type (WT) and SULT4A1(Δ8/Δ8) fish over 48 hours of a normal light/dark cycle. SULT4A1(Δ8/Δ8) fish were shown to exhibit increased inactivity bout length and frequency as well as a general decrease in daytime activity levels when compared with their WT counterparts.


Asunto(s)
Mutación del Sistema de Lectura/genética , Actividad Motora/genética , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Ansiedad/genética , Ansiedad/psicología , Secuencia de Bases , Desoxirribonucleasas/administración & dosificación , Desoxirribonucleasas/farmacología , Embrión no Mamífero , Exones , Microinyecciones , Datos de Secuencia Molecular , Mutación , Conducta Social
5.
PLoS One ; 9(12): e114632, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25503746

RESUMEN

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.


Asunto(s)
Ingeniería Genética/métodos , Genómica , Técnicas de Genotipaje/métodos , Fenotipo , Temperatura de Transición , Pez Cebra/genética , Animales , Composición de Base , Secuencia de Bases , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Daño del ADN , Análisis Mutacional de ADN , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Hibridación Genética , Mutación , Desnaturalización de Ácido Nucleico , Dedos de Zinc
6.
Pharmacogenet Genomics ; 18(1): 25-35, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18216719

RESUMEN

OBJECTIVE: Approximately 30-40% of grade III-IV toxicity to 5-FU has been associated with partial or profound deficiency in dihydropyrimidine dehydrogenase (DPD), the first of three enzymes in the catabolic pathway of fluoropyrimidines. There remains, however, a subset of patients presenting with 5-FU-associated toxicity despite normal DPD activity, suggesting possible deficiencies in enzymes downstream of DPD: dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or beta-ureidopropionase (BUP-1), encoded by the UPB1 gene. Previously, we reported the identification of inactivating mutations in the DPYS gene that could potentially alter the uracil catabolic pathway in healthy individuals with normal DPD enzyme activity. This study investigates the possible role of UPB1 genetic variations in the regulation of the uracil catabolic pathway in individuals presenting with a deficient uracil breath test (13C-UraBT) despite normal DPD enzyme activity. METHODS: This study included 219 healthy asymptomatic volunteers with known DPD enzyme activity and [2-(13)C]-uracil breath test (UraBT). All samples were genotyped for sequence variations in the UPB1 gene using denaturing high performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation of detected sequence variants by direct sequencing. RESULTS: Seven novel and six previously reported sequence variations were identified, including one nonconservative mutation, which demonstrated 97.3% reduction in BUP-1 activity when expressed in the RKO cell line. CONCLUSION: Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD and/or DHP deficiency and that inactivating mutations in the UPB1 gene might impair uracil catabolism.


Asunto(s)
Amidohidrolasas/genética , Regulación Enzimológica de la Expresión Génica , Uracilo/metabolismo , Amidohidrolasas/metabolismo , Pruebas Respiratorias , Línea Celular , Cromatografía Líquida de Alta Presión , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , beta-Galactosidasa/metabolismo
7.
Pharmacogenet Genomics ; 17(11): 973-87, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18075467

RESUMEN

OBJECTIVE: Dihydropyrimidine dehydrogenase (DPD) deficiency accounts for approximately 43% of grade 3-4 toxicity to 5-fluorouracil. There, however, remain a number of patients presenting with 5-fluorouracil-associated toxicity despite normal DPD enzyme activity, suggesting possible deficiencies in dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or beta-ureidopropionase (BUP-1), encoded by the UPB1 gene. This study investigates the role of DPYS sequence variations in individuals with unexplained molecular basis of altered uracil catabolism. METHODS: This study included 219 asymptomatic healthy volunteers with known DPD enzyme activity and [2-13C]-uracil breath test (UraBT) profiles. All samples were genotyped for sequence variations in the DPYS gene using denaturing high-performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation by direct sequencing. Site-directed mutagenesis and expression analysis were performed to determine the effect of the identified nonconservative mutations on DHP enzyme activity. RESULTS: Seven previously reported and 11 novel sequence variations were identified, including three nonconservative mutations; two of which (L7V and 1635delC) demonstrated decreased DHP activity when expressed in the RKO cell line (P=0.25). The P values were not significant due to the small sample size (n=3); however, a modified [2-13C]-uracil breath test, the 13C-dihydrouracil breath test, was administered to four volunteers to confirm that the 1635delC mutation does in fact reduce in-vivo DHP activity. CONCLUSION: Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD deficiency, and that inactivating mutations in DHP might impair uracil catabolism in cases of normal DPD activity.


Asunto(s)
Antineoplásicos/metabolismo , Dihidrouracilo Deshidrogenasa (NADP)/genética , Regulación de la Expresión Génica , Uracilo/metabolismo , Adulto , Secuencia de Aminoácidos , Pruebas Respiratorias , Cromatografía Líquida de Alta Presión , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Pruebas Diagnósticas de Rutina , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Femenino , Genotipo , Haplotipos , Humanos , Desequilibrio de Ligamiento , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Fenotipo , Homología de Secuencia de Aminoácido
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