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[This corrects the article DOI: http://dx.doi.org/10.1289/ehp.1408202.].
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Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney.
Assuntos
Desaminase APOBEC-1/genética , Apolipoproteínas B/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Glomérulos Renais/metabolismo , Edição de RNA , RNA Mensageiro/genética , Desaminase APOBEC-1/metabolismo , Animais , Apolipoproteínas B/metabolismo , Sequência de Bases , Feminino , Ribonucleoproteínas Nucleares Heterogêneas/deficiência , Intestino Delgado/metabolismo , Glomérulos Renais/patologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Fatores Sexuais , Desequilíbrio HidroeletrolíticoRESUMO
RNA editing refers to post-transcriptional processes that alter the base sequence of RNA. Recently, hundreds of new RNA editing targets have been reported. However, the mechanisms that determine the specificity and degree of editing are not well understood. We examined quantitative variation of site-specific editing in a genetically diverse multiparent population, Diversity Outbred mice, and mapped polymorphic loci that alter editing ratios globally for C-to-U editing and at specific sites for A-to-I editing. An allelic series in the C-to-U editing enzyme Apobec1 influences the editing efficiency of Apob and 58 additional C-to-U editing targets. We identified 49 A-to-I editing sites with polymorphisms in the edited transcript that alter editing efficiency. In contrast to the shared genetic control of C-to-U editing, most of the variable A-to-I editing sites were determined by local nucleotide polymorphisms in proximity to the editing site in the RNA secondary structure. Our results indicate that RNA editing is a quantitative trait subject to genetic variation and that evolutionary constraints have given rise to distinct genetic architectures in the two canonical types of RNA editing.
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Variação Genética , Herança Multifatorial , Locos de Características Quantitativas , Edição de RNA , Desaminase APOBEC-1 , Animais , Mapeamento Cromossômico , Citidina Desaminase/química , Citidina Desaminase/genética , Feminino , Perfilação da Expressão Gênica , Genoma , Masculino , CamundongosRESUMO
BACKGROUND: Inhalation of benzene at levels below the current exposure limit values leads to hematotoxicity in occupationally exposed workers. OBJECTIVE: We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure threshold assessment and to identify genetic factors that influence benzene-induced genotoxicity. METHODS: We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was repeated using two independent cohorts of 300 animals each. We measured micronuclei frequency in reticulocytes from peripheral blood and bone marrow and applied benchmark concentration modeling to estimate exposure thresholds. We genotyped the mice and performed linkage analysis. RESULTS: We observed a dose-dependent increase in benzene-induced chromosomal damage and estimated a benchmark concentration limit of 0.205 ppm benzene using DO mice. This estimate is an order of magnitude below the value estimated using B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of overexpressed sulfotransferases that were inversely correlated with genotoxicity. CONCLUSIONS: The genetically diverse DO mice provided a reproducible response to benzene exposure. The DO mice display interindividual variation in toxicity response and, as such, may more accurately reflect the range of response that is observed in human populations. Studies using DO mice can localize genetic associations with high precision. The identification of sulfotransferases as candidate genes suggests that DO mice may provide additional insight into benzene-induced genotoxicity.
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Benzeno/toxicidade , Substâncias Perigosas/toxicidade , Animais , Animais não Endogâmicos , Células da Medula Óssea/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Relação Dose-Resposta a Droga , Ligação Genética/efeitos dos fármacos , Exposição por Inalação , Camundongos , Testes para Micronúcleos , Reticulócitos/efeitos dos fármacos , Medição de Risco , Sulfotransferases/genéticaRESUMO
Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: for line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 21 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3' splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism.
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HDL-Colesterol/metabolismo , Etilnitrosoureia , Variação Genética , Modelos Animais , Fosfolipases A2/genética , Serina Endopeptidases/genética , Fosfatase Alcalina/sangue , Análise de Variância , Animais , Elementos Antissenso (Genética)/genética , Northern Blotting , Western Blotting , Mapeamento Cromossômico , Cruzamentos Genéticos , Potenciais Evocados Auditivos do Tronco Encefálico , Sequenciamento de Nucleotídeos em Larga Escala , Lipídeos/sangue , Escore Lod , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Mutagênese/genética , Especificidade da Espécie , Tiroxina/sangueRESUMO
In an effort to discover new mouse models of cardiovascular disease using N-ethyl-N-nitrosourea (ENU) mutagenesis followed by high-throughput phenotyping, we have identified a new mouse mutation, C699Y, in the LDL receptor (Ldlr), named wicked high cholesterol (WHC). When WHC was compared with the widely used Ldlr knockout (KO) mouse, notable phenotypic differences between strains were observed, such as accelerated atherosclerotic lesion formation and reduced hepatosteatosis in the ENU mutant after a short exposure to an atherogenic diet. This loss-of-function mouse model carries a single base mutation in the Ldlr gene on an otherwise pure C57BL/6J (B6) genetic background, making it a useful new tool for understanding the pathophysiology of atherosclerosis and for evaluating additional genetic modifiers regulating hyperlipidemia and atherogenesis. Further investigation of genomic differences between the ENU mutant and KO strains may reveal previously unappreciated sequence functionality.
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Etilnitrosoureia/administração & dosagem , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Mutagênicos/administração & dosagem , Mutação de Sentido Incorreto/genética , Receptores de LDL/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Modelos Animais de Doenças , Feminino , Hiperlipoproteinemia Tipo II/induzido quimicamente , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Dados de Sequência Molecular , Receptores de LDL/deficiênciaRESUMO
The mouse has been a powerful force in elucidating the genetic basis of human physiology and pathophysiology. From its beginnings as the model organism for cancer research and transplantation biology to the present, when dissection of the genetic basis of complex disease is at the forefront of genomics research, an enormous and remarkable mouse resource infrastructure has accumulated. This review summarizes those resources and provides practical guidelines for their use, particularly in the analysis of quantitative traits.