RESUMO
Interactions or epistasis between genetic factors may contribute to "missing heritability." While linkage analyses detect epistasis, defining the limits of the interacting segments poses a significant challenge especially when the interactions are between loci in close proximity. The goal of the present study was to isolate two such epistatic blood pressure (BP) loci on rat chromosome 5. A panel of S.LEW bicongenic strains along with the corresponding monocongenic strains was constructed. BP of each set comprising of one bicongenic and two corresponding monocongenic strains were determined along with the parental Salt-sensitive (S) strain. Epistasis was observed in one out of four sets of congenic strains, wherein systolic blood pressures (SBP) of the two monocongenic strains S.LEW(5)x6Bx9x5a and S.LEW(5)x6Bx9x5b were comparable to that of S, but the SBP of the bicongenic strain S.LEW(5)x6Bx9x5 (157 ± 4.3 mmHg) was significantly lower than that of S (196 ± 6.8 mmHg, P < 0.001). A two-way ANOVA indicated significant interactions between the LEW alleles at the two loci. The interacting loci were 2.02 Mb apart and located within genomic segments spanning 7.77 and 4.18 Mb containing 7,360 and 2,753 candidate variants, respectively. The current study demonstrates definitive evidence for epistasis and provides genetic tools for further dissection of the isolated epistatic BP loci.
Assuntos
Pressão Sanguínea/fisiologia , Epistasia Genética/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Hipertensão/fisiopatologia , Animais , Pressão Sanguínea/genética , Epistasia Genética/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Genótipo , Hipertensão/genética , Locos de Características Quantitativas/genética , RatosRESUMO
The common marmoset (Callithrix jacchus) is an omnivorous New World primate whose diet in the wild includes large amounts of fruit, seeds, flowers, and a variety of lizards and invertebrates. Marmosets also feed heavily on tree gums and exudates, and they have evolved unique morphological and anatomical characteristics to facilitate gum feeding (gummivory). In this study, we characterized the fecal microbiomes of adult and infant animals from a captive population of common marmosets at the Callitrichid Research Center at the University of Nebraska at Omaha under their normal dietary and environmental conditions. The microbiomes of adult animals were dominated by species of Bifidobacterium, Bacteroides, Prevotella, Phascolarctobacterium, Megamonas, and Megasphaera. Culturing and genomic analysis of the Bifidobacterium populations from adult animals identified four known marmoset-associated species (B. reuteri, B. aesculapii, B. myosotis, and B. hapali) and three unclassified taxa of Bifidobacterium that are phylogenetically distinct. Species-specific quantitative PCR (qPCR) confirmed that these same species of Bifidobacterium are abundant members of the microbiome throughout the lives of the animals. Genomic loci in each Bifidobacterium species encode enzymes to support growth and major marmoset milk oligosaccharides during breastfeeding; however, metabolic islands that can support growth on complex polysaccharide substrates in the diets of captive adults (pectin, xyloglucan, and xylan), including loci in B. aesculapii that can support its unique ability to grow on arabinogalactan-rich tree gums, were species-specific. IMPORTANCEBifidobacterium species are recognized as important, beneficial microbes in the human gut microbiome, and their ability colonize individuals at different stages of life is influenced by host, dietary, environmental, and ecological factors, which is poorly understood. The common marmoset is an emerging nonhuman primate model with a short maturation period, making this model amenable to study the microbiome throughout a life history. Features of the microbiome in captive marmosets are also shared with human gut microbiomes, including abundant populations of Bifidobacterium species. Our studies show that several species of Bifidobacterium are dominant members of the captive marmoset microbiome throughout their life history. Metabolic capacities in genomes of the marmoset Bifidobacterium species suggest species-specific adaptations to different components of the captive marmoset diet, including the unique capacity in B. aesculapii for degradation of gum arabic, suggesting that regular dietary exposure in captivity may be important for preserving gum-degrading species in the microbiome.
Assuntos
Adaptação Fisiológica/genética , Bifidobacterium/genética , Bifidobacterium/fisiologia , Callithrix/microbiologia , Microbioma Gastrointestinal/genética , Genoma Bacteriano , Especificidade da Espécie , Animais , Bactérias/classificação , Bactérias/genética , DNA Bacteriano/genética , Dieta , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/fisiologia , Goma Arábica/metabolismo , Masculino , FilogeniaRESUMO
BACKGROUND: Previously, using linkage analysis and substitution mapping, two closely-linked interactive blood pressure quantitative trait loci (QTLs), BP QTL1 and BP QTL2, were located within a 13.96 Mb region from 117894038 to 131853815 bp (RGSC 3.4 version) on rat chromosome 5 (RNO5). This was done by using a series of congenic strains consisting of genomic segments of the Dahl salt-sensitive (S) rat substituted with that of the normotensive Lewis (LEW) rat. The interactive nature of the two loci was further confirmed by the construction and characterization of a panel of S.LEW bicongenic strains and corresponding S.LEW monocongenic strains, which provided definitive evidence of epistasis (genetic interaction) between BP QTL1 (7.77 Mb) and BP QTL2 (4.18 Mb). The purpose of this work was to further map these interacting QTLs. METHOD: A new panel of seven new S.LEW bicongenic strains was constructed and characterized for BP. RESULTS: The data obtained from these new strains further resolved BP QTL1 from 7.77 to 2.93 Mb. Further, BP QTL2 was traceable as not being a single QTL, but a composite of at least three QTLs, LEW alleles at two of which located within 2.26 Mb and 175 kb lowered BP but the third one located within 1.31 Mb increased BP. CONCLUSION: Lack of coding variation within any of the regions further mapped within the previous QTL2 suggests noncoding variation as likely responsible for the observed epistasis.
Assuntos
Pressão Sanguínea/genética , Epistasia Genética , Hipertensão/genética , Locos de Características Quantitativas , Alelos , Animais , Animais Congênicos , Mapeamento Cromossômico , Ratos , Ratos Endogâmicos Dahl , Ratos Endogâmicos LewRESUMO
Genetic dissection of blood pressure (BP) quantitative trait loci (QTLs) in rats has facilitated the fine-mapping of regions linked to the inheritance of hypertension. The goal of the current study was to further fine-map one such genomic region on rat chromosome 1 (BPQTL1b1), the homologous region of which on human chromosome 15 harbors BP QTLs, as reported by four independent studies. Of the six substrains constructed and studied, the systolic BP of two of the congenic strains were significantly lower by 36 and 27 mm Hg than that of the salt-sensitive (S) rat (P < 0.0001, P = 0.0003, respectively). The congenic segments of these two strains overlapped between 135.12 and 138.78 Mb and contained eight genes and two predicted miRNAs. None of the annotations had variants within expressed sequences. These data taken together with the previous localization resolved QTL1b1 with a 70% improvement from the original 7.39 Mb to the current 2.247 Mb interval. Furthermore, the systolic BP of one of the congenic substrains was significantly higher by 20 mm Hg (P < 0.0001) than the BP of the S rat. The limits of this newly identified QTL with a BP increasing effect (QTL1b1a) were between 134.12 and 135.76 Mb, spanning 1.64 Mb, containing two protein-coding genes, Mctp2 and Rgma, and a predicted miRNA. There were four synonymous variants within Mctp2. These data provide evidence for two independent BP QTLs with opposing BP effects within the previously identified BP QTL1b1 region. Additionally, these findings illustrate the complexity underlying the genetic mechanisms of BP regulation, wherein inherited elements beyond protein-coding sequences or known regulatory regions could be operational.
Assuntos
Pressão Sanguínea/genética , Hipertensão/genética , Locos de Características Quantitativas , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 15 , Humanos , Masculino , Ratos Endogâmicos Dahl , Ratos Endogâmicos LewRESUMO
DNA double-strand breaks (DSBs) in embryonic stem (ES) cells are repaired primarily by homologous recombination (HR). The mechanism by which HR is regulated in these cells, however, remains enigmatic. To gain insight into such regulatory mechanisms, we have asked how protein levels of Rad51, a key component of HR, are controlled in mouse ES cells and mouse embryo fibroblasts (MEFs). The Rad51 protein level is about 15-fold higher in ES cells than in MEFs. The level of Rad51 mRNA, however, is only ~2-fold higher, indicating that the differences in mRNA levels due to rates of transcription or mRNA stability are not sufficient to account for the large difference in the abundance of Rad51 protein. Comparison of Rad51 half-lives between ES cells and MEFs also did not explain the elevated level of Rad51 protein in the ES cells. A comparative assessment of the Rad51 translation level demonstrated that it is translated with much greater efficacy in ES cells than in MEFs. To determine whether this high level of translation in ES cells is a general phenomenon in these cells or whether it is a characteristic of specific proteins, such as those involved with recombination and cell cycle progression, we compared mechanisms that regulate the level of Pcna in ES cells with those that regulate Rad51. The half-life of Pcna and its rate of synthesis were considerably different from those of Rad51 in ES cells, demonstrating that regulation of Rad51 abundance cannot be generalized to other ES cell proteins and not to proteins involved in DNA replication and cell cycle control. Finally, we show that only a small proportion of the abundant Rad51 protein population is activated under basal conditions in ES cells and recruited to DNA DSBs and/or stalled replication forks.
Assuntos
Células-Tronco Embrionárias/metabolismo , Rad51 Recombinase/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , Fatores de Transcrição E2F/metabolismo , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Camundongos , Antígeno Nuclear de Célula em Proliferação/biossíntese , Biossíntese de Proteínas , Estabilidade Proteica , Rad51 Recombinase/genéticaRESUMO
Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.