101 research outputs found
Chromosome-scale and haplotype-resolved genome assembly of a tetraploid potato cultivar
Potato is the most widely produced tuber crop worldwide. However, reconstructing the four haplotypes of its autotetraploid genome remained an unsolved challenge. Here, we report the 3.1 Gb haplotype-resolved (at 99.6% precision), chromosome-scale assembly of the potato cultivar ‘Otava’ based on high-quality long reads, single-cell sequencing of 717 pollen genomes and Hi-C data. Unexpectedly, ~50% of the genome was identical-by-descent due to recent inbreeding, which was contrasted by highly abundant structural rearrangements involving ~20% of the genome. Among 38,214 genes, only 54% were present in all four haplotypes with an average of 3.2 copies per gene. Taking the leaf transcriptome as an example, 11% of the genes were differently expressed in at least one haplotype, where 25% of them were likely regulated through allele-specific DNA methylation. Our work sheds light on the recent breeding history of potato, the functional organization of its tetraploid genome and has the potential to strengthen the future of genomics-assisted breeding
Unraveling structural rearrangements of the CFH gene cluster in atypical hemolytic uremic syndrome patients using molecular combing and long-fragment targeted sequencing
Complement factor H (CFH) and its related proteins have an essential role in regulating the alternative pathway of the complement system. Mutations and structural variants (SVs) of the CFH gene cluster, consisting of CFH and its five related genes (CFHR1-5), have been reported in renal pathologies as well as in complex immune diseases like age-related macular degeneration and systemic lupus erythematosus. SV analysis of this cluster is challenging due to its high degree of sequence homology. Following first-line NGS gene panel sequencing, we applied Genomic Vision's Molecular Combing Technology, to detect and visualize SVs within the CFH gene cluster and resolve its structural haplotypes completely. This approach was tested in three patients with atypical hemolytic uremic syndrome (aHUS) and known SVs, and 18 patients with aHUS or complement factor 3 glomerulopathy with unknown CFH gene cluster haplotypes. Three SVs, a CFH/CFHR1 hybrid gene in two patients and a rare heterozygous CFHR4/CFHR1 deletion in trans with the common CFHR3/CFHR1 deletion in a third patient were newly identified. For the latter, the breakpoints were determined using a targeted enrichment approach for long DNA fragments (Samplix Xdrop) in combination with Oxford Nanopore sequencing. Molecular combing in addition to NGS was able to improve the molecular genetic yield in this pilot study. This (cost-)effective approach warrants validation in larger cohorts with CFH/CFHR-associated disease
Reduction in the levels of CoQ biosynthetic proteins is related to an increase in lifespan without evidence of hepatic mitohormesis
Mitohormesis is an adaptive response induced by a mild mitochondrial stress that promotes longevity
and metabolic health in different organisms. This mechanism has been proposed as the cause of the
increase in the survival in Coq7+/− (Mclk1+/−) mice, which show hepatic reduction of COQ7, early
mitochondrial dysfunction and increased oxidative stress. Our study shows that the lack of COQ9 in
Coq9Q95X mice triggers the reduction of COQ7, COQ6 and COQ5, which results in an increase in life
expectancy. However, our results reveal that the hepatic CoQ levels are not decreased and, therefore,
neither mitochondrial dysfunction or increased oxidative stress are observed in liver of Coq9Q95X mice.
These data point out the tissue specific differences in CoQ biosynthesis. Moreover, our results suggest
that the effect of reduced levels of COQ7 on the increased survival in Coq9Q95X mice may be due to
mitochondrial mechanisms in non-liver tissues or to other unknown mechanisms.This work was supported by grants from Ministerio de
Economía Competitividad, Spain, and the ERDF (Grant Number SAF2015-65786-R), from the Consejería de
Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (grant number P10-CTS-6133) and from the
University of Granada (grant reference “UNETE”, UCE-PP2017-06). AHG is a “FPU fellow” from the Ministerio
de Educación Cultura y Deporte, Spain. MLS was a predoctoral fellow from the Consejería de Economía,
Innovación, Ciencia y Empleo, Junta de Andalucía. LCL was supported by the “Ramón y Cajal” National
Programme, Ministerio de Economía y Competitividad, Spain (RYC-2011-07643)
The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster
The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation
Methods for Monitoring Matrix-Induced Autophagy.
A growing body of research demonstrates modulation of autophagy by a variety of matrix constituents, including decorin, endorepellin, and endostatin. These matrix proteins are both pro-autophagic and anti-angiogenic. Here, we detail a series of methods to monitor matrix-induced autophagy and its concurrent effects on angiogenesis. We first discuss cloning and purifying proteoglycan fragment and core proteins in the laboratory and review relevant techniques spanning from cell culture to treatment with these purified proteoglycans in vitro and ex vivo. Further, we cover protocols in monitoring autophagic progression via morphological and microscopic characterization, biochemical western blot analysis, and signaling pathway investigation. Downstream angiogenic effects using in vivo approaches are then discussed using wild-type mice and the GFP-LC3 transgenic mouse model. Finally, we explore matrix-induced mitophagy via monitoring changes in mitochondrial DNA and permeability
Choosing proper fluorescent dyes, proteins, and imaging techniques to study mitochondrial dynamics in mammalian cells
QUAREP-LiMi:A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy
mtDNA makes a U-turn for the mitochondrial nucleoid
Mitochondria contain mtDNA derived from the ancestral endosymbiont genome. Important subunits of the oxidative phosphorylation system, which supplies cells with the energy currency ATP, are encoded by mtDNA. A naked mtDNA molecule is longer than a typical mitochondrion and is therefore compacted in vivo to form a nucleoprotein complex, denoted the mitochondrial nucleoid. Mitochondrial transcription factor A (TFAM) is the main factor packaging mtDNA into nucleoids and is also essential for mtDNA transcription initiation. The crystal structure of TFAM shows that it bends mtDNA in a sharp U-turn, which likely provides the structural basis for its dual functions. Super-resolution imaging studies have revealed that the nucleoid has an average diameter of approximately 100nm and frequently contains a single copy of mtDNA. In this review the structure of the mitochondrial nucleoid and its possible regulatory roles in mtDNA expression will be discussed
Tight control of mitochondrial membrane potential by cytochrome c oxidase
In the present work we have critically examined the use of the KCN-titration technique in the study of the control of the cellular respiration by cytochrome c oxidase (COX) in the presence of the mitochondrial membrane potential (Δψ(mito)) in HepG2 cells. We clearly show that the apparent high inhibition threshold of COX in the presence of maximal Δψ(mito) is due to the KCN-induced decrease of Δψ(mito) and not to a low control of COX on the mitochondrial respiration. The tight control exerted by COX on the Δψ(mito) provides further insights for understanding the pathogenetic mechanisms associated with mitochondrial defects in human neuromuscular degenerative disorders
Imaging Reporter Strategy to Monitor Gene Activation of Microglia Polarisation States under Stimulation
Microglial cells as innate immune key players have a critical and unique role in neurodegenerative disorders. They strongly interact with their microenvironment in a complex manner and react to changes by switching their phenotype and functional activation states. In order to understand the development of brain diseases, it is imperative to elucidate up- or down-regulation of genes involved in microglia polarisation in time-profile by a simple-to-use strategy. Here, we present a new imaging strategy to follow promoter activity of genes involved in microglia polarisation. We lentivirally transduced BV-2 microglia cells in culture with constructs consisting of the induced nitric oxide synthase (iNOS), Fc gamma receptor III (Fcgr3) (both resembling the pro-inflammatory M1-like phenotype) or Chitinase-like 3 (Chil3/Ym1) (resembling the anti-inflammatory M2-like phenotype) promoters and stimulated transgenic cells with potent activators for pro- or anti-inflammatory response, such as lipopolysaccharide (LPS) + interferon gamma (IFN-gamma) or interleukin (IL)-4, respectively. Promoter activities upon polarisation phases were quantitatively assessed by the two imaging reporters Luc2 for bioluminescence and eGFP for fluorescence
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