A large single nucleotide polymorphism (SNP) dataset was used to analyze genome-wide diversity in a diverse collection of watermelon cultivars representing globally cultivated, watermelon genetic diversity. The marker density required for conducting successful association mapping depends on the extent of linkage disequilibrium (LD) within a population. Use of genotyping by sequencing reveals large numbers of SNPs that in turn generate opportunities in genome-wide association mapping and marker-assisted selection, even in crops such as watermelon for which few genomic resources are available. In this paper, we used genome-wide genetic diversity to study LD, selective sweeps, and pairwise FST distributions among worldwide cultivated watermelons to track signals of domestication.
Knowledge of population structure and linkage disequilibrium among the worldwide collections of peppers currently classified as hot, mild, sweet and ornamental types is indispensable for applying association mapping and genomic selection to improve pepper. The current study aimed to resolve the genetic diversity and relatedness of Capsicum annuum germplasm by use of simple sequence repeat (SSR) loci across all chromosomes in samples collected in 2011 and 2012. The physical distance covered by the entire set of SSRs used was 2,265.9 Mb from the 3.48-Gb hot-pepper genome size. The model-based program STRUCTURE was used to infer five clusters, which was further confirmed by classical molecular-genetic diversity analysis. Mean heterozygosity of various loci was estimated to be 0.15. Linkage disequilibrium (LD) was used to identify 17 LD blocks across various chromosomes with sizes from 0.154 Kb to 126.28 Mb. CAMS-142 of chromosome 1 was significantly associated with both capsaicin (CA) and dihydrocapsaicin (DCA) levels. Further, CAMS-142 was located in an LD block of 98.18 Mb. CAMS-142 amplified bands of 244, 268, 283 and 326 bp. Alleles 268 and 283 bp had positive effects on both CA and DCA levels, with an average R(2) of 12.15 % (CA) and 12.3 % (DCA). Eight markers from seven different chromosomes were significantly associated with fruit weight, contributing an average effect of 15 %. CAMS-199, HpmsE082 and CAMS-190 are the three major quantitative trait loci located on chromosomes 8, 9, and 10, respectively, and were associated with fruit weight in samples from both years of the study. This research demonstrates the effectiveness of using genome-wide SSR-based markers to assess features of LD and genetic diversity within C. annuum.
Previous studies suggest that the number of proteins containing covalently bound biotin is larger than previously thought. Here, we report the identity of some of these proteins. Using mass spectrometry, we discovered 108 novel biotinylation sites in the human embryonic kidney HEK293 cell proteome; members of the heat shock protein (HSP) superfamily were overrepresented among the novel biotinylated proteins. About half of the biotinylated proteins also displayed various degrees of methionine oxidation, which is known to play an important role in the defense against reactive oxygen species; for biotinylated HSPs, the percent of methionine sulfoxidation approached 100%. Protein structure analysis suggests that methionine sulfoxides localize in close physical proximity to the biotinylated lysines on the protein surface. Mass spectrometric analysis revealed that between one and five of the methionine residues in the C-terminal KEEKDPGMGAMGGMGGGMGGGMF motif are oxidized in HSP60. The likelihood of methionine sulfoxidation is higher if one of the adjacent lysine residues is biotinylated. Knockdown of HSP60 caused a 60% increase in the level of reactive oxygen species in fibroblasts cultured in biotin-sufficient medium. When HEK293 cells were transferred from biotin-sufficient medium to biotin-free medium, the level of reactive oxygen species increased by >9 times compared with baseline controls and a time-response relationship was evident. High levels of methionine sulfoxidation coincided with cell cycle arrest in the G0/G1 and S phases in biotin-depleted cells. We conclude that biotinylation of lysines synergizes with sulfoxidation of methionines in heat shock proteins such as HSP60 in the defense against reactive oxygen species.
Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2 and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon and kidney cell lines; activities consistently followed the pattern P1>P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.
Holocarboxylase synthetase (HLCS) is a biotin protein ligase, which has a pivotal role in biotin-dependent metabolic pathways and epigenetic phenomena in humans. Knockdown of HLCS produces phenotypes such as heat susceptibility and decreased life span in Drosophila melanogaster, whereas knockout of HLCS appears to be embryonic lethal. HLCS comprises 726 amino acids in four domains. More than 2500 single-nucleotide polymorphisms (SNPs) have been identified in human HLCS. Here, we tested the hypotheses that HLCS SNPs impair enzyme activity, and that biotin supplementation restores the activities of HLCS variants to wild-type levels. We used an in silico approach to identify five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96?L SNP did not alter enzyme kinetics. Our findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.
The molecular mechanisms of genome reprogramming during transcriptional responses to stress are associated with specific chromatin modifications. Available data, however, describe histone modifications only at individual plant genes induced by stress. We have no knowledge of chromatin modifications taking place at genes whose transcription has been down-regulated or on the genome-wide chromatin modification patterns that occur during the plants response to dehydration stress.
Transcription is affected by nucleosomal resistance against polymerase passage. In turn, nucleosomal resistance is determined by DNA sequence, histone chaperones and remodeling enzymes. The contributions of these factors are widely debated: one recent title claims "… DNA-encoded nucleosome organization…" while another title states that "histone-DNA interactions are not the major determinant of nucleosome positions." These opposing conclusions were drawn from similar experiments analyzed by idealized methods. We attempt to resolve this controversy to reveal nucleosomal competency for transcription.
About 40% of the hotspots for meiotic recombination contain the degenerate consensus sequence 5-CCNCCNTNNCCNC-3. Here we present a novel protocol for enriching hotspot sequences from digested genomic DNA by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. The captured hotspots can be released by simple digestion with restriction enzymes for subsequent characterization by second generation sequencing or PCR. The capture protocol specifically enriches hotspot sequences, judged by using fluorophore-conjugated synthetic oligonucleotides and synthetic double-stranded oligonucleotides in combination with PCR. The capture protocol enriches single-stranded DNA, denatured double-stranded DNA, and large fragments (>3000 bp) of digested plasmid DNA with good efficacy. No false positive and false negatives were detected when enriching digested DNA from human cell cultures and primary human cells. The protocol can probably be adapted to enriching sequences other than the hotspot sequence by altering the sequence in the capture oligonucleotide. We intend to apply this protocol in studies assessing effects of micronutrient status on meiotic recombination events in human sperm.
Recent advances in "omics" research have resulted in the creation of large datasets that were generated by consortiums and centers, small datasets that were generated by individual investigators, and bioinformatics tools for mining these datasets. It is important for nutrition laboratories to take full advantage of the analysis tools to interrogate datasets for information relevant to genomics, epigenomics, transcriptomics, proteomics, and metabolomics. This review provides guidance regarding bioinformatics resources that are currently available in the public domain, with the intent to provide a starting point for investigators who want to take advantage of the opportunities provided by the bioinformatics field.
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