Quantification of rare somatic single nucleotide variants by droplet digital PCR using SuperSelective primers.

Somatic single-nucleotide variants (SNVs) occur every time a cell divides, appearing even in healthy tissues at low frequencies. These mutations may accumulate as neutral variants during aging, or eventually, promote the development of neoplasia. Here, we present the SP-ddPCR, a droplet digital PCR (ddPCR) based approach that utilizes customized SuperSelective primers aiming at quantifying the proportion of rare SNVs. For that purpose, we selected five potentially pathogenic variants identified by whole-exome sequencing (WES) occurring at low variant allele frequency (VAF) in at-risk colon healthy mucosa of patients diagnosed with colorectal cancer or advanced adenoma. Additionally, two APC SNVs detected in two cancer lesions were added to the study for WES-VAF validation. SuperSelective primers were designed to quantify SNVs at low VAFs both in silico and in clinical samples. In addition to the two APC SNVs in colonic lesions, SP-ddPCR confirmed the presence of three out of five selected SNVs in the normal colonic mucosa with allelic frequencies ≤ 5%. Moreover, SP-ddPCR showed the presence of two potentially pathogenic variants in the distal normal mucosa of patients with colorectal carcinoma. In summary, SP-ddPCR offers a rapid and feasible methodology to validate next-generation sequencing data and accurately quantify rare SNVs, thus providing a potential tool for diagnosis and stratification of at-risk patients based on their mutational profiling.

Germ line variants in patients with acute myeloid leukemia without a suspicion of hereditary hematologic malignancy syndrome.

Germ line predisposition in acute myeloid leukemia (AML) has gained attention in recent years because of a nonnegligible frequency and an impact on management of patients and their relatives. Risk alleles for AML development may be present in patients without a clinical suspicion of hereditary hematologic malignancy syndrome. In this study we investigated the presence of germ line variants (GVs) in 288 genes related to cancer predisposition in 47 patients with available paired, tumor-normal material, namely bone marrow stroma cells (n = 29), postremission bone marrow (n = 17), and saliva (n = 1). These patients correspond to 2 broad AML categories with heterogeneous genetic background (AML myelodysplasia related and AML defined by differentiation) and none of them had phenotypic abnormalities, previous history of cytopenia, or strong cancer aggregation. We found 11 pathogenic or likely pathogenic variants, 6 affecting genes related to autosomal dominant cancer predisposition syndromes (ATM, DDX41, and CHEK2) and 5 related to autosomal recessive bone marrow failure syndromes (FANCA, FANCM, SBDS, DNAJC21, and CSF3R). We did not find differences in clinical characteristics nor outcome between carriers of GVs vs noncarriers. Further studies in unselected AML cohorts are needed to determine GV incidence and penetrance and, in particular, to clarify the role of ATM nonsense mutations in AML predisposition.

Copy Number Variation on ABCC2-DNMBP Loci Affects the Diversity and Composition of the Fecal Microbiota in Pigs.

Genetic variation in the pig genome partially modulates the composition of porcine gut microbial communities. Previous studies have been focused on the association between single nucleotide polymorphisms (SNPs) and the gut microbiota, but little is known about the relationship between structural variants and fecal microbial traits. The main goal of this study was to explore the association between porcine genome copy number variants (CNVs) and the diversity and composition of pig fecal microbiota. For this purpose, we used whole-genome sequencing data to undertake a comprehensive identification of CNVs followed by a genome-wide association analysis between the estimated CNV status and the fecal bacterial diversity in a commercial Duroc pig population. A CNV predicted as gain (DUP) partially harboring ABCC2-DNMBP loci was associated with richness (P = 5.41 × 10-5, false discovery rate [FDR] = 0.022) and Shannon α-diversity (P = 1.42 × 10-4, FDR = 0.057). The in silico predicted gain of copies was validated by real-time quantitative PCR (qPCR), and its segregation, and positive association with the richness and Shannon α-diversity of the porcine fecal bacterial ecosystem was confirmed in an unrelated F1 (Duroc × Iberian) cross. Our results advise the relevance of considering the role of host-genome structural variants as potential modulators of microbial ecosystems and suggest the ABCC2-DNMBP CNV as a host-genetic factor for the modulation of the diversity and composition of the fecal microbiota in pigs. IMPORTANCE A better understanding of the environmental and host factors modulating gut microbiomes is a topic of greatest interest. Recent evidence suggests that genetic variation in the pig genome partially controls the composition of porcine gut microbiota. However, since previous studies have been focused on the association between single nucleotide polymorphisms and the fecal microbiota, little is known about the relationship between other sources of genetic variation, like the structural variants and microbial traits. Here, we identified, experimentally validated, and replicated in an independent population a positive link between the gain of copies of ABCC2-DNMBP loci and the diversity and composition of pig fecal microbiota. Our results advise the relevance of considering the role of host-genome structural variants as putative modulators of microbial ecosystems and open the possibility of implementing novel holobiont-based management strategies in breeding programs for the simultaneous improvement of microbial traits and host performance.

Antimicrobial Peptides Can Generate Tolerance by Lag and Interfere with Antimicrobial Therapy.

Antimicrobial peptides (AMPs) are widely distributed molecules secreted mostly by cells of the innate immune system to prevent bacterial proliferation at the site of infection. As with classic antibiotics, continued treatment with AMPs can create resistance in bacteria. However, whether AMPs can generate tolerance as an intermediate stage towards resistance is not known. Here, we show that the treatment of Escherichia coli with different AMPs induces tolerance by lag, particularly for those peptides that have internal targets. This tolerance can be detected as different morphological and physiological changes, which depend on the type of peptide molecule the bacterium has been exposed to. In addition, we show that AMP tolerance can also affect antibiotic treatment. The genomic sequencing of AMP-tolerant strains shows that different mutations alter membrane composition, DNA replication, and translation. Some of these mutations have also been observed in antibiotic-resistant strains, suggesting that AMP tolerance could be a relevant step in the development of antibiotic resistance. Monitoring AMP tolerance is relevant vis-á-vis the eventual therapeutic use of AMPs and because cross-tolerance might favor the emergence of resistance against conventional antibiotic treatments.

PrecisionFDA Truth Challenge V2: Calling variants from short and long reads in difficult-to-map regions.

The precisionFDA Truth Challenge V2 aimed to assess the state of the art of variant calling in challenging genomic regions. Starting with FASTQs, 20 challenge participants applied their variant-calling pipelines and submitted 64 variant call sets for one or more sequencing technologies (Illumina, PacBio HiFi, and Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with updated Genome in a Bottle benchmark sets and genome stratifications. Challenge submissions included numerous innovative methods, with graph-based and machine learning methods scoring best for short-read and long-read datasets, respectively. With machine learning approaches, combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants.

Use of patient derived orthotopic xenograft models for real-time therapy guidance in a pediatric sporadic malignant peripheral nerve sheath tumor.

BACKGROUND: The aim of this study was to test the feasibility and utility of developing patient-derived orthotopic xenograft (PDOX) models for patients with malignant peripheral nerve sheath tumors (MPNSTs) to aid therapeutic interventions in real time. PATIENT & METHODS: A sporadic relapsed MPNST developed in a 14-year-old boy was engrafted in mice, generating a PDOX model for use in co-clinical trials after informed consent. SNP-array and exome sequencing was performed on the relapsed tumor. Genomics, drug availability, and published literature guided PDOX treatments. RESULTS: A MPNST PDOX model was generated and expanded. Analysis of the patient's relapsed tumor revealed mutations in the MAPK1, EED, and CDK2NA/B genes. First, the PDOX model was treated with the same therapeutic regimen as received by the patient (everolimus and trametinib); after observing partial response, tumors were left to regrow. Regrown tumors were treated based on mutations (palbociclib and JQ1), drug availability, and published literature (nab-paclitaxel; bevacizumab; sorafenib plus doxorubicin; and gemcitabine plus docetaxel). The patient had a lung metastatic relapse and was treated according to PDOX results, first with nab-paclitaxel, second with sorafenib plus doxorubicin after progression, although a complete response was not achieved and multiple metastasectomies were performed. The patient is currently disease free 46 months after first relapse. CONCLUSION: Our results indicate the feasibility of generating MPNST-PDOX and genomic characterization to guide treatment in real time. Although the treatment responses observed in our model did not fully recapitulate the patient's response, this pilot study identify key aspects to improve our co-clinical testing approach in real time.

Transposons played a major role in the diversification between the closely related almond and peach genomes: results from the almond genome sequence.

We sequenced the genome of the highly heterozygous almond Prunus dulcis cv. Texas combining short- and long-read sequencing. We obtained a genome assembly totaling 227.6 Mb of the estimated almond genome size of 238 Mb, of which 91% is anchored to eight pseudomolecules corresponding to its haploid chromosome complement, and annotated 27 969 protein-coding genes and 6747 non-coding transcripts. By phylogenomic comparison with the genomes of 16 additional close and distant species we estimated that almond and peach (Prunus persica) diverged around 5.88 million years ago. These two genomes are highly syntenic and show a high degree of sequence conservation (20 nucleotide substitutions per kb). However, they also exhibit a high number of presence/absence variants, many attributable to the movement of transposable elements (TEs). Transposable elements have generated an important number of presence/absence variants between almond and peach, and we show that the recent history of TE movement seems markedly different between them. Transposable elements may also be at the origin of important phenotypic differences between both species, and in particular for the sweet kernel phenotype, a key agronomic and domestication character for almond. Here we show that in sweet almond cultivars, highly methylated TE insertions surround a gene involved in the biosynthesis of amygdalin, whose reduced expression has been correlated with the sweet almond phenotype. Altogether, our results suggest a key role of TEs in the recent history and diversification of almond and its close relative peach.

Differences in firing efficiency, chromatin, and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins.

Eukaryotic genome replication depends on thousands of DNA replication origins (ORIs). A major challenge is to learn ORI biology in multicellular organisms in the context of growing organs to understand their developmental plasticity. We have identified a set of ORIs of Arabidopsis thaliana and their chromatin landscape at two stages of post-embryonic development. ORIs associate with multiple chromatin signatures including transcription start sites (TSS) but also proximal and distal regulatory regions and heterochromatin, where ORIs colocalize with retrotransposons. In addition, quantitative analysis of ORI activity led us to conclude that strong ORIs have high GC content and clusters of GGN trinucleotides. Development primarily influences ORI firing strength rather than ORI location. ORIs that preferentially fire at early developmental stages colocalize with GC-rich heterochromatin, but at later stages with transcribed genes, perhaps as a consequence of changes in chromatin features associated with developmental processes. Our study provides the set of ORIs active in an organism at the post-embryo stage that should allow us to study ORI biology in response to development, environment, and mutations with a quantitative approach. In a wider scope, the computational strategies developed here can be transferred to other eukaryotic systems.

Novel Class of Viral Ankyrin Proteins Targeting the Host E3 Ubiquitin Ligase Cullin-2.

Ankyrin repeat (ANK) domains are among the most abundant motifs in eukaryotic proteins. ANK proteins are rare amongst viruses, with the exception of poxviruses, which presumably acquired them from the host via horizontal gene transfer. The architecture of poxvirus ANK proteins is, however, different from that of their cellular counterparts, and this precludes a direct acquisition event. Here we combine bioinformatics analysis and quantitative proteomics to discover a new class of viral ANK proteins with a domain organization that relates to cellular ANK proteins. These noncanonical viral ANK proteins, termed ANK/BC, interact with host Cullin-2 via a C-terminal BC box resembling that of cellular Cullin-2 substrate adaptors such as the von Hippel-Lindau protein. Mutagenesis of the BC box-like sequence abrogates binding to Cullin-2, whereas fusion of this motif to an ANK-only protein confers Cullin-2 association. We demonstrated that these viral ANK/BC proteins are potent immunomodulatory proteins suppressing the activation of the proinflammatory transcription factors NF-κB and interferon (IFN)-responsive factor 3 (IRF-3) and the production of cytokines and chemokines, including interferon, and that association with Cullin-2 is required for optimal inhibitory activity. ANK/BC proteins exist in several orthopoxviruses and cluster into 2 closely related orthologue groups in a phylogenetic lineage that is separate from that of canonical ANK/F-box proteins. Given the existence of cellular proteins with similar architecture, viral ANK/BC proteins may be closely related to the original ANK gene acquired by an ancestral orthopoxvirus. These findings uncover a novel viral strategy to antagonize innate immunity and shed light on the origin of the poxviral ANK protein family.IMPORTANCE Viruses encode multiple proteins aimed at modulating cellular homeostasis and antagonizing the host antiviral response. Most of these genes were originally acquired from the host and subsequently adapted to benefit the virus. ANK proteins are common in eukaryotes but are unusual amongst viruses, with the exception of poxviruses, where they represent one of the largest protein families. We report here the existence of a new class of viral ANK proteins, termed ANK/BC, that provide new insights into the origin of poxvirus ANK proteins. ANK/BC proteins target the host E3 ubiquitin ligase Cullin-2 via a C-terminal BC box domain and are potent suppressors of the production of inflammatory cytokines, including interferon. The existence of cellular ANK proteins whose architecture is similar suggests the acquisition of a host ANK/BC gene by an ancestral orthopoxvirus and its subsequent duplication and adaptation to widen the repertoire of immune evasion strategies.

The Evolutionary Consequences of Transposon-Related Pericentromer Expansion in Melon.

Transposable elements (TEs) are a major driver of plant genome evolution. A part from being a rich source of new genes and regulatory sequences, TEs can also affect plant genome evolution by modifying genome size and shaping chromosome structure. TEs tend to concentrate in heterochromatic pericentromeric regions and their proliferation may expand these regions. Here, we show that after the split of melon and cucumber, TEs have expanded the pericentromeric regions of melon chromosomes that, probably as a consequence, show a very low recombination frequency. In contrast, TEs have not proliferated to a high extent in cucumber, which has small TE-dense pericentromeric regions and shows a relatively constant recombination rate along chromosomes. These differences in chromosome structure also translate in differences in gene nucleotide diversity. Although gene nucleotide diversity is essentially constant along cucumber chromosomes, melon chromosomes show a bimodal pattern of genetic variability, with a gene-poor region where variability is negatively correlated with gene density. Interestingly, genes are not homogeneously distributed in melon, and the high variable low-recombining pericentromeric regions show a higher concentration of melon-specific genes whereas genes shared with cucumber and other plants are essentially found in gene-rich chromosomal arms. The results presented here suggest that melon pericentromeric regions may allow gene sequences to evolve more freely than in other chromosomal compartments which may allow new ORFs to arise and eventually be selected. These results show that TEs can drastically change the structure of chromosomes creating different chromosomal compartments imposing different constraints for gene evolution.

An improved assembly and annotation of the melon (Cucumis melo L.) reference genome.

We report an improved assembly (v3.6.1) of the melon (Cucumis melo L.) genome and a new genome annotation (v4.0). The optical mapping approach allowed correcting the order and the orientation of 21 previous scaffolds and permitted to correctly define the gap-size extension along the 12 pseudomolecules. A new comprehensive annotation was also built in order to update the previous annotation v3.5.1, released more than six years ago. Using an integrative annotation pipeline, based on exhaustive RNA-Seq collections and ad-hoc transposable element annotation, we identified 29,980 protein-coding loci. Compared to the previous version, the v4.0 annotation improved gene models in terms of completeness of gene structure, UTR regions definition, intron-exon junctions and reduction of fragmented genes. More than 8,000 new genes were identified, one third of them being well supported by RNA-Seq data. To make all the new resources easily exploitable and completely available for the scientific community, a redesigned Melonomics genomic platform was released at http://melonomics.net . The resources produced in this work considerably increase the reliability of the melon genome assembly and resolution of the gene models paving the way for further studies in melon and related species.

Plant Lineage-Specific Amplification of Transcription Factor Binding Motifs by Miniature Inverted-Repeat Transposable Elements (MITEs).

Transposable elements are one of the main drivers of plant genome evolution. Transposon insertions can modify the gene coding capacity or the regulation of their expression, the latter being a more subtle effect, and therefore particularly useful for evolution. Transposons have been show to contain transcription factor binding sites that can be mobilized upon transposition with the potential to integrate new genes into transcriptional networks. Miniature inverted-repeat transposable elements (MITEs) are a type of noncoding DNA transposons that could be particularly suited as a vector to mobilize transcription factor binding sites and modify transcriptional networks during evolution. MITEs are small in comparison to other transposons and can be excised, which should make them less mutagenic when inserting into promoters. On the other hand, in spite of their cut-and-paste mechanisms of transposition, they can reach very high copy numbers in genomes. We have previously shown that MITEs have amplified and redistributed the binding motif of the E2F transcription factor in different Brassicas. Here, we show that MITEs have amplified and mobilized the binding motifs of the bZIP60 and PIF3 transcription factors in peach and Prunus mume, and the TCP15/23 binding motif in tomato. Our results suggest that MITEs could have rewired new genes into transcriptional regulatory networks that are responsible for important adaptive responses and breeding traits in plants, such as stress responses, flowering time, or fruit ripening. The results presented here therefore suggest a general impact of MITEs in the evolution of transcriptional regulatory networks in plants.

The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.

Retrotransposons are specified as DNA replication origins in the gene-poor regions of Arabidopsis heterochromatin.

Genomic stability depends on faithful genome replication. This is achieved by the concerted activity of thousands of DNA replication origins (ORIs) scattered throughout the genome. The DNA and chromatin features determining ORI specification are not presently known. We have generated a high-resolution genome-wide map of 3230 ORIs in cultured Arabidopsis thaliana cells. Here, we focused on defining the features associated with ORIs in heterochromatin. In pericentromeric gene-poor domains ORIs associate almost exclusively with the retrotransposon class of transposable elements (TEs), in particular of the Gypsy family. ORI activity in retrotransposons occurs independently of TE expression and while maintaining high levels of H3K9me2 and H3K27me1, typical marks of repressed heterochromatin. ORI-TEs largely colocalize with chromatin signatures defining GC-rich heterochromatin. Importantly, TEs with active ORIs contain a local GC content higher than the TEs lacking them. Our results lead us to conclude that ORI colocalization with retrotransposons is determined by their transposition mechanism based on transcription, and a specific chromatin landscape. Our detailed analysis of ORIs responsible for heterochromatin replication has implications on the mechanisms of ORI specification in other multicellular organisms in which retrotransposons are major components of heterochromatin and of the entire genome.

Variability among Cucurbitaceae species (melon, cucumber and watermelon) in a genomic region containing a cluster of NBS-LRR genes.

BACKGROUND: Cucurbitaceae species contain a significantly lower number of genes coding for proteins with similarity to plant resistance genes belonging to the NBS-LRR family than other plant species of similar genome size. A large proportion of these genes are organized in clusters that appear to be hotspots of variability. The genomes of the Cucurbitaceae species measured until now are intermediate in size (between 350 and 450 Mb) and they apparently have not undergone any genome duplications beside those at the origin of eudicots. The cluster containing the largest number of NBS-LRR genes has previously been analyzed in melon and related species and showed a high degree of interspecific and intraspecific variability. It was of interest to study whether similar behavior occurred in other cluster of the same family of genes. RESULTS: The cluster of NBS-LRR genes located in melon chromosome 9 was analyzed and compared with the syntenic regions in other cucurbit genomes. This is the second cluster in number within this species and it contains nine sequences with a NBS-LRR annotation including two genes, Fom1 and Prv, providing resistance against Fusarium and Ppapaya ring-spot virus (PRSV). The variability within the melon species appears to consist essentially of single nucleotide polymorphisms. Clusters of similar genes are present in the syntenic regions of the two species of Cucurbitaceae that were sequenced, cucumber and watermelon. Most of the genes in the syntenic clusters can be aligned between species and a hypothesis of generation of the cluster is proposed. The number of genes in the watermelon cluster is similar to that in melon while a higher number of genes (12) is present in cucumber, a species with a smaller genome than melon. After comparing genome resequencing data of 115 cucumber varieties, deletion of a group of genes is observed in a group of varieties of Indian origin. CONCLUSIONS: Clusters of genes coding for NBS-LRR proteins in cucurbits appear to have specific variability in different regions of the genome and between different species. This observation is in favour of considering that the adaptation of plant species to changing environments is based upon the variability that may occur at any location in the genome and that has been produced by specific mechanisms of sequence variation acting on plant genomes. This information could be useful both to understand the evolution of species and for plant breeding.

Molecular dynamics study of naturally existing cavity couplings in proteins.

Couplings between protein sub-structures are a common property of protein dynamics. Some of these couplings are especially interesting since they relate to function and its regulation. In this article we have studied the case of cavity couplings because cavities can host functional sites, allosteric sites, and are the locus of interactions with the cell milieu. We have divided this problem into two parts. In the first part, we have explored the presence of cavity couplings in the natural dynamics of 75 proteins, using 20 ns molecular dynamics simulations. For each of these proteins, we have obtained two trajectories around their native state. After applying a stringent filtering procedure, we found significant cavity correlations in 60% of the proteins. We analyze and discuss the structure origins of these correlations, including neighbourhood, cavity distance, etc. In the second part of our study, we have used longer simulations (≥100 ns) from the MoDEL project, to obtain a broader view of cavity couplings, particularly about their dependence on time. Using moving window computations we explored the fluctuations of cavity couplings along time, finding that these couplings could fluctuate substantially during the trajectory, reaching in several cases correlations above 0.25/0.5. In summary, we describe the structural origin and the variations with time of cavity couplings. We complete our work with a brief discussion of the biological implications of these results.

Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly.

BACKGROUND: The genome of the melon (Cucumis melo L.) double-haploid line DHL92 was recently sequenced, with 87.5 and 80.8% of the scaffold assembly anchored and oriented to the 12 linkage groups, respectively. However, insufficient marker coverage and a lack of recombination left several large, gene rich scaffolds unanchored, and some anchored scaffolds unoriented. To improve the anchoring and orientation of the melon genome assembly, we used resequencing data between the parental lines of DHL92 to develop a new set of SNP markers from unanchored scaffolds. RESULTS: A high-resolution genetic map composed of 580 SNPs was used to anchor 354.8 Mb of sequence, contained in 141 scaffolds (average size 2.5 Mb) and corresponding to 98.2% of the scaffold assembly, to the 12 melon chromosomes. Over 325.4 Mb (90%) of the assembly was oriented. The genetic map revealed regions of segregation distortion favoring SC alleles as well as recombination suppression regions coinciding with putative centromere, 45S, and 5S rDNA sites. New chromosome-scale pseudomolecules were created by incorporating to the previous v3.5 version an additional 38.3 Mb of anchored sequence representing 1,837 predicted genes contained in 55 scaffolds. Using fluorescent in situ hybridization (FISH) with BACs that produced chromosome-specific signals, melon chromosomes that correspond to the twelve linkage groups were identified, and a standardized karyotype of melon inbred line T111 was developed. CONCLUSIONS: By utilizing resequencing data and targeted SNP selection combined with a large F2 mapping population, we significantly improved the quantity of anchored and oriented melon scaffold genome assembly. Using genome information combined with FISH mapping provided the first cytogenetic map of an inodorus melon type. With these results it was possible to make inferences on melon chromosome structure by relating zones of recombination suppression to centromeres and 45S and 5S heterochromatic regions. This study represents the first steps towards the integration of the high-resolution genetic and cytogenetic maps with the genomic sequence in melon that will provide more information on genome organization and allow for the improvement of the melon genome draft sequence.

Correction: Mechanisms Regulating GLUT4 Transcription in Skeletal Muscle Cells Are Highly Conserved across Vertebrates.

[This corrects the article DOI: 10.1371/journal.pone.0080628.].

Mechanisms regulating GLUT4 transcription in skeletal muscle cells are highly conserved across vertebrates.

The glucose transporter 4 (GLUT4) plays a key role in glucose uptake in insulin target tissues. This transporter has been extensively studied in many species in terms of its function, expression and cellular traffic and complex mechanisms are involved in its regulation at many different levels. However, studies investigating the transcription of the GLUT4 gene and its regulation are scarce. In this study, we have identified the GLUT4 gene in a teleost fish, the Fugu (Takifugu rubripes), and have cloned and characterized a functional promoter of this gene for the first time in a non-mammalian vertebrate. In silico analysis of the Fugu GLUT4 promoter identified potential binding sites for transcription factors such as SP1, C/EBP, MEF2, KLF, SREBP-1c and GC-boxes, as well as a CpG island, but failed to identify a TATA box. In vitro analysis revealed three transcription start sites, with the main residing 307 bp upstream of the ATG codon. Deletion analysis determined that the core promoter was located between nucleotides -132/+94. By transfecting a variety of 5´deletion constructs into L6 muscle cells we have determined that Fugu GLUT4 promoter transcription is regulated by insulin, PG-J2, a PPARγ agonist, and electrical pulse stimulation. Furthermore, our results suggest the implication of motifs such as PPARγ/RXR and HIF-1α in the regulation of Fugu GLUT4 promoter activity by PPARγ and contractile activity, respectively. These data suggest that the characteristics and regulation of the GLUT4 promoter have been remarkably conserved during the evolution from fish to mammals, further evidencing the important role of GLUT4 in metabolic regulation in vertebrates.

The relationship between gene isoform multiplicity, number of exons and protein divergence.

At present we know that phenotypic differences between organisms arise from a variety of sources, like protein sequence divergence, regulatory sequence divergence, alternative splicing, etc. However, we do not have yet a complete view of how these sources are related. Here we address this problem, studying the relationship between protein divergence and the ability of genes to express multiple isoforms. We used three genome-wide datasets of human-mouse orthologs to study the relationship between isoform multiplicity co-occurrence between orthologs (the fact that two orthologs have more than one isoform) and protein divergence. In all cases our results showed that there was a monotonic dependence between these two properties. We could explain this relationship in terms of a more fundamental one, between exon number of the largest isoform and protein divergence. We found that this last relationship was present, although with variations, in other species (chimpanzee, cow, rat, chicken, zebrafish and fruit fly). In summary, we have identified a relationship between protein divergence and isoform multiplicity co-occurrence and explained its origin in terms of a simple gene-level property. Finally, we discuss the biological implications of these findings for our understanding of inter-species phenotypic differences.