Functional and evolutionary genomic inferences in Populus through genome and population sequencing of American and European aspen.

The Populus genus is one of the major plant model systems, but genomic resources have thus far primarily been available for poplar species, and primarily Populus trichocarpa (Torr. & Gray), which was the first tree with a whole-genome assembly. To further advance evolutionary and functional genomic analyses in Populus, we produced genome assemblies and population genetics resources of two aspen species, Populus tremula L. and Populus tremuloides Michx. The two aspen species have distributions spanning the Northern Hemisphere, where they are keystone species supporting a wide variety of dependent communities and produce a diverse array of secondary metabolites. Our analyses show that the two aspens share a similar genome structure and a highly conserved gene content with P. trichocarpa but display substantially higher levels of heterozygosity. Based on population resequencing data, we observed widespread positive and negative selection acting on both coding and noncoding regions. Furthermore, patterns of genetic diversity and molecular evolution in aspen are influenced by a number of features, such as expression level, coexpression network connectivity, and regulatory variation. To maximize the community utility of these resources, we have integrated all presented data within the PopGenIE web resource (PopGenIE.org).

ACES: a machine learning toolbox for clustering analysis and visualization.

BACKGROUND: Studies that aim at explaining phenotypes or disease susceptibility by genetic or epigenetic variants often rely on clustering methods to stratify individuals or samples. While statistical associations may point at increased risk for certain parts of the population, the ultimate goal is to make precise predictions for each individual. This necessitates tools that allow for the rapid inspection of each data point, in particular to find explanations for outliers. RESULTS: ACES is an integrative cluster- and phenotype-browser, which implements standard clustering methods, as well as multiple visualization methods in which all sample information can be displayed quickly. In addition, ACES can automatically mine a list of phenotypes for cluster enrichment, whereby the number of clusters and their boundaries are estimated by a novel method. For visual data browsing, ACES provides a 2D or 3D PCA or Heat Map view. ACES is implemented in Java, with a focus on a user-friendly, interactive, graphical interface. CONCLUSIONS: ACES has been proven an invaluable tool for analyzing large, pre-filtered DNA methylation data sets and RNA-Sequencing data, due to its ease to link molecular markers to complex phenotypes. The source code is available from https://github.com/GrabherrGroup/ACES .

Whiteboard: a framework for the programmatic visualization of complex biological analyses.

UNLABELLED: Whiteboard is a class library implemented in C++ that enables visualization to be tightly coupled with computation when analyzing large and complex datasets. AVAILABILITY AND IMPLEMENTATION: the C++ source code, coding samples and documentation are freely available under the Lesser General Public License from http://whiteboard-class.sourceforge.net/.

Neuropeptide Y family receptors Y1 and Y2 from sea lamprey, Petromyzon marinus.

The vertebrate gene family for neuropeptide Y (NPY) receptors expanded by duplication of the chromosome carrying the ancestral Y1-Y2-Y5 gene triplet. After loss of some duplicates, the ancestral jawed vertebrate had seven receptor subtypes forming the Y1 (including Y1, Y4, Y6, Y8), Y2 (including Y2, Y7) and Y5 (only Y5) subfamilies. Lampreys are considered to have experienced the same chromosome duplications as gnathostomes and should also be expected to have multiple receptor genes. However, previously only a Y4-like and a Y5 receptor have been cloned and characterized. Here we report the cloning and characterization of two additional receptors from the sea lamprey Petromyzon marinus. Sequence phylogeny alone could not with certainty assign their identity, but based on synteny comparisons of P. marinus and the Arctic lamprey, Lethenteron camtschaticum, with jawed vertebrates, the two receptors most likely are Y1 and Y2. Both receptors were expressed in human HEK293 cells and inositol phosphate assays were performed to determine the response to the three native lamprey peptides NPY, PYY and PMY. The three peptides have similar potencies in the nanomolar range for Y1. No obvious response to the three peptides was detected for Y2. Synteny analysis supports identification of the previously cloned receptor as Y4. No additional NPY receptor genes could be identified in the presently available lamprey genome assemblies. Thus, four NPY-family receptors have been identified in lampreys, orthologs of the same subtypes as in humans (Y1, Y2, Y4 and Y5), whereas many other vertebrate lineages have retained additional ancestral subtypes.

Characterization of peptide QRFP (26RFa) and its receptor from amphioxus, Branchiostoma floridae.

A peptide ending with RFamide (Arg-Phe-amide) was discovered independently by three different laboratories in 2003 and named 26RFa or QRFP. In mammals, a longer version of the peptide, 43 amino acids, was identified and found to bind to the orphan G protein-coupled receptor GPR103. We searched the genome database of Branchiostoma floridae (Bfl) for receptor sequences related to those that bind peptides ending with RFa or RYa (including receptors for NPFF, PRLH, GnIH, and NPY). One receptor clustered in phylogenetic analyses with mammalian QRFP receptors. The gene has 3 introns in Bfl and 5 in human, but all intron positions differ, implying that the introns were inserted independently. A QRFP-like peptide consisting of 25 amino acids and ending with RFa was identified in the amphioxus genome. Eight of the ten last amino acids are identical between Bfl and human. The prepro-QRFP gene in Bfl has one intron in the propeptide whereas the human gene lacks introns. The Bfl QRFP peptide was synthesized and the receptor was functionally expressed in human cells. The response was measured as inositol phosphate (IP) turnover. The Bfl QRFP peptide was found to potently stimulate the receptor's ability to induce IP turnover with an EC50 of 0.28nM. Also the human QRFP peptides with 26 and 43 amino acids were found to stimulate the receptor (1.9 and 5.1nM, respectively). Human QRFP with 26 amino acids without the carboxyterminal amide had dramatically lower potency at 1.3µM. Thus, we have identified an amphioxus QRFP-related peptide and a corresponding receptor and shown that they interact to give a functional response.

Population-scale sequencing reveals genetic differentiation due to local adaptation in Atlantic herring.

The Atlantic herring (Clupea harengus), one of the most abundant marine fishes in the world, has historically been a critical food source in Northern Europe. It is one of the few marine species that can reproduce throughout the brackish salinity gradient of the Baltic Sea. Previous studies based on few genetic markers have revealed a conspicuous lack of genetic differentiation between geographic regions, consistent with huge population sizes and minute genetic drift. Here, we present a cost-effective genome-wide study in a species that lacks a genome sequence. We first assembled a muscle transcriptome and then aligned genomic reads to the transcripts, creating an "exome assembly," capturing both exons and flanking sequences. We then resequenced pools of fish from a wide geographic range, including the Northeast Atlantic, as well as different regions in the Baltic Sea, aligned the reads to the exome assembly, and identified 440,817 SNPs. The great majority of SNPs showed no appreciable differences in allele frequency among populations; however, several thousand SNPs showed striking differences, some approaching fixation for different alleles. The contrast between low genetic differentiation at most loci and striking differences at others implies that the latter category primarily reflects natural selection. A simulation study confirmed that the distribution of the fixation index F(ST) deviated significantly from expectation for selectively neutral loci. This study provides insights concerning the population structure of an important marine fish and establishes the Atlantic herring as a model for population genetic studies of adaptation and natural selection.

A universal genomic coordinate translator for comparative genomics.

BACKGROUND: Genomic duplications constitute major events in the evolution of species, allowing paralogous copies of genes to take on fine-tuned biological roles. Unambiguously identifying the orthology relationship between copies across multiple genomes can be resolved by synteny, i.e. the conserved order of genomic sequences. However, a comprehensive analysis of duplication events and their contributions to evolution would require all-to-all genome alignments, which increases at N2 with the number of available genomes, N. RESULTS: Here, we introduce Kraken, software that omits the all-to-all requirement by recursively traversing a graph of pairwise alignments and dynamically re-computing orthology. Kraken scales linearly with the number of targeted genomes, N, which allows for including large numbers of genomes in analyses. We first evaluated the method on the set of 12 Drosophila genomes, finding that orthologous correspondence computed indirectly through a graph of multiple synteny maps comes at minimal cost in terms of sensitivity, but reduces overall computational runtime by an order of magnitude. We then used the method on three well-annotated mammalian genomes, human, mouse, and rat, and show that up to 93% of protein coding transcripts have unambiguous pairwise orthologous relationships across the genomes. On a nucleotide level, 70 to 83% of exons match exactly at both splice junctions, and up to 97% on at least one junction. We last applied Kraken to an RNA-sequencing dataset from multiple vertebrates and diverse tissues, where we confirmed that brain-specific gene family members, i.e. one-to-many or many-to-many homologs, are more highly correlated across species than single-copy (i.e. one-to-one homologous) genes. Not limited to protein coding genes, Kraken also identifies thousands of newly identified transcribed loci, likely non-coding RNAs that are consistently transcribed in human, chimpanzee and gorilla, and maintain significant correlation of expression levels across species. CONCLUSIONS: Kraken is a computational genome coordinate translator that facilitates cross-species comparisons, distinguishes orthologs from paralogs, and does not require costly all-to-all whole genome mappings. Kraken is freely available under LPGL from http://github.com/nedaz/kraken.

The vertebrate ancestral repertoire of visual opsins, transducin alpha subunits and oxytocin/vasopressin receptors was established by duplication of their shared genomic region in the two rounds of early vertebrate genome duplications.

BACKGROUND: Vertebrate color vision is dependent on four major color opsin subtypes: RH2 (green opsin), SWS1 (ultraviolet opsin), SWS2 (blue opsin), and LWS (red opsin). Together with the dim-light receptor rhodopsin (RH1), these form the family of vertebrate visual opsins. Vertebrate genomes contain many multi-membered gene families that can largely be explained by the two rounds of whole genome duplication (WGD) in the vertebrate ancestor (2R) followed by a third round in the teleost ancestor (3R). Related chromosome regions resulting from WGD or block duplications are said to form a paralogon. We describe here a paralogon containing the genes for visual opsins, the G-protein alpha subunit families for transducin (GNAT) and adenylyl cyclase inhibition (GNAI), the oxytocin and vasopressin receptors (OT/VP-R), and the L-type voltage-gated calcium channels (CACNA1-L). RESULTS: Sequence-based phylogenies and analyses of conserved synteny show that the above-mentioned gene families, and many neighboring gene families, expanded in the early vertebrate WGDs. This allows us to deduce the following evolutionary scenario: The vertebrate ancestor had a chromosome containing the genes for two visual opsins, one GNAT, one GNAI, two OT/VP-Rs and one CACNA1-L gene. This chromosome was quadrupled in 2R. Subsequent gene losses resulted in a set of five visual opsin genes, three GNAT and GNAI genes, six OT/VP-R genes and four CACNA1-L genes. These regions were duplicated again in 3R resulting in additional teleost genes for some of the families. Major chromosomal rearrangements have taken place in the teleost genomes. By comparison with the corresponding chromosomal regions in the spotted gar, which diverged prior to 3R, we could time these rearrangements to post-3R. CONCLUSIONS: We present an extensive analysis of the paralogon housing the visual opsin, GNAT and GNAI, OT/VP-R, and CACNA1-L gene families. The combined data imply that the early vertebrate WGD events contributed to the evolution of vision and the other neuronal and neuroendocrine functions exerted by the proteins encoded by these gene families. In pouched lamprey all five visual opsin genes have previously been identified, suggesting that lampreys diverged from the jawed vertebrates after 2R.

Expansion of transducin subunit gene families in early vertebrate tetraploidizations.

Hundreds of gene families expanded in the early vertebrate tetraploidizations including many gene families in the phototransduction cascade. We have investigated the evolution of the heterotrimeric G-proteins of photoreceptors, the transducins, in relation to these events using both phylogenetic analyses and synteny comparisons. Three alpha subunit genes were identified in amniotes and the coelacanth, GNAT1-3; two of these were identified in amphibians and teleost fish, GNAT1 and GNAT2. Most tetrapods have four beta genes, GNB1-4, and teleosts have additional duplicates. Finally, three gamma genes were identified in mammals, GNGT1, GNG11 and GNGT2. Of these, GNGT1 and GNGT2 were found in the other vertebrates. In frog and zebrafish additional duplicates of GNGT2 were identified. Our analyses show all three transducin families expanded during the early vertebrate tetraploidizations and the beta and gamma families gained additional copies in the teleost-specific genome duplication. This suggests that the tetraploidizations contributed to visual specialisations.

The evolution of vertebrate somatostatin receptors and their gene regions involves extensive chromosomal rearrangements.

BACKGROUND: Somatostatin and its related neuroendocrine peptides have a wide variety of physiological functions that are mediated by five somatostatin receptors with gene names SSTR1-5 in mammals. To resolve their evolution in vertebrates we have investigated the SSTR genes and a large number of adjacent gene families by phylogeny and conserved synteny analyses in a broad range of vertebrate species. RESULTS: We find that the SSTRs form two families that belong to distinct paralogons. We observe not only chromosomal similarities reflecting the paralogy relationships between the SSTR-bearing chromosome regions, but also extensive rearrangements between these regions in teleost fish genomes, including fusions and translocations followed by reshuffling through intrachromosomal rearrangements. These events obscure the paralogy relationships but are still tractable thanks to the many genomes now available. We have identified a previously unrecognized SSTR subtype, SSTR6, previously misidentified as either SSTR1 or SSTR4. CONCLUSIONS: Two ancestral SSTR-bearing chromosome regions were duplicated in the two basal vertebrate tetraploidizations (2R). One of these ancestral SSTR genes generated SSTR2, -3 and -5, the other gave rise to SSTR1, -4 and -6. Subsequently SSTR6 was lost in tetrapods and SSTR4 in teleosts. Our study shows that extensive chromosomal rearrangements have taken place between related chromosome regions in teleosts, but that these events can be resolved by investigating several distantly related species.

Differential evolution of voltage-gated sodium channels in tetrapods and teleost fishes.

The voltage-gated sodium channel (SCN) alpha subunits are large proteins with central roles in the generation of action potentials. They consist of approximately 2,000 amino acids encoded by 24-27 exons. Previous evolutionary studies have been unable to reconcile the proposed gene duplication schemes with the species distribution and molecular phylogeny of the genes. We have carefully annotated the complete SCN gene sequences, correcting numerous database errors, for a broad range of vertebrate species and analyzed their phylogenetic relationships. We have also compared the chromosomal positions of the SCN genes relative to adjacent gene families. Our studies show that the ancestor of the vertebrates probably had a single sodium channel gene with two characteristic AT-AC introns, the second of which is unique to vertebrate SCN genes. This ancestral gene, located close to a HOX gene cluster, was quadrupled along with HOX in the two rounds of basal vertebrate tetraploidizations to generate the ancestors of the four channels SCN1A, SCN4A, SCN5A, and SCN8A. The third tetraploidization in the teleost fish ancestor doubled this set of genes and all eight are still present in at least three of four investigated teleost fish genomes. In tetrapods, the gene family expanded by local duplications before the radiation of amniotes, generating the cluster SCN5A, SCN10A, and SCN11A on one chromosome and the cluster SCN1A, SCN2A, SCN3A, and SCN9A on a different chromosome. In eutherian mammals, a tenth gene, SCN7A, arose in a local duplication in the SCN1A gene cluster. The SCN7A gene has undergone rapid evolution and has lost the ability to cause action potentials-instead, it functions as a sodium sensor. The three genes in the SCN5A cluster were translocated from the HOX-bearing chromosome in a mammalian ancestor along with several adjacent genes. This evolutionary scenario is supported by the adjacent TGF-ß receptor superfamily (comprised of five distinct families) and the cysteine-serine-rich nuclear protein gene family as well as the HOX clusters. The independent expansions of the SCN repertoires in tetrapods and teleosts suggest that the functional diversification may differ between the two lineages.

Evolution of vertebrate opioid receptors.

The opioid peptides and receptors have prominent roles in pain transmission and reward mechanisms in mammals. The evolution of the opioid receptors has so far been little studied, with only a few reports on species other than tetrapods. We have investigated species representing a broader range of vertebrates and found that the four opioid receptor types (delta, kappa, mu, and NOP) are present in most of the species. The gene relationships were deduced by using both phylogenetic analyses and chromosomal location relative to 20 neighboring gene families in databases of assembled genomes. The combined results show that the vertebrate opioid receptor gene family arose by quadruplication of a large chromosomal block containing at least 14 other gene families. The quadruplication seems to coincide with, and, therefore, probably resulted from, the two proposed genome duplications in early vertebrate evolution. We conclude that the quartet of opioid receptors was already present at the origin of jawed vertebrates approximately 450 million years ago. A few additional opioid receptor gene duplications have occurred in bony fishes. Interestingly, the ancestral receptor gene duplications coincide with the origin of the four opioid peptide precursor genes. Thus, the complete vertebrate opioid system was already established in the first jawed vertebrates.

Comparative Fungal Community Analyses Using Metatranscriptomics and Internal Transcribed Spacer Amplicon Sequencing from Norway Spruce.

The health, growth, and fitness of boreal forest trees are impacted and improved by their associated microbiomes. Microbial gene expression and functional activity can be assayed with RNA sequencing (RNA-Seq) data from host samples. In contrast, phylogenetic marker gene amplicon sequencing data are used to assess taxonomic composition and community structure of the microbiome. Few studies have considered how much of this structural and taxonomic information is included in transcriptomic data from matched samples. Here, we described fungal communities using both host-derived RNA-Seq and fungal ITS1 DNA amplicon sequencing to compare the outcomes between the methods. We used a panel of root and needle samples from the coniferous tree species Picea abies (Norway spruce) growing in untreated (nutrient-deficient) and nutrient-enriched plots at the Flakaliden forest research site in boreal northern Sweden. We show that the relationship between samples and alpha and beta diversity indicated by the fungal transcriptome is in agreement with that generated by the ITS data, while also identifying a lack of taxonomic overlap due to limitations imposed by current database coverage. Furthermore, we demonstrate how metatranscriptomics data additionally provide biologically informative functional insights. At the community level, there were changes in starch and sucrose metabolism, biosynthesis of amino acids, and pentose and glucuronate interconversions, while processing of organic macromolecules, including aromatic and heterocyclic compounds, was enriched in transcripts assigned to the genus Cortinarius IMPORTANCE A deeper understanding of microbial communities associated with plants is revealing their importance for plant health and productivity. RNA extracted from plant field samples represents the host and other organisms present. Typically, gene expression studies focus on the plant component or, in a limited number of studies, expression in one or more associated organisms. However, metatranscriptomic data are rarely used for taxonomic profiling, which is currently performed using amplicon approaches. We created an assembly-based, reproducible, and hardware-agnostic workflow to taxonomically and functionally annotate fungal RNA-Seq data obtained from Norway spruce roots, which we compared to matching ITS amplicon sequencing data. While we identified some limitations and caveats, we show that functional, taxonomic, and compositional insights can all be obtained from RNA-Seq data. These findings highlight the potential of metatranscriptomics to advance our understanding of interaction, response, and effect between host plants and their associated microbial communities.

Neuropeptide Y-family peptides and receptors in the elephant shark, Callorhinchus milii confirm gene duplications before the gnathostome radiation.

We describe here the repertoire of neuropeptide Y (NPY) peptides and receptors in the elephant shark Callorhinchus milii, belonging to the chondrichthyans that diverged from the rest of the gnathostome (jawed vertebrate) lineage about 450 million years ago and the first chondrichthyan with a genome project. We have identified two peptide genes that are orthologous to NPY and PYY (peptide YY) in other vertebrates, and seven receptor genes orthologous to the Y1, Y2, Y4, Y5, Y6, Y7 and Y8 subtypes found in tetrapods and teleost fishes. The repertoire of peptides and receptors seems to reflect the ancestral configuration in the predecessor of all gnathostomes, whereas other lineages such as mammals and teleosts have lost one or more receptor genes or have acquired 1-2 additional peptide genes. Both the peptides and receptors showed broad and overlapping mRNA expression which may explain why some receptor gene losses could take place in some lineages, but leaves open the question why all the known ancestral receptors have been retained in the elephant shark.

Phylogenetic and chromosomal analyses of multiple gene families syntenic with vertebrate Hox clusters.

BACKGROUND: Ever since the theory about two rounds of genome duplication (2R) in the vertebrate lineage was proposed, the Hox gene clusters have served as the prime example of quadruplicate paralogy in mammalian genomes. In teleost fishes, the observation of additional Hox clusters absent in other vertebrate lineages suggested a third tetraploidization (3R). Because the Hox clusters occupy a quite limited part of each chromosome, and are special in having position-dependent regulation within the multi-gene cluster, studies of syntenic gene families are needed to determine the extent of the duplicated chromosome segments. We have analyzed in detail 14 gene families that are syntenic with the Hox clusters to see if their phylogenies are compatible with the Hox duplications and the 2R/3R scenario. Our starting point was the gene family for the NPY family of peptides located near the Hox clusters in the pufferfish Takifugu rubripes, the zebrafish Danio rerio, and human. RESULTS: Seven of the gene families have members on at least three of the human Hox chromosomes and two families are present on all four. Using both neighbor-joining and quartet-puzzling maximum likelihood methods we found that 13 families have a phylogeny that supports duplications coinciding with the Hox cluster duplications. One additional family also has a topology consistent with 2R but due to lack of urochordate or cephalochordate sequences the time window when these duplications could have occurred is wider. All but two gene families also show teleost-specific duplicates. CONCLUSION: Based on this analysis we conclude that the Hox cluster duplications involved a large number of adjacent gene families, supporting expansion of these families in the 2R, as well as in the teleost 3R tetraploidization. The gene duplicates presumably provided raw material in early vertebrate evolution for neofunctionalization and subfunctionalization.

Early vertebrate chromosome duplications and the evolution of the neuropeptide Y receptor gene regions.

BACKGROUND: One of the many gene families that expanded in early vertebrate evolution is the neuropeptide (NPY) receptor family of G-protein coupled receptors. Earlier work by our lab suggested that several of the NPY receptor genes found in extant vertebrates resulted from two genome duplications before the origin of jawed vertebrates (gnathostomes) and one additional genome duplication in the actinopterygian lineage, based on their location on chromosomes sharing several gene families. In this study we have investigated, in five vertebrate genomes, 45 gene families with members close to the NPY receptor genes in the compact genomes of the teleost fishes Tetraodon nigroviridis and Takifugu rubripes. These correspond to Homo sapiens chromosomes 4, 5, 8 and 10. RESULTS: Chromosome regions with conserved synteny were identified and confirmed by phylogenetic analyses in H. sapiens, M. musculus, D. rerio, T. rubripes and T. nigroviridis. 26 gene families, including the NPY receptor genes, (plus 3 described recently by other labs) showed a tree topology consistent with duplications in early vertebrate evolution and in the actinopterygian lineage, thereby supporting expansion through block duplications. Eight gene families had complications that precluded analysis (such as short sequence length or variable number of repeated domains) and another eight families did not support block duplications (because the paralogs in these families seem to have originated in another time window than the proposed genome duplication events). RT-PCR carried out with several tissues in T. rubripes revealed that all five NPY receptors were expressed in the brain and subtypes Y2, Y4 and Y8 were also expressed in peripheral organs. CONCLUSION: We conclude that the phylogenetic analyses and chromosomal locations of these gene families support duplications of large blocks of genes or even entire chromosomes. Thus, these results are consistent with two early vertebrate tetraploidizations forming a paralogon comprising human chromosomes 4, 5, 8 and 10 and one teleost tetraploidization. The combination of positional and phylogenetic data further strengthens the identification of orthologs and paralogs in the NPY receptor family.

Ray-fin fish tetraploidization gave rise to pufferfish duplicates of NPY and PYY, but zebrafish NPY duplicate was lost.

We have used sequence information and gene location to identify NPY family genes in the pufferfish, Takifugu rubripes (fugu), and zebrafish. Fugu has two copies of NPY, presumably resulting from the ray-fin fish tetraploidization. Zebrafish has probably lost one of the copies. Both species have two copies of PYY, the second of which was previously named PY. The two fugu NPY genes are predominantly expressed in brain. The two PYY genes are expressed in a broad range of tissues including brain and gonads. Thus, the NPY system appears to be more complex in teleosts than in tetrapods.

Pufferfish and zebrafish have five distinct NPY receptor subtypes, but have lost appetite receptors Y1 and Y5.

The two neuropeptide Y (NPY) receptors Y1 and Y5 stimulate feeding in mammals, but are missing in the euteleosts, zebrafish and pufferfish (Takifugu rubripes). Both species have five other subtypes called Y2, Y7, Ya, Yb, and Yc. RT-PCR studies in pufferfish show that all five are expressed in the brain and may mediate NPY effects on feeding. Y2, Ya, and Yb are also broadly expressed in peripheral organs. These results reveal interesting differences in the NPY system of teleosts and mammals that may have arisen in the genetic turmoil involving the basal ray-fin fish tetraploidization.

Ancestral vertebrate complexity of the opioid system.

The evolution of the opioid peptides and nociceptin/orphanin as well as their receptors has been difficult to resolve due to variable evolutionary rates. By combining sequence comparisons with information on the chromosomal locations of the genes, we have deduced the following evolutionary scenario: The vertebrate predecessor had one opioid precursor gene and one receptor gene. The two genome doublings before the vertebrate radiation resulted in three peptide precursor genes whereupon a fourth copy arose by a local gene duplication. These four precursors diverged to become the prepropeptides for endorphin (POMC), enkephalins, dynorphins, and nociceptin, respectively. The ancestral receptor gene was quadrupled in the genome doublings leading to delta, kappa, and mu and the nociceptin/orphanin receptor. This scenario is corroborated by new data presented here for coelacanth and spotted gar, representing two basal branches in the vertebrate tree. A third genome doubling in the ancestor of teleost fishes generated additional gene copies. These results show that the opioid system was quite complex already in the first vertebrates and that it has more components in teleost fishes than in mammals. From an evolutionary point of view, nociceptin and its receptor can be considered full-fledged members of the opioid system.

Function of Isolated Pancreatic Islets From Patients at Onset of Type 1 Diabetes: Insulin Secretion Can Be Restored After Some Days in a Nondiabetogenic Environment In Vitro: Results From the DiViD Study.

The understanding of the etiology of type 1 diabetes (T1D) remains limited. One objective of the Diabetes Virus Detection (DiViD) study was to collect pancreatic tissue from living subjects shortly after the diagnosis of T1D. Here we report the insulin secretion ability by in vitro glucose perifusion and explore the expression of insulin pathway genes in isolated islets of Langerhans from these patients. Whole-genome RNA sequencing was performed on islets from six DiViD study patients and two organ donors who died at the onset of T1D, and the findings were compared with those from three nondiabetic organ donors. All human transcripts involved in the insulin pathway were present in the islets at the onset of T1D. Glucose-induced insulin secretion was present in some patients at the onset of T1D, and a perfectly normalized biphasic insulin release was obtained after some days in a nondiabetogenic environment in vitro. This indicates that the potential for endogenous insulin production is good, which could be taken advantage of if the disease process was reversed at diagnosis.