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Paralogs arise by duplications and belong to families. Ten paralogs (insulin; IGF-1 and -2; INSL3-6 and 3-relaxins) constitute the human insulin-relaxin family. The aim of this study was to outline the duplications that gave rise to the vertebrate insulin-relaxin genes and the chromosomal regions in which they reside. Neurotrophin and Trk-receptor families with more than 300, otherwise unrelated, families had paralogs in the regions hosting insulin/relaxin genes, defining two quadruplicate paralogy-regions, namely: insulin/IGF and INSL/relaxin paralogons. Thereby, the localization of insulin/relaxins in human shows that these regions were formed during two genome duplications at the stem of the vertebrates.We characterized insulin-like genes (INS-L1, -L2 and -L3) in the Ciona intestinalis genome, a species that split from the chordate lineage before the genome duplications. Conserved synteny between the Ciona region hosting the INS-Ls and two human paralogons as well as linkage of the actual paralogons, suggest that a segmental duplication gave rise to the entire region prior to the genome duplications. Synteny together with gene and protein structures demonstrate that INS-L1 is orthologous to the vertebrate INSLs/relaxins, INS-L2 to insulins and INS-L3 to IGFs. This indicates that pro-orthologs of the insulin-relaxin family were formed before Ciona. Our analysis also implies that the INSL/relaxin ancestor switched receptor from tyrosine kinase- to GPCR-type. This probably occurred after the Ciona-stage, but before the genome duplications.Using genes residing within the analyzed human paralogons that were present in a chromosomal region in the Ciona-human ancestor, we identified 37 segments with conserved synteny between the Drosophila melanogaster and human genomes. Orthologs residing in Ciona-, sea urchin- and the fly syntenic segments imply that such segments approximate an ancestral region from which the human paralogons originated.To conclude, the human paralogons are remnants of genome duplications that in addition to segmental- and single duplications, shaped the extant vertebrate genomes. Using the quadruplicate paralogy-regions we were able to deduce duplication events of the insulin-relaxin genes and their chromosomal regions.

The superfamily of G-protein-coupled receptors (GPCRs) is very diverse in structure and function and its members are among the most pursued targets for drug development. We identified more than 800 human GPCR sequences and simultaneously analyzed 342 unique functional nonolfactory human GPCR sequences with phylogenetic analyses. Our results show, with high bootstrap support, five main families, named glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin, forming the GRAFS classification system. The rhodopsin family is the largest and forms four main groups with 13 sub-branches. Positions of the GPCRs in chromosomal paralogons regions indicate the importance of tetraploidizations or local gene duplication events for their creation. We also searched for "fingerprint" motifs using Hidden Markov Models delineating the putative inter-relationship of the GRAFS families. We show several common structural features indicating that the human GPCRs in the GRAFS families share a common ancestor. This study represents the first overall map of the GPCRs in a single mammalian genome. Our novel approach of analyzing such large and diverse sequence sets may be useful for studies on GPCRs in other genomes and divergent protein families.

The appearance of the vertebrates demarcates some of the most far-reaching changes of structure and function seen during the evolution of the metazoans. These drastic changes of body plan and expansion of the central nervous system among other organs coincide with increased gene numbers. The presence of several groups of paralogous chromosomal regions in the human genome is a reflection of this increase. The simplest explanation for the existence of these paralogies would be two genome doublings with subsequent silencing of many genes. It is argued that gene localization data and the delineation of paralogous chromosomal regions give more reliable information about these types of events than dendrograms of gene families as gene relationships are often obscured by uneven replacement rates as well as other factors. Furthermore, the topographical relations of some paralogy groups are discussed.

We have isolated a neurotrophin from the lamprey that permitted us to perform a phylogenetic analysis of the neurotrophin gene family that dates back more than 460 million years to the early vertebrate ancestors. The results show that the neurotrophin gene family was originally formed by two subsequent duplications. The duplication that formed nerve growth factor, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 occurred after the split of lampreys but before the split of cartilaginous fish from the main vertebrate lineage. Compilation of chromosomal gene maps around the neurotrophins shows that they are located in paralogous regions, suggesting that the genes were formed at major duplication events possibly by complete genome doubling. Analysis of two isolated Trk receptor sequences shows similar results as for the lamprey neurotrophin. Multiple neurotrophin and Trk genes, including neurotrophin-6 and -7, have been found in bony fish, and we suggest that the extra genes were formed by an additional duplication in the bony fish lineage. Analysis of lamprey Trk mRNA expression in the adult brain shows that the genes are expressed in all regions analyzed so far. Together, the results suggest that the duplications of ancestral neurotrophin and Trk genes at an early vertebrate stage have permitted evolution to bring about differential neurotrophin and Trk expression, thereby allowing the formation of specific functions in selective neuronal populations.

The aim of this study was to outline the sequence of events that gave rise to the members of the insulin gene family in chordates. As part of our research, we looked for the chromosomal localization of insulin family members in the human genome.

Polyploidisations, that is, genome doublings, have taken place on numerous occasions during the evolution of animals and plants. The resulting duplicated chromosome regions can be recognised by their similar repertoires of gene families. Duplicated genes or regions within a genome are referred to as paralogous, and each set of related chromosome regions comprise a paralogon. In the ancestor of vertebrates, before the origin of the gnathostomes (jawed vertebrates), two tetraploidisations took place, abbreviated 2R for two rounds of tetraploidisation. A third tetraploidisation, 3R, happened before the radiation of the true bony fishes, the teleosts. Paralogues resulting from tetraploidisations are called ohnologues in honour of Susumu Ohno, who proposed the vertebrate tetraploidisations. Paralogous genes can undergo either subfunctionalisation (become more specialised) or neofunctionalisation (evolve novel functions). The vertebrate tetraploidisations seem to have paved the way for many gnathostome innovations such as jaws, limbs, an advanced nervous system and a complex adaptive immune system. Key Concepts: Polyploidisations have happened on numerous occasions in plant evolution and several times in vertebrate evolution. A paralogon is a set of related chromosome regions resulting from duplications, usually tetraploidizations. Paralogous genes can undergo either subfunctionalisation (become more specialised), neofunctionalisation (evolve novel functions) or loss. The tetraploidisations in vertebrate evolution are thought to have facilitated evolution of new functions and structures. After tetraploidisations, rearrangements scramble the chromosome regions, thereby obscuring the relationships. The tetraploidisations do not only generate copies of single genes. Since all genes are duplicated, whole networks of genes or pathways are duplicated, such as the phototransduction pathway. The total number of genes are doubled after a tetraploidisation event. However, the number of genes is later reduced due to different mutations such as deletions. Keywords: paralogue; orthologue; Ohnologue; gene duplication; genome duplication; polyploidy; polyploidisation; tetraploidy; tetraploidisation

Paralogous genes exist within the genome of a particular species and have a common ancestry as a result of gene duplications. These can take place by a variety of mechanisms and may involve individual genes, chromosome regions, chromosomes or even entire genomes (tetraploidization). Gene duplications have facilitated evolution of more complex organisms. Keywords: paralogues; orthologues; regional duplications; genome duplications; gene maps

BackgroundOne 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 fishesTetraodon nigroviridisandTakifugu rubripes. These correspond toHomo sapienschromosomes 4, 5, 8 and 10.ResultsChromosome regions with conserved synteny were identified and confirmed by phylogenetic analyses inH. sapiens, M. musculus, D. rerio, T. rubripesandT. 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 inT. rubripesrevealed that all five NPY receptors were expressed in the brain and subtypes Y2, Y4 and Y8 were also expressed in peripheral organs.ConclusionWe 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.

We present seven new vertebrate homologs of the prolactin-releasing hormone receptor (PRLHR) and show that these are found as two separate subtypes, PRLHR1 and PRLHR2. Analysis of a number of vertebrate sequences using phylogeny, pharmacology, and paralogon analysis indicates that the PRLHRs are likely to share a common ancestry with the neuropeptide Y (NPY) receptors. Moreover, a micromolar level of NPY was able to bind and inhibit completely the PRLH-evoked response in PRLHR1-expressing cells. We suggest that an ancestral PRLH peptide started coevolving with a redundant NPY binding receptor, which then became PRLHR, approximately 500 million years ago. The PRLHR1 subtype was shown to have a relatively high evolutionary rate compared to receptors with fixed peptide preference, which could indicate a drastic change in binding preference, thus supporting this hypothesis. This report suggests how gene duplication events can lead to novel peptide ligand/receptor interactions and hence spur the evolution of new physiological functions.

The appearance of the vertebrates demarcates some of the most far-reaching changes of structure and function seen during the evolution of the metazoans. These drastic changes of body plan and expansion of the central nervous system among other organs coincide with increased gene numbers. The presence of several groups of paralogous chromosomal regions in the human genome is a reflection of this increase. The simplest explanation for the existence of these paralogies would be two genome doublings with subsequent silencing of many genes. It is argued that gene localization data and the delineation of paralogous chromosomal regions give more reliable information about these types of events than dendrograms of gene families as gene relationships are often obscured by uneven replacement rates as well as other factors. Furthermore, the topographical relations of some paralogy groups are discussed.

Many chromosome regions in the human genome exist in four similar copies, suggesting that the entire genome was duplicated twice in early vertebrate evolution, a concept called the 2R hypothesis. Forty-two gene families on the four Hox-bearing chromosomes were recently analyzed by others, and 32 of these were reported to have evolutionary histories incompatible with duplications concomitant with the Hox clusters, thereby contradicting the 2R hypothesis. However, we show here that nine of the families have probably been translocated to the Hox-bearing chromosomes more recently, and that three of these belong to other chromosome quartets where they actually support the 2R hypothesis. We consider 13 families too complex to shed light on the chromosome duplication hypothesis. Among the remaining 20 families, 14 display phylogenies that support or are at least consistent with the Hox-cluster duplications. Only six families seem to have other phylogenies, but these trees are highly uncertain due to shortage of sequence information. We conclude that all relevant and analyzable families support or are consistent with block/chromosome duplications and that none clearly contradicts the 2R hypothesis.

Neuropeptide Y (NPY) receptors mediate a variety of physiological responses including feeding and vasoconstriction. To investigate the evolutionary events that have generated this receptor family, we have sequenced and determined the chromosomal localizations of all five presently known mammalian NPY receptor subtype genes in the domestic pig, Sus scrofa (SSC). The orthologs of the Y1 and Y2 subtypes display high amino acid sequence identities between pig, human, and mouse (92%–94%), whereas the Y4, Y5, and y6 subtypes display lower identities (76%–87%). The lower identity of Y5 is due to high sequence divergence in the large third intracellular loop. TheNPY1R, NPY2R, and NPY5R receptor genes were localized to SSC8, the NPY4R to SSC14, andNPY6R to SSC2. Our comparisons strongly suggest that the tight cluster of NPY1R, NPY2R, and NPY5R on human chromosome 4 (HSA4) represents the ancestral configuration, whereas the porcine cluster has been split by two inversions on SSC8. These 3 genes, along with adjacent genes from 14 other gene families, form a cluster on HSA4 with extensive similarities to a cluster on HSA5, whereNPY6R and >13 other paralogs reside, as well as another large cluster on HSA10 that includes NPY4R. Thus, these gene families have expanded through large-scale duplications. The sequence comparisons show that the NPY receptor tripletNPY1R–NPY2R–NPY5R existed before these large-scale duplications.[Sequence data for this article were deposited with the GenBank data library under accession nos.AF106081, PID g6457648 (for Pig Y1 sequence); accession nos. AF106082, PID g4249727 (for Pig Y2 sequence); accession no. AF227955 (for Pig Y4 sequence); accession nos. AF106083, PID g4249729 (for Pig Y5 sequence); accession no. AF227956 (for Pig Y6 sequence).]

A series of recombinant inbred strains called BXD [produced from a cross between C57BL/6J (B6) and DBA/2J (D2)] were given single i.p. doses of 0.6 mg/kg 2,3,7,8-tetrachlorodibenzofuran (TCDBF) on day 12 of gestation. The uteri were examined in late gestation with respect to resorptions and fetal death, and fetal malformations. The strains of the B6-type with respect to Ah -locus (Nos. 5, 6, 8, 11, 12, 14, 16 and 29) that are Ah -responsive, exhibited cleft palates in 80–100% of all fetuses, while hydronephrosis occurred at a rate of 20–70%. These two types of malformation are well recognized from earlier experiments with 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) and its structural analogues, including TCDBF. In the strains of D2-type with respect to Ah -locus (Nos. 2, 15, 19, 21, 22, 24, and 31), which are Ah -nonresponsive, no cleft palates occurred. One strain (No. 2) had a few (17%) fetuses with hydronephrosis. The frequency of fetal deaths and resorptions were relatively low, but slightly higher among B6-strains than D2-strains. The results indicate an association between the genes producing malformations by TCDBF and the Ah -locus.

The glucuronidation of morphine and naloxone was demonstrated in the brain and liver in 2 inbred strains of mice, C57BL/6J (B6) and DBA/2J (D2) and their F1 hybrid generation. These strains showed a significant difference in latency of withdrawal in the tail-immersion test, the B6 strain being the most sensitive. The rate of naloxone glucuronidation in the brain was 5 times higher in the B6 than in the D2 strain. In the liver the UDP-glucuronosyl transferase (UDPGT) activity was slightly higher in the D2 strain. The naloxone- and morphine-3′-glucuronide (N3G, M3G) formation rate ratio was close to 1 in both the brain and liver in all except the B6 strain, where it was 2.6 in the brain. There was a correlation between formation rate of M3G and N3G ( r = 0.65 brain and r = 0.73 liver ). Our results indicate a common glucuronidation pathway for morphine and naloxone.