An evolutionary model for the duplication and divergence of esterase genes in Drosophila

Summary The esterase 5 (Est-5 = gene, EST 5 = protein) enzyme in Drosophila pseudoobscura is encoded by one of three paralogous genes, Est-5A, Est5B, and Est-5C, that are tightly clustered on the right arm of the X chromosome. The homologous Est-6 locus in Drosophila melanogaster has only one paralogous neighbor, Est-P. Comparisons of coding and flanking DNA sequences among the three D. pseudoobscura and two D. melanogaster genes suggest that two paralogous genes were present before the divergence of D. pseudoobscura from D. melanogaster and that, later, a second duplication occurred in D. pseudoobscura. Nucleotide sequences of the coding regions of the three D. pseudoobscura genes showed 78–85% similarity in pairwise comparisons, whereas the relatedness between Est-6 and Est-P was only 67%. The higher degree of conservation in D. pseudoobscura likely results from the comparatively recent divergence of Est-5B and Est-5C and from possible gene conversion events between Est-5A and Est-5B. Analyses of silent and replacement site differences in the two exons of the paralogous and orthologous genes in each species indicate that common selective forces are acting on all five loci. Further evidence for common purifying selective constraints comes from the conservation of hydropathy profiles and proposed catalytic residues. However, different levels of amino acid substitution between the paralogous genes in D. melanogaster relative to those in D. pseudoobscura suggest that interspecific differences in selection also exist.Offprint requests to: R.C. Richmond     

Repetitive sequence involvement in the duplication and divergence of mouse lysozyme genes.

Mouse M and P lysozymes are the products of separate genes, are specifically expressed in separate tissues, and are adapted to different functions. The lysozyme genes have assumed these markedly different characteristics following their generation by gene duplication 30-50 million years ago. The discovery of the lysozyme P gene only 5 kb upstream from the M gene in tandem repeat has enabled an investigation of the molecular basis of their duplication and subsequent divergence. The duplication is shown to have involved recombination between two B2 repeat sequences flanking the original gene. The resulting downstream copy has retained the myeloid specificity of expression along with just 1.7 kb of upstream sequences, while the upstream copy is inactive in macrophages and has become expressed instead in the small intestine. Although multiple gene conversion events have served to maintain a generally high homology between the genes, certain regions have been found to be specific for either one of the gene pair: two repetitive sequences peculiar to the P region may serve to protect the coding regions from gene conversion, while sequences unique to the M gene may be more directly involved in differential regulation.     

Conservation and divergence in the control of yolk protein genes in dipteran insects

 We have investigated the conservation of regulatory elements for sex- and tissue-specific gene expression in three dipteran species, Drosophila melanogaster, Musca domestica and Calliphora erythrocephala, using the yolk protein (yp) genes. Yolk proteins of the fruitfly, medfly, housefly and blowfly are very well conserved both in their sequence and their expression in ovarian follicle cells and in fat bodies of adult females. Furthermore, yp regulation by both hormonal and nutritional factors shows similar features in all four species. To study conservation of yp regulation in dipteran insects, we tested 5′ flanking regions from one Musca yp gene and one Calliphora yp gene for enhancer functions in D. melanogaster. Two fragments of 823 and 1046 bp isolated from Musca and Calliphora yp genes, respectively, are able to direct correct expression of a reporter gene in the ovarian follicle cells of transformed Drosophila at specific stages during oogenesis. Surprisingly, these enhancers do not confer sex-specific reporter gene expression in the fat body, as expression was found in both sexes of the transformed flies. None-the-less by in vitro DNA/protein interaction assays, a 284-bp DNA region from the Musca yp enhancer was able to bind the Drosophila DOUBLESEX (DSX) protein, which in D.melanogaster confers sex-specific expression of yp. We speculate that the sex-determining pathway is not directly involved in yp regulation in Musca or Calliphora adult females, but depends instead on hormonal controls to achieve sex-specific expression of yp genes in the adult. Received: 17 April 1997 / Accepted: 12 July 1997     

Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish

Background  

The zebrafish has become a widely used model to study disease resistance and immunity. Although the genes encoding many components of immune signaling pathways have been found in teleost fish, it is not clear whether all components are present or whether the complexity of the signaling mechanisms employed by mammals is similar in fish.     

Conservation and divergence of autonomous pathway genes in the flowering regulatory network of Beta vulgaris

The transition from vegetative growth to reproductive development is a complex process that requires an integrated response to multiple environmental cues and endogenous signals. In Arabidopsis thaliana, which has a facultative requirement for vernalization and long days, the genes of the autonomous pathway function as floral promoters by repressing the central repressor and vernalization-regulatory gene FLC. Environmental regulation by seasonal changes in daylength is under control of the photoperiod pathway and its key gene CO. The root and leaf crop species Beta vulgaris in the caryophyllid clade of core eudicots, which is only very distantly related to Arabidopsis, is an obligate long-day plant and includes forms with or without vernalization requirement. FLC and CO homologues with related functions in beet have been identified, but the presence of autonomous pathway genes which function in parallel to the vernalization and photoperiod pathways has not yet been reported. Here, this begins to be addressed by the identification and genetic mapping of full-length homologues of the RNA-regulatory gene FLK and the chromatin-regulatory genes FVE, LD, and LDL1. When overexpressed in A. thaliana, BvFLK accelerates bolting in the Col-0 background and fully complements the late-bolting phenotype of an flk mutant through repression of FLC. In contrast, complementation analysis of BvFVE1 and the presence of a putative paralogue in beet suggest evolutionary divergence of FVE homologues. It is further shown that BvFVE1, unlike FVE in Arabidopsis, is under circadian clock control. Together, the data provide first evidence for evolutionary conservation of components of the autonomous pathway in B. vulgaris, while also suggesting divergence or subfunctionalization of one gene. The results are likely to be of broader relevance because B. vulgaris expands the spectrum of evolutionarily diverse species which are subject to differential developmental and/or environmental regulation of floral transition.     

Allelic divergence precedes and promotes gene duplication

One of the striking observations from recent whole-genome comparisons is that changes in the number of specialized genes in existing gene families, as opposed to novel taxon-specific gene families, are responsible for the majority of the difference in genome composition between major taxa. Previous models of duplicate gene evolution focused primarily on the role that neutral processes can play in evolutionary divergence after the duplicates are already fixed in the population. By instead including the entire cycle of duplication and divergence, we show that specialized functions are most likely to evolve through strong selection acting on segregating alleles at a single locus, even before the duplicate arises. We show that the fitness relationships that allow divergent alleles to evolve at a single locus largely overlap with the conditions that allow divergence of previously duplicated genes. Thus, a solution to the paradox of the origin of organismal complexity via the expansion of gene families exists in the form of the deterministic spread of novel duplicates via natural selection.     

The role of stat1b in zebrafish hematopoiesis

STAT1 mediates response to interferons and regulates immunity, cell proliferation, apoptosis, and sensitivity of Fanconi Anemia cells to apoptosis after interferon signaling; the roles of STAT1 in embryos, however, are not understood. To explore embryonic functions of STAT1, we investigated stat1b, an unstudied zebrafish co-ortholog of human STAT1. Zebrafish stat1a encodes all five domains of the human STAT1-alpha splice form but, like the human STAT1-beta splice variant, stat1b lacks a complete transactivation domain; thus, two unlinked zebrafish paralogs encode protein forms translated from two splice variants of a single human gene, as expected by sub-functionalization after genome duplication. Phylogenetic and conserved synteny studies showed that stat1b and stat1a arose as duplicates in the teleost genome duplication (TGD) and clarified the evolutionary origin of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 by tandem and genome duplication. RT-PCR revealed maternal expression of stat1a and stat1b. In situ hybridization detected stat1b but not stat1a expression in embryonic hematopoietic tissues. Morpholino knockdown of stat1b, but not stat1a, decreased expression of the myeloid and granulocyte markers spi and mpo and increased expression of the hematopoietic progenitor marker scl, the erythrocyte marker gata1, and hemoglobin. These results suggest that zebrafish Stat1b promotes myeloid development at the expense of erythroid development.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Gene duplication and divergence in the early evolution of vertebrates

The duplication-degeneration-complementation model of duplicate gene preservation by subfunctionalisation is currently the best explanation for the high level of retention of duplicate genes in early vertebrate evolution. But a direct test of the applicability of this model to such ancient evolutionary events may be difficult. More likely, recent duplications in other lineages will allow us to establish general principles concerning the fate of genes of different types that are duplicated in different ways. These principles may be then extrapolated to understanding the early evolution of the vertebrates.     

Opsin gene duplication and divergence in ray-finned fish

Opsin gene sequences were first reported in the 1980s. The goal of that research was to test the hypothesis that human opsins were members of a single gene family and that variation in human color vision was mediated by mutations in these genes. While the new data supported both hypotheses, the greatest contribution of this work was, arguably, that it provided the data necessary for PCR-based surveys in a diversity of other species. Such studies, and recent whole genome sequencing projects, have uncovered exceptionally large opsin gene repertoires in ray-finned fishes (taxon, Actinopterygii). Guppies and zebrafish, for example, have 10 visual opsin genes each. Here we review the duplication and divergence events that have generated these gene collections. Phylogenetic analyses revealed that large opsin gene repertories in fish have been generated by gene duplication and divergence events that span the age of the ray-finned fishes. Data from whole genome sequencing projects and from large-insert clones show that tandem duplication is the primary mode of opsin gene family expansion in fishes. In some instances gene conversion between tandem duplicates has obscured evolutionary relationships among genes and generated unique key-site haplotypes. We mapped amino acid substitutions at so-called key-sites onto phylogenies and this exposed many examples of convergence. We found that dN/dS values were higher on the branches of our trees that followed gene duplication than on branches that followed speciation events, suggesting that duplication relaxes constraints on opsin sequence evolution. Though the focus of the review is opsin sequence evolution, we also note that there are few clear connections between opsin gene repertoires and variation in spectral environment, morphological traits, or life history traits.     

Conservation and divergence in plant microRNAs

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression in eukaryotic cells. The past decade has seen an explosion in our understanding of the sets of miRNA genes encoded in the genomes in different species of plants and the mechanisms by which miRNAs interact with target RNAs. A subset of miRNA families (and their binding sites in target RNAs) are conserved between angiosperms and basal plants, suggesting they predate the divergence of existing lineages of plants. However, the majority of miRNA families expressed by any given plant species have a narrow phylogenetic distribution. As a group, these "young" miRNAs genes appear to be evolutionarily fluid and lack clearly understood biological function. The goal of this review is to summarize our understanding of the sets of miRNA genes and miRNA targets that exist in various plant species and to discuss hypotheses that explain the patterns of conservation and divergence observed among microRNAs in plants.     

Characterization of duplicated Dunaliella viridis SPT1 genes provides insights into early gene divergence after duplication

The sodium-dependent phosphate transporter gene from unicellular green algae Dunaliella viridis, DvSPT1, shares similarity with members of Pi transporter family. Sequencing analysis of D. viridis BAC clone containing the DvSPT1 gene revealed two inverted duplicated copies of this gene (DvSPT1 and DvSPT1-2 respectively). The duplication covered most of both genes except for their 3' downstream region. The duplicated genomic sequences exhibited 97.9% identity with a synonymous divergence of Ks=0.0126 in the coding region. This data indicated very recent gene duplication in D. viridis genome, providing an excellent opportunity to investigate sequence and expression divergence of duplicated genes at an early stage. Scatted point mutations and length polymorphism of simple sequence repeats (SSRs) were predominant among the sequence divergence soon after gene duplication. Due to sequence divergence in the 5' regulatory regions and a swap of the entire 3' downstream regions (3'-UTR), DvSPT1 and DvSPT1-2 showed expression divergence in response to extra-cellular NaCl concentration changes. According to their expression patterns, the two diverged gene copies would provide better adaptation to a broader range of extra-cellular NaCl concentration. Furthermore, Southern blot analysis indicated that there might be a large phosphate transporter gene family in D. viridis.     

Conservation and divergence in the AGAMOUS subfamily of MADS-box genes: evidence of independent sub- and neofunctionalization events

The MADS-box gene AGAMOUS (AG) plays a key role in determining floral meristem and organ identities. We identified three AG homologs, EScaAG1, EScaAG2, and EScaAGL11 from the basal eudicot Eschscholzia californica (California poppy). Phylogenetic analyses indicate that EScaAG1 and EScaAG2 are recent paralogs within the AG clade, independent of the duplication in ancestral core eudicots that gave rise to the euAG and PLENA (PLE) orthologs. EScaAGL11 is basal to core eudicot AGL11 orthologs in a clade representing an older duplication event after the divergence of the angiosperm and gymnosperm lineages. Detailed in situ hybridization experiments show that expression of EScaAG1 and EScaAG2 is similar to AG; however, both genes appear to be expressed earlier in floral development than described in the core eudicots. A thorough examination of available expression and functional data in a phylogenetic context for members of the AG and AGL11 clades reveals that gene expression has been quite variable throughout the evolutionary history of the AG subfamily and that ovule-specific expression might have evolved more than twice. Although sub- and neofunctionalization are inferred to have occurred following gene duplication, functional divergence among orthologs is evident, as is convergence, among paralogs sampled from different species. We propose that retention of multiple AG homologs in several paralogous lineages can be explained by the conservation of ancestral protein activity combined with evolutionarily labile regulation of expression in the AG and AGL11 clades such that the collective functions of the AG subfamily in stamen and carpel development are maintained following gene duplication.     

Conserved functions of yeast genes support the duplication, degeneration and complementation model for gene duplication

Gene duplication is often cited as a potential mechanism for the evolution of new traits, but this hypothesis has not been thoroughly tested experimentally. A classical model of gene duplication states that after gene duplication one copy of the gene preserves the ancestral function, while the other copy is free to evolve a new function. In an alternative duplication, divergence, and complementation model, duplicated genes are preserved because each copy of the gene loses some, but not all, of its functions through degenerating mutations. This results in the degenerating mutations in one gene being complemented by the other and vice versa. These two models make very different predictions about the function of the preduplication orthologs in closely related species. These predictions have been tested here for several duplicated yeast genes that appeared to be the leading candidates to fit the classical model. Surprisingly, the results show that duplicated genes are maintained because each copy carries out a subset of the conserved functions that were already present in the preduplication gene. Therefore, the results are not consistent with the classical model, but instead fit the duplication, divergence, and complementation model.