RESUMEN
RNase A is the prototype of an extensive family of divergent proteins whose members share a unique disulfide-bonded tertiary structure, conserved catalytic motifs, and the ability to hydrolyze polymeric RNA. Several members of this family maintain independent roles as ribonucleases and modulators of innate immunity. Here we characterize mouse eosinophil-associated RNase (Ear) 11, a divergent member of the eosinophil ribonuclease cluster, and the only known RNase A ribonuclease expressed specifically in response to Th2 cytokine stimulation. Mouse Ear 11 is differentially expressed in somatic tissues at baseline (brain ⪠liver < lung < spleen); systemic stimulation with IL-33 results in 10-5000-fold increased expression in lung and spleen, respectively. Ear 11 is also expressed in response to protective priming of the respiratory mucosa with Lactobacillus plantarum; transcripts are detected both locally in lung as well as systemically in bone marrow and spleen. Mouse Ear 11 is enzymatically active, although substantially less so than mEar 1 and mEar 2; the relative catalytic efficiency (kcat/Km) of mEar 11 is diminished â¼1000-1500-fold. However, in contrast to RNase 2/EDN and mEar 2, which have been characterized as selective chemoattractants for CD11c(+) dendritic cells, mEar 11 has prominent chemoattractant activity for F4/80(+)CD11c(-) tissue macrophages. Chemoattractant activity is not dependent on full enzymatic activity, and requires no interaction with the pattern recognition receptor, Toll-like receptor 2 (TLR2). Taken together, this work characterizes a divergent RNase A ribonuclease with a unique expression pattern and function, and highlights the versatility of this family in promoting innate immunity.
Asunto(s)
Proteína Catiónica del Eosinófilo/metabolismo , Macrófagos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Proteína Catiónica del Eosinófilo/química , Proteína Catiónica del Eosinófilo/genética , Inmunidad Innata , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Bazo/citologíaRESUMEN
Sex-determination mechanisms vary greatly among taxa. It has been proposed that genetic sex-determination pathways evolve in reverse order from the final step in the pathway to the first step. Consistent with this hypothesis, doublesex (dsx), the most downstream gene in the Drosophila sex-determination cascade that determines most sexual phenotypes also determines sex in other dipterans and the silk moth, while the upstream genes vary among these species. However, it is unknown when dsx was recruited to the sex-determination pathway during insect evolution. Furthermore, sex-specific splicing of dsx, by which dsx determines sex, is different in pattern and mechanism between the moth and the fly, raising an interesting question of how these insects have kept the executor of sex determination while allowing flexibility in the means of execution. To address these questions, here we study the dsx gene of the honeybee Apis mellifera, a member of the most basal lineage of holometabolous insects. We report that honeybee dsx is sex-specifically spliced and that it produces both the fly-type and moth-type splicing forms, indicating that the use of different splicing forms of Dsx in controlling sexual differentiation was present in the common ancestor of holometabolous insects. Our data suggest that in ancestral holometabolous insects the female Dsx form is the default and the male form is generated by suppressing the splicing of the female form. Thus, it is likely that the dsx splicing activator system in flies, where the male form is the default, arose during early dipteran evolution.
Asunto(s)
Empalme Alternativo , Abejas/genética , Evolución Molecular , Genes de Insecto , Procesos de Determinación del Sexo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Abejas/embriología , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Diferenciación Sexual/genética , Factores de TiempoRESUMEN
Members of the ribonuclease (RNase) A superfamily participate in a diverse array of biological processes, including digestion, angiogenesis, innate immunity, and possibly male reproduction. The superfamily is vertebrate-specific, with 13-20 highly divergent members in primates and rodents, but only a few members in chicken and fish. This has led to the proposal that the superfamily started off from a progenitor with structural similarities to angiogenin and that the superfamily underwent a dramatic expansion during mammalian evolution. To date this evolutionary expansion and understand the functional diversification of the superfamily, we here determine its entire repertoire in the sequenced genomes of dog, cow, and opossum. We identified 7, 20, and 21 putatively functional RNase genes from these three species, respectively. Many of the identified genes are highly divergent from all previously known RNase genes, thus representing new lineages within the superfamily. Phylogenetic analysis indicates that the superfamily expansion predated the separation of placental and marsupial mammals and that differential gene loss and duplication occurred in different species, generating a great variation in gene number and content among extant mammals.
Asunto(s)
Evolución Molecular , Marsupiales/genética , Ribonucleasa Pancreática/genética , Secuencia de Aminoácidos , Animales , Bovinos , Perros , Duplicación de Gen , Zarigüeyas , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Pancreatic ribonuclease (RNASE1) is a digestive enzyme that has been one of the key models in studies of evolutionary innovation and functional diversification. It has been believed that the RNASE1 gene duplications are correlated with the plant-feeding adaptation of foregut-fermenting herbivores. Here, we characterized RNASE1 genes from Caniformia, which has a simple digestive system and lacks microbial digestion typical of herbivores, in an unprecedented scope based on both gene sequence and tissue expression analyses. Remarkably, the results yielded new hypotheses regarding the evolution and the function of Caniformia RNASE1 genes. Four independent gene duplication events in the families of superfamily Musteloidea, including Procyonidae, Ailuridae, Mephitidae and Mustelidae, were recovered, rejecting previous Mustelidae-specific duplication hypothesis, but supporting Musteloidea duplication hypothesis. Moreover, our analyses revealed pronounced differences among the RNASE1 gene copies regarding their selection pressures, pI values and tissue expression patterns, suggesting the differences in their physiological functions. Notably, the expression analyses detected the transcription of a RNASE1 pseudogene in several tissues, raising the possibility that pseudogenes are also a potential source during the RNase functional diversification. In sum, the present work demonstrated a far more complex and intriguing evolutionary pattern and functional diversity of mammalian ribonuclease than previously thought.
Asunto(s)
Evolución Molecular , Mustelidae/genética , Filogenia , Ribonucleasa Pancreática/genética , Animales , Carnívoros , Mamíferos , Ribonucleasa Pancreática/metabolismoRESUMEN
The ribonuclease (RNase) A superfamily is a vertebrate-specific gene family. Because of a massive expansion that occurred during the early mammalian evolution, extant mammals in general have much more RNase genes than nonmammalian vertebrates. Mammalian RNases have been associated with diverse physiological functions including digestion, cytotoxicity, angiogenesis, male reproduction, and host defense. However, it is still uncertain when their expansion occurred and how a wide array of functions arose during their evolution. To answer these questions, we generate a compendium of all RNase genes identified in 20 complete mammalian genomes including the platypus, Ornithorhynchus anatinus. Using this, we delineate 13 ancient RNase gene lineages that arose before the divergence between the monotreme and the other mammals (â¼220 Ma). These 13 ancient gene lineages are differentially retained in the 20 mammals, and the rate of protein sequence evolution is highly variable among them, which suggest that they have undergone extensive functional diversification. In addition, we identify 22 episodes of recent expansion of RNase genes, many of which have signatures of adaptive functional differentiation. Exemplifying this, bursts of gene duplication occurred for the RNase1, RNase4, and RNase5 genes of the little brown bat (Myotis lucifugus), which might have contributed to the species' effective defense against heavier pathogen loads caused by its communal roosting behavior. Our study illustrates how host-defense systems can generate new functions efficiently by employing a multigene family, which is crucial for a host organism to adapt to its ever-changing pathogen environment.
Asunto(s)
Expansión de las Repeticiones de ADN , Evolución Molecular , Mamíferos/genética , Familia de Multigenes , Ribonucleasa Pancreática/genética , Animales , Duplicación de Gen , Humanos , Mamíferos/clasificación , Filogenia , Selección GenéticaRESUMEN
The silks of arthropods have an elementary role in the natural history of the organisms that spin them, yet they are coded by rapidly evolving genes leading some authors to speculate that silk proteins are non-homologous proteins co-opted multiple times independently for similar functions. However, some general structural patterns are emerging. In this work we identified three major silk gland proteins using a combined biochemical, proteomic, next-generation sequencing and bioinformatic approach. Biochemical characterization determined that they were phosphorylated with multiple isoforms and potentially differential phosphorylation. Structural characterization showed that their structure was more similar to silk proteins from distantly related aquatic Trichopteran species than more closely related terrestrial or aquatic Diptera. Overall, our approach is easily transferable to any non-model species and if used across a larger number of aquatic species, we will be able to better understand the processes involved in linking the secondary structure of silk proteins with their function between in an organisms and its habitat.
Asunto(s)
Proteómica , Seda/química , Animales , Fosforilación , Estructura Secundaria de Proteína , Seda/genética , Simuliidae/química , Simuliidae/genéticaRESUMEN
The leptin gene has received intensive attention and scientific investigation for its importance in energy homeostasis and reproductive regulation in mammals. Furthermore, study of the leptin gene is of crucial importance for public health, particularly for its role in obesity, as well as for other numerous physiological roles that it plays in mammals. In the present work, we report the identification of novel leptin genes in 4 species of Cetacea, and a comparison with 55 publicly available leptin sequences from mammalian genome assemblies and previous studies. Our study provides evidence for positive selection in the suborder Odontoceti (toothed whales) of the Cetacea and the family Phocidae (earless seals) of the Pinnipedia. We also detected positive selection in several leptin gene residues in these two lineages. To test whether leptin and its receptor evolved in a coordinated manner, we analyzed 24 leptin receptor gene (LPR) sequences from available mammalian genome assemblies and other published data. Unlike the case of leptin, our analyses did not find evidence of positive selection for LPR across the Cetacea and Pinnipedia lineages. In line with this, positively selected sites identified in the leptin genes of these two lineages were located outside of leptin receptor binding sites, which at least partially explains why co-evolution of leptin and its receptor was not observed in the present study. Our study provides interesting insights into current understanding of the evolution of mammalian leptin genes in response to selective pressures from life in an aquatic environment, and leads to a hypothesis that new tissue specificity or novel physiologic functions of leptin genes may have arisen in both odontocetes and phocids. Additional data from other species encompassing varying life histories and functional tests of the adaptive role of the amino acid changes identified in this study will help determine the factors that promote the adaptive evolution of the leptin genes in marine mammals.
Asunto(s)
Caniformia/genética , Cetáceos/genética , Leptina/genética , Selección Genética/fisiología , Animales , Evolución Biológica , Evolución Molecular , Receptores de Leptina/genética , Análisis de Secuencia de ADNRESUMEN
Although the nematode Caenorhabditis elegans produces self-fertile hermaphrodites, it descended from a male/female species, so hermaphroditism provides a model for the origin of novel traits. In the related species C. remanei, which has only male and female sexes, lowering the activity of tra-2 by RNA interference created XX animals that made spermatids as well as oocytes, but their spermatids could not activate without the addition of male seminal fluid. However, by lowering the expression of both tra-2 and swm-1, a gene that regulates sperm activation in C. elegans, we produced XX animals with active sperm that were self-fertile. Thus, the evolution of hermaphroditism in Caenorhabditis probably required two steps: a mutation in the sex-determination pathway that caused XX spermatogenesis and a mutation that allowed these spermatids to self-activate.
Asunto(s)
Evolución Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Caenorhabditis/genética , Caenorhabditis/fisiología , Mutación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis/anatomía & histología , Caenorhabditis/clasificación , Caenorhabditis elegans/anatomía & histología , Caenorhabditis elegans/clasificación , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Cruzamientos Genéticos , Trastornos del Desarrollo Sexual/genética , Femenino , Genes de Helminto , Células Germinativas/fisiología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Oogénesis , Ovulación , Filogenia , Reproducción , Selección Genética , Procesos de Determinación del Sexo , Espermátides/fisiología , EspermatogénesisRESUMEN
Understanding the evolutionary origin of the ribonuclease (RNase) A superfamily is of great interest because the superfamily is the sole vertebrate-specific enzyme family known to date. Although mammalian RNases have a diverse array of biochemical and physiological functions, the original function of the superfamily at its birth is enigmatic. Such information may be obtained by studying basal lineages of the vertebrate phylogeny and is necessary for discerning how and why this superfamily originated. Here, we clone and characterize 3 RNase genes from the zebrafish, the most basal vertebrate examined for RNases. We report 1) that all the 3 zebrafish RNases are ribonucleolytically active, with one of them having an RNase activity comparable to that of bovine RNase A, the prototype of the superfamily; 2) that 2 zebrafish RNases have prominent expressions in adult liver and gut, whereas the 3rd is expressed in adult eye and heart; and 3) that all 3 RNases have antibacterial activities in vitro. These results, together with the presence of antibacterial and/or antiviral activities in multiple distantly related mammalian RNases, strongly suggest that the superfamily started as a host-defense mechanism in vertebrate evolution.
Asunto(s)
Antibacterianos/metabolismo , Evolución Molecular , Ribonucleasa Pancreática , Ribonucleasas/fisiología , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Peces/genética , Peces/metabolismo , Expresión Génica , Genoma , Humanos , Hígado/enzimología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , ARN de Transferencia/metabolismo , Ribonucleasa Pancreática/genética , Ribonucleasas/clasificación , Ribonucleasas/genética , Distribución Tisular , Vertebrados , Pez Cebra/genética , Pez Cebra/microbiologíaRESUMEN
The mechanism of sex determination varies substantively among evolutionary lineages. One important mode of genetic sex determination is haplodiploidy, which is used by approximately 20% of all animal species, including >200,000 species of the entire insect order Hymenoptera. In the honey bee Apis mellifera, a hymenopteran model organism, females are heterozygous at the csd (complementary sex determination) locus, whereas males are hemizygous (from unfertilized eggs). Fertilized homozygotes develop into sterile males that are eaten before maturity. Because homozygotes have zero fitness and because common alleles are more likely than rare ones to form homozygotes, csd should be subject to strong overdominant selection and negative frequency-dependent selection. Under these selective forces, together known as balancing selection, csd is expected to exhibit a high degree of intraspecific polymorphism, with long-lived alleles that may be even older than the species. Here we sequence the csd genes as well as randomly selected neutral genomic regions from individuals of three closely related species, A. mellifera, Apis cerana, and Apis dorsata. The polymorphic level is approximately seven times higher in csd than in the neutral regions. Gene genealogies reveal trans-species polymorphisms at csd but not at any neutral regions. Consistent with the prediction of rare-allele advantage, nonsynonymous mutations are found to be positively selected in csd only in early stages after their appearances. Surprisingly, three different hypervariable repetitive regions in csd are present in the three species, suggesting variable mechanisms underlying allelic specificities. Our results provide a definitive demonstration of balancing selection acting at the honey bee csd gene, offer insights into the molecular determinants of csd allelic specificities, and help avoid homozygosity in bee breeding.
Asunto(s)
Abejas/genética , Evolución Biológica , Genes de Insecto , Procesos de Determinación del Sexo , Alelos , Secuencia de Aminoácidos , Animales , ADN/genética , Femenino , Proteínas de Insectos/genética , Masculino , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Polimorfismo Genético , Secuencias Repetitivas de Aminoácido , Selección Genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
The RNase A superfamily has been important in biochemical, structural, and evolutionary studies and is believed to be the sole vertebrate-specific enzyme family. To understand the origin and diversification of the superfamily, we here determine its entire repertoire in the sequenced genomes of human, mouse, rat, and chicken. We report a previously unnoticed gene cluster in mouse chromosome 10 and a number of new genes, including mammalian RNases 11-13, which are close relatives of the recently identified RNases 9 and 10. Gene expression data imply male-reproductive functions for RNases 9-13, although their sequences suggest the lack of ribonucleolytic activities. In contrast to the presence of 13-20 functional genes in mammals, chicken has only 3 RNase genes, which are evolutionarily close to mammalian RNase 5, like other nonmammalian RNases. This and other evidence suggests that the RNase A superfamily originated from an RNase 5-like gene and expanded in mammals. Together with the fact that multiple lineages of the superfamily, including RNases 2, 3, 5, and 7, have antipathogenic activities, we suggest that the superfamily started off as a host-defense mechanism in vertebrates. Consistent with this hypothesis, all members of the superfamily exhibit high rates of amino acid substitution as is commonly observed in immunity genes.
Asunto(s)
Aves/genética , Evolución Molecular , Mamíferos/genética , Filogenia , Ribonucleasa Pancreática/genética , Secuencia de Aminoácidos , Animales , Pollos/genética , Proteína Catiónica del Eosinófilo/genética , Duplicación de Gen , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Since introns were discovered 26 years ago, people have wondered how changes in intron/exon structure occur, and what role these changes play in evolution. To answer these questions, we have begun studying gene structure in nematodes related to Caenorhabditis elegans. As a first step, we cloned a set of five genes from six different Caenorhabditis species, and used their amino acid sequences to construct the first detailed phylogeny of this genus. Our data indicate that nematode introns are lost at a very high rate during evolution, almost 400-fold higher than in mammals. These losses do not occur randomly, but instead, favor some introns and do not affect others. In contrast, intron gains are far less common than losses in these genes. On the basis of the sequences at each intron site, we suggest that several distinct mechanisms can cause introns to be lost. The small size of C. elegans introns should increase the rate at which each of these types of loss can occur, and might account for the dramatic difference in loss rate between nematodes and mammals.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis/genética , Evolución Molecular , Intrones/genética , Nematodos/genética , Filogenia , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Proteínas de Caenorhabditis elegans/genética , Deleción Cromosómica , Secuencia Conservada/genética , Exones/genética , Genes de Helminto/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Carácter Cuantitativo Heredable , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Especificidad de la Especie , Factores de Transcripción/genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Wingless directs many developmental processes in Drosophila by regulating expression of specific target genes through a conserved signaling pathway. Although many nuclear factors have been implicated in mediating Wingless-induced transcription, the mechanism of how Wingless regulates different targets in different tissues remains poorly understood. We report here that the split ends gene is required for Wingless signaling in the eye, wing and leg imaginal discs. Expression of a dominant-negative version of split ends resulted in more dramatic reductions in Wingless signaling than split ends-null alleles, suggesting that it may have a redundant partner. However, removal of split ends or expression of the dominant-negative had no effect on several Wingless signaling readouts in the embryo. The expression pattern of Split ends cannot explain this tissue-specific requirement, as the protein is predominantly nuclear and present throughout embryogenesis and larval tissues. Consistent with its nuclear location, the split ends dominant-negative acts downstream of Armadillo stabilization. Our data indicate that Split ends is an important positive regulator of Wingless signaling in larval tissues. However, it has no detectable role in the embryonic Wingless pathway, suggesting that it is a tissue or promoter-specific factor.