RESUMEN
Adaptive immune systems are present only in vertebrates. How do all the remaining animals withstand continuous attacks of permanently evolving pathogens? Even in the absence of adaptive immunity, every organism must be able to unambiguously distinguish "self" cells from any imaginable "nonself." Here, we analyzed the function of highly polymorphic gene vCRL1, which is expressed in follicle and blood cells of Ciona intestinalis, pointing to possible recognition roles either during fertilization or in immune reactions. By using segregation analysis, we demonstrate that vCRL1 locus is not involved in the control of self-sterility. Interestingly, genetic knockdown of vCRL1 in all tissues or specifically in hemocytes results in a drastic developmental arrest during metamorphosis exactly when blood system formation in Ciona normally occurs. Our data demonstrate that vCRL1 gene might be essential for the establishment of a functional blood system in Ciona. Presumably, presence of the vCRL1 receptor on the surface of blood cells renders them as self, whereas any cell lacking it is referred to as nonself and will be consequently destroyed. We propose that individual-specific receptor vCRL1 might be utilized to facilitate somatic self/nonself discrimination.
Asunto(s)
Ciona intestinalis/metabolismo , Hemocitos/metabolismo , Polimorfismo Genético , Receptores de Superficie Celular/metabolismo , Alelos , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Membrana Celular/metabolismo , Cruzamientos Genéticos , Femenino , Fertilización/genética , Técnicas de Silenciamiento del Gen , Sitios Genéticos/genética , Genotipo , Hemocitos/citología , Infertilidad/genética , Masculino , Metamorfosis Biológica/genética , Modelos Biológicos , Datos de Secuencia Molecular , Oocitos/citología , Oocitos/metabolismo , Fenotipo , Transporte de ProteínasRESUMEN
Achievement of transposon mediated germline transgenesis in a basal chordate, Ciona intestinalis, is discussed. A Tc1/mariner superfamily transposon, Minos, has excision and transposition activities in Ciona. Minos enables the creation of stable transgenic lines, enhancer detection, and insertional mutagenesis.
Asunto(s)
Ciona intestinalis/genética , Elementos Transponibles de ADN/genética , Técnicas de Transferencia de Gen , Animales , Elementos de Facilitación Genéticos , Genoma , Mutagénesis InsercionalRESUMEN
The enhancer trap approach utilizing transposons yields us information about gene functions and gene expression patterns. In the ascidian Ciona intestinalis, transposon-based transgenesis and insertional mutagenesis were achieved with a Tc1/mariner transposon Minos. We report development of a novel technique for enhancer trap in C. intestinalis. This technique uses remobilization of Minos in the Ciona genome. A Minos vector for enhancer trap was constructed and a tandem array insertion of the vector was introduced into the Ciona genome to create a mutator line. Minos was remobilized in Ciona chromosomes to create new insertions by providing transposases. These transposase-introduced animals were crossed with wild-type animals. Nearly 80% of F1 families showed novel GFP expression patterns. This high-throughput enhancer trap screen will be useful to create new marker transgenic lines showing reporter gene expression in specific tissues and to identify novel patterns of gene expression.
Asunto(s)
Ciona intestinalis/genética , Elementos Transponibles de ADN , Elementos de Facilitación Genéticos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , ADN/genética , Expresión Génica , Genes Reporteros , Técnicas Genéticas , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Hibridación Fluorescente in Situ , Proteínas Recombinantes/genética , Transposasas/genéticaRESUMEN
Improvements in closed-system culturing methods for marine invertebrates are important prerequisites for the generalized use of transgenic lines. We discuss here the effects of several closed-system conditions on the growth and survival of the solitary ascidian, Ciona intestinalis. In Shimoda, close to the sea, a small-tank system was used to ensure that tanks and systems were reasonably equipped, water exchange was rapid, and animals separated to minimize the risk of infection. In Gif-sur-Yvette, an inland site, we tried to determine the optimal conditions to limit handling operations, and to save artificial seawater by avoiding water pollution. A mixture of at least two types of live algae was better than any single-organism diet. With these maintenance protocols, we were able to obtain several generations of Ciona intestinalis, including several transgenic lines. Because these systems make it easier to rear Ciona intestinalis in laboratories, they increase the potentialities of this model organism for research.
Asunto(s)
Acuicultura , Ciona intestinalis , Animales , Animales Modificados Genéticamente , Ciona intestinalis/genética , Agua de MarRESUMEN
Tunicates are the only animals that perform cellulose biosynthesis. The tunicate gene for cellulose synthase, Ci-CesA, was likely acquired by horizontal transfer from bacteria and was a key innovation in the evolution of tunicates. Transposon-based mutagenesis in an ascidian, Ciona intestinalis, has generated a mutant, swimming juvenile (sj). Ci-CesA is the gene responsible for the sj mutant, in which a drastic reduction in cellulose was observed in the tunic. Furthermore, during metamorphosis, which in ascidians convert the vertebrate-like larva into a sessile filter feeder, sj showed abnormalities in the order of metamorphic events. In normal larvae, the metamorphic events in the trunk region are initiated after tail resorption. In contrast, sj mutant larvae initiated the metamorphic events in the trunk without tail resorption. Thus, sj larvae show a "swimming juvenile" phenotype, the juvenile-like trunk structure with a complete tail and the ability to swim. It is likely that ascidian cellulose synthase is required for the coordination of the metamorphic events in the trunk and tail in addition to cellulose biosynthesis.
Asunto(s)
Ciona intestinalis/enzimología , Elementos Transponibles de ADN , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Mutagénesis Insercional , Animales , Celulosa/biosíntesis , Ciona intestinalis/anatomía & histología , Ciona intestinalis/fisiología , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Metamorfosis Biológica , Fenotipo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Microinjection of the Minos transposon is the only reported technique for generating stable transgenic lines in the cosmopolitan ascidian, Ciona intestinalis. To establish a more amenable method for generating stable transgenic Ciona, we examined the possibility of using electroporation of DNA into eggs. From 0-44.4% of electroporated individuals transmitted transgenes to the next generation. The transgene was integrated into one chromosome and multiple copies of the transgene were inserted into one site of the chromosome, indicating that electroporation is an easy and powerful technique for achieving stable transgenesis in C. intestinalis. Together with possible inland culture of this ascidian, this technique will be useful for generating stable lines which have reporter gene expression in a specific tissue or organ and the generation of transposase-expressing stable transgenic (jump-starter) lines and mutator lines which contain a lot of Minos transposons in an insertion position.
Asunto(s)
Ciona intestinalis/genética , Electroporación/métodos , Técnicas Genéticas , Animales , Animales Modificados Genéticamente , Elementos Transponibles de ADN/genética , Femenino , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Hibridación Fluorescente in Situ , Masculino , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The enhancer trap technique, established in Drosophila melanogaster, is a very sophisticated tool. Despite its usefulness, however, there have been very few reports on enhancer traps in other animals. The ascidian Ciona intestinalis, a splendid experimental system for developmental biology, provides good material for developmental genetics. Recently, germline transgenesis of C. intestinalis has been achieved using the Tc1/mariner superfamily transposon Minos. During the course of that study, one Minos insertion line that showed a different GFP expression pattern from other lines was isolated. One fascinating possibility is that an enhancer trap event occurred in this line. Here we show that a Minos insertion in the Ci-Musashi gene was responsible for the altered GFP expression. Ci-Musashi showed a similar expression pattern to GFP. In addition, introns of Ci-Musashi have enhancer activity that can alter the expression pattern of nearby genes to resemble that of GFP in this line. These results clearly demonstrate that an enhancer trap event that entrapped enhancers of Ci-Musashi occurred in C. intestinalis.
Asunto(s)
Ciona intestinalis/genética , Elementos Transponibles de ADN/genética , Elementos de Facilitación Genéticos/genética , Proteínas de Unión al ARN/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Análisis por Conglomerados , Componentes del Gen , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Japón , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
An ascidian, Ciona savignyi, is regarded as a good experimental animal for genetics because of its small and compact genome for which a draft sequence is available, its short generation time and its interesting phylogenic position. ENU-based mutagenesis has been carried out using this animal. However, insertional mutagenesis using transposable elements (transposons) has not yet been introduced. Recently, one of the Tc1/mariner superfamily transposons, Minos, was demonstrated to cause germline transgenesis in the related species Ciona intestinalis. In this report, we show that Minos has the ability to transpose from DNA to DNA in Ciona savignyi in transposition assays. Although the activity was slightly weaker than in Ciona intestinalis, Minos still caused germline transgenesis in Ciona savignyi. In addition, one insertion seemed to have caused an enhancer trapping. These results indicate that Minos provides a potential tool for transgenic techniques such as insertional mutagenesis in Ciona savignyi.
Asunto(s)
Elementos Transponibles de ADN , Urocordados/genética , Animales , Mutación de Línea Germinal , Mutagénesis Insercional , Plásmidos , Transformación GenéticaRESUMEN
The tadpole larva of the basal chordate Ciona intestinalis has the most simplified, basic body-plan of chordates. Because it has a compact genome with a complete draft sequence, a large quantity of EST/cDNA information, and a short generation time, Ciona is a suitable model for future genetics. We establish here a transgenic technique in Ciona that uses the Tc1/mariner superfamily transposon Minos. Minos was integrated efficiently into the genome of germ cells and transmitted stably to subsequent generations. In addition, an enhancer-trap line was obtained. This is a demonstration of efficient, Minos-mediated transgenesis in marine invertebrates.
Asunto(s)
Animales Modificados Genéticamente , Ciona intestinalis/genética , Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Técnicas Genéticas , Transposasas/genética , Animales , Southern Blotting , ADN Complementario/metabolismo , Etiquetas de Secuencia Expresada , Vectores Genéticos , Genoma , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Modelos Genéticos , Reacción en Cadena de la PolimerasaRESUMEN
Cell growth and cell divisions are two fundamental biological processes for cells in multi-cellular organisms. The molecules involved in these biological processes are highly conserved within eukaryotes, including plants and unicellular organisms such as yeast. However, some regulatory molecules seem to be innovated during animal evolution. Therefore, to understand how the ubiquitous systems have evolved or have been conserved, we examined genes for the phosphoinositide 3-kinase (PI3K) pathway that is important for cell growth, and genes for cell cycle regulation in the genome of Ciona intestinalis. It was found that the Ciona intestinalis genome contains all the essential constituents of the PI3K pathway. In addition, the class IB PI3K catalytic and regulatory subunits, which had not previously been known in animals other than mammals, were found in the Ciona genome. Similarly, all essential cyclins and CDKs were found in the Ciona genome, while cyclin G and cyclin L were likely to be independently lost in the ascidian lineage, which may be dispensable for the cell cycle. Cyclin F, which was previously known only in vertebrates, was not found in the Ciona genome. Therefore, this gene was probably innovated during the evolution of vertebrates to be involved in vertebrate-specific cell cycle regulation. Since Ciona is regarded as one of the most primitive extant chordates, the present analysis gives us an insight into how these fundamental biological genes are evolved or are conserved during chordate evolution.
Asunto(s)
Ciclo Celular/genética , Ciona intestinalis/genética , Genoma , Fosfatidilinositol 3-Quinasas/genética , Filogenia , Transducción de Señal/genética , Animales , Ciona intestinalis/embriología , Análisis por Conglomerados , Bases de Datos GenéticasRESUMEN
Ascidians are simple chordates that are related to, and may resemble, vertebrate ancestors. Comparison of ascidian and vertebrate genomes is expected to provide insight into the molecular genetic basis of chordate/vertebrate evolution. We annotated muscle structural (contractile protein) genes in the completely determined genome sequence of the ascidian Ciona intestinalis, and examined gene expression patterns through extensive EST analysis. Ascidian muscle protein isoform families are generally of similar, or lesser, complexity in comparison with the corresponding vertebrate isoform families, and are based on gene duplication histories and alternative splicing mechanisms that are largely or entirely distinct from those responsible for generating the vertebrate isoforms. Although each of the three ascidian muscle types - larval tail muscle, adult body-wall muscle and heart - expresses a distinct profile of contractile protein isoforms, none of these isoforms are strictly orthologous to the smooth-muscle-specific, fast or slow skeletal muscle-specific, or heart-specific isoforms of vertebrates. Many isoform families showed larval-versus-adult differential expression and in several cases numerous very similar genes were expressed specifically in larval muscle. This may reflect different functional requirements of the locomotor larval muscle as opposed to the non-locomotor muscles of the sessile adult, and/or the biosynthetic demands of extremely rapid larval development.
Asunto(s)
Ciona intestinalis/genética , Proteínas Contráctiles/genética , Genoma , Filogenia , Secuencia de Aminoácidos , Animales , Ciona intestinalis/embriología , Análisis por Conglomerados , Bases de Datos Genéticas , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
As it has a simple genome structure, Ciona intestinalis is a good chordate species for studying the function of genes. To this end, it is a key requirement to introduce insertional mutagenesis using a transposable element to the ascidian system. The present study focuses on Minos, one of the Tc1/mariner superfamily transposons that is already used in a human cell line. By extrachromosomal excision and transposition assays, we found that Minos activity is very high in C. intestinalis. We also demonstrated the nuclear localization activity of Minos transposase in Ciona embryos. From these tests, we concluded that Minos transposase has complete activity when it is expressed in C. intestinalis, suggesting that Minos has the potential to be used for genome-wide insertional mutagenesis of C. intestinalis.
Asunto(s)
Ciona intestinalis/genética , Elementos Transponibles de ADN/genética , Embrión no Mamífero/metabolismo , Mutagénesis Insercional/métodos , Animales , Núcleo Celular/metabolismo , Ciona intestinalis/embriología , Femenino , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Microinyecciones , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transposasas/genética , Transposasas/metabolismoRESUMEN
The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.
Asunto(s)
Ciona intestinalis/genética , Genoma , Análisis de Secuencia de ADN , Alelos , Animales , Apoptosis , Secuencia de Bases , Celulosa/metabolismo , Sistema Nervioso Central/fisiología , Ciona intestinalis/anatomía & histología , Ciona intestinalis/clasificación , Ciona intestinalis/fisiología , Biología Computacional , Sistema Endocrino/fisiología , Dosificación de Gen , Duplicación de Gen , Genes , Genes Homeobox , Corazón/embriología , Corazón/fisiología , Inmunidad/genética , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Musculares/genética , Organizadores Embrionarios/fisiología , Filogenia , Polimorfismo Genético , Proteínas/genética , Proteínas/fisiología , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Glándula Tiroides/fisiología , Urocordados/genética , Vertebrados/anatomía & histología , Vertebrados/clasificación , Vertebrados/genética , Vertebrados/fisiologíaRESUMEN
The genome of the basal choradate Ciona intestinalis contains a basic set of genes with less redundancy compared to the vertebrate genome. Extensive EST analyses, cDNA sequencing, and clustering yielded "Ciona intestinalis Gene Collection Release 1," which contains cDNA clones for 13,464 genes, covering nearly 85% of the Ciona mRNA species. This release is ready for use in cDNA cloning, micro/macroarray analysis, and other comprehensive genome-wide analyses for further molecular studies of basal chordates.