RESUMEN
Historically, mutations have had a significant impact on the study of developmental processes and phenotypic evolution. Lesions in DNA are created by artificial methods or detected by natural genetic variation. Random mutations are then ascribed to genetic change by direct sequencing or positional cloning. Tunicate species of the ascidian genus Ciona represent nearly fully realized model systems in which gene function can be investigated in depth. Additionally, tunicates are valuable organisms for the study of naturally occurring mutations due to the capability to exploit genetic variation down to the molecular level. Here, we summarize the available information about how mutations are studied in ascidians with examples of insights that have resulted from these applications. We also describe notions and methodologies that might be useful for the implementation of easy and tight procedures for mutations studies in Ciona.
Asunto(s)
Ciona intestinalis/genética , Mutación , Animales , ADN/genética , Evolución Molecular , Técnicas Genéticas , Variación Genética , FenotipoRESUMEN
The expression pattern of Onecut genes in the central and peripheral nervous systems is highly conserved in invertebrates and vertebrates but the regulatory networks in which they are involved are still largely unknown. The presence of three gene copies in vertebrates has revealed the functional roles of the Onecut genes in liver, pancreas and some populations of motor neurons. Urochordates have only one Onecut gene and are the closest living relatives of vertebrates and thus represent a good model system to understand its regulatory network and involvement in nervous system formation. In order to define the Onecut genetic cascade, we extensively characterized the Onecut upstream cis-regulatory DNA in the ascidian Ciona intestinalis. Electroporation experiments using a 2.5kb genomic fragment and of a series of deletion constructs identified a small region of 262bp able to reproduce most of the Onecut expression profile during embryonic development. Further analyses, both bioinformatic and in vivo using transient transgenes, permitted the identification of transcription factors responsible for Onecut endogenous expression. We provide evidence that Neurogenin is a direct activator of Onecut and that an autoregulatory loop is responsible for the maintenance of its expression. Furthermore, for the first time we propose the existence of a direct connection among Neurogenin, Onecut and Rx transcription factors in photoreceptor cell formation.
Asunto(s)
Regulación de la Expresión Génica/genética , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/metabolismo , Factores de Transcripción Onecut/metabolismo , Células Fotorreceptoras/fisiología , Elementos Reguladores de la Transcripción/genética , Urocordados/genética , Animales , Electroporación , Histocitoquímica , Hibridación in Situ , Italia , Mar Mediterráneo , Proteínas del Tejido Nervioso/genética , Sistema Nervioso/embriología , Factores de Transcripción Onecut/genética , Células Fotorreceptoras/metabolismo , TranscriptomaRESUMEN
The sperm nuclear basic proteins (SNBPs) of the marine annelid worm Chaetopterus variopedatus have been shown previously to consist of a mixture of two SNBPs: histone H1-like (CvH1) and C.variopedatus protamine-like (CvPL). Here, we report the structural characterization of CvPL. The protein has a molecular weight of 8370.5 Da, a K/R ratio of 0.34, and a secondary structure, which are intermediate between those of protamine (P) and protamine-like (PL) SNBPs. The N-terminal sequence of CvPL shows a high extent of similarity with the arginine-rich C-terminal domain of chordate PL-type SNBPs. Furthermore, the protein binds to DNA in a similar fashion as vertebrate PLs and their own CvH1, but in a way that is different from that of the lysine-rich somatic H1 histones. We have experimentally determined the molar ratio CvH1:CvPL to be â¼1:6 in C. variopedatus sperm. Based on all of these, a model is proposed for the organization of the sperm chromatin by CvH1 and CvPL.
Asunto(s)
Proteínas Nucleares/química , Poliquetos/química , Homología de Secuencia de Aminoácido , Espermatozoides/química , Secuencia de Aminoácidos , Animales , Masculino , Datos de Secuencia Molecular , Protaminas/química , Alineación de SecuenciaRESUMEN
BACKGROUND: The Hox family of transcription factors has a fundamental role in segmentation pathways and axial patterning of embryonic development and their clustered organization is linked with the regulatory mechanisms governing their coordinated expression along embryonic axes. Among chordates, of particular interest are the Hox paralogous genes in groups 1-4 since their expression is coupled to the control of regional identity in the anterior nervous system, where the highest structural diversity is observed. RESULTS: To investigate the degree of conservation in cis-regulatory components that form the basis of Hox expression in the anterior nervous system, we have used assays for transcriptional activity in ascidians and vertebrates to compare and contrast regulatory potential. We identified four regulatory sequences located near the CiHox1, CiHox2 and CiHox4 genes of the ascidian Ciona intestinalis which direct neural specific domains of expression. Using functional assays in Ciona and vertebrate embryos in combination with sequence analyses of enhancer fragments located in similar positions adjacent to Hox paralogy group genes, we compared the activity of these four Ciona cis-elements with a series of neural specific enhancers from the amphioxus Hox1-3 genes and from mouse Hox paralogous groups 1-4. CONCLUSIONS: This analysis revealed that Kreisler and Krox20 dependent enhancers critical in segmental regulation of the hindbrain appear to be specific for the vertebrate lineage. In contrast, neural enhancers that function as Hox response elements through the action of Hox/Pbx binding motifs have been conserved during chordate evolution. The functional assays reveal that these Hox response cis-elements are recognized by the regulatory components of different and extant species. Together, our results indicate that during chordate evolution, cis-elements dependent upon Hox/Pbx regulatory complexes, are responsible for key aspects of segmental Hox expression in neural tissue and appeared with urochordates after cephalochordate divergence.
Asunto(s)
Ciona intestinalis/embriología , Ciona intestinalis/genética , Elementos de Facilitación Genéticos , Evolución Molecular , Genes Homeobox , Proteínas de Homeodominio/genética , Animales , Embrión de Pollo , Pollos , Regulación del Desarrollo de la Expresión Génica , RatonesRESUMEN
Retinal homeobox (Rx) genes play a crucial and conserved role in the development of the anterior neural plate of metazoans. During chordate evolution, they have also acquired a novel function in the control of eye formation and neurogenesis. To characterize the Rx genetic cascade and shed light on the mechanisms that led to the acquisition of this new role in eye development, we studied Rx transcriptional regulation using the ascidian, Ciona intestinalis. Through deletion analysis of the Ci-Rx promoter, we have identified two distinct enhancer elements able to induce Ci-Rx specific expression in the anterior part of the CNS and in the photosensory organ at tailbud and larva stages. Bioinformatic analysis highlighted the presence of two Onecut binding sites contained in these enhancers, so we explored the role of this transcription factor in the regulation of Ci-Rx. By in situ hybridization, we first confirmed that these genes are co-expressed in the same cells. Through a series of in vivo and in vitro experiments, we then demonstrated that the two Onecut sites are responsible for enhancer activation in Ci-Rx endogenous territories. We also demonstrated in vivo that Onecut misexpression is able to induce ectopic activation of the Rx promoter. Finally, we demonstrated that Ci-Onecut is able to promote Ci-Rx expression in the sensory vesicle. Together, these results support the conclusion that in Ciona embryogenesis, Ci-Rx expression is under the control of the Onecut transcription factor and that this factor is necessary and sufficient to specifically activate Ci-Rx through two enhancer elements.
Asunto(s)
Ciona intestinalis/embriología , Proteínas del Ojo/metabolismo , Ojo/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Onecut/metabolismo , Animales , Sistema Nervioso Central/metabolismo , Ciona intestinalis/genética , Biología Computacional , Ensayo de Cambio de Movilidad Electroforética , Electroporación , Proteínas de Homeodominio/genética , Hibridación in Situ , Regiones Promotoras Genéticas/genética , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. RESULTS: We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. CONCLUSIONS: The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.
Asunto(s)
Proteínas Arqueales/genética , Proteínas Bacterianas/genética , Eucariontes/genética , Animales , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Clonación Molecular , Biología Computacional , Flavobacteriaceae/genética , Ensayos Analíticos de Alto Rendimiento , Plásmidos/genética , Plásmidos/metabolismo , Pyrococcus abyssi/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Dorada/genéticaRESUMEN
BACKGROUND: The study of ascidians (Chordata, Tunicata) has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. CONCLUSIONS/SIGNIFICANCE: Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity.
Asunto(s)
Ciona intestinalis/fisiología , Variación Genética , Animales , Ciona intestinalis/genética , Genética de Población , Repeticiones de Microsatélite/genética , Mutación , FenotipoRESUMEN
Metamorphosis in the ascidian Ciona intestinalis is a very complex process which converts a swimming tadpole to an adult. The process involves reorganisation of the body plan and a remarkable regression of the tail, which is controlled by caspase-dependent apoptosis. However, the endogenous signals triggering apoptosis and metamorphosis are little explored. Herein, we report evidence that nitric oxide (NO) regulates tail regression in a dose-dependent manner, acting on caspase-dependent apoptosis. An increase or decrease of NO levels resulted in a delay or acceleration of tail resorption, without affecting subsequent juvenile development. A similar hastening effect was induced by suppression of cGMP-dependent NO signalling. Inhibition of NO production resulted in an increase in caspase-3-like activity with respect to untreated larvae. Detection of endogenously activated caspase-3 and NO revealed the existence of a spatial correlation between the diminution of the NO signal and caspase-3 activation during the last phases of tail regression. Real-time PCR during development, from early larva to early juveniles, showed that during all stages examined, NO synthase (NOS) is always more expressed than arginase and it reaches the maximum value at late larva, the stage immediately preceding tail resorption. The spatial expression pattern of NOS is very dynamic, moving rapidly along the body in very few hours, from the anterior part of the trunk to central nervous system (CNS), tail and new forming juvenile digestive organs. NO detection revealed free diffusion from the production sites to other cellular districts. Overall, the results of this study provide a new important link between NO signalling and apoptosis during metamorphosis in C. intestinalis and hint at novel roles for the NO signalling system in other developmental and metamorphosis-related events preceding and following tail resorption.
Asunto(s)
Apoptosis , Ciona intestinalis/embriología , Ciona intestinalis/genética , Regulación del Desarrollo de la Expresión Génica , Óxido Nítrico/metabolismo , Secuencia de Aminoácidos , Animales , Caspasa 3/metabolismo , Caspasas/metabolismo , GMP Cíclico/metabolismo , ADN Complementario/metabolismo , Hibridación in Situ , Larva/metabolismo , Metamorfosis Biológica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Frog relaxin (fRLX) belongs to the relaxin/insulin gene family present in the testis of Rana esculenta and is specifically expressed by Leydig cells. Since the expression of fRLX transcript changes during the reproductive cycle and is more abundant when circulating levels of androgens are relatively high, we investigated the effect(s) of testosterone and its antagonist (cyproterone acetate, CPA) on its expression pattern, in the testis of the frog Rana esculenta. Results from in vivo and in vitro experiments demonstrate that testosterone strongly induces a significant increase of fRLX mRNA expression in frog testes and, this effect is counteracted by CPA, supporting the existence of intratesticular (autocrine/paracrine) mechanisms of action. Interestingly, in both the control and testosterone-treated testes, fRLX mRNA expression was markedly decreased 24 h post-treatment, as compared to that measured at 2 h and 8 h post-treatment, suggesting that factor(s), other than testosterone, may act(s) in controlling its expression. In addition, RT-PCR analysis and in situ hybridization performed on frog testis injected with CPA for 15 days, on alternate days, showed a strong decrease of fRLX expression, suggesting that CPA counteracts the effect of testosterone on fRLX expression. Taken together our results strongly indicate that changes in the production, by the Leydig cells, of both testosterone and fRLX may represent a marker for the study of Leydig cell activity in the testis of the frog Rana esculenta.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , ARN Mensajero/metabolismo , Rana esculenta/metabolismo , Relaxina/metabolismo , Testosterona/farmacología , Animales , Northern Blotting , Acetato de Ciproterona/farmacología , Hibridación in Situ , Italia , Masculino , Rana esculenta/genética , Relaxina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testosterona/antagonistas & inhibidoresRESUMEN
Methylation of histones is one of the important "epigenetic" mechanisms associated with the transcriptional silencing and/or activating of tumor suppressor genes. To assess whether epigenetic phenomena could be involved in salivary gland carcinogenesis, the expression levels of four histone lysine-methyltransferases (HMT) were investigated, in both pleomorphic adenoma and the adjacent normal tissue of the parotid glands. The expression levels of three HMTs, SETB1, Eu-HMTase and SET08, were higher in tumor tissues. On the contrary, DOTL1 presented a lower expression level in the tumor tissues than in the corresponding normal tissues. These data suggest that the HMTs may be involved in the differentiation of pleomorphic adenoma, probably through chromatin structural changes, and indicates that the study of the epigenetic mechanism which modulates the variation in the methylation profile of histones may be useful to obtain information concerning those genes involved in tumor transformation in human parotid glands.
Asunto(s)
Adenoma Pleomórfico/enzimología , N-Metiltransferasa de Histona-Lisina/biosíntesis , Neoplasias de la Parótida/enzimología , Adenoma Pleomórfico/genética , Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/biosíntesis , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Neoplasias de la Parótida/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The tadpole larvae prosencephalon of the ascidian Ciona intestinalis contains a single large ventricle, along the inner walls of which lie two sensory organs: the otolith (a gravity-sensing organ) and the ocellus (a photo-sensing organ composed of a single cup-shaped pigment cell, about 20 photoreceptor cells, and three lens cells). Comparison has been drawn between the morphology and physiology of photoreceptor cells in the ascidian ocellus and the vertebrate eye. The development of vertebrate and invertebrate eyes requires the activity of several conserved genes and it is regulated by precise expression patterns and cell fate decisions common to several species. We have isolated a Ciona homeobox gene (Ci-Rx) that belongs to the paired-like class of homeobox genes. Rx genes have been identified from a variety of organisms and have been demonstrated to have a role in vertebrate eye formation. Ci-Rx is expressed in the anterior neural plate in the middle tailbud stage and subsequently in the larval stage in the sensory vesicle around the ocellus. Loss of Ci-Rx function leads to an ocellus-less phenotype that shows a loss of photosensitive swimming behavior, suggesting the important role played by Ci-Rx in basal chordate photoreceptor cell differentiation and ocellus formation. Furthermore, studies on Ci-Rx regulatory elements electroporated into Ciona embryos using LacZ or GFP as reporter genes indicate the presence of Ci-Rx in pigment cells, photoreceptors, and neurons surrounding the sensory vesicle. In Ci-Rx knocked-down larvae, neither basal swimming activity nor shadow responses develop. Thus, Rx has a role not only in pigment cells and photoreceptor formation but also in the correct development of the neuronal circuit that controls larval photosensitivity and swimming behavior. The results suggest that a Ci-Rx "retinal" territory exists, which consists of pigment cells, photoreceptors, and neurons involved in transducing the photoreceptor signals.
Asunto(s)
Ciona intestinalis/genética , Ojo/embriología , Genes Homeobox , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Electroporación , Hibridación in Situ , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
In the marine mollusk Aplysia limacina, a substantial amount of endogenous D-aspartic acid (D-Asp) was found following its synthesis from L-aspartate by an aspartate racemase. Concentrations of D-Asp between 3.9 and 4.6 micromol/g tissue were found in the cerebral, abdominal, buccal, pleural, and pedal ganglia. In non nervous tissues, D-Asp occurred at a very low concentration compared to the nervous system. Immunohistochemical studies conducted on cultured Aplysia neurons using an anti-D-aspartate antibody demonstrated that D-Asp occurs in the soma, dendrites, and in synaptic varicosities. Synaptosomes and synaptic vesicles from cerebral ganglia were prepared and characterized by electron microscopy. HPLC analysis revealed high concentrations of D-Asp together with L-aspartate and L-glutamate in isolated synaptosomes In addition, D-Asp was released from synaptosomes by K+ depolarization or by ionomycin. D-Asp was one of the principal amino acids present in synaptic vesicles representing about the 25% of total amino acids present in these cellular organelles. Injection of D-Asp into live animals or addition to the incubation media of cultured neurons, caused an increase in cAMP content. Taken as a whole, these findings suggest a possible role of D-Asp in neurotransmission in the nervous system of Aplysia limacina.
Asunto(s)
Aplysia/metabolismo , Ácido D-Aspártico/metabolismo , Sistema Nervioso/metabolismo , Animales , Aplysia/fisiología , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Inmunohistoquímica , Ionomicina/farmacología , Microscopía Electrónica , Modelos Biológicos , Sistema Nervioso/enzimología , Sistemas Neurosecretores/fisiología , Potasio/farmacología , Racemasas y Epimerasas/metabolismo , Transducción de SeñalRESUMEN
Several lines of evidence suggest that members of the POU domain gene family may regulate invertebrate and vertebrate neurogenesis. In particular, POU IV genes appear to be neural genes involved in differentiation of sensory neurons, as demonstrated in mollusc, Drosophila, Caenorhabditis elegans and vertebrates. In the present work, we describe the developmental expression of a homologue of POU IV genes, Ci-POU-IV, in the ascidian Ciona intestinalis. Ci-POU-IV is expressed in the precursor cells of the neural system during development and in the neural system of the larva. In particular, transcripts are prevalent in the peripheral nervous system (PNS), with expression in the central nervous system (CNS) restricted to the posterior sensory vesicle. Therefore, the evolution of a complex sensory system seems to be under the control of a common genetic mechanism.
Asunto(s)
Ciona intestinalis/embriología , Ciona intestinalis/genética , Regulación del Desarrollo de la Expresión Génica , Neuronas/metabolismo , Sistema Nervioso Periférico/citología , Secuencia de Aminoácidos , Animales , Evolución Biológica , Diferenciación Celular , Sistema Nervioso Central/citología , Ciona intestinalis/citología , Ciona intestinalis/metabolismo , Secuencia Conservada , Embrión no Mamífero , Proteínas de Homeodominio/química , Proteínas de Homeodominio/clasificación , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Larva/citología , Larva/fisiología , Metamorfosis Biológica , Datos de Secuencia Molecular , Neuronas/citología , Filogenia , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The msh/Msx family is a subclass of homeobox-containing genes suggested to perform a conserved function in the patterning of the early embryo. We had already isolated a member of this gene family (Ci-msxb) in Ciona intestinalis, which has a very complex expression pattern during embryogenesis. To identify the regulatory elements controlling its tissue-specific expression, we have characterized the gene structure and the regulatory upstream region. By electroporation experiments, we demonstrated that a 3.8-kb region located upstream of the gene contains all the regulatory elements able to reproduce its spatial expression pattern. Analyzing progressively truncated fragments of this region, three discrete and separate regions driving LacZ reporter gene expression in the ventral epidermis, primordial pharynx and neural territories have been identified. We further investigated the element(s) necessary for Ci-msxb activation in the nervous system during embryonic development by in vivo and in vitro experiments. Both electroporation and gel-shift assays of overlapping wild type and mutated oligonucleotides demonstrated that a unique sequence of 30 bp is involved in Ci-msxb neural activation from neurula to larva stage. This sequence contains consensus binding sites for various ubiquitous transcription factors such as TCF11 whose possible implication in formation of the regulatory complexes is discussed.
Asunto(s)
Ciona intestinalis/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Secuencia de Bases , Southern Blotting , Ensayo de Cambio de Movilidad Electroforética , Electroporación , Genes Reporteros/genética , Italia , Operón Lac/genética , Datos de Secuencia Molecular , Factores de Transcripción/metabolismoRESUMEN
Mos is a MAPK kinase kinase with an expression that is highly restricted to the gonads. Its function is mainly associated to the meiotic metaphase II arrest occurring during female gametogenesis, whereas to our knowledge, its role during spermatogenesis has not yet clarified. In the present paper, we report the isolation of c-mos cDNA and the identification of a 60-kDa Mos protein from the testis of the anuran amphibian, Rana esculenta. Both the transcript and the protein are always present at low levels in the testis during the frog annual sexual cycle, with single significant peaks of expression in March and May, respectively. Mos is mainly localized in the cytoplasm of primary and secondary spermatogonia (SPG). Therefore, we have used treatments with ethane-dimethane sulphonate (EDS), which blocks spermatogonial mitosis in frogs. Four days after a single EDS injection, Mos expression in SPG highly increases concomitantly with the temporary arrest of mitosis. From 8 to 28 days after the injection, the normal proliferative activity of SPG is restored, and Mos expression gradually decreases to control levels. These results strongly indicate that the c-mos proto-oncogene exerts a new role associated to the regulation of spermatogonial proliferation.
Asunto(s)
Proteínas Proto-Oncogénicas c-mos/metabolismo , Rana esculenta/anatomía & histología , Rana esculenta/metabolismo , Espermatogonias/citología , Espermatogonias/enzimología , Testículo/enzimología , Secuencia de Aminoácidos , Animales , División Celular , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Expresión Génica/efectos de los fármacos , Genes mos , Masculino , Mesilatos/farmacología , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-mos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estaciones del Año , Homología de Secuencia de Aminoácido , Testículo/citología , Testículo/efectos de los fármacosRESUMEN
Probes for the occurrence of endogenous D-aspartic acid (D-Asp) and N-methyl-D-aspartic acid (NMDA) in the neural complex and gonads of a protochordate, the ascidian Ciona intestinalis, have confirmed the presence of these two excitatory amino acids and their involvement in hormonal activity. A hormonal pathway similar to that which occurs in vertebrates has been discovered. In the cerebral ganglion D-Asp is synthesized from L-Asp by an aspartate racemase. Then, D-Asp is transferred through the blood stream into the neural gland where it gives rise to NMDA by means of an NMDA synthase. NMDA, in turn, passes from the neuronal gland into the gonads where it induces the synthesis and release of a gonadotropin-releasing hormone (GnRH). The GnRH in turn modulates the release and synthesis of testosterone and progesterone in the gonads, which are implicated in reproduction.
Asunto(s)
Ácido Aspártico/fisiología , Ciona intestinalis/metabolismo , N-Metilaspartato/fisiología , Animales , Ácido Aspártico/química , Ciona intestinalis/fisiología , Femenino , Hormona Liberadora de Gonadotropina/fisiología , Gónadas/metabolismo , Técnicas In Vitro , Hormonas de Invertebrados/química , Hormonas de Invertebrados/fisiología , Masculino , N-Metilaspartato/química , Sistemas Neurosecretores/fisiología , Progesterona/biosíntesis , Reproducción , Estereoisomerismo , Testosterona/biosíntesisRESUMEN
Homeobox-containing genes play crucial roles in various developmental processes, including body-plan specification, pattern formation and cell-type specification. The present study searched the draft genome sequence and cDNA/EST database of the basal chordate Ciona intestinalis to identify 83 homeobox-containing genes in this animal. This number of homeobox genes in the Ciona genome is smaller than that in the Caenorhabditis elegans, Drosophila melanogaster, human and mouse genomes. Of the 83 genes, 76 have possible human orthologues and 7 may be unique to Ciona. The ascidian homeobox genes were classified into 11 classes, including Hox class, NK class, Paired class, POU class, LIM class, TALE class, SIX class, Prox class, Cut class, ZFH class and HNF1 class, according to the classification scheme devised for known homeobox genes. As to the Hox cluster, the Ciona genome contains single copies of each of the paralogous groups, suggesting that there is a single Hox cluster, if any, but genes orthologous to Hox7, 8, 9 and 11 were not found in the genome. In addition, loss of genes had occurred independently in the Ciona lineage and was noticed in Gbx of the EHGbox subclass, Sax, NK3, Vax and vent of the NK class, Cart, Og9, Anf and Mix of the Paired class, POU-I, III, V and VI of the POU class, Lhx6/7 of the LIM class, TGIF of the TALE class, Cux and SATB of the Cut class, and ZFH1 of the ZFH class, which might have reduced the number of Ciona homeobox genes. Interestingly, one of the newly identified Ciona intestinalis genes and its vertebrate counterparts constitute a novel subclass of HNF1 class homeobox genes. Furthermore, evidence for the gene structures and expression of 54 of the 83 homeobox genes was provided by analysis of ESTs, suggesting that cDNAs for these 54 genes are available. The present data thus reveal the repertoire of homeodomain-containing transcription factors in the Ciona genome, which will be useful for future research on the development and evolution of chordates.
Asunto(s)
Ciona intestinalis/genética , Genes Homeobox/genética , Genoma , Filogenia , Animales , Ciona intestinalis/embriología , Análisis por Conglomerados , Bases de Datos GenéticasRESUMEN
Hox genes are organized in genomic clusters. In all organisms where their role has been studied, Hox genes determine developmental fate along the antero-posterior axis. Hence, these genes represent an ideal system for the understanding of relationships between the number and expression of genes and body organization. We report in this paper that the ascidian Ciona intestinalis genome appears to contain a single Hox gene complex which shows absence of some of the members found in all chordates investigated up to now. Furthermore, the complex appears to be either unusually long or split in different subunits. We speculate that such an arrangement of Hox genes does not correspond to the chordate primordial cluster but occurred independently in the ascidian lineage.
Asunto(s)
Ciona intestinalis/genética , Proteínas de Homeodominio/genética , Familia de Multigenes/genética , Secuencia de Aminoácidos , Animales , Mapeo Contig , ADN/química , ADN/genética , ADN Complementario/química , ADN Complementario/genética , Orden Génico , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Sea urchin DNA (cytosine-5)-methyltransferase (Dnmt1) that is responsible for maintenance of DNA methylation patterns clearly shares similarity with various Dnmt1s identified in vertebrates. In this study, we determined the structure of the sea urchin Dnmt1 gene by screening a genomic library of the sea urchin Paracentrotus lividus with the complementary DNA (cDNA) as probe. Analysis of the positive clones demonstrated that the Dnmt1 gene consists of 34 exons and 33 introns spanning a distance of 35 kb. All exon-intron junction sequences agree with the GT/AG consensus with the exception of the 3' acceptor site of intron 8 where CT replaces AG consensus. The differences in the total number of exons between sea urchin and mouse genes reside mainly in the N-terminal region of the protein (exons 5-7 of the sea urchin, exons 5-12 of the mouse) where there is very low similarity in the amino acid sequence. By reverse transcription-polymerase chain reaction using oligonucleotides spanning different regions of the cDNA we carried out a comprehensive analysis of alternative splicing of the Dnmt1 messenger RNA (mRNA) in sea urchin embryos at different stages of development. We demonstrated the presence of at least five alternative spliced mRNAs that are regulated during development and are translated in truncated or deleted proteins.