Nonmethylated transposable elements and methylated genes in a chordate genome.
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The genome of the invertebrate chordate Ciona intestinalis was found to be a stable mosaic of methylated and nonmethylated domains. Multiple copies of an apparently active long terminal repeat retrotransposon and a long interspersed element are nonmethylated and a large fraction of abundant short interspersed elements are also methylation free. Genes, by contrast, are predominantly methylated. These data are incompatible with the genome defense model, which proposes that DNA methylation in animals is primarily targeted to endogenous transposable elements. Cytosine methylation in this urochordate may be preferentially directed to genes. (+info)
A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis.
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A trypsin inhibitor from Ciona intestinalis, present throughout the animal, was purified by ion-exchange chromatography followed by four HPLC steps. By MS the molecular mass of the native form was determined to be 6675 Da. The N-terminal amino acid sequence was determined by protein sequencing, but appeared to be partial because the theoretical molecular mass of the protein was 1101 Da too low. Thermolysin treatment gave rise to several fragments each containing a single disulphide bridge. By sequence analysis and MS intramolecular disulphide bridges could unequivocally be assigned to connect the pairs Cys4-Cys37, Cys8-Cys30 and Cys16-Cys51. The structure of the inhibitor is homologous to Kazal-type trypsin inhibitors. The inhibitor constant, KI, for trypsin inhibition was 0.05 nM whereas chymotrypsin and elastase were not inhibited. To reveal the complete sequence the cDNA encoding the trypsin inhibitor was isolated. This cDNA of 454 bp predicts a protein of 82 amino acid residues including a 20 amino acid signal peptide. Moreover, the cDNA predicts a C-terminal extension of 11 amino acids compared to the part identified by protein sequencing. The molecular mass calculated for this predicted protein is in accordance with the measured value. This C-terminal sequence is unusual for Kazal-type trypsin inhibitors and has apparently been lost early in evolution. The high degree of conservation around the active site strongly supports the importance of the Kazal-type inhibitors. (+info)
Developmental expression of Pax1/9 genes in urochordate and hemichordate gills: insight into function and evolution of the pharyngeal epithelium.
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The epithelium of the pharynx contributes to the formation of gills in hemichordates, urochordates, cephalochordates and primitive vertebrates, and is therefore a key structure for understanding developmental mechanisms underlying the establishment of chordate body plans. Pax1- and Pax9-related genes encode transcription factors which are expressed in the pharyngeal region of cephalochordates as well as in the vertebrate pharyngeal pouch epithelium that forms the thymus and parathyroid glands. To explore the molecular basis underlying the occurrence and modifications of the pharyngeal epithelium during evolution, we isolated cDNA clones for Pax1- and Pax9-related genes of urochordates (HrPax1/9 of Halocynthia roretzi and CiPax1/9 of Ciona intestinalis) and a hemichordate (PfPax1/9 of Ptychodera flava) from gill cDNA libraries. Each gene is present as a single copy per haploid genome. All of the cDNAs encode typical paired domains and octapeptides but not a homeodomain, as is also true of other Pax1- and Pax9-related genes. Molecular phylogenetic analysis based on comparison of the paired domain amino-acid sequences suggests that HrPax1/9, CiPax1/9 and PfPax1/9 belong to the Pax1/9 subfamily, and that they are descendants of a single precursor of Pax1/Pax9. Screening of HrPax1/9 cDNA clones yielded six different types of transcripts which were generated by alternative splicing. Northern blot, RT-PCR/Southern and in situ hybridization analyses revealed that HrPax1/9, CiPax1/9 and PfPax1/9 are not expressed during early embryogenesis but are expressed in the epithelia of differentiating gills, suggesting that these genes encode gill-specific transcription factors. The Pax1/9 genes therefore might provide the first developmental genetic corroboration of hypotheses of organ-level homology that unifies hemichordates, urochordates and cephalochordates. (+info)
Brachyury downstream notochord differentiation in the ascidian embryo.
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The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential Ci-Bra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation. (+info)
Evolutionary alterations of the minimal promoter for notochord-specific Brachyury expression in ascidian embryos.
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The Brachyury genes of two divergent ascidians, As-T of Halocynthia roretzi and Ci-Bra of Ciona intestinalis, are expressed exclusively in notochord precursor cells. A previous study showed that the notochord-specific expression of Ci-Bra is controlled by a minimal promoter that is composed of three distinct regions: a region responsible for repression of expression in non-notochord mesoderm cells, a region for activation of expression in notochord cells, and a region for activation of expression in non-notochord mesoderm cells, distal to proximal to the transcription initiation site, respectively. We examined various deletion constructs of the As-T/lacZ fusion gene and demonstrate that a module between -289 and -250 bp of the 5'-flanking region is responsible for notochord-specific expression of the reporter gene. Gel-shift assays suggested the binding of nuclear protein(s) to this module. The 5'-flanking region of As-T contains a potential T-binding motif (-ACCTAGGT-) around -160 bp. Deletion of this motif from the p(-289)As-T/lacZ diminished the reporter gene expression. In addition, coinjection of p(-289)As-T/lacZ and synthetic As-T mRNA resulted in ectopic expression of lacZ in non-notochord cells, suggesting that the T-binding motif is responsible for autoactivation of the gene. These findings revealed striking differences between the minimal promoters of As-T and Ci-Bra so far revealed, with respect to their notochord-specific expression. Furthermore, reciprocal injections of reporter gene constructs, namely As-T/lacZ into Ciona eggs and Ci-Bra/lacZ into Halocynthia eggs, suggest alterations in the cis-regulatory elements and trans-activation factors that have occurred during evolution of the two ascidian species. (+info)
Follicle cell proteasome activity and acid extract from the egg vitelline coat prompt the onset of self-sterility in Ciona intestinalis oocytes.
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In the hermaphrodite ascidian Ciona intestinalis, the egg vitelline coat (VC) controls gamete self-nonself discrimination. Oocytes, after germinal vesicle breakdown, can be fertilized by both self and nonself sperm. However, a barrier to fertilization by self sperm progressively develops in the VC in the 3 hours after germinal vesicle breakdown. During this period, follicle cells attached to the outer surface of the VC release self-sterility factors that bind to the VC. Within the follicle cells, these factors (possibly peptides) are thought to be shuttled to the cell membrane by an hsp70 homolog (Cihsp70). In fact, antibodies to hsp70 block the development of self-sterility. Proteasomes are central to the production of antigen peptides. Specific inhibition of proteasome activity with clasto-lactacystin beta-lactone (CLbetaL) prevented the onset of self-sterility, but had no effect once this process had started. CLbetaL did not block fertilization by nonself sperm. The self-sterility factors were removed from mature oocytes by exposure to acidified media, and their biological activity was transferred to immature oocytes treated with CLbetaL. The obvious high multiplicity of self-nonself recognition alleles involved in fertilization, and the involvement of an hsp70 and a proteasome in processing self-sterility factors, suggests that this system may be evolutionarily related to the vertebrate immune system. (+info)
Membrane hyperpolarization by sperm-activating and -attracting factor increases cAMP level and activates sperm motility in the ascidian Ciona intestinalis.
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In the ascidian Ciona intestinalis (and C. savignyi), sperm-activating and -attracting factor (SAAF) is released from the egg at fertilization and stimulates both Ca(2+) influx and a transient increase in cAMP level of the sperm, leading to the activation of sperm motility (M. Yoshida et al., 1994, Dev. Growth Differ. 36, 589-595). In this paper we show in C. intestinalis that valinomycin, a potassium-selective ionophore, as well as SAAF, activated sperm motility, and this activation was suppressed by extracellular high K(+). Membrane potential measurements showed that both SAAF and valinomycin increase K(+) permeability of sperm and induce membrane hyperpolarization, the amplitude of which depends on the external K(+) concentration. The membrane potential and intracellular K(+) concentration of Ciona sperm without SAAF were estimated to be about -50 mV and 560 +/- 40 mM, respectively. After treatment with SAAF or valinomycin the membrane potential became almost equal to the equilibrium potential of K(+) (-100 mV), and the cAMP level increased in artificial seawater. A potent voltage-dependent K(+) channel blocker, MCD peptide, at the concentration of 10 microM blocked SAAF-induced hyperpolarization of the cells, increase in cAMP, and sperm motility. These results suggest that membrane hyperpolarization produced by the opening of K(+) channels elevates cAMP synthesis and leads to the activation of sperm motility in Ciona. (+info)
Patterning the ascidian nervous system: structure, expression and transgenic analysis of the CiHox3 gene.
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Hox genes play a fundamental role in the establishment of chordate body plan, especially in the anteroposterior patterning of the nervous system. Particularly interesting are the anterior groups of Hox genes (Hox1-Hox4) since their expression is coupled to the control of regional identity in the anterior regions of the nervous system, where the highest structural diversity is observed. Ascidians, among chordates, are considered a good model to investigate evolution of Hox gene, organisation, regulation and function. We report here the cloning and the expression pattern of CiHox3, a Ciona intestinalis anterior Hox gene homologous to the paralogy group 3 genes. In situ hybridization at the larva stage revealed that CiHox3 expression was restricted to the visceral ganglion of the central nervous system. The presence of a sharp posterior boundary and the absence of transcript in mesodermal tissues are distinctive features of CiHox3 expression when compared to the paralogy group 3 in other chordates. We have investigated the regulatory elements underlying CiHox3 neural-specific expression and, using transgenic analysis, we were able to isolate an 80 bp enhancer responsible of CiHox3 activation in the central nervous system (CNS). A comparative study between mouse and Ciona Hox3 promoters demonstrated that divergent mechanisms are involved in the regulation of these genes in vertebrates and ascidians. (+info)