The caudal gene of the barnacle Sacculina carcini is not expressed in its vestigial abdomen.

We report the characterization of a caudal gene from the rhizocephalan cirripede Sacculina carcini and its embryonic and larval expression patterns. Cirripedes are maxillopodan crustaceans that are devoid of any complete abdominal segment at the adult stage. We currently explore the genetic basis of this peculiar body plan. In a previous study we have shown that they probably lack the abdominalA gene, while possessing the other Hox genes shared by arthropods. However, at least a part of the genetic program might be conserved, since the engrailed.a and engrailed.b genes are expressed in a posterior region that we interpret as a relic of an ancestral abdomen. Here we show first that the Sacculina caudal gene is expressed early in embryogenesis, which makes it the earliest genetic marker evidenced in the development of Sacculina and of any other crustacean species. It is expressed later in the embryo in the caudal papilla, a posterior proliferating zone of cells. During the larval stages, the caudal gene is first expressed in the whole thoracic region; then its expression regresses to the posterior end of the larva. Surprisingly, it is never expressed in the vestigial abdomen. This lack of expression of the Sacculina caudal gene in a posterior region, at odds with what is known in all other studied metazoan species, might be correlated with the defective development of the abdomen.

Barnacle duplicate engrailed genes: divergent expression patterns and evidence for a vestigial abdomen.

Cirripedes (barnacles) are crustaceans that are characterized by a very peculiar body plan, in particular by the lack of an abdomen. To study their body plan, we searched for their engrailed gene. We found two engrailed (en.a/en.b) genes in cirripedes. The two engrailed genes of the rhizocephalan barnacle Sacculina carcini are expressed in the posterior compartment of developing segments and appendages. When the neuroectoderm differentiates into epidermis and neuroderm the expression patterns of en.a and en.b diverge dramatically. en.a expression fades in segment epidermis whereas it is subsequently detected ventrally in reiterated putative neural cells. At the same time, en.b expression increases in the epidermis, which makes it a very good segmentation marker. Five tiny en.b stripes are observed between the sixth thoracic segment and the telson. We interpret these stripes as the molecular definition of vestigial abdominal segments, being the remnant of an ancestral state in keeping with the bodyplan of maxillopod crustaceans. engrailedexpression is the first molecular evidence for a segmented abdomen in barnacles.

Cloning and expression of the engrailed.a gene of the barnacle Sacculina carcini.

Cirripedia (barnacles) constitute a crustacean monophyletic taxon which is very well defined by several synapomorphies. In particular, all cirripedes are composed of six thoracic segments, but are devoid of any complete abdominal segment. This body plan is preserved in the adult in non-parasitic groups, while the parasitic rhizocephalan cirripedes completely lose arthropodian segmentation at the adult stage. These traits make them a particularly favourable model for studying the formation and maintenance of segmental identity. For the above reasons, it seemed worthwhile to look at the segmentation gene engrailed in a cirripede. A complete engrailed.a cDNA was isolated from larvae of the rhizocephalan cirripede Sacculina carcini. Its expression was monitored during larval development by use of the monoclonal antibody MAb4D9 directed against the Drosophila homologous proteins. The Sacculina engrailed.a gene is expressed during the second and third larval stages in stripes within a posterior area corresponding to the presumptive trunk segments. Surprisingly, these stripes appear in a posterior to anterior sequence. Six engrailed.a stripes characterize the thoracic segments of the cirripedean ground plan.

Molecules and the body plan: the Hox genes of Cirripedes (Crustacea).

Among arthropods, Cirripedia (barnacles) are remarkable in that they completely lack abdominal segments. This feature prompted us to study the Hox genes of three cirripede species, representing a wide array of the diversity of these organisms, a segmented sessile barnacle, Elminius modestus (Thoracica), the parasite of a crab, Sacculina carcini (Rhizocephala), and the burrowing barnacle Trypetesa lampas (Acrothoracica). Using PCR amplification of genomic DNA and cDNA and library probing, we have found seven clear cirripedian homologues of the eight homeotic Hox genes known in insects, including labial and proboscipedia homologues, that were not previously reported in crustaceans. In addition we have isolated a divergent Antp-like gene, named Diva, that we homologize to the ftz gene of insects. The homeotic gene abdominalA (abdA) was not retrieved from any of these three cirripede species. By contrast, we have found all eight homeotic homologue genes, including abdA, in Ulophysema oeresundense, a crustacean possessing a well-developed abdomen, belonging to the Ascothoracica, generally thought to be the sister group of Cirripedia. Since we have found in barnacles homeobox-containing genes that are more divergent from the Antennapedia type than the typical abdA, we believe that a bona fide abdA gene would not have escaped our search. Hence, the abdA gene has been lost or is profoundly derived in sequence during the evolution leading to the cirripedian lineage. If confirmed, the lack of abdA would represent the first case in which the loss of a homeotic gene is correlated with a change in body plan during the evolution of metazoans.

engrailed duplication events during the evolution of barnacles.

Barnacles (Cirripedia) are a subclass of Crustacea. Their peculiar segmentation pattern (few segments, absence of abdominal segments, and, in the parasitic rhizocephalan, loss of segmentation at the adult stage) prompted us to study the engrailed gene, which encodes a homeodomain transcription factor and is expressed in arthropods in the posterior half of each segment. We searched for engrailed genes by PCR in a representative cross section of the Cirripedia. Eight unambiguous engrailed genes were cloned from five species, three genes belonging to the same species (Elminius modestus). This implies two duplication events. Molecular phylogenies were constructed and a cladistic approach was applied to the most informative sites. The results indicate that the older duplication (en.a/en.b) is probably very ancient and concerns the whole cirripedean subclass, whereas the other (en.a1/en.a2) is specific to the Elminius lineage.

Domains of interaction between alpha interferon and its receptor components.

We describe how constraints on the binding of human interferons (IFNs), alpha1 and alpha2 and alpha8 on mouse cells are partially relieved by the expression of the bovine (Bo) or human (Hu) IFN alpha/beta receptor (IFNAR) component in these cells. We show that, while the binding of all three is substantially increased by the transfection of Bo IFNAR, it is accompanied by an increase in activity only in the case of alpha2 and alpha8 (IFNs that otherwise have little activity on mouse cells). IFN alpha1, which shows some partial activity on mouse cells, responds to the presence of Bo IFNAR by acting, at low concentrations, as a competitive antagonist to IFNs alpha2 and alpha8. A review of published results on IFN hybrid scanning and on the effects of expressing Bo IFNAR in human cells led us to propose that an N-terminal segment of the IFN molecule interacts directly with IFNAR. Applying site-directed mutagenesis to an IFN hybrid; alpha8[60]alpha1[92]alpha8, we show that the point mutations K84 to E84 and Y90 to D90 act synergistically to cause the hybrid to behave as the parental IFN alpha8, switching the preference from Mu to Hu IFNAR in transfected mouse cells. The published structural models for IFN reveal that positions 84 and 90 span the exposed residues of the alpha-helix C of the IFN molecule. We derive a model of IFN-receptor interaction in which the A helix and the C helix of IFN interact with IFNAR and in which a binding phase can be distinguished from a binding/activity phase. We propose that the so-called "hot" domains of the IFN molecule (the AB loop and the D helix) are presented by IFNAR to interact with an additional component of the functional receptor.

Specific antiviral activities of the human alpha interferons are determined at the level of receptor (IFNAR) structure.

Differences in activity among the family of human IFNs alpha are much reduced if these ligands are assayed on bovine cells. In particular, the activity of IFN alpha D is much higher on bovine than on human cells. To examine these differences, the bovine counterpart of the human IFNAR has been cloned and expressed in a human cell line. The transfected cell line now recognizes the human IFN alpha D as a high-specific-activity IFN subtype, indicating that the differences in sensitivity between the bovine and human cells to the human IFN alpha lie in the structure of the IFNAR chain rather than in the other components of the functional receptor.

The structure of the human interferon alpha/beta receptor gene.

Using the cDNA coding for the human interferon alpha/beta receptor (IFNAR), the IFNAR gene has been physically mapped relative to the other loci of the chromosome 21q22.1 region. 32,906 base pairs covering the IFNAR gene have been cloned and sequenced. Primer extension and solution hybridization-ribonuclease protection have been used to determine that the transcription of the gene is initiated in a broad region of 20 base pairs. Some aspects of the polymorphism of the gene, including noncoding sequences, have been analyzed; some are allelic differences in the coding sequence that induce amino acid variations in the resulting protein. The exon structure of the IFNAR gene and of that of the available genes for the receptors of the cytokine/growth hormone/prolactin/interferon receptor family have been compared with the predictions for the secondary structure of those receptors. From this analysis, we postulate a common origin and propose an hypothesis for the divergence from the immunoglobulin superfamily.

Biochemical properties of the YPT-related rab1B protein. Comparison with rab1A.

We recently identified a novel rat cDNA: rab1B, closely related to the rab1A cDNA and to the yeast YPT1 gene. The rab1B cDNA encodes a 202 amino acid protein (22.1 kDa) that was produced in Escherichia coli under the control of the phi 10 promoter for the T7 RNA polymerase. The rab1B protein was purified in large amounts to near homogeneity in a simplified procedure. We studied the biochemical properties of rab1B and rab1A proteins. They both bind specifically GTP and GDP and possess intrinsic GTPase activities. The rab1B Lys21----Met mutant protein does not bind GTP, whereas the Ala65----Thr mutant has a reduced GTPase activity and is competent for autophosphorylation in the presence of GTP.