Formation of the chordamesoderm in the amphioxus embryo: Analysis with Brachyury and fork head/HNF-3 genes

 The embryonic development of amphioxus (cephalochordates) has much in common with that of vertebrates, suggesting a close phylogenetic relationship between the two chordate groups. To gain insight into alterations in the genetic cascade that accompanied the evolution of vertebrate embryogenesis, we investigated the formation of the chordamesoderm in amphioxus embryos using the genes Brachyury and fork head/HNF-3 as probes. Am(Bb)Bra1 and Am(Bb)Bra2 are homologues of the mouse Brachyury gene isolated from Branchiostoma belcheri. Molecular phylogenetic analysis suggests that the genes are independently duplicated in the amphioxus lineage. Both genes are initially expressed in the involuting mesoderm of the gastrula, then in the differentiating somites of neurulae, followed by the differentiating notochord and finally in the tail bud of ten-somite stage embryos. On the other hand, Am(Bb)fkh/HNF3-1, an amphioxus (B. belcheri) homologue of the fork head/HNF-3 gene, is initially expressed in the invaginating endoderm and mesoderm, then later in the differentiating notochord and in the tail bud. With respect to these two types of genes, the formation of the notochord and tail bud in amphioxus embryos shows similarity and dissimilarity with that of the notochord and tail bud in vertebrate embryos. Received: 21 November 1996 / Accepted: 24 January 1997     

Exon duplication from a fork head to a homeodomain protein

The evolution of complex organisms such as animals requires a large expansion of the number of genes controlling developmental events. In addition, it is thought that domains are shuffled between genes to further increase the complexity and generate new types of genes and functions. Working with the Caenorhabditis elegans homeobox gene ceh-43, the orthologue of fly Distal-less (Dll), we observed sequence similarity to the C. elegans gene fkh-1. Now, with the complete genomic sequence available, we examined this similarity in detail. The region of similarity is confined essentially to one exon in the carboxy terminus of the two genes. Based on the gene structure, we think that an exon of fkh-1 was duplicated to the carboxy terminus of ceh-43, where it was incorporated as the last exon. This duplication event seems to have happened recently since the similarity on the nucleotide level is higher than the sequence similarity between fkh-1 of C. elegans and C. briggsae. Potentially the duplication event was mediated via a short region of sequence similarity between the two open reading frames of the genes. This duplication event clear shows that a part of a gene can successfully be juxtaposed to another gene. These events may perhaps not be rare. Received: 28 March 1999 / Accepted: 8 June 1999     

Mutant HNF-1alpha and mutant HNF-1beta identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1alpha and mutant HNF-1beta in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1alpha and 13 mutant HNF-1alpha, as well as wild HNF-1beta and 2 mutant HNF-1beta, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1alpha and wild HNF-1beta significantly transactivated DPP-IV promoter, but mutant HNF-1alpha and mutant HNF-1beta exhibited low transactivation activity. Moreover, to study whether mutant HNF-1alpha and mutant HNF-1beta change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1alpha or wild HNF-1beta, or else respective dominant-negative mutant HNF-1alphaT539fsdelC or dominant-negative mutant HNF-1betaR177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1alpha cells and wild HNF-1beta cells, whereas they decreased in HNF-1alphaT539fsdelC cells and HNF-1betaR177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1alpha and wild HNF-1beta have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1alpha and mutant HNF-1beta attenuate the stimulatory effect.     

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. Received: 18 June 1998 / Accepted: 24 October 1998     

Molecular characterization of a LMW-GS gene located on chromosome 1B and the development of primers specific for the Glu-B3 complex locus in durum wheat

 Low-molecular-weight glutenin subunits (LMW-GS) represent a specific class of wheat storage proteins encoded at the Glu-3 loci. Particularly interesting are the LMW-GS encoded at the Glu-B3 locus because they have been shown to play an important role in determining the pasta-making properties of durum wheat. Genes encoding LMW-GS have been characterized but only a few of them have been assigned to specific loci. Notably, no complete LMW-GS gene encoded at the Glu-B3 locus has yet been described. The present paper reports the isolation and characterization of a lmw-gs gene located at the Glu-B3 locus. The clone involved, designated pLDNLMW1B, contains the entire coding region and 524 bp of the 5′ upstream region. A nucleotide comparison between the pLDNLMW1B clone and other LMW-GS genes showed the presence of some peculiar structural characteristics, such as short insertions in the promoter region, the presence of a cysteine codon in the repetitive domain, and a more regular structure of this region, which could be important for its tissue-specific expression and for the functional properties of the encoded protein, respectively. Received : 30 May 1997 / Accepted : 29 July 1997