Search PubMed⌕ Search

Biomedical subjects

C Desplan

Publications and source records attributed to C Desplan.

At least 55 records · Page 3Linked to original sources
PubMed1992

The homeodomain: a new face for the helix-turn-helix?

The discovery of conserved protein domains found in many Drosophila and mammalian developmental gene products suggests that fundamental developmental processes are conserved throughout evolution. Our understanding of development has been enhanced by the discovery of the widespread role of the homeodomain (HD). The action of HD-containing proteins as transcriptional regulators is mediated through a helix-turn-helix motif which confers sequence specific DNA binding. Unexpectedly, the well conserved structural homology between the HD and the prokaryotic helix-turn-helix proteins contrasts with their divergent types of physical interaction with DNA. A C-terminal extension of the HD recognition helix has assumed the role that the N-terminus of the prokaryotic helix plays for specification of DNA binding preference. However, the HD appears also capable of recognizing DNA in an alternative way and its specificity in vivo may be modified by regions outside the helix-turn-helix motif. We propose that this intrinsic complexity of the HD, as well as its frequent association with other DNA binding domains, explains the functional specificity achieved by genes encoding highly related HDs.

Amino Acid Sequence↗
PubMed1991

The paired box encodes a second DNA-binding domain in the paired homeo domain protein.

The homeo box, which encodes the DNA-binding homeo domain, is a DNA sequence motif present in several Drosophila developmental genes; it has been used to identify many homologous genes involved in mammalian development. The paired box is another conserved sequence motif, first identified in the paired (prd) and gooseberry (gsb) Drosophila homeo domain genes. It encodes a 128-amino-acid domain, the paired domain, which has since been found in other fly and mouse gene products, in association with the homeo domain or in its absence. We show that the paired box of the prd gene encodes a DNA-binding activity, independent of the DNA-binding activity of the Paired (Prd) homeo domain and with a different sequence specificity. The amino-terminal region of the paired domain, including one of the three predicted alpha-helices, is necessary and sufficient for binding. We investigate the binding of the Prd protein to two sites in the even-skipped promoter, which are composed of overlapping sequences bound by the homeo domain and by the paired domain. We also show that a mutation in the paired box of Prd, corresponding to the mutation in the paired box of the mouse Pax-1 gene thought to cause the undulated skeletal phenotype, destroys the ability of the Prd protein to bind to the paired domain-specific site. This supports the view that the undulated phenotype results from the inactivation of the DNA-binding activity of the paired domain of Pax-1.

Animals↗
PubMed1990

Binding site-dependent direct activation and repression of in vitro transcription by Drosophila homeodomain proteins.

Fushi tarazu and engrailed are two of the genes required for proper segmentation of the Drosophila embryo. Their protein products Fushi tarazu and Engrailed (Ftz and En) each contain a homeodomain and have been shown to act as transcriptional regulators in transient expression experiments in a Drosophila cell culture system. We used an in vitro transcription system to test whether the effects of Ftz and En on transcription were direct or indirect. Purified Ftz directly activates in vitro transcription by binding to homeodomain binding sites inserted upstream of the TATA box of the Drosophila hsp70 promoter. Equimolar amounts of purified En repress this activation by competition with Ftz for binding to these sites. These results indicate that Ftz and En act directly as transcription factors and suggest that such homeodomain proteins regulate development by combinatorial transcriptional control.

Animals↗
PubMed1990

Engrailed, a homeodomain protein, can repress in vitro transcription by competition with the TATA box-binding protein transcription factor IID.

Engrailed (En) is a homeodomain protein that binds to a consensus sequence (NP) and plays an important role during Drosophila development. Purified En, which is produced in Escherichia coli, binds not only to this consensus sequence but also to the TATA box of the Drosophila Hsp70 promoter and of other eukaryotic promoters. Interestingly, En represses transcription of these promoters in an in vitro-reconstituted mammalian transcription system and footprint analyses show that En competes with the TATA box-binding protein transcription factor IID for binding to the TATA box. In contrast, a stable template-committed complex formed by preincubation of transcription factor IID with the promoter is not disrupted by addition of En, and in this case transcription is not repressed. These in vitro studies suggest a transcriptional repression mechanism, involving competition between En and transcription factor IID for TATA box binding, that may be involved in En-mediated repression in vivo.

Animals↗
PubMed1989

A single amino acid can determine the DNA binding specificity of homeodomain proteins.

Many Drosophila developmental genes contain a DNA binding domain encoded by the homeobox. This homeodomain contains a region distantly homologous to the helix-turn-helix motif present in several prokaryotic DNA binding proteins. We investigated the nature of homeodomain-DNA interactions by making a series of mutations in the helix-turn-helix motif of the Drosophila homeodomain protein Paired (Prd). This protein does not recognize sequences bound by the homeodomain proteins Fushi tarazu (Ftz) or Bicoid (Bcd). We show that changing a single amino acid at the C-terminus of the recognition helix is both necessary and sufficient to confer the DNA binding specificity of either Ftz or Bcd on Prd. This simple rule indicates that the amino acids that determine the specificity of homeodomains are different from those mediating protein-DNA contacts in prokaryotic proteins. We further show that Prd contains two DNA binding activities. The Prd homeodomain is responsible for one of them while the other is not dependent on the recognition helix.

Amino Acid Sequence↗
PubMed1989

The products of the Drosophila gap genes hunchback and Krüppel bind to the hunchback promoters.

The first zygotic genes to be expressed during early Drosophila development are the gap genes. Their role is to read and interpret coarse positional information deposited in the egg by the mother and to refine it by cross-regulatory interactions and by controlling a class of pair-rule genes. Little is known about the molecular mechanisms by which the three cloned gap genes carry out their genetically defined functions. Here we report that the Krüppel (Kr) gene product (Kr) binds to the sequence AAGGGGTTAA, whereas the hunchback (hb) gene product (Hb) recognizes the consensus ACNCAAAAAANTA. We have identified binding sites for these proteins upstream of the two hb promoters, which we suggest could mediate the repression of hb by Kr and perhaps allow hb to influence its own expression.

Animals↗
PubMed1989

Evolutionary conservation of homeodomain-binding sites and other sequences upstream and within the major transcription unit of the Drosophila segmentation gene engrailed.

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.

Animals↗
PubMed1988

The sequence specificity of homeodomain-DNA interaction.

The Drosophila developmental gene, engrailed, encodes a sequence-specific DNA binding activity. Using deletion constructs expressed as fusion proteins in E. coli, we localized this activity to the conserved homeodomain (HD). The binding site consensus, TCAATTAAAT, is found in clusters in the engrailed regulatory region. Weak binding of the En HD to one copy of a synthetic consensus is enhanced by adjacent copies. The distantly related HD encoded by fushi tarazu binds to the same sites as the En HD, but differs in its preference for related sites. Both HDs bind a second type of sequence, a repeat of TAA. The similarity in sequence specificity of En and Ftz HDs suggests that, within families of DNA binding proteins, close relatives will exhibit similar specificities. Competition among related regulatory proteins might govern which protein occupies a given binding site and consequently determine the ultimate effect of cis-acting regulatory sites.

Allosteric Regulation↗
PubMed1986

In situ detection of vitamin D-induced calcium-binding protein (9-kDa CaBP) messenger RNA in rat duodenum.

We have previously described the molecular cloning of a cDNA fragment synthesized from rat duodenal mRNA coding for a 9000-dalton vitamin D-induced calcium-binding protein (9-kDa CaBP) (3). We now report the use of this cloned cDNA to study the cytological distribution of 9-kDa CaBP mRNA in rat duodenum by in situ hybridization. Tissue sections, fixed in ethanol:acetic acid, were hybridized to the 3H-cDNA probe and processed for autoradiography. The specificity of the CaBP mRNA-DNA hybrid formation was checked using 3H-labeled plasmid pBR322 DNA as a control probe. 9k-Da CaBP mRNA, visualized by silver grains, was found only in the absorptive epithelial cells, and the concentration was greater in the cells at the villous tips than in those of the crypts. The 9k-Da CaBP mRNA was observed mainly in the cytoplasm of the columnar cells and less frequently in the nucleus. Labeling was not seen in the brush border and goblet cells. The submucosa, with Brunner's glands and muscularis, also showed no specific 9-kDa CaBP mRNA concentration. This demonstration of 9-kDa CaBP gene activity in the columnar cells of the rat duodenum illustrates the usefulness of in situ hybridization for characterization of specific cells involved in the expression of 1,25(OH)2 D3 activity.

Animals↗
PubMed1985

Cholecalcin (a 9-kDa cholecalciferol-induced calcium-binding protein) messenger RNA. Distribution and induction by calcitriol in the rat digestive tract.

In view of the possible physiological importance of the 9-kDa cholecalcin (a 9000-Mr cholecalciferol-induced calcium-binding protein) in the intestinal transport of calcium in mammals, the gene expression of this protein has been analysed. Its regulation in the digestive tract of the growing rat by calcitriol (1,25-dihydroxycholecalciferol) was studied using a specific cloned [32P]cDNA to 9-kDa cholecalcin. Northern hybridisation studies show that the cDNA sequence hybridises to a single 500-600-nucleotide species throughout the digestive tract and therefore demonstrate identical 9-kDa-cholecalcin mRNA processing in the whole of the intestine and caecum. The highest concentrations of cholecalcin mRNA occur in the duodenum, proximal jejunum and caecum. The observed differences in 9-kDa-cholecalcin mRNA levels correlate well with both the in vivo variations in cholecalcin itself and with the known intestinal sites of calcium absorption. The whole intestine is able to respond to exogenous calcitriol but the response of the distal intestine and caecum, as measured by the increase in cholecalcin mRNA and corresponding protein, was proportionally higher than in the duodenum. The rapid production of fully functional cholecalcin mRNA, which was detectable as early as 1 h after a single dose of calcitriol to vitamin-D-deficient rats, provides convincing evidence that calcitriol increases 9-kDa cholecalcin production by increasing cholecalcin gene expression at the transcriptional level.

Animals↗
PubMed1985

Characterisation of rat 9-kDa cholecalcin (CaBP) messenger RNA using a complementary DNA. Absence of homology with 28-kDa cholecalcin mRNA.

The rat possesses two cholecalciferol-induced calcium-binding proteins, the cholecalcins (CaBP). The 9-kDa CaBP is mainly concentrated in the duodenum while 28-kDa CaBP is located in the kidney and cerebellum. The mRNA encoding 9-kDa CaBP has been characterised using the cloned cDNA, pC109, synthesised from rat duodenal 9-kDa CaBP mRNA [Desplan et al. (1983) J. Biol. Chem. 258, 13502-13505]. Nucleotide sequence analysis of this cDNA shows the presence of two stop codons, TGA and TAG, at positions 207 and 271, respectively, of the 3' untranslated region. The cDNA-hybridised mRNA, isolated from rat duodenum, directs the cell-free synthesis of two proteins precipitable by antisera to 9-kDa intestinal CaBP. A major protein comigrates with 9-kDa CaBP whereas a minor product corresponds to a protein which is larger by 2000 Da. The minor protein appears to result from read-through of the 'leaky' UGA stop signal. No protein band which was immunoprecipitable with 28-kDa CaBP antiserum was detected when cDNA-hybridized mRNA from rat kidney and cerebellum was translated in a cell-free system. Northern blots show that the cDNA pC109 sequence hybridizes to a homogeneous mRNA species 500-600 nucleotides long from rat duodenum. Larger mRNA species encoding 28-kDa CaBP are undetectable in rat kidney and cerebellum even under low stringency conditions. All these findings demonstrate that there is no cross-hybridisation between 9-kDa and 28-kDa CaBP mRNAs. Southern blot analysis of rat genomic DNA, that shows only one homologous 9-kDa gene, is consistent with these findings. Thus, all our data indicate that there are distinct genes coding for each rat cholecalcin.

Animals↗
PubMed1984

Rapid increase of calcitonin-specific mRNA after acute hypercalcemia.

The regulation of calcitonin (CT) secretion by calcium was studied by measuring CT mRNA extracted from thyroids of normal rats subjected to acute calcium stimulation in vivo. The 15000-Mr primary translation product of CT mRNA was identified by immunoprecipitation using specific antibodies. While total mRNA and total radioactivity incorporated after translation of total mRNA remained unaffected by the calcium stimulation, a fourfold increase in radioactivity incorporated in CT primary translation product occurred as early as 2 min after calcium administration. This peak coincided with a rise in plasma levels of the hormone and preceded a detectable decrease in tissue stores. These results suggest that calcium ion, either directly or indirectly via its action on intracellular stores of the hormone or its precursors, causes a rapid increase in cell levels of translatable CT mRNA. In view of the extremely short time (2 min) in which this increase occurs, the action is probably at the post-transcriptional level as no increase in CT mRNA levels could be detected by hybridization assay using a specific cDNA probe for human CT mRNA.

Acute Disease↗
PubMed1983

Sequence of rat intestinal vitamin D-dependent calcium-binding protein derived from a cDNA clone. Evolutionary implications.

We have recently reported molecular cloning of the cDNA synthesized from rat duodenal mRNA-encoding intestinal calcium-binding protein (ICaBP), a vitamin D3-induced protein (Desplan, C., Thomasset, M., and Moukhtar, M. S. (1983) J. Biol. Chem. 258, 2762-2765). Nucleotide sequence analysis of the longest cDNA insert (375 base pairs) permitted the assignment of 207 nucleotides of the coding region and 104 nucleotides of the entire 3'-noncoding region of the mRNA. Although the derived amino acid sequence for rat ICaBP differed from the bovine and porcine sequences by 16 and 14 residues, respectively, all the residues of each calcium-binding site met the proposed requirements of the "EF hand" theory. In contrast, several differences found in the linker regions might explain the absence of cross-immunoreactivity between rat and porcine ICaBPs. Analysis of nucleotide sequence homologies between the coding and noncoding regions showed that the region coding for the two calcium-binding sites (I and II) was immediately followed in the noncoding region by a sequence very similar to the sequence coding for site I. This suggests that rat ICaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure and that low Mr ICaBP could result in early termination of the translation of a larger molecule containing four sites.

Amino Acid Sequence↗
PubMed1983

Cell free translation of chicken calcitonin messenger RNA.

The primary step of calcitonin biosynthesis was studied in a normal organ: chicken ultimobranchial gland, a tissue particularly rich in calcitonin secretory cells. Poly(A)-rich RNA was extracted and purified from ultimobranchial organs and translated in a reticulocyte lysate in the presence of labelled methionine. Polyacrylamide gel electrophoresis of specific immunoprecipitates revealed a major band of Mr 14 500 and a band of Mr 13 300. Thus, in chicken the precursor of calcitonin is a Mr 14 500 polypeptide. The minor component of Mr 13 300 could represent limited processing by the reticulocyte lysate.

Animals↗