Biomedical subjects

J Delachambre

Publications and source records attributed to J Delachambre.

At least 19 recordsLinked to original sources

PubMed2004

A functional analysis of ACP-20, an adult-specific cuticular protein gene from the beetle Tenebrio: role of an intronic sequence in transcriptional activation during the late metamorphic period.

A gene encoding the adult cuticular protein ACP-20 was isolated in Tenebrio. It consists of three exons interspersed by two introns, intron 1 interrupting the signal peptide. To understand the regulatory mechanisms of ACP-20 expression, ACP-20 promoter-luciferase reporter gene constructs were transfected into cultured pharate adult wing epidermis. Transfection assays needed the presence of 20-hydroxyecdysone, confirming that ACP-20 is up-regulated by ecdysteroids. Analysis of 5' deletion constructs revealed that three regions are necessary for high levels of transcription. Interaction experiments between intronic fragments and epidermal nuclear proteins confirmed the importance of intron 1 in ACP-20 transcriptional control, which results from the combined activity of regulatory cis-acting elements of the promoter and those of intron 1.

Amino Acid Sequence↗

PubMed2000

A novel chitin-binding protein from the vestimentiferan Riftia pachyptila interacts specifically with beta-chitin. Cloning, expression, and characterization.

A cDNA from Riftia pachyptila was cloned. It encodes a novel 21.3-kDa protein from the worm protective tube, named RCBP (for Riftia chitin-binding protein). On the basis of partial tube-peptide sequences previously obtained, experiments using reverse transcriptase-mediated polymerase chain reaction and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of its deduced amino acid sequence shows the presence of two chitin-binding domains. These domains are closely related to type 2 chitin-binding domains that are restricted to the animal kingdom. We showed by affinity assay and immunogold labeling that RCBP is the first protein so far known that binds specifically beta-chitin and that is unable to bind the most common alpha-form found in chitin secreting animals. The RCBP mRNA was found to be present in specific epidermal cells from the worm body wall, but never in the chitin-secreting gland cells. This unexpected result clearly indicates that this tube protein is synthesized in specialized areas of the outer epithelium and that at least two different tissues are involved in this exoskeleton synthesis.

Amino Acid Sequence↗

PubMed2000

Characterization of a cDNA encoding RP43, a CUB-domain-containing protein from the tube of Riftia pachyptila (Vestimentifera), and distribution of its transcript.

A major 43 kDa protein from the protective tube of Riftia pachyptila (Vestimentifera), named RP43, was partly microsequenced after isolation by SDS/PAGE from the protein fraction of tubes collected around the hydrothermal vents at the East Pacific Rise. On the basis of the partial peptide sequences obtained, experiments using reverse-transcriptase-mediated PCR and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of deduced amino acid sequence of RP43 showed the presence of CUB domains (100-110-residue-spanning domains first reported in the complement subcomponents C1r/C1s, epidermal-growth-factor-related sea urchin protein and bone morphogenetic protein 1) that seem to be involved in protein-protein and glycosaminoglycan-protein interactions. This peculiarity strongly suggests that RP43 might have a crucial role in tightening the different elements of the worm tube. However, the absence of chitin-binding motifs inclines us to favour a role in protein-protein interactions during assembly of the tube components. The RP43 mRNA was found to be present in specific epidermal cells from the worm body wall but never in the chitin-synthesizing gland cells. This unexpected result clearly indicates that the major tube protein is synthesized in specialized areas of the outer epithelium and that at least two different tissues are involved in the synthesis of the exoskeleton.

Amino Acid Sequence↗

PubMed2000

Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysone.

Using a RT-PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by lambda-phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20-hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post-translational modification may play a role in the 20-hydroxyecdysone response.

Amino Acid Sequence↗

PubMed1999

cDNA cloning of an adult male putative lipocalin specific to tergal gland aphrodisiac secretion in an insect (Leucophaea maderae).

Lma-P22 is a cuticular surface protein specific to the tergal gland secretion of Leucophaea maderae adult males which is ingested by females just before copulation. The complete Lma-P22 cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. The recombinant protein expressed in Escherichia coli was recognized by an anti-Lma-P22 antibody. Northern blot analysis indicates that the corresponding mRNA is transcribed only in the epidermis of male tergites. Sequence analysis indicated that Lma-P22 deduced protein belongs to the lipocalin family. Lipocalins are extracellular proteins which carry hydrophobic compounds and some of them can bind sexual pheromone in vertebrates. Lma-P22 is the first example of a lipocalin-like protein involved in insect sexual behavior.

Amino Acid Sequence↗

PubMed1999

Is tubulin the sole antigen recognized by a putative anti-bursicon antibody?

A 56-kDa polypeptide suspected to be the tanning hormone 'bursicon' was analyzed using the monoclonal antibody (mAb) 01C10 of Song and Ma. We studied the beetle Tenebrio molitor, for which data on bursicon have been recently published. After purification by two-dimensional gel electrophoresis of brain proteins, the immunoreactive 56-kDa polypeptide was trypsinated and microsequenced. The obtained sequences revealed a high homology with alpha- and beta-tubulins. In a complementary study, immunoreactive clones were isolated, using the 01C10 mAb, from a library in expression vector obtained from Drosophila melanogaster head cDNAs. Again, the isolated clones were found, after cDNA sequencing, to correspond to tubulin. Our results suggest that, although the 01C10 mAb could possibly still have a great affinity for a polypeptide present in very low quantities in a few brain neurosecretory cells, it also proved to have an artefactual affinity for a 56-kDa polypeptide, identified as tubulin, which is not involved in tanning control.

Animals↗

PubMed1999

Cloning and characterization of new orphan nuclear receptors and their developmental profiles during Tenebrio metamorphosis.

Five PCR fragments corresponding to a part of the DNA-binding domain of different hormone nuclear receptors were isolated from Tenebrio molitor mRNAs. The sequence identity of three of them with known Drosophila nuclear receptors strongly suggests that they are the Tenebrio orthologs of seven-up, DHR3 and beta-FTZ-F1, and thus named Tmsvp, TmHR3 and TmFTZ-F1. The full-length sequences of the other two were established. TmHR78 is either a new receptor of the DHR78 family or the same gene which has evolved rapidly, particularly in the E domain. TmGRF belongs to the GCNF1 family and its in vitro translated product binds to the extended half site TCAAGGTCA with high affinity. The periods of expression of the corresponding transcripts in epidermal cells during Tenebrio metamorphosis were analyzed as a function of 20-hydroxyecdysone titers measured in the hemolymph of the animals taken for RNA extraction. Comparison of the expression profiles of these nuclear receptors with those observed during Drosophila metamorphosis revealed similar temporal correlations as a function of ecdysteroid variations, which further supported the sequence identity data for TmSVP, TmHR3, TmFTZ-F1 and TmHR78.

Amino Acid Sequence↗

PubMed1998

Structure, organization and expression of two clustered cuticle protein genes during the metamorphosis of an insect, Tenebrio molitor.

A 4-kb DNA segment of Tenebrio molitor (Insecta, Coleoptera) genomic DNA containing two larval-pupal cuticular genes has been cloned and sequenced. These genes, transcribed in opposite directions, are related in DNA sequence and the proteins encoded are very similar. Each of them contains a single intron located inside the sequence encoding the signal peptide, and a conserved sequence at -200 bp from the mRNA start position. These similarities in sequence suggest that these genes have evolved by duplication followed by diversification and that they are members of a family of genes with a common ancestry. They are the first example of clustered genes in Tenebrio molitor.

Amino Acid Sequence↗

PubMed1998

Characterization of two new cuticular genes specifically expressed during the post-ecdysial molting period in Tenebrio molitor.

In a previous study, we have isolated a cDNA, TM-ACP17, coding for a post-ecdysial adult protein of Tenebrio molitor. After screening of a genomic library with TM-ACP17, we report isolation and sequencing of TM-ACP17 gene and a new gene, TM-LPCP29, coding for a larval-pupal protein. These two genes exhibit a common sequence of 15 nucleotides and a characteristic of most cuticular protein genes so far described: an intron interrupting the signal peptide. The deduced aa sequence of TM-LPCP29 exhibits a high percentage of Ala (26.5%) and Val (17.5%) and is highly hydrophobic. In the N-terminal part, the motif VAAPV is repeated ten times. Numerous histidine residues are present in the C- and N-terminal regions. A comparison is made with other cuticle protein sequences. Northern hybridization analysis showed that TM-LPCP29 is present during larval and mainly pupal post-ecdysial cuticle secretion. In-situ hybridization revealed that TM-LPCP29 mRNA is expressed in epidermis and not in muscles or fat body.

Amino Acid Sequence↗

PubMed1997

Cloning of two putative ecdysteroid receptor isoforms from Tenebrio molitor and their developmental expression in the epidermis during metamorphosis.

Using the Drosophila EcR-B1 cDNA as a probe, we have cloned the putative ecdysteroid receptor from the mealworm Tenebrio molitor. We have isolated two cDNAs with different 5' termini that contain a complete open reading frame. These two cDNAs encode two proteins with distinct N-terminal regions corresponding to two isoforms. The coleopteran receptor is obviously related to the ecdysteroid receptor of other insects, but shares only 89% and 61% amino acid identities with the DNA-binding and ligand-binding domains of the Drosophila receptor, respectively. Its expression pattern has been examined in the epidermis during the last larval instar and pupal stage of T. molitor, in correlation with the hemolymph ecdysteroid titer. Hybridizations revealed two transcripts of 7 kb and 6.5 kb detected in most stages during metamorphosis and corresponding to the A and B1 isoforms. These two mRNAs are highly evident just before the rise of each ecdysteroid peak both in prepupae and in pupae. They show almost the same expression pattern in epidermis except for the second part of the pupal stage, during which only the A isoform is detected.

Amino Acid Sequence↗

PubMed1996

Up-regulation of an adult cuticular gene by 20-hydroxyecdysone in insect metamorphosing epidermis cultured in vitro.

Pupal forewing epidermis of the Coleoptera, Tenebrio molitor, was used to develop an in vitro system to study the hormonal control of metamorphosis at the cellular and molecular levels. Exposure to 1 microM 20-hydroxyecdysone for 48 h caused the formation of a typical adult cuticle. Under these conditions the expression of ACP-20, an adult-specific cuticular gene, was fivefold higher than in absence of exogenous hormone. This stimulation was also observed when a higher level of 20-hydroxyecdysone was maintained, and prevented by protein inhibitors, indicating that 20-hydroxyecdysone does not act directly on this gene. Exposure to 20-hydroxyecdysone followed by exposure in hormone-free medium caused the cessation of this stimulation, showing the requirement of the 20-hydroxyecdysone continuous presence for stimulating ACP-20 gene expression. Thus, unlike the other cuticular protein genes so far studied, its expression is not repressed by 20-hydroxyecdysone, and does not need the decline in ecdysteroids titer.

Animals↗

PubMed1996

Cloning and sequencing of a cDNA encoding a larval-pupal-specific cuticular protein in Tenebrio molitor (Insecta, Coleoptera). Developmental expression and effect of a juvenile hormone analogue.

A cDNA clone encoding a larval-pupal cuticular protein, named TMLPCP-22, has been isolated by screening a library in expression vector with a monoclonal antibody made against pupal cuticular proteins of Tenebrio molitor. Northern-blot and in situ hybridization analyses showed that the expression of TMLPCP-22 is regulated in a stage-specific and tissue-specific manner; the transcript was present during the secretion of preecdysial larval and pupal cuticles and was restricted to epidermal cells. No expression was observed during adult cuticle deposition. In supernumerary pupae obtained after application of a juvenile hormone analogue, which is known to inhibit the adult programme, TMLPCP-22 mRNA was expressed again, confirming its larval-pupal specificity.

Amino Acid Sequence↗

PubMed1995

Identification, sequence and mRNA expression pattern during metamorphosis of a cDNA encoding a glycine-rich cuticular protein in Tenebrio molitor.

The study of insect cuticular proteins and their sequences is of interest because they are involved in protein-protein and protein-chitin interactions which confer the mechanical properties and fine architecture of the cuticle. Moreover, in the coleopteran Tenebrio molitor there is a dramatic change in cuticular architecture between pre- and postecdysial secretion. We report the isolation, by differential screening, and the sequence characterization of a cDNA clone encoding a cuticular protein of T. molitor, ACP17. After insertion in the expression vector pEX1, the recognition of the fusion protein by an anti-cuticular monoclonal antibody confirmed the cuticular nature of ACP17. Northern hybridization analysis showed that ACP17 mRNA expression begins weakly 3 days before adult ecdysis and strongly increases during the secretion of postecdysial adult cuticle, with a maximum just after ecdysis. In situ hybridization revealed that the ACP17 mRNA is only present in the epidermis which secretes hard cuticle. The deduced amino acid (aa) composition exhibits a high content of Gly (28%) and Ala (20%) and, particularly, two poly(Gx) stretches separated by repetitive motifs with proline AAPVA. A comparison is made with other cuticle aa sequences.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence of an adult-specific cuticular protein gene from the beetle Tenebrio molitor: effects of 20-hydroxyecdysone on mRNA accumulation.

The accumulation of transcripts from two adult-specific cuticular genes (ACP-20 and ACP-22) is shown to be modified after addition of exogenous 20-hydroxyecdysone. In the continuous presence of high levels of the hormone, the expression of ACP-20 gene is significantly weaker than that of untreated controls, while ACP-22 expression is 2.5-fold increased. During active synthesis of the ACP messages, a 0.5 microg 20-hydroxyecdysone injection causes a rapid 2-fold increase in ACP-22 mRNA and is not able to repress ACP-20 mRNA accumulation. We conclude that these genes whose transcripts appear in an almost coordinated manner in epidermal cells during the moulting cycle are regulated by ecdysteroids in a different way. In order to undertake a functional dissection of the promoter regions of ACP-22 gene, we have isolated and sequenced a genomic clone. The sequence similarities with other cuticular protein genes are discussed.

Amino Acid Sequence↗

PubMed1992

cDNA cloning and deduced amino acid sequence of a major, glycine-rich cuticular protein from the coleopteran Tenebrio molitor. Temporal and spatial distribution of the transcript during metamorphosis.

In Coleoptera, the elytra (forewings), with a very hard and thick cuticle, protect the membranous and delicate hindwings against mechanical stress. We have isolated and characterized a cDNA encoding a major cuticle protein in Tenebrio molitor, named ACP-20. The deduced amino acid sequence is roughly tripartite, with two terminal glycine-rich domains and a central region showing pronounced similarities with some other hard cuticle proteins. Northern blot and in situ hybridization analyses reveal that ACP-20 gene expression is developmentally regulated since transcript accumulation occurs only in epidermal regions synthesizing hard cuticle and is restricted to the period of preecdysial adult cuticle deposition. Moreover, application of a juvenile hormone analogue prevents the appearance of the transcript, indicating that juvenile hormone, a key molecule involved in the control of insect metamorphosis, negatively regulates the expression of the ACP-20 gene.

Amino Acid Sequence↗

PubMed1992

Developmental profiles of epidermal mRNAs during the pupal-adult molt of Tenebrio molitor and isolation of a cDNA clone encoding an adult cuticular protein: effects of a juvenile hormone analogue.

Changes in translatable mRNAs from the wing epidermis of the Coleoptera Tenebrio molitor have been investigated during metamorphosis by analysis of in vitro translated products. Striking differences between the patterns obtained from mRNAs extracted during pupal and adult cuticle secretion indicated that a drastic change in gene expression occurs during the pupal-adult transition. In addition to these stage-specific modifications, the mRNA patterns changed within each cuticular synthesis program (pupal or adult), especially at ecdysis. After tritiated leucine incorporation, some of the major radiolabeled cuticular proteins showed similar changes suggesting that the sequential appearance of mRNAs corresponds to sequential deposition of cuticular proteins. In supernumerary pupae obtained after juvenile hormone analogue (JHA) application on newly ecdysed pupae, translatable mRNA were very similar to those of pharate pupae. The JHA seemed, therefore, to prevent the expression of the adult program. By immunoblotting in vitro translated products with a monoclonal antibody recognizing an adult-specific cuticular protein, the developmental profile of the corresponding mRNA was studied. This mRNA was detected in anterior wing epidermis during the first 80 hr of the pharate adult stage. Using the same antibody, a cDNA clone was isolated from epidermal mRNA. The hybrid selected mRNA coded for only one protein with an apparent MW of 22 kDa which was, furthermore, recognized by the antibody. The Northern blot analysis performed with the clone confirmed the Western blot analysis of the in vitro translation products. JHA application at the beginning of the pupal-adult reprograming prevented the appearance of this mRNA; however, this transcript was present during the following molting cycle. This reversibility of the JHA action was confirmed by immunogold labeling of the cuticles formed in treated animals.

Animals↗

PubMed1992

Characterization of a cDNA clone encoding a glycine-rich cuticular protein of Tenebrio molitor: developmental expression and effect of a juvenile hormone analogue.

The complete sequence of a cDNA clone, isolated from epidermal mRNA of Tenebrio molitor using a monoclonal antibody raised against an adult-specific cuticular antigen only present in the hard cuticle, was obtained after primer extension at the 5' end. From this cDNA sequence, the deduced protein encompasses 199 amino acids (including a signal peptide) with a total molecular weight of 20.7 kDa. The protein exhibits a bipartite structure: glycine-rich region located in its NH2-terminal part and a carboxy-terminal domain sharing homologies with other cuticular proteins of Orthoptera, Diptera and Lepidoptera. In-situ hybridization analysis shows that the corresponding mRNA is present only in epidermal cells secreting the adult fibrous cuticle destined to become heavily sclerotized. In supernumerary pupae obtained after the application of the juvenile hormone analogue (JHA) to newly ecdysed pupae, the mRNA was undetectable, indicating that JHA can prevent the switch to the adult programme. However, in pupal-adult intermediates, obtained when JHA is applied later, the mRNA is detected.

Amino Acid Sequence↗

PubMed1989

A monoclonal antibody against an adult-specific cuticular protein of Tenebrio molitor (Insecta, Coleoptera).

To study the sequential expression of the epidermal program in the mealworm Tenebrio molitor, monoclonal antibodies were prepared against the water-soluble proteins from preecdysial adult cuticle. Among the 16 clones obtained, one of them (named K2F6) recognized a 20-kDa antigen, found only in adult extracts but not in the larval or pupal ones, as revealed by immunoblot analysis. Our results strongly suggest an epidermal origin for this protein. The monoclonal antibody K2F6 fails to react with water-soluble proteins from fat body and hemolymph taken during the deposition of the 20-kDa antigen. Electron microscopic immunogold localization of this antigen showed that it is secreted, just after epicuticle deposition, in the 30 first-deposited preecdysial lamellae of sternal and elytral cuticles only. The sclerotizing process, which modifies the physicochemical properties of these cuticles, does not prevent the immunoreaction. When the expression of the adult program was inhibited by application of a juvenile hormone analog (ZR 515), the water-soluble proteins from different pupal-adult intermediates were never recognized by the monoclonal antibody K2F6 using immunoblot analysis. These results support the conclusion that this 20-kDa antigen is a protein specific for the sclerotized cuticle of the adult stage.

Animals↗