Cuticle Protein Genes Research Articles

Glycine-rich protein genes, which encode a major component of the cuticle, have different developmental profiles from other cuticle protein genes in Bombyx mori

Three types of GRP (glycine-rich proteins) cDNAs were identified in the EST database of Bombyx mori. These came from 21 ESTs in the W3-stage wing disc EST library. We named them BmGRP1, BmGRP2 and BmGRP3. BmGRP1 and BmGRP2 had 57% identity in deduced amino acid sequences. Expression of all BmGRPs was observed in the epidermis at the fourth molting stage, and in the wing at pupation and mid-pupal stage. It is suggested that BmGRPs contribute to larval, pupal and adult cuticles together with other cuticle proteins. Transcripts of BmGRP2 increased after 7 days of pupal stage. BmGRP2 is suggested to construct adult trachea in the wing. Hormonal response of BmGRPs was compared with that of another group of cuticular protein genes, BMWCPs. BmGRPs were induced by a pulse of 20E. Induction of BmGRP3 was observed in W1 wing discs in the presence of JHA which was added with 20E, whereas that of BMWCP2 was inhibited in the presence of JHA. Induction of BmGRPs was observed in the wing discs of V3 and W1 stages, while that of BMWCP2 was not observed in the V3 wing discs. These differences between BMWCPs and BmGRPs in response to hormones at different developmental stages are discussed.

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein from the Chinese oak Silkmoth,Antheraea pernyi

In our research to identify gene involved in the cuticle protein, we cloned a novel cuticle protein gene, ApCP13, from the Chinese oak silkmoth, Antheraea pernyi, larvae cDNA library. The ApCP13 gene encodes a 120 amino acid polypeptide with a predicted molecular mass of 13 kDa and a pI of 4.01, and is intron-less gene. The ApCP13 contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the ApCP13 cDNA is most homologous to another wild silkmoth, A. yamamai CP12 (86% protein sequence identity), followed by Bombyx mori LCP18 (35% protein sequence identity). Northern blot analysis revealed that the ApCP13 showed the epidermis-specific expression. This is the first report of cuticle protein gene in the wild silkmoth, A. pernyi.

Isolation and comparison of different ecdysone-responsive cuticle protein genes in wing discs of Bombyx mori

Microarray analysis was used to isolate an ecdysone up-regulated cuticle protein gene from wing discs of Bombyx mori. Transcripts of isolated cDNAs were identified by Northern blot analysis. Expression of the BMWCP10 gene was observed during the W0-W3 stages with the strongest signal being at the W2 stage. In contrast, expression of the BMWCP2 gene was observed at the W3and P0 stages. Expression of BMWCP10 was identified after exposure to 20E in vitro, while that of BMWCP2 was identified after exposure to 20E followed by its removal. Induction of BMWCP10 by 20E was observed in 30 min and was not inhibited by cycloheximide. Expression of BMWCP2 was observed in wing discs cultured for more than 18 h in a hormone-free medium after 20E removal. At least 4 h exposure to 20E was required before removal for induction of BMWCP2. Induction of BMWCP2 required protein synthesis. Thus, different ecdysone-responsive cuticle protein genes in wing discs of Bombyx mori were isolated.

Comparison of newly isolated cuticular protein genes from six aphid species

This paper reports on the first aphids’ cuticular proteins. One gene ( Mpcp1) was obtained by screening a cDNA library of Myzus persicae with antibodies to a lepidopteran cuticle protein. MpCP1 presents a putative signal peptide, a central extended R&R domain, flanked by N- and C-terminal repeats of alanine, tyrosine and proline. The mRNA of Mpcp1 could be detected in a larval and in adult stages. Primers based on Mpcp1 allowed isolating and comparing cuticle protein genes from five aphid species, but not from whitefly or thrips. Comparison revealed a high degree of similarity. Data from this paper suggest that this cuticle protein family is typical and predominant to aphids. The conformation of these cuticle proteins and the significance on particular properties of aphid cuticle is discussed.

Ecdysteroid-dependent expression of a novel cuticle protein gene BMCPG1 in the silkworm, Bombyx mori

When insects molt, the exoskeleton is renewed under the controls of insect hormones via the biosynthesis and degradation of cuticle proteins. To understand the hormonal control of cuticle formation, we used the differential display method to look for stage-specific cuticle genes, and identified a novel cDNA named Bombyx mori Cuticle Protein GlyGlyTyr-repeat 1 ( BMCPG1). Expression of BMCPG1 mRNA peaked sharply immediately after a pulse of ecdysteroid during the fourth molt and pre-pupal stages, concurrent with the expression of genes for FTZF1 and dopa decarboxylase. BMCPG1 was expressed only in the epidermis, but not in any other tissue. We cultured the larval epidermis and found that BMCPG1 expression is not induced by the continuous presence of ecdysteroid. Removal of ecdysteroid from the medium, which constitutes a pulse treatment, is required for the induction of BMCPG1 transcription. These results explain well the stage-specific expression of BMCPG1 by ecdysteroid in vivo. Based on its expression patterns and unique structure, we propose that BMCPG1 may be a novel component of epicuticle of B. mori, and is probably involved in cross-linking of proteins via its GGY repeats.

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.

Structural analysis of gene encoding cuticle protein BMCP18, and characterization of its putative transcription factor in the silkworm, Bombyx mori

BMCP18 2 This protein was previously referred to as LCP18 ( Shofuda et al., 1999). Since its expression is not restricted to larval stages, the nomenclature has been changed to BMCP18, Mr=18 k Bombyx mori cuticle protein. In the same way, other cuticle proteins, LCP30 ( Nakato et al., 1994), 22, and 17 ( Nakato et al., 1997), have been renamed as BMCP30, 22, and 17, respectively. 2 is one of the major cuticle proteins identified in the larval cuticle of the silkworm, Bombyx mori. A genomic clone coding for BMCP18 was isolated from a B. mori genomic library, and its structure was analyzed. The BMCP18 gene consists of three exons interspersed by two introns. Bm1 element-like sequences were identified around this gene, suggesting possible involvement of this retroposon in the duplication of B. mori cuticle protein genes during evolution. A structural comparison of the BMCP18 gene and related cuticle protein genes of other lepidopteran species (MSCP14.6 and HCCP12) showed that the 5′ upstream region of the BMCP18, MSCP14.6, and HCCP12 genes has a 12-bp identical sequence matching the recognition sequence for transcription factors COUP-TF and HNF-4. This implies that molecular mechanisms regulating expression of these cuticle protein genes are also conserved. mRNAs coding for Bmsvp, the B. mori homolog of Drosophila Seven-up, which is known as a homolog of vertebrate COUP-TF, and BmHNF-4, a homolog of vertebrate HNF-4, were detected in the larval epidermis. Bmsvp bound to the 12-bp sequence in vitro, suggesting that Bmsvp regulates the BMCP18 gene expression.

Down-regulation of expression of a pupal cuticle protein gene by transcriptional and posttranscriptional control mechanisms during metamorphosis in Galleria.

Down-regulation of expression of a pupal cuticle protein gene (GmPCP52) was investigated during metamorphosis in Galleria during normal development and in response to 20-hydroxyecdysone (20 E) and a juvenile hormone analogue (epofenonane). The developmental profile of GmPCP52 transcription was traced by nuclear run-on transcription assays. Transcription of the GmPCP52 gene is highest shortly after pupal ecdysis. There is a rapid decline between a pupal age of 12 and 18 h. Transcription becomes undetectable at 24 h. 20 E accelerates cessation of transcription, but induces a short second period of GmPCP52 transcriptional activity. Epofenonane prolongs transcription and induces a second round of transcriptional activity in relation to the synthesis of a second pupal cuticle. Analysis of changes in poly(A) tail length of GmPCP52 mRNA demonstrated control of expression at the level of mRNA translatability and stability. At 6 to 9 h poly(A) tails of GmPCP52 mRNA have lengths of 70 to 170 A-residues. From 9 h on mRNA with about 50 As accumulates. This material is regarded as translationally inactive. From 18 h on, further poly(A) shortening and degradation of the transcript occurs. Again, 20 E has an accelerating effect. In accordance with the results of the run-on experiments, there is a second increase in GmPCP52 mRNA with poly(A) tail lengths greater than 50 A. Epofenonane causes delay but does not prevent the changes observed in untreated animals. The results demonstrate, that expression of the GmPCP52 gene is regulated at the level of transcription, translation, as well as transcript accumulation and degradation. Targets of hormonal action are discussed.

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.

Identification of proteins and developmental expression of RNAs encoded by the 65A cuticle protein gene cluster in Drosophila melanogaster

Proteins of the third instar larval cuticle of Drosophila melanogaster, LCP5–LCP9, were purified and their N-terminal sequences determined. Three of these proteins (LCP5, 6, and 8) were found to be encoded by two multicopy genes previously mapped to the gene cluster at 65A 5–6 on the left arm of the third chromosome. The analysis of the patterns of developmental expression of the 8 distinct genes at this site showed that all but two were expressed during larval life. The patterns fell into three groups: one where expression was all through larval life, one where expression was primarily in the third instar, and one only during the production of the adult cuticle. One duplicated gene was not expressed in the Canton S strain at any time from the embryo to adult ecdysis. These findings indicate that there is not a unique set of cuticle proteins in the third versus the first and second instar larval cuticles and indicates that overlapping gene sets in several different gene clusters encode the proteins of the cuticle of different developmental stages.

Purification and cDNA cloning of evolutionally conserved larval cuticle proteins of the silkworm, Bombyx mori

A specific set of structural proteins termed larval cuticle proteins (LCPs) accumulates in integuments during larval development of the silkworm, Bombyx mori. Two major larval cuticle proteins, LCP17 and LCP22, were purified from the guanidine hydrochloride extract of the larval cuticle, and specific antibodies were raised against these proteins. Immunoblot analysis revealed that both LCPs are actively synthesized during larval intermolt stages, whereas the LCP17 epitope is also slightly but significantly detectable in pupal integuments. cDNA clones for LCPs were isolated by immunoscreening of the cDNA expression library constructed from larval epidermal mRNA. Predicted amino acid sequences of LCP17 and LCP22 are homologous to cuticle proteins from other insect species, including Manduca sexta, Drosophila melanogaster and Locusta migratoria. This fact suggests that these cuticle protein genes originated from a common ancestral gene and have been conserved during evolution. Northern blot hybridization demonstrated that the expression of LCP17 as well as LCP22 mRNA is controlled in a stage-specific manner in the epidermis of the final instar larvae, suggesting a common regulatory mechanism for transcription of these two intermolt genes.

Evolution of the Larval Cuticle Proteins Coded by the Secondary Sex Chromosome Pair: X2 and Neo-Y of Drosophila miranda: I. Comparison at the DNA Sequence Level

The larval cuticle protein genes (Lcps) represent a multigene family located at the right arm of the metacentric autosome 2 (2R) in Drosophila melanogaster. Due to a chromosome fusion the Lcp locus of Drosophila miranda is situated on a pair of secondary sex chromosomes, the X2 and neo-Y chromosome. Comparing the DNA sequences from D. miranda and D. melanogaster organization and the gene arrangement of Lcp1-Lcp4 are similar, although the intergene distances vary considerably. The greatest difference between Lcp1 and Lcp2 is due to the occurrence of a pseudogene in D. melanogaster which is not present in D. miranda. Thus the cluster of the four Lcp genes existed already before the separation of the melanogaster and obscura group. Intraspecific homogenizations of different cluster units must have occurred repeatedly between the Lcp1/Lcp2 and Lcp3/Lcp4 sequence types. The most obvious example is exon 2 of the Lcp3 gene in D. miranda, which has been substituted by the corresponding section of the Lcp4 gene rather recently. The homogenization must have occurred before the translocation which generated the neo-Y chromosome. Lcp3 of D. melanogaster has therefore no orthologous partner in D. miranda. Rearrangements in the promoter regions of the D. miranda Lcp genes have generated new, potentially functional CAAT-box motifs. Since three of the Lcp alleles on the neo-Y are not expressed and Lcp3 is expressed only at a reduced level, it is suggestive to speculate that the rearrangements might be involved as cis-regulatory elements in the up-regulation of the X2-chromosomal Lcp alleles, in Drosophila an essential process for dosage compensation. The Lcp genes on the neo-Y chromosome have accumulated more base substitutions than the corresponding alleles on the X2.

Temporal and Spatial Expression of an Adult Cuticle Protein Gene from Drosophila Suggests That Its Protein Product May Impart Some Specialized Cuticle Function

An adult cuticle protein gene (Dacp-1) from Drosophila melanogaster has been isolated and characterized. This gene was classified as an adult cuticle protein gene because it maintains the conserved structure of other cuticle protein genes, the sequence of its conceptual translation product contains a repeated motif that is found almost exclusively in a subset of adult cuticle proteins from Locust migratoria, and the gene is expressed in the epidermis underlying the head and thoracic cuticle. The bulk of Dacp-1 expression starts ∼72 hr after pupariation, peaks approximately 12 hr after eclosion, and decreases thereafter to undetectable levels by 3 days after eclosion, The stage specificity and spatial restriction of Dacp-1 expression as well as the physical properties of its conceptual translation product suggest that it may be involved in some specialized function such as thickening of the adult cuticle.

The fat body cell-free system for tissue-specific transcription of plasma protein gene of Bombyx mori.

A nuclear extract was prepared for the larval fat body of the silkworm, Bombyx mori, and a homologous in vitro system was developed for the transcription of major plasma protein gene of B.mori. The gene for SP1, a storage protein of B.mori, and adenovirus 2 major late (AdML) gene were faithfully transcribed under relatively high template concentrations in the nuclear extract prepared from the fat body of female fifth instar larvae. Complete inhibition of gene transcription by a low concentration of alpha-amanitin indicated that the reaction is catalyzed by RNA polymerase II. At low template concentration (0.6 nM) the fat body nuclear extract transcribed the homologous SP1 gene with high efficiency, while AdML gene and larval cuticle protein gene were only barely transcribed in the same extract. The SP1 gene deleted upstream of the TATA box sequence showed little effect on transcription, whereas mutations that destroy TATA sequence totally abolished the gene transcription. These results suggested that the core promoter region of SP1 gene spanning between positions -44 and +16 is essential for the fat body specific transcription in vitro.

Structure and developmental expression of a larval cuticle protein gene of the silkworm, Bombyx mori

Structure and expression of the gene for a larval cuticle protein of the silkworm, Bombyx mori were studied. A major cuticle protein, termed ‘LCP30’ was purified from the urea extract of integuments of the fifth (final) instar larvae. Immunoblot analysis by use of the anti-LCP30 antibody revealed that LCP30 begins to accumulate in larvae as early as 10 h after hatch and is present throughout the larval stages. The LCP30 epitope is also detectable in the adult abdominal integument but is absent from pupal integument and adult wing. Screening of an epidermal cDNA expression library with the antibody probe yielded a cDNA clone for LCP30. Primary structure deduced from the cDNA sequence showed that LCP30 bears an arginine-glycine-aspartate (RGD) sequence. The region around this domain exhibits striking similarity with the amino acid sequences found in vertebrate collagens. The genomic DNA clone coding for LCP30 was isolated by screening a B. mori gene library with the LCP30 cDNA probe. The gene consists of five exons interspersed by four introns spanning over 2.7 kb region of chromosomal DNA. The LCP30 mRNA is detectable at high levels at larval intermolt stages, gradually declines after the fourth molt and totally disappears at mid-fifth larval instar, indicating that the expression of LCP30 gene is regulated in a stage-specific fashion in the epidermal cells. Topical application of a juvenile hormone analogue (methoprene) to the fifth instar larvae followed by RNA blot and S1 nuclease mapping analyses of the epidermal RNA proved that juvenile hormone activates transcription of the LCP30 gene.

Characterization of a cDNA and gene encoding a cuticular protein from rigid cuticles of the giant silkmoth, Hyalophora cecropia

We have isolated a cDNA and gene encoding a protein (HCCP 66) found in the rigid cuticles of both larvae and pupae of the silkmoth, Hyalophora cecropia. The cDNA encoded a protein similar to cuticle proteins isolated from several other insects and contained a sequence motif similar to one present in a “family” of cuticular proteins from flexible cuticles. The gene had a structure similar to that of cuticle protein genes isolated from Drosophila melanogaster, albeit with a much larger intron that contained three copies of a transposable element-like sequence similar to short interspersed repeated DNA elements (SINEs). A sequence found 5′ to the transcription start site matched the Octamer (Oct) cis-acting element. This sequence was capable of binding protein(s) from whole cell extracts of wing epidermis with high affinity and sequence specificity suggesting a role in transcriptional regulation.

How Y chromosomes become genetically inert.

We have investigated the mechanistic aspects of inactivation of the major larval cuticle protein genes (Lcp1-4) in Drosophila miranda during Y chromosome evolution. The Lcp genes are located on the X2 and neo-Y chromosomes in D. miranda but are autosomally inherited in all other Drosophila species investigated so far. In the neo-Y chromosome all four Lcp loci are embedded within a dense cluster of transposable elements. The X2 Lcp1-4 loci are expressed, while the Y chromosomal Lcp3 locus shows only reduced activity and the Lcp1, Lcp2, and Lcp4 are completely inactive. Our results suggest that Lcp1 and Lcp3 loci on the degenerating Y chromosome of D. miranda are silenced by neighboring transposable elements. These observations support our assumption that the first step in Y chromosome degeneration is the successive silencing of Y chromosomal loci caused by trapping and accumulation of transposons.

20-Hydroxyecdysone is required for, and negatively regulates, transcription of Drosophila pupal cuticle protein genes

Transcripts of ecdysone-dependent genes (EDGs) accumulate in isolated imaginal discs within 8 hr after exposure to a pulse of the steroid hormone 20-hydroxyecdysone (20-HE; 1 μg/ml for 6 hr) but not in discs cultured in the continuous presence or absence of the hormone. Sequence analyses show that two of the EDGs are members of gene families encoding insect cuticle proteins. We conclude that a third EDG encodes a cuticle protein because the conceptual glycine-rich protein contains sequence motifs similar to those found in insect egg shell proteins and vertebrate cytokeratins and because expression of this gene is limited to tissues that deposit the pupal cuticle. Nuclear run-on assays show that the hormone-dependent expression of each of these EDGs is due to transcriptional regulation. Readdition of hormone to imaginal discs actively synthesizing the EDG messages causes rapid repression of EDG transcription. Thus, 20-HE acts as both a positive and a negative regulator of EDG transcription. Sequences in the promoter regions of two of the EDGs are similar to an ecdysone response element and may play a role in negative regulation.

Evolutionary changes in the organization of the major LCP gene cluster during sex chromosomal differentiation in the sibling speciesDrosophila persimilis, D. pseudoobscura andD. miranda

The major larval cuticle protein (LCP) genes I–IV ofDrosophila melanogaster are clustered on the right arm of the second chromosome. By cross-hybridization we cloned the corresponding genes from three different members of theobscura group:D. persimilis, D. pseudoobscura andD. miranda. InD. pseudoobscura andD. persimilis the gene cluster maps to autosome3. In contrast, inD. miranda it was found on theX2 andY sex chromosome. Hence, this exceptional karyotypic situation offers a unique opportunity to analyse the molecular processes underlying the phenomenon of chromosome degeneration. Comparison of LCP genes I–IV in theX2 andY chromosomal region inD. miranda revealed extensive DNA rearrangements at the latter. TheY chromosomal LCP cluster is characterized by DNA insertions which are absent in the correspondingX2 chromosomal DNA, suggesting that these DNA sequences must have invaded this area. In addition, part of the analysedY chromosomal region is duplicated.

Evolution of nested genes with special reference to cuticle proteins inDrosophila melanogaster

One of the pupal cuticle protein (PCP) genes has been found within an intron of a Drosophila housekeeping gene (the Gart locus) that encodes three enzymes involved in the purine pathway. This intronic gene has been described as a gene within a gene, and the gene is now called a "nested" gene. Because the intronic PCP gene has sequence similarity with the larval cuticle protein (LCP) gene, it may have been derived from one of the LCP genes or their ancestral gene. We have studied possible phylogenetic relationships among these five genes by comparing nucleotide sequences of four LCP genes with that of the PCP gene. The results obtained suggest that the PCP gene may have originated from an ancestral gene before duplication of the LCP genes occurred. Using the number of synonymous (silent) substitutions, we then estimated the divergence time between the PCP gene and the LCP genes to be about 70 million years (Myr). The divergence time estimated is much larger than that for the sibling species of D. melanogaster (about 2.5 Myr), indicating that the "nested" gene structure can be seen not only in Drosophila melanogaster, but also in other distantly related Drosophila species.