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

BMCP18(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.

Biosynthesis of major plasma proteins in the primary culture of fat body cells from the silkworm, Bombyx mori.

Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, Bombyx mori, in a sex- and stage-specific manner during larval development. We successfully established a primary culture of the fat body cells in order to investigate the regulatory mechanisms of plasma protein gene expression. The primary cultures of fat body cells contained at least two cell types: small oval cells, and large spherical cells. The cells adhered to and migrated on the cultured dish after plating. By the 7th day of cultivation, the cells clustered to form fat body-like structures, which were maintained for at least 3 months. Plasma proteins were actively synthesized in the primary cultures of the fat body cells isolated from the final instar larvae only when the cells tightly adhered to and clustered on the cultured dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system, indicating that the primary culture comprises heterogeneous cells that are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced their sex-dependency in vivo.

Molecular cloning and characterization of a cDNA encoding a larval cuticle protein of Bombyx mori.

Cuticle proteins termed LCPs are the major protein components of the larval integument of the silkworm, Bombyx mori. We purified an 18 kDa LCP (LCP18) from the guanidine hydrochloride extract of the larval cuticle and identified an LCP18 cDNA clone. The deduced primary structure and mRNA expression pattern of LCP18 are similar to those of other Bombyx LCPs and to several cuticle proteins identified in other insect species. RNA blot analysis demonstrated that the biosynthesis of LCP18 is regulated in a stage-dependent manner at the level of mRNA in epidermal cells. An in vivo study using a juvenile hormone analogue suggested that juvenile hormone positively regulates expression of LCP18 mRNA during larval intermolt stages.

Structure and expression of a cyst specific protein of Acanthamoeba castellanii.

The life cycle of Acanthamoeba is divided into a growth-division phase and two distinctive processes of cellular differentiation, termed encystment and excystment. Polyacrylamide gel electrophoresis revealed that a specific protein of 21 kDa in molecular weight occurs in the cyst, but not in the trophozoite stages of A. castellanii Neff strain. This cyst-specific protein, designated as CSP21, was purified from guanidine-HCl extract of cyst wall and anti-CSP21 antibody was produced. Immunoblotting of proteins extracted from a variety of species of Acanthamoeba genus suggested that the antibody is specific for group II amoebae, therefore, providing a useful tool for Acanthamoebae taxonomy. A cDNA clone for A. castellanii CSP21 was isolated by immunoscreening of a cDNA expression library constructed from mRNA of amoebae at encysting stage. The deduced primary structure indicated that CSP21 is a hydrophilic protein showing no significant homology with peptides thus far published. RNA blot analysis showed that the expression of CSP21 mRNA was restricted within early stages of encystment, suggesting that the biosynthesis of CSP21 is regulated at mRNA level.

Chromosome size polymorphisms in the genus acanthamoeba electrokaryotype by pulsed-field gel electrophoresis.

Twenty-eight strains from 12 species from the genus Acanthamoeba, including five isolates from amoebic keratitis patients, were subjected to molecular karyotyping by pulsed-field gel electrophoresis. 9 to 21 chromosome-sized DNA bands ranging from 200 kb to 3 Mb in size were detected. Molecular karyotypes also showed a wide multifariousness, i.e. there existed inter- and intraspecific heterogeneity. The five isolates from amoebic keratitis patients did not exhibit characteristic molecular karyotypes distinguishable from environmental isolates. Although karyotypic heterogeneity was observed within group I amoeba, they are distinguishable from those of group II and III. Strains having identical restriction fragment length polymorphism profiles of mtDNA did not have an identical molecular karyotype, i.e. weak correlation was found between molecular karyotypes and mtDNA restriction fragment length polymorphism profiles.

In vitro transcription systems from cultured cells and fat-body tissue of the silkworm, Bombyx mori.

This article describes the development of cell-free transcription systems from the cultured cells and fat body tissues of a Lepidpteran insect, the silkworm, Bombyx mori. Detailed protocols are provided for the culture of a B. mori cell line, rearing larvae, preparation of whole cell as well as nuclear extract and conditions for in vitro transcription of cloned plasma protein gene templates.

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.

Molecular cloning and characterization of cDNA for insect biogenic peptide, growth-blocking peptide.

Growth-blocking peptide (GBP) is an insect biogenic peptide that prevents the onset of metamorphosis from larva to pupa. A cDNA coding for GBP is described. Mixed oligonucleotides derived from a GBP peptide sequence were used to generate amplified DNA by the polymerase chain reaction (PCR). Based on the sequence of the amplified DNA, a 41 bases oligonucleotide was designed for screening a cDNA library which was constructed from the armyworm Pseudaletia separata larvae parasitized with the parasitic wasp Cotesia kariyai. The cloned cDNA for GBP was 809 base pairs in length. An open reading frame of 429 base pairs encodes a pre-pro-peptide of 143 amino acid residues in which GBP is localized at the C-terminal region, and other three peptides including a putative signal peptide and appropriate processing sites for endoproteolytic cleavage precede the GBP sequence. Northern blot analyses demonstrate the presence of a 800-base mRNA transcript in fat body and 2.5-kilobase transcript in brain and nerve cord, suggesting the possibility that the transcription of GBP gene is regulated in a tissue-dependent manner. This interpretation was supported by isolating a GBP cDNA fragment from cDNA pool of brain-nerve cords. GBP mRNA is constantly expressed in both parasitized and non-parasitized last instar larvae and there is no difference in the levels of the mRNA between both larvae, thus indicating that parasitism may effect on translational or posttranslational level to elevate plasma GBP concentration.

Structure of two cecropin B-encoding genes and bacteria-inducible DNA-binding proteins which bind to the 5'-upstream regulatory region in the silkworm, Bombyx mori.

Two genomic DNAs encoding cecropin B (CecB), an antibacterial protein from Bombyx mori, were cloned and sequenced. The number of CecB genes was estimated to be more than four copies per haploid by genomic Southern blotting. Two genes, CecB1 and CecB2, were located tandemly within 12 kb in the same orientation. These two genes encoded identical amino acids, though 15 nucleotides (nt) were different in the coding region and the intron size varied. About 90% of the nt spanning 800 bp in the 5'-untranslated region (UTR) were identical between the two genes. This 5'-flanking region contained characteristic sequences such as a repetitive element of B. mori (Bm1), an interleukin-6 response element (IL-6 RE), and two putative lipopolysaccharide (LPS) response elements (LPS RE). An electrophoretic mobility shift assay (EMSA) showed that the fat body contains at least three different nuclear proteins inducible by bacteria which bind to the 5'-UTR, suggesting that these proteins may be involved in CecB expression triggered by bacteria.

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.

Vitellogenin gene of the silkworm, Bombyx mori: structure and sex-dependent expression.

Vitellogenin of Bombyx mori is a precursor of major yolk protein synthesized in the female fat body at larval-pupal ecdysis. The gene for B. mori vitellogenin is composed of seven exons interspersed by six introns. Developmental profile of the primary transcript of the gene indicated that the biosynthesis of B. mori vitellogenin is regulated transcriptionally in a sex- and stage-dependent manner in the fat body. The Arg-X-Arg-Arg sequence, which conforms to the recognition site of mammalian furin, occurs in a region just upstream of the putative proteolytic cleavage site of B. mori previtellogenin.

A defective non-LTR retrotransposon is dispersed throughout the genome of the silkworm, Bombyx mori.

The presence of long repetitive sequences is demonstrated in the genome of the silkworm, Bombyx mori. Members of this BMC1 family reveal several features typical of the L1 (long interspersed sequence one) family of mammals, except for species specific elements. The number of BMC1 elements is estimated to be approximately 3500 per haploid genome. Elements containing the full length unit of 5.1 kb are dispersed throughout the genome and their restriction sites are conserved, although most members are preferentially truncated to varying extents at their 5' ends. DNA sequencing indicates that this element contains six tandem repeats of 15 bp CpG-rich sequence in the 5' proximal region. It terminates with a 3' oligo(A) stretch, and is flanked at both ends by a 7-10 bp target sequence duplication. In addition, there is significant evidence for amino acid sequence homology with reverse transcriptase domains of other L1 families, especially F, Doc and Jockey of Drosophila melanogaster. No large open reading frame is present. The BMC1 element is suggested to be dispersed in the genome by a transposition mechanism involving RNA intermediates.

Structure and expression of mRNA for vitellogenin in Bombyx mori.

Vitellogenin, a precursor of major yolk protein of the silkworm, Bombyx mori is a tetramer composed of each two molecules of heavy and light subunits. We cloned mRNA sequence for the B. mori vitellogenin and analyzed its structure. Sequence alignment of several overlapping cDNA clones indicated that the vitellogenin mRNA is approx. 5.7 kb, containing an open reading frame for a peptide with 1782 amino acid residues. By comparing the deduced amino acid sequence with the amino-terminal primary structures of vitellogenin subunits, it is suggested that the heavy and light subunits of the B. mori vitellogenin are encoded by a single contiguous mRNA. The primary translation product of the vitellogenin mRNA was detected in the microsomal fraction prepared from the fat body of vitellogenic females. Northern blot analysis of the fat body RNA demonstrated that the biosynthesis of vitellogenin in B. mori is regulated in a tissue-, sex- and stage-specific manner at the level of mRNA. Possible cause for discrepancy between the present results and our previous proposal (Izumi, S. and Tomino, S. (1983) Insect Biochem. 13, 81-85) on the biosynthesis of B. mori vitellogenin is also discussed.

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.

Characterizations of sea urchin fibrillar collagen and its cDNA clone.

Collagens were isolated from the adult test of the sea urchin species, Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus, and their molecular properties were compared with those of Asthenosoma ijimai collagen. Collagens from H. pulcherrimus and S. purpuratus comprised two major alpha-chains (alpha 120 and alpha 90) and a minor chain (alpha 140), while collagen from A. ijimai contained four alpha-chains (alpha 1, alpha 2, alpha 3 and alpha 4). Based on their molecular and immunological properties, the alpha 90 chain of H. pulcherrimus and S. purpuratus, and the alpha 2 and alpha 4 chains of A. ijimai are grouped together, while the alpha 120 and alpha 140 chains of H. pulcherrimus and S. purpuratus, and the alpha 1 and alpha 3 chains of A. ijimai are classified into another group. It is likely that collagen molecules of sea urchins are heterotrimers composed of these two types of alpha-chains. A cDNA of collagen was cloned from the cDNA library prepared from mRNA of H. pulcherrimus test and denoted as Hpcol1. This clone contained sequences for uninterrupted triple helical domain (378 amino acids), carboxyl telopeptide (28 amino acids) and carboxyl propeptide (225 amino acids). This structure is characteristic for fibril-forming collagens and was shown to encode alpha 120 and alpha 140 chains of H. pulcherrimus collagen. Hpcol1-mRNA was expressed in embryos as early as the prism stage.

Structure and expression of gene coding for a pupal cuticle protein of Bombyx mori.

A specific protein termed as PCP accumulates in the newly synthesized pupal cuticle of the silkworm, Bombyx mori. We have cloned the genomic sequence encoding PCP and analyzed its structure. The PCP gene comprises two exons interspersed by a single intron approx. 5.8 kb in length. Transcription initiation sites of the PCP gene were located at nucleotide level. The 5' flanking region of the gene contains a sequence homologous to the Pit-1 DNA recognition element of the rat prolactin and growth hormone genes. The developmental profile of the PCP precursor RNA in epidermal cells showed that the biosynthesis of PCP is regulated at the transcriptional level in a stage- and tissue-specific fashion during post-embryonic development. Administration of 20-hydroxyecdysone to the isolated abdomens prepared from the early fifth instar larvae provoked the accumulation of PCP mRNA in epidermis, suggesting that the molting hormone triggers the expression of PCP gene.

Cloning and sequence analysis of membrane-bound alkaline phosphatase cDNA of the silkworm, Bombyx mori.

The nucleotide sequence (1974 bp) of cDNA coding for membrane-bound alkaline phosphatases (m-ALP) of Bombyx mori was isolated. The cDNA clone contained an open reading frame encoding a polypeptide (547 amino acids), which contains a hydrophobic signal peptide of 36 amino acids and the mature protein of 511 amino acids (Mr = 56,163). We found a highly hydrophobic domain presumed to be a membrane anchoring region at the C-terminus. Comparing analysis between Bombyx m-ALP and mammalian and Escherichia coli ALPs suggested an evolutionary relationship of sharing a common ancestral gene.

Complete nucleotide sequences of major plasma protein genes of Bombyx mori.

In the silkworm, Bombyx mori, a family of hormone-sensitive genes encodes major plasma proteins, termed '30K proteins'. We reported the organization of the 30K protein gene family together with the structure and expression of a member of the gene family (Mori, S. et al. (1991) J. Mol. Biol. 218, 7-12). In this communication, we present the complete structures of two other 30K protein genes in the family. Sequence analyses demonstrated that all three genes are similar to each other; a short first exon and protein-coding second exon are interspersed by a single intron. Structures homologous to the putative regulatory elements for the 30K protein gene expression are also found around each gene.

Structures and organization of major plasma protein genes of the silkworm Bombyx mori.

In the silkworm, Bombyx mori, a group of structurally related proteins, termed 30K proteins, accumulate in the hemolymph of the last instar larvae. We have isolated and characterized three genes, each of which encodes a distinct 30K protein component. Each 30K protein gene is composed of a short first exon and a protein-coding second exon interspersed by a single intron. The transcription initiation site of the 30K protein mRNA was identified at the nucleotide level. A typical TATA box exists some 30 base-pairs upstream from the transcription initiation site. The 5'-flanking region of each gene also contains octamer-like sequences. Restriction mapping analyses revealed that the cloned 46 x 10(3) base-pair region of the chromosomal DNA bears three 30K protein genes. Several copies of highly reiterated retrotransposon-like sequences are present around the 30K protein genes. S1 nuclease protection analysis provided evidence that the biosynthesis of 30K protein is regulated in a stage-specific manner at the transcriptional level in the fat body.