Kazuhiro Okano

The construction of an EST database for Bombyx mori and its application

To build a foundation for the complete genome analysis of Bombyx mori, we have constructed an EST database. Because gene expression patterns deeply depend on tissues as well as developmental stages, we analyzed many cDNA libraries prepared from various tissues and different developmental stages to cover the entire set of Bombyx genes. So far, the Bombyx EST database contains 35,000 ESTs from 36 cDNA libraries, which are grouped into ≈11,000 nonredundant ESTs with the average length of 1.25 kb. The comparison with FlyBase suggests that the present EST database, SilkBase, covers >55% of all genes of Bombyx. The fraction of library-specific ESTs in each cDNA library indicates that we have not yet reached saturation, showing the validity of our strategy for constructing an EST database to cover all genes. To tackle the coming saturation problem, we have checked two methods, subtraction and normalization, to increase coverage and decrease the number of housekeeping genes, resulting in a 5–11% increase of library-specific ESTs. The identification of a number of genes and comprehensive cloning of gene families have already emerged from the SilkBase search. Direct links of SilkBase with FlyBase and WormBase provide ready identification of candidate Lepidoptera-specific genes.

Pheromone gland-specific fatty-acyl reductase of the silkmoth, Bombyx mori

The C10-C18 unsaturated, acyclic, aliphatic compounds that contain an oxygenated functional group (alcohol, aldehyde, or acetate ester) are a major class of sex pheromones produced by female moths. In the biosynthesis of these pheromone components, the key enzyme required to produce the oxygenated functional groups is fatty-acyl reductase (FAR). This enzyme converts fatty-acyl pheromone precursors to their corresponding alcohols, which, depending on the moth species, can then be acetylated or oxidized to the corresponding aldehydes. Despite the significant role this enzyme has in generating the species-specific oxygenated constituents of lepidopteran sex pheromones, the enzyme has yet to be fully characterized and identified. In experiments designed to characterize a pheromone-gland-specific FAR in the silkmoth, Bombyx mori, we have isolated a cDNA clone encoding a protein homologous to a FAR from the desert shrub, Simmondsia chinensis, commonly known as jojoba. The deduced amino acid sequence of this clone predicts a 460-aa protein with a consensus NAD(P)H binding motif within the amino terminus. Northern blot analysis indicated that 2-kb transcripts of this gene were specifically expressed in the pheromone gland at 1 day before adult eclosion. Functional expression of this gene in the yeast Saccharomyces cerevisiae not only confirmed the long-chain FAR activity, but also indicated a distinct substrate specificity. Finally, the transformed yeast cells evoked typical mating behavior in male moths when cultured with the pheromone precursor fatty acid, (E,Z)-10,12-hexadecadienoic acid.

Characterization of the Role of Very Late Expression Factor 1 in Baculovirus Capsid Structure and DNA Processing

ABSTRACT Very late expression factor 1 (VLF-1) of Autographa californica multiple nucleopolyhedrovirus is a putative tyrosine recombinase and is required for both very late gene expression and budded virus production. In this report, we show that a vlf-1 knockout bacmid was able to synthesize viral DNA at levels similar to that detected for a gp64 knockout bacmid that served as a noninfectious control virus. Additionally, analysis of replicated bacmid DNA by field-inversion gel electrophoresis indicated that VLF-1 is not required for synthesizing high-molecular-weight intermediates that could be resolved into unit-length genomes when cut at a unique restriction site. However, immunoelectron microscopic analysis revealed that in cells transfected with a vlf-1 knockout bacmid, aberrant tubular structures containing the capsid protein vp39 were observed, suggesting that this virus construct was defective in producing mature capsids. In contrast, rescuing the vlf-1 knockout bacmid construct with a copy of VLF-1 that carries a mutation of a highly conserved tyrosine (Y355F) was sufficient to restore the production of nucleocapsids with a normal appearance, but not infectious virus production. Furthermore, the results of a DNase I protection assay indicated that the DNA packaging efficiency of the VLF-1(Y355F) virus construct was similar to that of the gp64 knockout control. Finally, a recombinant virus containing a functional hemagglutinin epitope-tagged version of VLF-1 was constructed to investigate the association of VLF-1 with the nucleocapsid. Analysis by immunoelectron microscopy of Sf-9 cells infected with this virus showed that VLF-1 localized to an end region of the nucleocapsid. Collectively, these results indicate that VLF-1 is required for normal capsid assembly and serves an essential function during the final stages of the DNA packaging process.

A homologue of the Drosophila doublesex gene is transcribed into sex-specific mRNA isoforms in the silkworm, Bombyx mori

The doublesex (dsx) gene is known as the final gene of the sex-determining cascade in Drosophila melanogaster. We have isolated a homologue of dsx in the silkworm, Bombyx mori, which has an epistatic feminizing gene located on the W chromosome. RT-PCR analysis indicated that B. mori dsx (Bmdsx) was transcribed in all the examined tissues, and the size of the amplified products was different between males and females. In Northern blot hybridization of poly(A)(+) RNA, the Bmdsx probe also detected a band with a sex-specific size difference. The male-specific cDNA lacked the sequence between 713 and 961nt of the female-specific cDNA. An RNase protection assay indicated that this sequence was male-specifically removed from the Bmdsx pre-mRNA. Southern blot analysis showed that Bmdsx is present at a single copy in the genome. These results suggested that the primary Bmdsx transcript is alternatively spliced to yield male- and female-specific mRNA isoforms. These sex-specific isoforms encode polypeptides with a common amino-terminal sequence but sex-specific carboxyl termini. DNA binding domain (DM domain) of BmDSX has 80% identity with D. melanogaster DSX proteins. These results suggest the Bmdsx would also regulate sexual differentiation, as does the Drosophila dsx gene.

Baculovirus Alkaline Nuclease Possesses a 5′→3′ Exonuclease Activity and Associates with the DNA-Binding Protein LEF-3

ABSTRACT Alkaline nuclease (AN) of the Autographa californica multiple-capsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133) was expressed in recombinant baculovirus as a His6-tagged fusion and purified by sequential chromatography on Ni-NTA-agarose, DEAE-Toyopearl, and heparin-Sepharose. At all stages of purification, AcMNPV AN was found to copurify with a 44-kDa polypeptide which was identified as the baculovirus single-stranded DNA (ssDNA)-binding (SSB) protein, LEF-3. Sedimentation analysis in glycerol gradients of highly purified samples suggested that AN and LEF-3 are associated in a complex (designated *AN/L3), predominantly as heterodimers, although oligomeric forms containing both proteins were evident. In reactions with single- or double-stranded 62-mer oligonucleotides that were labeled with 32P at the 5′ or 3′ ends, *AN/L3 carried out exonucleolytic hydrolysis of both substrates exclusively in a 5′→3′ direction. Saturation of ssDNA with an excess of LEF-3 prior to the addition of *AN/L3 resulted in a marked decrease in the rate of ssDNA hydrolysis. This suggests that excess LEF-3 may protect ssDNA from digestion by a AN-LEF-3 complex, thus regulating its activity in infected cells. The association of baculovirus AN with the viral SSB LEF-3 and the 5′→3′ exonuclease activity of this complex suggests that AN and LEF-3 may participate in homologous recombination of the baculovirus genome in a manner similar to that of exonuclease (Redα) and DNA-binding protein (Redβ) of the Red-mediated homologous recombination system of bacteriophage λ.

cDNA cloning of acyl-CoA desaturase homologs in the silkworm, Bombyx mori.

We have isolated two acyl-CoA desaturase clones from a pheromone gland cDNA library by using the EST (expressed sequence tag) database of Bombyx mori. The putative acyl-CoA desaturases encoded by the clones desat 1 (2029bp) and desat 2 (2341bp) have 98% identity, and both proteins show 61% identities to Trichoplusia ni acyl-CoA Delta(11) desaturase. The deduced amino acid sequences conserve well the histidine clusters that are catalytically essential for acyl-CoA desaturase activity. Northern blot and RT-PCR analyses revealed that both transcripts of desat 1 and desat 2 were expressed predominantly in the pheromone gland. Both transcripts detected 3days before adult eclosion dramatically increased a day before adult eclosion, keeping the mRNA levels high even after eclosion. These results, combined with the fact that Delta(11) and Delta(10, 12) desaturation of palmitate is a key step to synthesize pheromone in B. mori, suggest that the desaturases encoded by desat 1 and desat 2 are involved in either or both of the desaturation steps in the pheromone biosynthetic pathway of B. mori. The mRNA levels of desat 1 and desat 2 were not affected by decapitation or injection of the pheromone biosynthesis activating neuropeptide (PBAN) into the adult female moth, suggesting that the transcription of desat 1 and desat 2 is not regulated by PBAN. In addition to the clones in the pheromone gland, eight other clones encoding the same Delta(9) desaturase homolog were found in an embryonic cDNA library by searching from the EST database of B. mori. The deduced amino acid sequence from one of the clones (desat 3) shows 79% identity to T. ni Delta(9) desaturase but only 52% identity to the desaturases in the pheromone gland of B. mori. Northern blot analysis showed that the mRNA corresponding to the desat 3 was detected in the ovary and fat body, but not in the pheromone gland. Abundance of the Delta(9) desaturase clones (eight out of the 762 randomly sequenced clones) in the library prepared from diapause-destined embryos (40h after oviposition) suggests that the Delta(9) desaturase encoded by desat 3 plays an important role in embryonic development in B. mori.

Isolation and expression of the ecdysteroid-inducible angiotensin- converting enzyme-related gene in wing discs of Bombyx mori

We isolated a clone encoding a putative angiotensin-converting enzyme-related gene from the wing disc cDNA library of the silkworm, Bombyx mori (refer to as BmAcer). The predicted open reading frame encoded 648 amino acids with about 50% identities with the Drosophila melanogaster angiotensin-converting enzyme Ance and Acer. Northern analysis identified a 2.2-kilobase mRNA which was abundant in wing discs two days after the beginning of wandering. An accumulation of the transcript was observed approximately 2 h after 20-hydroxyecdysone (20E) exposure in vitro and was blocked slightly by a protein synthetic inhibitor. These data suggest that the transcription of the BmAcer gene is directly 20E-inducible.

Characterization of acyl-CoA-binding protein (ACBP) in the pheromone gland of the silkworm, Bombyx mori.

Various fatty acyl-CoAs are involved as intermediates or precursors of sex pheromone components in the biosynthetic pathway of the pheromones in many lepidopteran insects. We have purified a 10-kDa protein from the cytosolic fraction of Bombyx mori pheromone glands by using affinity chromatography with a palmitoyl-CoA-agarose column and reversed-phase HPLC. Amino acid sequence analysis of the fragment peptides obtained from the purified protein, and a homology search, revealed that this protein was a member of acyl-CoA-binding proteins (ACBPs). MALDI-TOF mass spectral analysis of the purified protein and cloning of the gene from a pheromone gland cDNA library confirmed B. mori ACBP to be a 90 amino acid protein with 78.9% identity to that of Manduca sexta ACBP. The secondary structure of the recombinant B. mori ACBP was determined by NMR spectroscopy. Northern blot analysis demonstrated that B. mori ACBP was predominantly expressed in the pheromone gland and the corresponding transcript was expressed from the day before adult eclosion. Present results suggest that ACBP plays a significant role in the production of sex pheromones regulated by the neurohormone, pheromone biosynthesis activating neuropeptide (PBAN).

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 and Functional Analysis of the Xestia c-nigrum Granulovirus Matrix Metalloproteinase†

ABSTRACT Sequence analysis of the Xestia c-nigrum granulovirus (XcGV) genome identified an open reading frame encoding a 469-amino-acid (54-kDa) protein with over 30% amino acid sequence identity to a region of about 150 amino acids that includes the catalytic domains of human stromelysin 1 (Str1)/matrix metalloproteinase 3 (MMP-3) (EC 3.4.24.17 ) and sea urchin hatching enzyme (HE). Stromelysin homologs have not been reported from baculoviruses or other viruses. Unlike human Str1 and sea urchin HE, the putative XcGV-MMP does not have a signal peptide and lacks the peptide motif involved in the cysteine switch that maintains other MMPs in an inactive form. The putative XcGV-MMP, however, possesses a conserved zinc-binding motif in its putative catalytic domain. The XcGV-MMP homolog was cloned, and a recombinant Bombyx morinucleopolyhedrovirus (BmNPV) that expresses XcGV-MMP under the polyhedrin promoter was constructed. A distinct pattern of melanization was observed in B. mori larvae infected with MMP-expressing BmNPV. Fat body extracts from larvae overexpressing the 54-kDa recombinant MMP digested dye-impregnated collagen (Azocoll). The enzymatic activity was inhibited by two metalloproteinase inhibitors, EDTA and 1,10-phenanthroline. These results suggest that the XcGV MMP-3 gene homolog encodes a functional metalloproteinase.