Toru Shimada

The sex chromosomes of the silkworm Bombyx mori are designated ZW (XY) for females and ZZ (XX) for males. Numerous long terminal repeat (LTR) and non-LTR retrotransposons, retroposons and DNA transposons have accumulated as strata on the W chromosome. However, there are nucleotide sequences that do not show the characteristics of typical transposable elements on the W chromosome. To analyse these uncharacterized nucleotide sequences on the W chromosome, we used whole-genome shotgun (WGS) data and assembled data that was obtained using male genome DNA. Through these analyses, we found that almost all of these uncharacterized sequences were non-autonomous transposable elements that do not fit into the conventional classification. It is notable that some of these transposable elements contained the Bombyx short interspersed element (Bm1) sequences in the elements. We designated them as secondary-Bm1 transposable elements (SBTEs). Because putative ancestral SBTE nucleotide sequences without Brill do not occur in the WGS data, we suggest that the Brill sequences of SBTEs are not carried on each element merely as a package but are components of each element. Therefore, we confirmed that SBTEs should be classified as a new group of transposable elements.

In Bombyx mori, the W chromosome determines the female sex. A few W chromosome-linked mutations that cause masculinization of the female genitalia have been found. In female antennae of a recently isolated mutant, both female-type and male-type Bmdsx mRNAs were expressed, and BmOr1 (bombykol receptor) and BmOr3 (bombykal receptor), which are predominantly expressed in the antennae of male moths, were expressed about 50 times more abundantly in the antennae of mutant females than in those of normal females. These mutants are valuable resources for the molecular analysis of the sex-determination system. Besides the Fern gene, the quantitative egg size-determining gene Esd is thought to be present on the W chromosome, based on the observation that ZWW triploid moths produce larger eggs than ZZW triploids. The most recently updated B. mori genome assembly comprises 20.5 Mb of Z chromosome sequence. Using these sequence data, responsible genes or candidate genes for four Z-linked mutants have been reported. The od (distinct oily) and spli (soft and pliable) are caused by mutation in BmBLOS2 and Bmacj6, respectively. Bmap is a candidate gene for Vg (vestigial). Similarly, Bmprm is a candidate gene for Md (muscle dystrophy), causing abnormal development of indirect flight muscle.

Recent studies have shown that dual mutations in fp25K and p35 of Autographa californica nucleopolyhedrovirus (AcMNPV) result in a typical apoptotic infection on Trichoplusia ni cells, suggesting the involvement of FP25K on NPV-induced apoptosis. To examine the effect of fp25K deletion on Bombyx mori NPV (BmNPV)-induced apoptosis, we generated a BmNPV mutant, fp-p35D, in which both fp25K and p35 genes are deleted from the genome, and compared its phenotype with wild-type (T3), fp25K-deleted (fp-null), and p35-deleted (p35D) BmNPVs. In BmN cells, p35D, but not T3 or fp-null, caused apoptosis with caspase-3 activation. Infection with fp-p35D also resulted in caspase-3 activation, but the level was comparable to that of p35D. Also, we did not observe any apoptotic responses in hemocytes from larvae infected with p35D or fp-p35D. These results indicate that unlike AcMNPV, deletion of fp25K does not affect the pathway of p35D-induced apoptosis of BmN cells and B. mori larvae.

On the W chromosome of the silkworm, Bombyx mori, we found a novel piggyBac-like DNA transposon that potentially encodes an intact transposase (610 amino acid residues), which is flanked by 16-bp perfect inverted terminal repeats and a duplicated TTAA target site. Interestingly, we also identified another intact copy of this transposon on an autosome (chromosome 21), which showed 99.6% identity in the DNA sequence of the transposase (99.3% amino acid identity). These features raised the possibility that this novel piggyBac-like DNA transposon, designated as yabusame, may retain transposition activity. Here we report the identification and characterization of yabusame transposons from the silkworm. We cloned the full length of the yabusame transposon on the W chromosome (yabusame-W) and its autosomal copy (yabusame-1). Southern blot analysis showed that there are interstrain polymorphisms in yabusame elements for their insertion sites and copy number. We also found strong evidence for the recent transposition of yabusame elements in the silkworm genome. Although our in vitro excision assays suggested that the transposition activity of yabusame-1 and yabusame- W has been lost almost entirely, our data will lead to a greater understanding of the characteristics of piggyBac superfamily elements.

In the silkworm Bombyx mori, dietary flavonoids are metabolized and accumulate in cocoons, thereby causing green coloration. Classical genetic studies suggest that more than seven independent loci are associated with this trait; however, because of the complex inheritance pattern, none of these loci have been characterized molecularly, and a plausible and comprehensive model for their action has not been proposed. Here, we report the identification of the gene responsible for the Green b (Gb) locus involving the green cocoon trait. In +(Gb) animals, glucosylation at the 5-O position of dietary quercetin did not occur, and the total amount of flavonoids in tissues and cocoons was dramatically reduced. We performed positional cloning of Gb and found a 38-kb deletion in a UDP-glucosyltransferase (UGT) gene cluster associated with the +(Gb) allele. RT-PCR and biochemical studies suggested that deletion of Bm-UGT10286 (UGT) is responsible for Gb and Bm-UGT10286 is virtually the sole source of UGT activity toward the 5-O position of quercetin. Our data show that the regiospecific glucosylation of flavonoids by the quercetin 5-O-glucosyltransferase can greatly affect the overall bioavailability of flavonoids in animals. Furthermore, we provide evidence that flavonoids increase the UV-shielding activity of cocoons and thus could confer an increased survival advantage to insects contained in these cocoons. This study will lead to greater understanding of mechanisms for metabolism, uptake, and transport of dietary flavonoids, which have a variety of biological activities in animals and beneficial effects on human health.

In insects, the precise timing of molting and metamorphosis is strictly guided by a principal steroid hormone, ecdysone. Among the multiple conversion steps for synthesizing ecdysone from dietary cholesterol, the conversion of 7-dehydrocholesterol to 5 beta-ketodiol, the so-called 'Black Box', is thought to be the important rate-limiting step. Although a number of genes essential for ecdysone synthesis have recently been revealed, much less is known about the genes that are crucial for functioning in the Black Box. Here we report on a novel ecdysteroidgenic gene, non-molting glossy (nm-g)/shroud (sro), which encodes a short-chain dehydrogenase/reductase. This gene was first isolated by positional cloning of the nm-g mutant of the silkworm Bombyx mori, which exhibits a low ecdysteroid titer and consequently causes a larval arrest phenotype. In the fruit fly, Drosophila melanogaster, the closest gene to nm-g is encoded by the sro locus, one of the Halloween mutant members that are characterized by embryonic ecdysone deficiency. The lethality of the sro mutant is rescued by the overexpression of either sro or nm-g genes, indicating that these two genes are orthologous. Both the nm-g and the sro genes are predominantly expressed in tissues producing ecdysone, such as the prothoracic glands and the ovaries. Furthermore, the phenotypes caused by the loss of function of these genes are restored by the application of ecdysteroids and their precursor 5 beta-ketodiol, but not by cholesterol or 7-dehydrocholesterol. Altogether, we conclude that the Nm-g/Sro family protein is an essential enzyme for ecdysteroidogenesis working in the Black Box.

Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.

Yellow proteins form a large family in insects. In Drosophila melanogaster, there are 14 yellow genes in the genome. Previous studies have shown that the yellow gene is necessary for normal pigmentation; however, the roles of other yellow genes in body coloration are not known. Here, we provide the first evidence that yellow-e is required for normal body color pattern in insect larvae. In two mutant strains, bts and its allele bts2, of the silkworm Bombyx mori, the larval head cuticle and anal plates are reddish brown instead of the white color found in the wild type. Positional cloning revealed that deletions in the Bombyx homolog of the Drosophila yellow-e gene (Bmyellow-e) were responsible for the bts/bts2 phenotype. Bmyellow-e mRNA was strongly expressed in the trachea, testis, and integument, and expression markedly increased at the molting stages. This profile is quite similar to that of Bmyellow, a regulator of neonatal body color and body markings in Bombyx. Quantitative reverse transcription-PCR analysis showed that Bmyellow-e mRNA was heavily expressed in the integument of the head and tail in which the bts phenotype is observed. The present results suggest that Yellow-e plays a crucial role in the pigmentation process of lepidopteran larvae.

Two types of thiolases are involved in the synthesis and catabolism of fatty acids; acetyl-CoA acetyltransferase (AT) which catalyzes the formation of acetoacetyl-CoA from acetyl-CoA by transferring an acetyl group from one acetyl-CoA molecule to another, and 3-ketoacyl-CoA thiolase which catalyzes a reversible thiolytic cleavage of 3-ketoacyl-CoA into acetyl-CoA and acyl-CoA. Although many mammalian thiolases have been characterized in detail, no thiolases from insects have been functionally characterized to date. Here we report first characterization of an insect AT gene, Osat1, from the pheromone gland of the adzuki bean borer moth Ostrinia scapulalis (Lepidoptera: Crambidae). Osat1 encodes a 41.2 kDa protein comprising 396 amino acid residues (OsAT1), which possesses structural features of the thiolase family. An Osat1 homologue of Bombyx mori (Bmat1) was cloned through exploration of an EST library of the silk-worm. Subsequent survey of the genome database revealed that B. mori has at least six Osat1 homologues, among which Bmat1 was most closely related to Osat1. We expressed recombinant OsAT1 using a baculovirus expression system, and verified that OsAT1 catalyzes the formation of acetoacetyl-CoA from acetyl-CoA. Osat1 was expressed in all adult tissues examined. These results indicate that OsAT1 is a functional AT ubiquitously expressed in O. scapulalis tissues. (C) 2009 Elsevier Ltd. All rights reserved.

We carried out genetic and cytogenetic analyses of X-ray-induced deleterious Z chromosomes that result in a soft and pliable (spli) phenotype in the silkworm, Bombyx mori. In a B. mori strain with a spli phenotype, we found the Z chromosome broken between the sch (1-21.5) and od (1-49.6) loci. We also found a chromosomal fragment bearing a fifth-chromosome locus for egg and eye pigmentation fused to a Z chromosome fragment. By means of fluorescence in situ hybridization using bacterial artificial chromosome clones as probes, we confirmed that the fused chromosome is composed of a fragment of chromosome 5 and a fragment of the Z chromosome. Moreover, a predicted gene, GA002017, the Bombyx ortholog of the Drosophila gene acj6 (Bmacj6), was completely deleted by the Z chromosome breakage event. The relationship between Bmacj6 and the spli phenotype is discussed.

Silkworms have played an important agricultural role in supporting Japan's modernization, and traditionally, Japan has led the world as a repository of silkworm bioresources. The silkworm is a small and highly domesticated insect, which is ideal as a laboratory tool, although it is a bioresource that is relatively infrequently used in experiments at present. In this review, we describe the potential for silkworm resources to contribute to life sciences.

Silkworms have played an important agricultural role in supporting Japan’s modernization, and traditionally, Japan has led the world as a repository of silkworm bioresources. The silkworm is a small and highly domesticated insect, which is ideal as a laboratory tool, although it is a bioresource that is relatively infrequently used in experiments at present. In this review, we describe the potential for silkworm resources to contribute to life sciences.

カイコのゲノムは、2004年に日本と中国のグループにより独立にドラフト配列が発表された後、その両国間で共同研究が進められ、2008年に精密なゲノム情報が公表された。カイコには、より高度な機構に依存し、かつカイコだけに特徴的にみられる現象、たとえば卵での越冬休眠やクワへの寄主特異性などがある。これらが、はたしてカイコだけが持つ遺伝子によって支配されているのか、あるいは他の昆虫と共有する遺伝子のマイナーチェンジによって実現しているのか、読者にとって興味のあるところであろう。本稿では、カイコのゲノムの特異性と、生物としてのカイコの特性の一つであるクワへの依存性を関連づける研究について紹介することにする。未開拓、未解明の研究領域であり、以下に述べる内容には筆者の推察が含まれていることを予めお断りしたい。

カイコの遺伝学は、外山亀太郎博士によってメンデルの法則の成立が動物で初めて確かめられてから、すでに100年を超える歴史を作ってきた。この間に数百の突然変異が発見され、それらは連関関係および組換え価に基づいて28の連関群に配置されている。一方で、Watson and CrickによるDNAの二重らせん構造の発見以降の分子生物学の興隆期には、真核生物の遺伝子のクローニングとしては最も早い成功例の一つである、鈴木義昭博士らによるフィブロイン遺伝子の単離をはじめ、富野士良博士らによる体液タンパク質遺伝子に関する研究や山下興亜博士らによる休眠ホルモン遺伝子の単離など、日本人がカイコを用いて世界の分子遺伝学をリードする成果を挙げてきた。

Homologs of Antographa colifornica nucleopolyhedrovirus ORF43 (Ac43) are found in all group I and most group II NPVs, but their functions remain unknown. In Bombyx mori NPV (BmNPV)-infected BmN cells, Bm34, a BmNPV homolog of Ac43, is expressed as a late gene and its product is localized in the nucleus. To examine the role of Bm34 during BmNPV infection, we constructed a Bm34 deletion mutant (BmORF34D) and characterized its infectivity in BmN cells and B. mori larvae. BmORF34D Produced far fewer occlusion bodies (OBs) in BmN cells as compared with wild-type BmNPV. This reduction is assumed to be mainly due to the transcriptional down-regulation of two genes, very late expression factor (vlf-1) and few polyhedra (fp25K), both of which are required for efficient polyhedrin expression. Larval bioassays also revealed that Bm34 accelerates death of B. mori larvae. These results Suggest that Bm34 is required for efficient late and very late gene expression. (C) 2009 Elsevier Inc. All rights reserved.

Genetic studies and large-scale sequencing experiments have revealed that the PIWI subfamily proteins and PIWI-interacting RNAs (piRNAs) play an important role in germ line development and transposon control. Biochemical studies in vitro have greatly contributed to the understanding of small interfering RNA ( siRNA) and microRNA ( miRNA) pathways. However, in vitro analyses of the piRNA pathway have been thus far quite challenging, because their expression is largely restricted to the germ line. Here we report that Bombyx mori ovary-derived cultured cell line, BmN4, endogenously expresses two PIWI subfamily proteins, silkworm Piwi (Siwi) and Ago3 (BmAgo3), and piRNAs associated with them. Siwi-bound piRNAs have a strong bias for uridine at their 59 end and BmAgo3-bound piRNAs are enriched for adenine at position 10. In addition, Siwi preferentially binds antisense piRNAs, whereas BmAgo3 binds sense piRNAs. Moreover, we identified many pairs in which Siwi-bound antisense and BmAgo3-bound sense piRNAs are overlapped by precisely 10 nt at their 59 ends. These signatures are known to be important for secondary piRNA biogenesis in other organisms. Taken together, BmN4 is a unique cell line in which both primary and secondary steps of piRNA biogenesis pathways are active. This cell line would provide useful tools for analysis of piRNA biogenesis and function.

Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. An inherited defect in BH4 biosynthesis is caused by the deficiency of sepiapterin reductase (SPR), which catalyzes the biosynthesis of BH4 from guanosine triphosphate at the terminal step. The human SPR gene has been mapped at the PARK3 locus, which is related to the onset of Parkinson disease. In this study, we report that mutant strains, lemon (lem) and its lethal allele lemon lethal (lem(1)) with yellow body coloration, of the silkworm Bombyx mori could be used as the first insect model for human SPR deficiency diseases. We demonstrated that mutations in the SPR gene (BmSpr) were responsible for the irregular body coloration of lem and lem(l). Moreover, biochemical analysis revealed that SPR activity in lem(l) larvae was almost completely diminished, resulting in a lethal phenotype that the larvae cannot feed and that die immediately after the first ecdysis. Oral administration of BH4 and dopamine to lem(l) larvae effectively increased their survival rates and feeding abilities. Our data demonstrate that BmSPR plays a crucial role in the generation of BH4, and monoamine neurotransmitters in silkworms and the lem (lem(l)) mutant strains will be an invaluable resource to address many questions regarding SPR and BH4 deficiencies.

In Bombyx mori, there are more than 35 mutant strains whose larval skin color is transparent. The waxy translucent strain ow is one of the oily mutants which lack accumulation of uric acid in the epidermis. Here we performed positional cloning of the ow gene using the Bombyx draft genome sequence. For fine structure mapping, we succeeded to narrow the ow linked region to approximately 150 kb. and identified the ow candidate gene by annotation analysis and DNA sequencing. The complete cDNA sequences of the ow gene from wild-type strains were 3501 bp-long and potentially encoded a protein of 920 amino acids. We found a 25 bp-long insertion in this gene in the ow mutant strain, resulting in a frame-shift mutation and generation of a premature stop codon. A BLAST search revealed that this protein had high homology to Varp, a recently identified protein containing a vacuolar sorting protein 9 domain and ankyrin repeats, and we termed the silkworm protein BmVarp. Varp has been shown to regulate endosome dynamics, suggesting that BmVarp may play an important role in the incorporation and/or accumulation of uric acid in the epidermis. (C) 2009 Elsevier Ltd. All rights reserved.

During the maintenance of the wild silkworm, Bombyx mandarina, a mutant phenotype exhibiting translucent skin was identified. Based on the crossing experiments with the domesticated silkworm, Bombyx mori, we found that the mutant was controlled by molybdenum cofactor sulfurase (MoCoS) gene. We designated the mutant "Ozaki's translucent" (og(Z)). We found a 2.1-kb deletion containing the transcription initiation site, exons 1 and 2, and the 5' end of exon 3 of the MoCoS gene. The transcript of the MoCoS gene was not detected in the og(Z) homozygote. We concluded that og(Z) is a complete loss-of-function allele generated by a disruption of the MoCoS gene.

It has been previously reported that the fp25K product of Bombyx man nucleopolyhedrovirus (BmNPV) is required for post-mortem host degradation, but the mechanism by which it regulates host degradation is still unknown. This study shows that disruption of BmNPV fp25K attenuates the expression of viral cathepsin gene (v-cath) at a late stage of infection, and thus reduces the secretion of its product V-CATH. Western blot analysis showed that secretion of V-CATH was severely reduced in BmN cells and B. mori larvae infected with Bm25KD, a BmNPV mutant lacking functional fp25K, compared to that of wild-type BmNPV. Also, reduced accumulation of pro-V-CATH in Bm25KD-infected cells was observed from 4 days postinfection (dpi), during which V-CATH was first detected in the medium of BmNPV-infected cells. qRT-PCR experiments showed that the expression levels of v-cath mRNA in wild-type- and Bm25KD-infected BmN cells were comparable at 3 dpi, but showed a marked decrease in Bm25KD-infected BmN cells at 4 dpi. Collectively, these results suggest that BmNPV FP25K is essential for the proper transcriptional regulation of v-cath and efficient secretion of V-CATH, and a steady-state level of v-cath expression during the period of V-CATH secretion (after 4 dpi) is required for post-mortem host degradation. (C) 2008 Elsevier B.V. All rights reserved.

We describe the characterization of hemoglobin-like genes from two lepidopteran insects, Bombyx mori (Bmglobin) and Samia cynthia ricini (Scglobin). Bmglobin and Scglobin are predicted to be intracellular proteins and contain amino acids required for heme and oxygen binding. Expression profiles of two lepidopteran globins, especially Bmglobin, were different from that of other insect globins. Although other insect globins are mainly associated with the tracheal system, Bmglobin was expressed almost exclusively in the Malpighian tubules, and the strongest signal for Scglobin was detected in the fat body. Furthermore, biochemical fractionation analysis revealed that both Bmglobin and Scglobin were localized in the cytoplasm. These results suggest that each lepidopteran globin has a distinct role in the tissues in which it is expressed and that the functions of insect globins are more divergent than previously thought. (C) 2008 Elsevier B.V. All rights reserved.

Larvae of the body color mutant red blood (rb) of the silkworm, Bombyx mori, display reddish skin whose hemolymph becomes red in air, whereas hemolymphs of normal strains become black during melanization. The irregular coloring was assumed to result from an abnormal accumulation of 3-hydroxykynurenine. However, the gene responsible for the rb phenotype is not yet known. Here, we provide evidence that the rb gene corresponds to a novel bacterial-type kynureninase gene, BmKynu. Kynureninase (KYNU) hydrolyzes kynurenine and 3-hydroxykynurenine to anthranilic acid and 3-hydroxyanthranilic acid, respectively. KYNU has been identified in microorganisms and animals but not in insects. Therefore, BmKynu is the first KYNU gene observed in insects. Our results clearly showed that a point mutation (T102I) in BmKYNU of the rb strain led to a marked decrease in KYNU activity, presumably resulting in abnormal accumulation of 3-hydroxykynurenine. Additionally, linkage analysis indicated that no recombination between rb and BmKynu was detected. We conclude that T102I in BmKYNU causes the red body coloration in the rb strain. Our study proves that B. mori has a unique side branch in the kynurenine pathway, distinctly different from other insects.

Post-mortem host degradation by infection of Bombyx mori nucleopolyhedrovirus (BmNPV) requires the synergistic activation of two virus-encoded genes, cathepsin (v-cath) and chitinase (v-chiA). Previous studies have suggested that V-CHIA is essential for the proper folding of the nascent V-CATH polypeptide in the endoplasmic reticulum, and that the putative V-CHIA-V-CATH interaction might be mediated by N-linked glycans of V-CATH. Sequence analysis shows that BmNPV V-CATH includes three consensus N-linked glycosylation sites (asparagine 38, 65 and 158). To clarify the role of N-linked glycans of V-CATH in its biological activity, we generated three recombinant BmNPVs; expressing mutant V-CATHs, and found that the two residues, asparagine 38 and 65, which are localized in the pro-region of V-CATH, are the glycosylation sites of BmNPV V-CATH. Western blot analysis also showed that removal of N-linked glycans from BmNPV V-CATH resulted in production of the insoluble forms of V-CATH and V-CHIA. These results demonstrate that N-linked glycans located in the pro-region of BmNPV V-CATH are essential for the proper folding of V-CATH and V-CHIA.

Selfish genetic elements called transposons can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Expansion of transposons can occur when novel transposition events are transmitted to subsequent generations after germline hopping. Therefore, organisms seem likely to have evolved defense mechanisms to silence transposons in the germline. Recently, small RNAs interacting with Piwi proteins (piwi-interacting RNAs: piRNAs) have been demonstrated to be involved in genomic defense mechanism against transposons. Here, we show that piRNA-like small RNAs are present abundantly in the Bombyx ovary. We cloned 38,493 kinds of Bombyx small RNA from the ovary and performed functional characterization. Bombyx small RNAs showed a unimodal length distribution with a peak at 28 nt and a strong bias for U at the 5' end. We found that 12,869 kinds of Bombyx small RNAs were associated with transposons or repetitive sequences. We classified them as repeat-associated small interfering RNAs (rasiRNAs), a subclass of piRNAs. Notably, antisense rasiRNAs have a strong bias toward U at 5' ends; in contrast, sense rasiRNAs have a strong bias toward A at nucleotide position 10, indicating that the piRNA amplification loop proposed in Drosophila is evolutionarily conserved in Bombyx. These results suggest that Bombyx small RNAs regulate transposon activity, (C) 2008 Elsevier Ltd. All rights reserved.

Many larval color mutants; have been obtained in the silkworm Bombyx mori. Mapping of melaninsynthesis genes oil the Bombyx linkage map revealed that yellow and ebony genes were located near the chocolate (ch) and sooly (so) loci, respectively. In the ch mutants, body color of neonate larvae and the body markings of elder instar larvae are reddish brown instead of normal black. Mutations at the so locus produce smoky larvae and black pupae. F-2 linkage analyses showed that. sequence polymorphisms of yellow and ebony genes perfectly cosegregated with the ch and so mutant phenotypes, respectively. Both yellow and ebony were expressed in the epidermis during the molting period when cuticular pigmentation occurred. The spatial expression pattern of yellow transcripts coincided with the larval black markings. In the ch mutants, nonsense mutations of the yellow gene were detected, whereas large deletions of the ebony ORF were detected in the so mutants. These results indicate that yellow and ebony are the responsible genes for the ch and so loci, respectively. Our findings suggest that Yellow promotes melanization, whereas Ebony inhibits melanization in Lepidoptera and that melanin-synthesis enzymes play a critical role in the lepiclopteran larval color pattern.

The SNF2 global transactivator gene homologue (Bm33) of Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the genes exclusive to group I NPVs, but its function remains unknown. This study describes the characterization of Bm33. Transcriptional analysis suggested that Bm33 is an early gene, as its transcript was observed at 4 h post-infection in BmNPV-infected BmN cells. To examine the role of Bm33 during BmNPV infection, a Bm33 deletion mutant (BmORF33D) was constructed and its infectivity was characterized in BmN cells and B. mori larvae. BmORF33D did not have any obvious defects in the production of budded viruses (BVs) or occlusion bodies (OBs) in BmN cells compared with wild-type BmNPV. Larval bioassays revealed that deletion of Bm33 did not reduce virus infectivity. However, BmORF33D took approximately 10-15 h longer than wild-type BmNPV to kill B. mori larvae when tested by either BV injection or OB ingestion. These results suggest that Bm33 is not essential for virus growth in vitro or in vivo, but that it accelerates the time of death of B. mori larvae.

Many strains of Bombyx mori carry chromosomal aberrations, and they are useful resources for integration between phenotypes and genomic sequences. We compared the molecular structures of three kinds of Z chromosomes, i.e., two strains with chromosome deletions and one strain with translocation involving the Z chromosome. Using polymerase chain reaction markers, we showed that: (1) the Z, chromosome lacks more than 6 Mb, including the proximal end; (2) the Z(Vg) chromosome lacks 1.5 Mb in the interstitial portion; and (3) the +(od)p(Sa)+(p)W carries a 0.6-Mb Z-derived fragment surrounding the +(od) gene. The breakpoint junctions of these deletions and a translocation were precisely determined. Through deletion mapping, we narrowed down the regions where distinct oily (od), vestigial (Vg), and muscle dystrophy (Md) are located and identified a candidate gene for ad. A retroposon-mediated deletion in BmBLOS2-the Bombyx gene homologous to human "biogenesis of lysosome-related organelles complex-1, subunit 2"-was detected in the ad mutant. Although the genes responsible for Vg and Md were not definitively identified, we propose the candidate genes on the basis of their locations and phenotypes. (C) 2008 Elsevier Ltd. All rights reserved.

The Baculoviridae is a large family of pathogens that are infectious for arthropods, particularly insects of the Lepidoptera. Nucleopolyhedroviruses (NPVs), a genus of Baculoviridae, have a large circular, super-coiled, and double-stranded DNA genome packaged into rod-shaped virions. The Bombyx mori NPV (BmNPV), an NPV pathogenic for B. mori, is known to potentially encode 136 proteins. Using the B. mori genome information, we found that 15 of 136 BmNPV proteins (11%) show significant similarity to the B. mori proteins. Among them, genes encoding nine proteins can be deleted in B. mori cultured cell line BmN by homologous recombination, indicating that these genes are dispensable for normal virus production. Interestingly, most of non-essential auxiliary genes encode proteins controlling host physiology at cellular and/or organismal levels: ecdysteroid UDP-glucosyltransferase inactivates an insect molting hormone ecdysone, protein tyrosine phosphatase is involved in wandering behavior at the late stage of infection, fibroblast growth factor induces host cell chemotaxis, and chitinase and cathepsin are required for postmortem host liquefaction. Deletion analysis of other non-essential genes also showed that three of them are viral pathogenicity factors for B. mori. These findings suggest that the modem lepidopteran baculovirus may have acquired auxiliary genes from an ancestral host insect to control host physiology and to increase the efficiency of virus transmission in nature. (C) 2008 Elsevier Ltd. All rights reserved.

カイコ生殖巣に存在するsmall RNAの特性と機能。多くの生物のゲノム解読が完了した昨今、「ゲノム上に存在するタンパク質非コード領域が持つ機能」は、ますます興味深い問題のひとつである。カイコゲノムのほぼ80％、ヒトゲノムでは実に98％が、反復配列をはじめとするタンパク質非コード領域であると認識されている。長い間、これらの領域は「ジャンクDNA」などと呼ばれ、生物にとって不要なものであると見なされてきた。しかしながら、最近になって、これら非コード領域が活発に転写され、転写された非コードRNAが重要な生物機能を有する、ということが分かってきた。我々が最もよく知っている重要な非コードRNAとしては、tRNAやrRNAが挙げられるが、大きな注目が集まっているのは、長さが21-30塩基程度の小さなRNA、small RNAの機能である。ここでは、動物のsmall RNAの基本的な機能に関して概説するとともに、筆者らが最近得た、カイコにおけるsmall RNA研究についての結果を紹介する。

Bombyx mori, the domesticated silkworm, is a major insect model for research, and the first lepidopteran for which draft genome sequences became available in 2004. Two independent data sets from whole-genome shotgun sequencing were merged and assembled together with newly obtained fosmid- and BAC-end sequences. The remarkably improved new assembly is presented here. The 8.5-fold sequence coverage of an estimated 432 Mb genome was assembled into scaffolds with an N50 size of ∼3.7 Mb; the largest scaffold was 14.5 million base pairs. With help of a high-density SNP linkage map, we anchored 87% of the scaffold sequences to all 28 chromosomes. A particular feature was the high repetitive sequence content estimated to be 43.6% and that consisted mainly of transposable elements. We predicted 14,623 gene models based on a GLEAN-based algorithm, a more accurate prediction than the previous gene models for this species. Over three thousand silkworm genes have no homologs in other insect or vertebrate genomes. Some insights into gene evolution and into characteristic biological processes are presented here and in other papers in this issue. The massive silk production correlates with the existence of specific tRNA clusters, and of several sericin genes assembled in a cluster. The silkworm's adaptation to feeding on mulberry leaves, which contain toxic alkaloids, is likely linked to the presence of new-type sucrase genes, apparently acquired from bacteria. The silkworm genome also revealed the cascade of genes involved in the juvenile hormone biosynthesis pathway, and a large number of cuticular protein genes. © 2008 Elsevier Ltd. All rights reserved.

All lepidopteran baculovirus genomes sequenced to date encode a viral fibroblast growth factor homolog (vFGF). Recently, we generated a Bombyx mori nucleopolyhedrovirus (BmNPV) mutant lacking, functional vfgf and found that BmNPV vfgf contributes to virus virulence in B. mori larvae. However, the steps at which BmNPVvFGF works during in vivo virus infection were unclear. To uncover the role of vFGF during systemic infection of silkworm larvae, we generated a BmNPV mutant, BmIEGFP, possessing an ie-1 promoter-driven green fluorescent protein gene, and its derivative BmIEGFP/FGFD, in which vfgf was partially deleted from the genome of BmIEGFP. Intrahemoccrelic and oral infection experiments using these viruses revealed that the loss of functional vFGF reduces viral infectivity in B. mori hemocytes. Our results suggest that BmNPV vFGF is required for efficient systemic infection, presumably by a chemotactic effect that allows budded virus to infect hemocytes efficiently. (C) 2008 Elsevier B.V. All rights reserved.

The 14-3-3 protein family consists of evolutionarily conserved, acidic 30 kDa proteins composed of seven isoforms named beta, gamma, epsilon, zeta, eta, theta, and sigma in mammalian cells. The dimeric complex of 14-3-3 isoforms, acting as a molecular adaptor, plays a central role in regulation of neuronal function. Since aberrant expression of 14-3-3 is identified in the brains of Alzheimer disease and Parkinson disease, a convenient insect model, if it is available, is highly valuable for studying a pathological role of 14-3-3 in neurodegeneration. Here, we identified the silkworm Bombyx mori 14-3-3 orthologs, zeta and epsilon isoforms highly homologous in amino acid sequences to the human 14-3-3 zeta and 14-3-3 epsilon. By Western blot, the expression of zeta and epsilon isoforms was identified at substantial levels in the first instar larva, markedly upregulated in the second instar larva, and the highest levels were maintained in the late stage of larva, the pupa, and the adult. Furthermore, by protein overlay and immunoprecipitation, we identified Hsp60 as a 14-3-3-binding partner. The 14-3-3 proteins interacted with the N-terminal fragment of Hsp60. The 14-3-3 and F isoforms, along with Hsp60, were expressed widely with overlapping distribution in larval and adult tissues, including brain, fat body, silk gland, Malpighian tube, midgut, ovary, testis, antenna, and pheromone gland. These observations suggest that a molecular adaptor 14-3-3 and a molecular chaperone Hsp60 cooperate to achieve a wide range of cellular functions in B. mori. (c) 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Background: Functional genomics has particular promise in silkworm biology for identifying genes involved in a variety of biological functions that include: synthesis and secretion of silk, sex determination pathways, insect-pathogen interactions, chorionogenesis, molecular clocks. Wild silkmoths have hardly been the subject of detailed scientific investigations, owing largely to nonavailability of molecular and genetic data on these species. As a first step, in the present study we generated large scale expressed sequence tags ( EST) in three economically important species of wild silkmoths. In order to make these resources available for the use of global scientific community, an EST database called 'WildSilkbase' was developed. Description: WildSilkbase is a catalogue of ESTs generated from several tissues at different developmental stages of 3 economically important saturniid silkmoths, an Indian golden silkmoth, Antheraea assama, an Indian tropical tasar silkmoth, A. mylitta and eri silkmoth, Samia cynthia ricini. Currently the database is provided with 57,113 ESTs which are clustered and assembled into 4,019 contigs and 10,019 singletons. Data can be browsed and downloaded using a standard web browser. Users can search the database either by BLAST query, keywords or Gene Ontology query. There are options to carry out searches for species, tissue and developmental stage specific ESTs in BLAST page. Other features of the WildSilkbase include cSNP discovery, GO viewer, homologue finder, SSR finder and links to all other related databases. The WildSilkbase is freely available from http://www.cdfd.org.in/wildsilkbase/. Conclusion: A total of 14,038 putative unigenes was identified in 3 species of wild silkmoths. These genes provide important resources to gain insight into the functional and evolutionary study of wild silkmoths. We believe that WildSilkbase will be extremely useful for all those researchers working in the areas of comparative genomics, functional genomics and molecular evolution in general, and gene discovery, gene organization, transposable elements and genome variability of insect species in particular.

Mulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1) and 1-deoxynojirimycin (DNJ). Although these compounds do not harm the silkworm, Bombyx mori, a mulberry specialist, they are highly toxic to insects that do not normally feed on mulberry leaves. D-AB1 and DNJ are strong inhibitors of alpha-glucosidases (EC 3.2.1.20); however, they do not affect the activity of beta-fructofuranosidases (EC 3.2.1.26). Although alpha-glucosidase genes are found in a wide range of organisms, beta-fructofuranosidase genes have not been identified in any animals so far. In this study, we report the identification and characterization of beta-fructofuranosidase genes (BmSuc1 and BmSuc2) from B. mod. The BmSuc1 gene was highly expressed in the midgut and silk gland, whereas the expression of BmSuc2 gene was not detected. BmSuc1 encodes a functional beta-fructofuranosidase, whose enzymatic activity was not inhibited by DNJ or D-AB1. We also showed that BmSUC1 protein localized within the midgut goblet cell cavities. Collectively, our data clearly demonstrated that BmSuc1 serves as a sugar-digesting enzyme in the silkworm physiology. This anomalous presence of the beta-fructofuranosidase gene in the B. mori genome may partly explain why the silkworm can circumvent the mulberry's defense system.

Bombyx mori densovirus type 2 (BmDNV-2), a parvo-like virus, replicates only in midgut columnar cells and causes fatal disease. The resistance expressed in some silkworm strains against the virus is determined by a single gone, nsd-2, which is characterized as nonsusceptibility irrespective of the viral dose. However, the responsible gene has been unknown. We isolated the nsd-2 gene by positional cloning. The virus resistance is caused by a 6-kb deletion in the ORF of a gene encoding a 12-pass transmembrane protein, a member of an amino acid transporter family, and expressed only in midgut. Germ-line transformation with a wildtype transgene expressed in the midgut restores susceptibility, showing that the defective membrane protein is responsible for resistance. Cumulatively, our data show that the membrane protein is a functional receptor for BmDNV-2. This is a previously undescribed report of positional cloning of a mutant gene in Bombyx and isolation of an absolute virus resistance gene in insects.

The Piwi subfamily proteins and their partner, Piwi-interacting RNAs (piRNAs), play an important roles in germ-line development and silencing of selfish DNA elements. To date, however, the developmental expression profiles of the Piwi subfamily genes are poorly known. In this study, we examined the expression profiles of two Bombyx mori Piwi subfamily genes, silkworm Piwi (SIWI) and BmAGO3, which are possible partners of Bombyx piRNA-like small RNAs we recently identified in B. mori germ-line cells. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that these two genes were abundantly expressed in the larval testis, pupal ovary, and adult eggs, suggesting that they might be involved in spermatogenesis and oogenesis in Bombyx. Notably, developmental expression patterns of SIWI and BmAGO3 were remarkably similar. Collectively, our results suggest that SIWI and BmAGO3 may cooperate in an unknown pathway during the development of B. mori germ-line cells. (c) 2008 Elsevier Inc. All rights reserved.

To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1-4, in the Bombyx genome. Northern blot and RTPCR analysis showed that BmGlov1-4 were induced in the larval fat body after an immune challenge by the injection of E. coli, however, less induction was observed after the injection of a yeast Candida albicons. In silico sequence analysis revealed the presence of a motif homologous to NF-kappa B binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1-4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1-4 significantly inhibited the growth of E. coli.

The Bombyx mori homolog of doublesex, Bmdsx, plays an essential role in silkworm sexual development. Exons 3 and 4 of Bmdsx pre-mRNA are specifically excluded in males. To explore how this occurs, we developed a novel in vivo sex-specific splicing assay system using sexually differentiated cultured cells. A series of mutation analyses using a Bmdsx minigene with this in vivo splicing assay system identified three distinct sequences (CE1, CE2, and CE3) positioned in exon 4 as exonic splicing silencers responsible for male-specific splicing. Gel shift analysis showed that CE1 binds to a nuclear protein from male cells but not that from female cells. Mutation of UAA repeats within CE1 inhibited the binding of the nuclear protein to the RNA and caused female-specific splicing in male cells. We have identified BmPSI, a Bombyx homolog of P-element somatic inhibitor (PSI), as the nuclear factor that specifically binds CE1. Down-regulation of endogenous BmPSI by RNA interference significantly increased female-specific splicing in male cells. This is the first report of a PSI homolog implicated in the regulated sex-specific splicing of dsx pre-mRNA.

We constructed two independent cDNA libraries from the fat body of Escherichia coli- or Candida albicans-challenged eri-silkworm Samia cynthia ricini larvae. We performed comparative expressed sequence tag (EST) analysis of the two cDNA libraries and found that two putative storage protein genes, ScSP1 and ScSP2, were markedly repressed by E. coli injection as compared with C. albicans injection. By quantitative real-time RT-PCR analysis, we showed that ScSP1 mRNA significantly reduced to 1/32-1/3 in the fat body of the female larvae, and ScSP2 mRNA reduced to 1/7-1/3 and 1/22-1/5 in the females and males, respectively, 12-36 h after E. coli injection as compared with PBS injection. In addition, SDS-PAGE analysis revealed that the accumulation of both the ScSP proteins in the larval hemolymph apparently decreased up to 36 h after E. coli injection. However, the amounts of the two ScSP proteins returned to the some level as those in the larvae injected with PBS by 48 h after injection, showing that the reduction in ScSPs caused by the bacterial challenge was transient. Moreover, potential binding sites for the Drosophila ReI/NF-kappa B protein Dorsal were found in the 5' upstream regulatory regions of ScSP1 and ScSP2, suggesting the participation of the ReI/NF-kappa B proteins in controlling the bacterial suppression of the ScSP genes. These results suggested the hypothesis that S. c. ricini has a genetic program to shut down temporarily dispensable gene expression in order to induce an acute and efficient expression of immune-related genes. These findings may provide new insight into the innate immune system in lepidopteran insects.

Ganciclovir, foscarnet, vidarabine and ribavirin, which are used to treat viral infections in humans, inhibited the proliferation of a baculovirus (Bombyx mori nucleopolyhedrovirus) in BmN4 cells, a cultured silkworm cell line. These antiviral agents inhibited the proliferation of baculovirus in silkworm body fluid and had therapeutic effects. Using the silkworm infection model, the antiviral activity of Kampo medicines was screened and it was found that cinnamon bark, a component of the traditional Japanese medicine Mao-to, had a therapeutic effect. Based on the therapeutic activity, the antiviral substance was purified. Nuclear magnetic resonance analysis of the purified fraction revealed that the antiviral activity was due to cinnzeylanine, which has previously been isolated from Cinnamomum zeylanicum. Cinnzeylanine inhibits the proliferation of herpes simplex virus type 1 in Vero cells. These results suggest that the silkworm-baculovirus infection model is useful for screening antiviral agents that are effective for treating humans infected with DNA viruses.

Mitogen-activated protein kinases (MAPKs) often play important roles in virus infection. To explore intracellular signaling pathways induced by baculovirus infection, we examined the involvement of MAPKs in Bombyx mori nucleopolyhedrovirus (BmNPV) infection of BmN cells. We found that specific inhibitors of extracellular signal-regulated kinase (ERK) kinase and c-Jun NH2-terminal kinase (JNK) significantly reduced occlusion body (OB) formation and budded virus (BV) production. Next, we quantified OB and BV production after applying the inhibitors at different times postinfection (p.i.). The inhibitors significantly reduced OB and BV production to various extents when applied at 12 h p.i., indicating that the reduction of BmNPV infectivity by these inhibitors occurs at the late stage of infection. Also, we observed that these inhibitors markedly repressed or deregulated the expression of delayed early, late, and very late gene products. Western blot analysis using phospho-MAPK-specific antibodies showed that ERK and JNK were activated at the late stage of BmNPV infection. In addition, the magnitude and pattern of MAPK activation were dependent on the multiplicity of infection. To verify the effects of the inhibitors on BmNPV infection, we also attempted to knock down the B. mori genes BmErk and BmJnk, which encode ERK and JNK, respectively. Knockdown of BmErk and BmJnk resulted in the reduced production of OBs and BVs, confirming that BmERK and BmJNK are involved in the BmNPV infection process. Taken together, these results indicate that the activation of MAPK signaling pathways is required for efficient infection by BmNPV.

Baculovirus chitinases (V-CHIAs) play a crucial role in the terminal liquefaction of virus-infected larvae after death. Although v-chiAs from nucleopolyhedroviruses (NPVs) have been well characterized, little is known about v-chiAs from granuloviruses (GVs). We characterized the v-chiA of Cydia pomonella GV (CpGV) by constructing a recombinant Bombyx mori NPV (BmNPV) in which BmNPV v-chiA was replaced by CpGV v-chiA (103CpGV virus). CpGV v-chiA encoded an approximately 70-kDa chitinase with an exo-type substrate preference. CpGV V-CHIA lacked a C-terminal KDEL endoplasmic reticulum retention motif and was suggested to be a secretory protein. Terminal host liquefaction of B. mori larvae and proper folding of BmNPV-encoded cysteine protease (BmNPV V-CATH) were observed following infection with 103CpGV, indicating that CpGV v-chiA is able to compensate for the absence of its BmNPV counterpart. Our data suggest that the molecular interaction between V-CHIA and V-CATH may be conserved across a broad range of lepidopteran GVs and NPVs.

In insects, the sex is determined completely by genetic mechanisms, which at least in somatic tissues, are cell autonomous. The sex of the silkworm, Bombyx mori, is strongly controlled by the presence of the W chromosome. Genetic studies using translocations and deletions of W suggested that a presumptive feminizing gene (Fem) is located in a limited region of the W chromosome. Recent genomic studies revealed a small number of potential candidates for the Fen? gene in this region. In addition, a Bombyx homologue of the Drosophila sex determining gene doublesex has been identified on an autosome and analyzed. Whereas the Drosophila doublesex gene is regulated by activation of splicing in females, the Bombyx doublesex gene (Bmdsx) encodes female- and male-specific mRNAs regulated via male-specific repression of splicing. The vitellogenin gene (Vg) is a target of the BmDSX protein, which directly binds to the Vg promoter. Furthermore, as ectopic expression of the rnale-type Bmdsx induces male-like transformation of the sexual organs, BmDSX may control sex-specific morphological characteristics in Bombyx. This suggests that although upstream events in Drosophila and Bombyx sex determination differ, similarities between the two species do exist in downstream genetic control of sex determination. (c) 2007 Elsevier Ltd. All rights reserved.

Infection of Bombyx mori larvae with B. mori nucleopolyhedrovirus (BmNPV) results in liquefaction of the host. This process is attributed to the synergistic action of two virus-encoded genes, chitinase (v-chiA) and cathepsin (v-cath). Previous studies have suggested that Autographa californica nucleopolyhedrovirus (AcMNPV) CATH cannot be processed within infected cells in the absence of AcMNPV CHIA. To investigate the interactions between V-CHIA and V-CATH, we generated a recombinant BmNPV (103ChiAmut) in which the residues of the active site of BmNPV chiA were mutated (D302NE306Q) and the gene was driven by its own promoter at the native locus. Mutation at the active site of Bn-tNPV CHIA resulted in complete loss of chitinolytic activity. Bombyx mori larvae infected with 103ChiAmut survived longer than larvae infected with wild-type BmNPV and did not undergo terminal liquefaction after death. Cysteine protease activity and Western blot analysis showed that, in cells infected with v-chiA-deleted BmNPV (ChiAD), BmNPV CATH was not processed properly and was accumulated as a detergent-insoluble form, suggesting that BmNPV CHIA plays a crucial role in V-CATH processing. In cells infected with 103ChiAmut, BtnNPV CATH formed insoluble aggregates, suggesting that active site-mutated BmNPV CHIA loses its additional role as a molecular chaperon during V-CATH processing. (c) 2006 Elsevier B.V. All rights reserved.

To identify genes involved in the differentiation of Bombyx cystoblast, we constructed two 3' long serial analysis, of gene expression (Long SAGE) libraries from stage 1-3 or stage 2-3 egg chambers and compared their gene expression profiles. In both libraries, the most frequent tags were derived from the same novel transcript. The transcript does not have any open reading frame capable of encoding a protein with over 100 amino acids in length. RNA blot analysis revealed that this transcript is specifically and abundantly expressed in the Bombyx ovary, mainly the germ line cells in the ovarioles. These results suggest that Bombyx oogenesis may be regulated by a previously unidentified non-coding RNA. Comparison of the gene expression profiles between the stage 1-3 and stage 2-3 egg chamber libraries revealed that 272 tags were significantly more abundant in stage 1-3 egg chambers (p < 0.05 and at least two-fold change) than in library 2. Among the differentially expressed transcripts were the sequences that correspond to ATP synthase subunit d (3.1-fold enriched) and ATP synthase coupling factor 6 (9.1-fold enriched), suggesting that they are involved in regulation of cell cycle of cystocytes. (c) 2006 Elsevier Ltd. All rights reserved.

Following baculovirus infection, the level of host messenger RNA (mRNA) declines and the synthesis of host proteins is shutoff in virus-infected cells. To comprehensively understand the regulation of host gene expression by Bombyx mori nucleopolyhedrovirus (BmNPV), we took a complementary DNA (cDNA) subtraction approach. Northern blot analysis was then performed to confirm whether obtained clones are differentially expressed. From these analyses, we obtained 4 down-, 7 up- and 2 non-regulated host gene candidates during the early stage of infection. Sequence analyses showed that these may encode proteins involved in transcription, cell cycle, cell adhesion, protein degradation, apoptosis or energy metabolism. We then measured the ADP/ATP ratio to address the response of host energy metabolism upon baculovirus infection. The ADP/ATP ratio showed cell death-like rapid increase following BmNPV infection with a transient decrease during 12-24 h post-infection (p.i.). Taken together, our results suggest that general events in host cells are controlled at the level of mRNA expression by BmNPV infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) fibroblast growth factor (BmFGF) is a glycosylated protein that is efficiently secreted into the medium. Here, we constructed mutant NPVs expressing His-tagged wild-type (wt) or mutant BmFGFs and showed that the two residues, asparagine 44 and 171, are the glycosylation sites of BmFGF. Also, removal of N-linked glycans from BmFGF resulted in almost complete inhibition of the secretion into the medium, suggesting that N-linked glycans of BmFGF are required for its secretion. These residues are not conserved in closely related Autographa californica NPV (AcMNPV)-encoded vFGF (AcFGF). Western blot analysis suggested that AcFGF is not glycosylated and is poorly secreted. A mutant AcFGF possessing two N-linked glycosylation sites was secreted into the medium more abundantly than that which occurred for wt AcFGF. This is the first direct evidence showing the role of N-linked glycans in the secretion process of a baculovirus protein. (c) 2006 Elsevier Inc. All rights reserved.