The ORC (origin recognition complex) binds to the DNA replication origin and recruits other replication factors to form the pre-replication complex. The cDNA and genomic sequences of all six subunits of ORC in Bombyx mori (BmORC1–6) were determined by RACE (rapid amplification of cDNA ends) and bioinformatic analysis. The conserved domains were identified in BmOrc1p–6p and the C-terminal of BmOrc6p features a short sequence that may be specific for Lepidoptera. As in other organisms, each of the six BmORC subunits had evolved individually from ancestral genes in early eukaryotes. During embryo development, the six genes were co-regulated, but different ratios of the abundance of mRNAs were observed in 13 tissues of the fifth instar day-6 larvae. Infection by BmNPV (B. mori nucleopolyhedrovirus) initially decreased and then increased the abundance of BmORC. We suggest that some of the BmOrc proteins may have additional functions and that BmOrc proteins participate in the replication of BmNPV.

INTRODUCTION

The ORC (origin recognition complex) was first discovered in budding yeast [1]. It contains six different protein subunits (Orc1p–6p) that are encoded by the genes ORC1–6, which are located on different yeast chromosomes. Homologues of the six Orc proteins have been identified in Arabidopsis thaliana [2], Drosophila melanogaster [3] and even in Homo sapiens [4]. ORCs from different species are identified by the initials of the genus and species name such as BmORC (Bombyx mori ORC) [5].

The heterohexameric ORC complex was shown to bind to the origin of DNA replication during the transformation from M phase to G1 phase of the cell cycle. ORC functions as a landing pad for additional replication factors, including Cdc6 (cell division cycle 6), Cdt1 (chromatin licensing and DNA replication factor 1) and MCM (mini-chromosome maintenance) to form the pre-RC (pre-replication complex), which initiated the start of DNA replication [5]. ScORC (Saccharomyces cerevisiae ORC) also participates in sister-chromatid cohesion [6].

ScOrc1p–5p contained an AAA+ domain that consisted of four motifs, including WA (Walker A), WB (Walker B), S1 (Sensor-1) and S2 (Sensor-2) [7,8]. Conservation of these motifs was strongest in Orc1p, 4p and 5p, and less in Orc2p and 3p. The C-terminals of Orc1p–5p also contained a WH (winged-helix) motif that mediated the binding between ORC and DNA replication origins [911]. Most of the Orc1p contained a BAH (bromo-adjacent homology) domain at the N-termini, which was similar to the Sir3 (silent information regulator 3) and it was also predicted to possess the same function [12,13]. Mutation analysis of the BAH domain in H. sapiens Orc1p (HsOrc1p) indicated that it mediated the interaction between HsOrc1p and the DNA replication origin during the transformation from M phase to G1 phase. The BAH domain was also involved in the binding of HsOrc1p to oriP, a replication origin of the Epstein–Barr virus, and it participated in the amplification of a plasmid containing oriP [14]. In addition, the BAH domain helped HsOrc2p bind to the origin of DNA replication [15,16]. In budding yeast and humans, the Orc1p–Orc5p but not Orc6p were essential for ORC to recognize the origin of DNA replication [17]. All the six subunits were, however, essential for origin recognition in D. melanogaster [18].

A mutation of D. melanogaster Orc3p caused a failure of cell proliferation during larval development, which affected the central nervous system including the brain [19]. The MmORC (Mus musculus ORC) core complex (containing Orc2p–5p) was highly expressed in adult mouse brain tissue, and knockdown using siRNA (small interfering RNA) reduced the dendrite and dendritic spine developments in postmitotic neurons [20].

ScOrc6 proteins contain a unique conserved domain named the Orc6 protein fold superfamily that was interrupted by a large disordered region [21]. Orc6p of the lepidopteran Choristoneura fumiferana contains approx. 100 C-terminal amino acids that were not found in other Orc6 proteins [22].

A two-hybrid experiment showed that DmOrc6p interacts with Pnut protein (Septin protein family, involved in cell division) [23]. Immunoprecipitation assays proved that the interaction was mediated by the C-terminal domain of DmOrc6p. RNA interference of DmORC6 (D. melanogaster ORC 6) abolished DNA replication and cytokinesis [23]. Imprecise splicing of DmOrc6p using the P element had a similar effect, which could be released by introducing a full-length DmORC6 or HsORC6 gene [24]. CfMNPV (C. fumiferana multiple nucleopolyhedrovirus) infection in Cf124T cells did not change the level of CfOrc6p (C. fumiferana Orc6p) expression until 26 hpi (h post-infection) [22]. The limited conservation of the individual eukaryotic ORC subunits might be indicative of their diverse functions [25].

So far, the genes for the six ORC subunits of three Diptera species have been described from the genomic sequences of Aedes aegypti, Culex quinquefasciatus and D. melanogaster.

Here we cloned the cDNA sequences and genomic sequences of all six ORC subunits of the lepidopteran B. mori. Expression of the BmORC subunit mRNAs was monitored by quantitative real-time PCR during embryo development, and in 13 tissues of fifth instar day-6 larvae and after BmNPV (B. mori nucleopolyhedrovirus) infection of fifth instar larvae.

MATERIALS AND METHODS

Insects and tissue dissection

The silkworm stock JY-1 was supplied by the Sericultural Research Institute of the Chinese Academy of Agricultural Sciences. The insects were reared using common methods [26]. The BV (budded virus) of BmNPV was maintained and titrated by our laboratory.

Database searching and sequence assembly

DmORC sequences and BLAST searches were used to identify homologous sequences in the EST (expressed sequence tag) library of B. mori (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Overlapping B. mori EST sequences (Table 1) were assembled into single contigs using the DNASTAR software package.

Table 1
Gene family encoding ORC proteins in the silkworm B. mori

aa, amino acid; Chr, chromosome.

GeneRelated ESTsNumber of intronsOrigin of mRNAscDNA length [without poly(A) tail]aa lengthConserved domain (aa position)GenBank® accession numberChr location
BmORC1 CK563734; CK512140; CK520558 Unfertilized embryo/fat body/haemocyte 2003 595 240–420 GQ214390 Not mapped; Bm_scaf146 
BmORC2 CK565003; CN212162; CK514845 Unfertilized/fertilized embryo/posterior silk gland 1835 549 229–549 GQ214391 Chr 9; Bm_scaf56 
BmORC3 BY938544; AV398523; DC439421; BP119933; BB983596; DC556000; DC554298; BY935502; AU000373; DC434830; BJ985480; CK537868; CK527963; CK556136; BP126248; BP119569; CK519175; CK526722; CK521977; CK542243; CK521782; CK506238; DC565627; DC567338 11 Embryo/ovary/epidermis/posterior silk gland/compound eye/wing disc/prothoracic gland/maxillary glae/haemocyte/testis/fat body 2750 722 35–330 GQ214392 Chr 22; Bm_scaf18 
BmORC4 CK559677 Unfertilized embryo 1791 496 40–215 GQ214393 Chr 2; Bm_scaf27 
BmORC5 AU003674; BW999349; BW999377; BY921684; BW998393; DC543047; BW999431; BY930190 Wing disc/ovary/embryo 1511 460 5–205 GQ214394 Not mapped; Bm_scaf286 
BmORC6 BP118659 Compound eye 1260 290 10–150 GQ214395 Chr 20; Bm_scaf79 
GeneRelated ESTsNumber of intronsOrigin of mRNAscDNA length [without poly(A) tail]aa lengthConserved domain (aa position)GenBank® accession numberChr location
BmORC1 CK563734; CK512140; CK520558 Unfertilized embryo/fat body/haemocyte 2003 595 240–420 GQ214390 Not mapped; Bm_scaf146 
BmORC2 CK565003; CN212162; CK514845 Unfertilized/fertilized embryo/posterior silk gland 1835 549 229–549 GQ214391 Chr 9; Bm_scaf56 
BmORC3 BY938544; AV398523; DC439421; BP119933; BB983596; DC556000; DC554298; BY935502; AU000373; DC434830; BJ985480; CK537868; CK527963; CK556136; BP126248; BP119569; CK519175; CK526722; CK521977; CK542243; CK521782; CK506238; DC565627; DC567338 11 Embryo/ovary/epidermis/posterior silk gland/compound eye/wing disc/prothoracic gland/maxillary glae/haemocyte/testis/fat body 2750 722 35–330 GQ214392 Chr 22; Bm_scaf18 
BmORC4 CK559677 Unfertilized embryo 1791 496 40–215 GQ214393 Chr 2; Bm_scaf27 
BmORC5 AU003674; BW999349; BW999377; BY921684; BW998393; DC543047; BW999431; BY930190 Wing disc/ovary/embryo 1511 460 5–205 GQ214394 Not mapped; Bm_scaf286 
BmORC6 BP118659 Compound eye 1260 290 10–150 GQ214395 Chr 20; Bm_scaf79 

Infection with BmNPV and RNA isolation

Hibernating eggs were incubated using the simplified method [26]. Total RNA was isolated daily until the larvae hatched. When the larvae had grown to the fourth moulting stage, feeding was stopped until 12 h after all of the larvae had moulted (fifth instar day-1 larvae). Feeding was resumed 1 h before the larvae were divided randomly into two groups. One group was injected BV [5.0×105 pfu (plaque-forming units) of BmNPV each larva], and the other group was mock-infected and used as a control. At 1 h postinfection was labelled as 0 hpi. At every time point, fat body total RNA was isolated from five larvae according to the standard protocol supplied with the Invitrogen RNA isolation kit. The remaining healthy larvae were kept fed to the fifth instar day-6. Then total RNA was isolated from the head, epidermis, anterior silk gland, middle silk gland, posterior silk gland, wing disc, testis, haemocyte, ovary, Malpighian tube, tracheal bush, midgut and fat body [31,32].

cDNA synthesis and RACE (rapid amplification of cDNA ends)

A 1 μg sample of total RNA extracted from each given condition was used for cDNA synthesis (BD SMART™ RACE cDNA Amplification Kit; Clontech), according to the supplied user manual. Primers (Supplementary Table S1 at http://www.bioscirep.org/bsr/031/bsr0310353add.htm) were designed according to the assembled sequences. PCR was performed using a specific primer 1 and NUP (nested universal primer), followed by nested PCR using primer 2 and NUP, and suitably diluted PCR product from the first round as the template. Each PCR reaction was carried out under the following conditions: after denaturing for 5 min at 95°C, subsequent cooling on ice and addition of Taq DNA polymerase, PCR was performed for 30 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 1–2 min, followed by a 10-min incubation at 72°C. The PCR products were separated by agarose-gel electrophoresis, purified and ligated into the pMD18-T vector (Takara). Several clones were sequenced by the dideoxynucleotide method using the ABI-3730 automatic sequencer.

DNA sequence analysis, protein sequence alignment and phylogenetic analysis

The silkworm genome program (http://kaikoblast.dna.affrc.go.jp) was used to identify the genomic DNA sequences corresponding to the assembled cDNA sequences for each BmORC subunit. The protein sequence alignments were established using ClustalX and the neighbour-joining tree was constructed using MEGA 4. Conserved protein sequence domains were identified using the NCBI conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), and the SignalIP 3.0 (http://www.cbs.dtu.dk/services/SignalP) server program was used to search for possible signal peptides.

Quantitative real-time PCR

A portion of total RNA (1 μg) was used to synthesize cDNAs according to the Reverse Transcription Kit's protocol (Promega; cat no.: A3500). The real-time–PCRs were carried out according to the user manual for the Toyobo SYBR Green Real-time PCR master mix. For each BmActin3 was used as an internal reference. Primers for real-time PCR are listed in Supplementary Table S1.

Standard curves and data analysis

The products of real-time PCR were separated by agarose-gel electrophoresis, purified using glass milk and the DNA concentrations were determined using a spectrophotometer (an A260 of 1.0 is equivalent to 50 μg/ml). The molecular mass of the DNA fragments was determined using the DNASTAR software package. Real-time PCR using dilutions from 109 to 104 copies/μl was carried out to obtain a standard curve, which was then used to calculate the absolute copy numbers of the relevant genes. Comparison with the internal reference gave the relative copy numbers [28].

RESULTS

Analysis of the six B. mori ORC subunit genes, their mRNAs and predicted protein sequences

EST sequences specifying the six B. mori ORC subunits were identified in the EST database of B. mori using BLAST searches with the six known D. melanogaster ORC protein subunit sequences. Sequence information missing from the EST sequences was obtained experimentally using 3′- or 5′-RACE. Typical polyadenylation (AAUAAA) signals were missing from all cDNAs except for BmORC4. Comparison of the cDNA sequences with the B. mori genome database revealed the intron structure of each gene (GenBank® accession nos. GQ214390–GQ214395; Table 1 and Figure 1). The assembled cDNAs predicted the protein sequences of the six B. mori ORC proteins, each of which contained an identifying homologous conserved domain (Figure 2) and no indication of a signal peptide.

Genomic structures of BmORC1–6 of B. mori

Figure 1
Genomic structures of BmORC1–6 of B. mori

Dark bands represent exons. BmORC5 consists of a single 1511 bp uninterrupted exon.

Figure 1
Genomic structures of BmORC1–6 of B. mori

Dark bands represent exons. BmORC5 consists of a single 1511 bp uninterrupted exon.

The predicted functional domains in BmOrc proteins

Figure 2
The predicted functional domains in BmOrc proteins

The locations of the predicted functional domains in the six BmOrc proteins are indicated as dark bars. The AAA+ domains were predicted in BmOrc1p and BmOrc4p. Conserved domain in Orc2p, Orc3p and Orc6p were named ORC2, ORC3 and ORC6 superfamily sequence motifs respectively. A partial CDC6 domain was identified at the N-terminus of BmOrc5p.

Figure 2
The predicted functional domains in BmOrc proteins

The locations of the predicted functional domains in the six BmOrc proteins are indicated as dark bars. The AAA+ domains were predicted in BmOrc1p and BmOrc4p. Conserved domain in Orc2p, Orc3p and Orc6p were named ORC2, ORC3 and ORC6 superfamily sequence motifs respectively. A partial CDC6 domain was identified at the N-terminus of BmOrc5p.

Phylogeny of the BmOrc1–6 protein subunits

The neighbour-joining tree in Figure 3 showed that each of the six BmOrc proteins was most similar to its DmOrc counterpart from Drosophila. They were also more similar to their mammalian counterparts (HsOrc and MmOrc) than to each other. ScOrc1p–6p were, as expected, most different from their animal counterparts, but they still grouped with the expected clades.

Evolutionary relationships of ORCs

Figure 3
Evolutionary relationships of ORCs

The evolutionary history was inferred using the neighbour-joining method. The bootstrap consensus tree inferred from 500 replicates was taken to represent the evolutionary history of the ORCs. The Orc1p–6p sequences were obtained from the NCBI protein database for H. sapiens (Hs), D. melanogaster (Dm), S. cerevisiae (Sc) and M. musculus (Mm). The Bombyx mori BmOrc1p–6p (GenBank® accession no. GQ214390–GQ214395) were cloned by our laboratory. ScOrc1p–6p (GenBank® accession nos. P54784, CAA79883, P54790, P54791, P50874 and P38826), DmOrc1p–6p (GenBank® accession nos. NP_477303, NP_731873, AAD39472, AAD39473, NP_477132 and NP_477319), MmOrc1p–6p (GenBank® accession nos. Q9Z1N2, Q60862, Q9JK30, O88708, Q9WUV0 and Q9WUJ8), HsOrc1p–6p (GenBank® accession nos. NP_004144, NP_006181, NP_862820, NP_002543, NP_002544 and NP_055136).

Figure 3
Evolutionary relationships of ORCs

The evolutionary history was inferred using the neighbour-joining method. The bootstrap consensus tree inferred from 500 replicates was taken to represent the evolutionary history of the ORCs. The Orc1p–6p sequences were obtained from the NCBI protein database for H. sapiens (Hs), D. melanogaster (Dm), S. cerevisiae (Sc) and M. musculus (Mm). The Bombyx mori BmOrc1p–6p (GenBank® accession no. GQ214390–GQ214395) were cloned by our laboratory. ScOrc1p–6p (GenBank® accession nos. P54784, CAA79883, P54790, P54791, P50874 and P38826), DmOrc1p–6p (GenBank® accession nos. NP_477303, NP_731873, AAD39472, AAD39473, NP_477132 and NP_477319), MmOrc1p–6p (GenBank® accession nos. Q9Z1N2, Q60862, Q9JK30, O88708, Q9WUV0 and Q9WUJ8), HsOrc1p–6p (GenBank® accession nos. NP_004144, NP_006181, NP_862820, NP_002543, NP_002544 and NP_055136).

The N-terminal sequences of Orc1p and Orc2p were generally less conserved than the C-terminal ones. All known Orc2p–5p homologues were similar to their counterparts in other species, and Orc3p–5p had high similarity along the entire sequences (Supplementary Figures S2–S5 http://www.bioscirep.org/bsr/031/bsr0310353add.htm). The N- and C-termini of BmOrc6p and CfOrc6p were similar but CfOrc6p had an approx. 80 amino acid insert near the C-terminus that was only found in lepidoptera (Supplementary Figure S6 at http://www.bioscirep.org/bsr/031/bsr0310353add.htm).

Expression of BmORC during embryo development

Hibernating eggs that were at the critical development stage II were incubated by the simplified method [26] and BmORC mRNA levels were measured for 11 days. Generally, the mRNA expression levels decreased until the sixth day, and then increased again. The copy number of BmORC2 peaked strongly on the second day. An appreciable increase of the amounts of BmORC6 arose at the second and seventh days (Figure 4).

Quantitative real-time PCR measurements of the expression of BmORC mRNA during embryo development in the eggs

Figure 4
Quantitative real-time PCR measurements of the expression of BmORC mRNA during embryo development in the eggs

The hibernating eggs were synchronized to the critical development stage II and stored at 5°C. At day zero, the eggs were warmed according to the simplified incubation method [26]. The larvae hatched on day 11. B. mori cytoplasmic actin (A3) was used as an internal reference. The inset shows the poorly expressed samples enlarged.

Figure 4
Quantitative real-time PCR measurements of the expression of BmORC mRNA during embryo development in the eggs

The hibernating eggs were synchronized to the critical development stage II and stored at 5°C. At day zero, the eggs were warmed according to the simplified incubation method [26]. The larvae hatched on day 11. B. mori cytoplasmic actin (A3) was used as an internal reference. The inset shows the poorly expressed samples enlarged.

The tissue expression pattern of BmORC in fifth instar day-6 larvae

In the posterior silk gland, ovary, Malpighian tube, fat body and testis, the BmORC mRNA copy number was higher than in most other tissues. Values were lowest in the haemocyte and midgut. The expression of BmORC1 was very low in most tissues. The lowest level was observed in the midgut, and a slightly elevated level in the fat body. BmORC2 and BmORC3 were most highly expressed in the posterior silk gland and least in the haemocyte. BmORC4 expression was highest in the Malpighian tube least in the haemocyte. BmORC5 expression was highest in the posterior silk gland and lowest in wing disc. BmORC6 expression was highest in the ovary and lowest in the epidermis (Figure 5).

Copy number change post BmNPV infection

Figure 5
Copy number change post BmNPV infection

The fifth instar day-1 larvae were injected with BV of BmNPV. The sample taken 1 h after BmNPV infection was labelled as 0 hpi, and the later time points were the corresponding time intervals. The relative change is the copy number of BmORC in BmNPV-infected larvae divided by the copy number of the same mRNA in non-infected larvae of the same stage.

Figure 5
Copy number change post BmNPV infection

The fifth instar day-1 larvae were injected with BV of BmNPV. The sample taken 1 h after BmNPV infection was labelled as 0 hpi, and the later time points were the corresponding time intervals. The relative change is the copy number of BmORC in BmNPV-infected larvae divided by the copy number of the same mRNA in non-infected larvae of the same stage.

The change of BmORCs with BmNPV infection

At 0 hpi, the relative copy numbers of all BmORCs were at a high level, followed by a rapid decrease reaching their lowest levels at 2–4 hpi. After this, the relative values increased to a moderate level until 36 hpi at which time the relative copy number declined again (Figure 5).

DISCUSSION

The structures of BmORC

Like ScORCs [27], BmORC2, 3, 4 and the 6 subunit genes are on different chromosomes (BmORC1 and BmORC5 have not yet been mapped to a chromosome). A total of 35 introns were identified in the six BmORCs and only 16 in DmORCs.

In the Silkworm Genome Database [http://silkworm.genomics.org.cn/silkdb/), only two sequences (BGIBMGA012496-TA and BGIBMGA009708-TA) were labelled as subunits of BmORC. Alignment using the protein database of NCBI showed an ORC2 superfamily conserved domain in BGIBMGA012496-TA and a TIGR02928 conserved domain (belonging to ORC/CDC6 superfamily) in BGIBMGA009708-TA. Comparison with our sequences showed that BGIBMGA012496-TA and BGIBMGA009708-TA corresponded to BmORC2 and BmORC4 respectively, but with different predicted start codons and some differences in the predicted introns.

Judging from the similarity of the protein structures, genomic sequences and the relevant ESTs, we concluded that BmORC1–6 were complete. The structures of the BmOrc proteins (Figure 2) and the alignment of ORC/Cdc6 domains of BmOrc1p–5p (BmOrc6p does not contain this domain) (Figure 6) revealed that the amino acid sequences were more varied than expected, even though they all contained an AAA+ domain [21]. There was a unique, highly similar fragment at the C-terminals of CfOrc6p and BmOrc6p that may be specific for lepidopterans (Supplementary Figure S6).

Alignment of conserved domains

Figure 6
Alignment of conserved domains

The domains of BmOrc1p–5p (indicated in Figure 2) were aligned. Similar amino acids were shaded using the same grey shading. The dashes (−) represent no amino acid.

Figure 6
Alignment of conserved domains

The domains of BmOrc1p–5p (indicated in Figure 2) were aligned. Similar amino acids were shaded using the same grey shading. The dashes (−) represent no amino acid.

The phylogenetic analysis (Figure 3) of the subunits of ScORC, DmORC, BmORC, MmORC and HsORC showed that BmOrc proteins grouped with the same clade as their counterparts. This was unlike the members of the Bm-YELLOW protein family [28] and the Bm-ASH protein family, [29] whose duplication must have occurred at the time or after the evolutionary separation of the insects from other animals. Consequently, the six BmORC subunits must have originated before the differentiation of eukaryotes and evolved individually ever since.

BmORC expression patterns during embryo development and in different tissues of fifth instar day-6 larvae

Embryos at the critical development stage II [26] were incubated and started cell division and organogenesis immediately, continuing to the sixth day when the main organs had formed and the embryo entered into the period of embryonic reversal. The expression level of BmORCs was high on the first day, and then decreased day by day until the sixth day when the expression levels were lowest (Figure 4). On the seventh day the reversal was completed, and the embryo became longer and formed the dorsal-side integument and digestive tube. Concurrently, the copy numbers of the BmORCs increased slightly. On day 8, the seta appeared, the ocellus and mandible became pigmented and the taenidia emerged in the trachea. With the stage of organogenesis finished and the embryo breathing using trachea, DNA replication became stabilized and consequently the copy number of BmORCs remained stable.

Two days before larvae hatched, the development of the embryo entered into the stage of embryogenesis perfection during which the head and other body parts became pigmented one after another. Until day 10 the expression of BmORCs stayed at a low level. Subsequently, the newly hatched larvae appeared and the BmORCs increased again, possibly as a result of the preparation for feeding and rapid growth of the newly hatched larvae. Among all subunits, the change of BmORC5 expression was the sharpest. Perhaps BmOrc5p played additional roles, and this will require further investigation.

The expression levels of BmORCs (Figure 7) varied from tissue to tissue according to the stage of development: as larvae grew to the fifth instar stage on day 6, the silk gland had nearly matured and large quantities of silk fibroin were produced. The fat body thickened rapidly for storing energy, and the organs of the reproductive system started to prepare for germ cell genesis. Consequently, the DNA replication in these tissues was vigorous, so that the copy number of BmORCs was higher than that in the haemocyte and midgut, which started to degenerate.

Expression of BmORC in different tissues of the fifth instar day-6 larvae

Figure 7
Expression of BmORC in different tissues of the fifth instar day-6 larvae

The tissues were collected from fifth instar day-6 larvae. The relative copy numbers were calculated as in Figure 5. H: head; Ep: epidermis; Asg: anterior silk gland; Msg: middle silk gland; Psg: posterior silk gland; Wd: wing disc; Te: testis; He: haemocyte; Ov: ovary; Mt: malpighian tube; Tb: tracheal bush; Mg: midgut; Fb: fat body.

Figure 7
Expression of BmORC in different tissues of the fifth instar day-6 larvae

The tissues were collected from fifth instar day-6 larvae. The relative copy numbers were calculated as in Figure 5. H: head; Ep: epidermis; Asg: anterior silk gland; Msg: middle silk gland; Psg: posterior silk gland; Wd: wing disc; Te: testis; He: haemocyte; Ov: ovary; Mt: malpighian tube; Tb: tracheal bush; Mg: midgut; Fb: fat body.

The different expression levels of different subunits of BmORCs might reflect their additional non-replication functions. Remarkably, the high-expression level and pronounced change in the intron-free BmORC5 during both embryo development and in different tissues might indicate that this subunit might play important additional roles. The relative copy numbers of BmORC2–5 were increased in head tissue, suggesting that BmOrc2–5 proteins are involved in the brain development in the silkworm.

The relevance of BmORC and BmNPV DNA replication

Baculovirus is the only virus with biphasic replication [30]. The period 0–4 hpi of baculovirus is called the eclipse phase when the copy number of virions decreased rapidly, and the relative copy numbers of BmORCs followed the same trend. From 8 to 12 hpi was the replication fastigium of BV virions, at which time the relative copy numbers of BmORCs rose again. At 18 hpi, the replication of BV was almost completed and the relative copy number of BmORCs declined slightly. The relative copy number of BmORCs reached a second peak at 24–36 hpi, when there was the period of occluded virus replication. After 48 hpi, the replication of virions was nearly completed, and the copy number of BmORCs decreased until 96 hpi (Figure 5). This suggested that the viral replication of baculovirus affected the expression of BmORCs, but the relevance and mechanism in detail remains to be discovered.

Abbreviations

     
  • BAH

    bromo-adjacent homology

  •  
  • BmNPV

    Bombyx mori nucleopolyhedrovirus

  •  
  • BV

    budded virus

  •  
  • ORC

    origin recognition complex

  •  
  • DmORC

    D. melanogaster ORC

  •  
  • EST

    expressed sequence tag

  •  
  • hpi

    h post-infection

  •  
  • MmORC

    Mus musculus ORC

  •  
  • NUP

    nested universal primer

  •  
  • RACE

    rapid amplification of cDNA ends

  •  
  • ScORC

    Saccharomyces cerevisiae ORC

AUTHOR CONTRIBUTION

Hui-Peng Yang, Su-Juan Luo and Yi-Nü Li carried out the silkworm rearing, tissue dissection, RNA extraction, cDNA synthesis and real-time PCR. Hui-Peng Yang carried out the data analysis and the drafting of the manuscript. Yao-Zhou Zhang and Zhi-Fang Zhang conceived the study and participated in its design. Zhi-Fang Zhang helped to draft the manuscript. All authors read and approved the final manuscript.

We thank Dr T. Kieser and Dr Q. Chen for helpful discussions and critical reading of the manuscript prior to submission.

FUNDING

This work was supported by the National Natural Sciences Foundation of China [grant number 30770279] and the National High Technology Research and Development Program of China (‘863’) [grant number 2006AA10A119].

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Supplementary data