Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori.

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.

Genomic sequence of a 320-kb segment of the Z chromosome of Bombyx mori containing a kettin ortholog.

The sex chromosome constitution of the silkworm, Bombyx mori, is ZW in the female and ZZ in the male. Very little molecular information is available about the Z chromosome in Lepidoptera, although the topic is interesting because of the absence of gene dosage compensation in this chromosome. We constructed a 320-kb BAC contig around the Bmkettin gene on the Z chromosome in Bombyx and determined its nucleotide sequence by the shotgun method. We found 13 novel protein-coding sequences in addition to Bmkettin. All the transposable elements detected in the region were truncated, and no LTR retrotransposons were found, in stark contrast to the situation on the W chromosome. In this 320-kb region, four genes for muscle proteins (Bmkettin, Bmtitin1, Bmtitin2, and Bmprojectin) are clustered, together with another gene (Bmmiple) on the Z chromosome in B. mori; their orthologs are also closely linked on chromosome 3 in Drosophila, suggesting a partial synteny. Real-time RT-PCR experiments demonstrated that transcripts of 13 genes of the 14 Z-linked genes found accumulated in larger amounts in males than in female moths, indicating the absence of gene dosage compensation. The implications of these findings for the evolution and function of the Z chromosome in Lepidoptera are discussed.

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori.

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3' splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.

An element downstream of the transcription start site is required for activation of Bombyx mori nucleopolyhedrovirus bro-c promoter.

Bombyx mori nucleopolyhedrovirus (BmNPV) contains five baculovirus repeated ORF (bro) genes. We have previously reported that all of these genes (bro-a, b, c, d and e) are transcribed as early genes and require viral factor(s) for their expression. In this study, we investigated the mechanism of promoter activation of the bro-c gene. Transient expression assays using genomic libraries of BmNPV indicated that the baculoviral trans-regulator IE-1 is responsible for activating the bro-c promoter. To identify essential site(s) for promoter activation, mutations were introduced to the promoter region of bro-c. Interestingly, it was shown that the pentanucleotide sequence CACGC located 30 nucleotides downstream of the RNA start site was essential for bro-c promoter activation. In addition, the RNA start site and the spacing between the RNA start site and CACGC were also required for promoter activation. By introducing a CACGC sequence into the corresponding region of the bro-b promoter, which is not normally trans-activated by IE-1, we demonstrated that this pentanucleotide motif has the ability to confer trans-activation by IE-1 on a promoter. Gel retardation experiments also showed a sequence-specific DNA binding protein induced by baculovirus infection interacts with the CACGC motif.

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.

Diapause-associated transcription of BmEts, a gene encoding an ETS transcription factor homolog in Bombyx mori.

To understand the molecular mechanism of diapause determination in early embryogenesis of the silkworm, Bombyx mori, mRNA from diapause and non-diapause eggs was compared using the differential display technique. A 1.2 kbp differentially amplified cDNA fragment was cloned and sequenced. Northern blot analysis confirmed that the mRNA corresponding to this clone, D1, was specifically induced in diapause eggs from 20 h after oviposition, and decreased gradually but was clearly detectable until 40 days after oviposition. On the other hand, if diapause eggs were activated by HCl treatment 20 h after oviposition, the mRNA corresponding to D1 vanished 72 h after HCl treatment. In pnd (pigmented and non-diapausing egg) homozygous embryos, which never enter into diapause, the RNA was not transcribed at any stage, whereas, in pnd-2 homozygous embryos which also have no diapause, similar results were obtained to those for HCl treated eggs. The deduced amino acid sequence of D1 was most highly related to the identified Drosophila and vertebrate ETS proteins, within the approximately 85 amino acid ETS domain. ETS proteins play an important role in transcription activation during a variety of biological processes and can be grouped into sub-families, based on sequence similarity in the ETS domain which has been shown to be a DNA-binding domain. Therefore, we have called the gene corresponding to D1 BmEts. These observations suggest that BmEts encodes a novel ETS family member which is strongly associated with the embryonic diapause. Moreover, BmEts probably acts downstream of the pnd gene in the regulatory hierarchy of diapause determination, alternatively BmEts itself might be the pnd gene.

Bm kettin, homologue of the Drosophila kettin gene, is located on the Z chromosome in Bombyx mori and is not dosage compensated.

In Bombyx mori, the female is the heterogametic sex and the sex determining system is referred to as ZZ/ZW. In a previous study, we found that this insect does not show dosage compensation at the transcriptional level. To confirm the validity of our conclusion, we investigated whether or not another sex-linked gene is dosage compensated. To identify new Z-linked genes, total RNA from reciprocal hybrid females between the silkworm strains p50 and C108 was compared using the differential display technique. Nine cDNA fragments corresponding to several differentially expressed mRNAs were cloned and sequenced. The analysis of nucleotide sequence polymorphisms confirmed that one of these cDNAs, ZDD4, originated from the Z chromosome. The amino acid sequence deduced from ZDD4 has homology with kettin, a modular protein in the Z-disc of Drosophila melanogaster muscles. On immunoblots of Bombyx larval muscle proteins a polypeptide of 380 kDa was labelled with antibody to the ZDD4 peptide. We considered that the gene corresponding to ZDD4 encodes a kettin homologue in the silkworm, and denote it as Bm kettin. By a three-point cross, Bm kettin was mapped at 40.0 CM on the Z chromosome. Southern blot analysis revealed that Bm kettin was present at one copy in the genome. Northern blot analysis showed that Bm Kettin mRNA was 9.1kb in length, and that the level of the mRNA in males was two times greater than that of females. Taken together with our previous observations, the present data suggest that lack of dosage compensation is a general rule in B. mori. Moreover, the twofold difference in Bm kettin expression between males and females may help explain the sexual difference in the wing flapping activity observed in some groups of Lepidoptera.

Absence of dosage compensation at the transcription level of a sex-linked gene in a female heterogametic insect, Bombyx mori.

To confirm whether sex-linked genes of the silkworm are dosage compensated or not, we investigated three RAPDs and one RFLP marker which have been mapped to the Z chromosome as candidate genes to test for dosage compensation. Northern blot analysis demonstrated that one of the RAPDs, T15.180a, is transcribed into several mRNAs in the embryo, midgut, Malpighian tubule, testis and ovary. Southern blot analysis revealed that T15.180a is present at one copy in the genome. Sequence data showed that T15.180a contained two open-reading frames (ORFs) separated by an intron of 605 bp. These ORFs encoded a possible polypeptide of 445 amino acids containing the proline cluster. These results suggested that T15.180a is part of a functional gene on the Z chromosome of the silkworm. We have called this gene T15, and compared its transcriptional level between males and females. Northern blot analysis showed that the level of T15 mRNA in males was twice that of females. For a more precise analysis, the levels of transcription from both alleles of T15 (T15P, T15C) were measured by the single-nucleotide primer extension (SNuPE) assay. The results indicated that the amount of transcription from each T15 allele in both sexes (male, T15P/T15C; female, T15P/W) was equivalent. All the results suggest that sex-linked genes in the silkworm are not dosage compensated.