Fumi Ohbayashi

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.

Molecular structure of a novel gypsy-Ty3-like retrotransposon (Kabuki) and nested retrotransposable elements on the W chromosome of the silkworm Bombyx mori

Abstract We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD), designated W-Kabuki, derived from the W chromosome of the silkworm, Bombyx mori. To further analyze the W chromosome of B. mori, we obtained a lambda phage clone which contains the W-Kabuki RAPD sequence and sequenced the 18.1-kb DNA insert. We found that this DNA comprises a nested structure of at least seven elements; three retrotransposons, two retroposons, one functionally unknown insertion, and one Bombyx repetitive sequence. The non-LTR retrotransposon BMC1, the retroposon Bm1, a functionally unknown inserted DNA (FUI), and a copia-like LTR retrotransposon (Yokozuna) are themselves inserted into a novel gypsy-Ty3-like LTR retrotransposon, named Kabuki. Furthermore, this Kabuki element is itself inserted into another copy of Bm1. The BMC1 and Yokozuna elements inserted in the Kabuki sequence are intact. Moreover, the Kabuki element is largely intact. These results suggest that many retrotransposable elements have accumulated on the W chromosome, and these elements are expected to evolve more slowly than those on other chromosomes.

Two novel Pao-like retrotransposons (Kamikaze and Yamato) from the silkworm species Bombyx mori and B. mandarina: common structural features of Pao-like elements.

Abstract. To characterize the structural features common to Pao-like retrotransposons, we analyzed two lambda phage clones which contain the Pao-like elements from the silkworm species Bombyx mori and B. mandarina, and copies of Pao itself and ninja of Drosophila simulans, amplified by PCR. We previously identified two randomly amplified polymorphic DNAs (RAPDs), W-Kamikaze and W-Yamato, from B. mori and B. mandarina, which are part of two novel Pao-like retrotransposons, Kamikaze and Yamato, respectively. Complete characterization of these and other elements of this group reported here shows that Pao-like elements have common features that distinguish them from the other groups of LTR-retrotransposons. While the elements of the Ty1-copia group encode only one cysteine and histidine (Cys) motif in their gag-like region, the Pao-like elements specify three Cys motifs. The highly conserved D(35)E motif in the integrase domain of the retrotransposon polyprotein seems to be conserved in Pao-like elements, but the number of amino acid residues between D and E varies and is greater than 35. A comparison of the deduced amino acid sequences of the reverse transcriptase domain revealed that the Pao-like elements are members of neither the Ty1-copia nor the gypsy-Ty3 groups. Therefore, we confirmed that the long-terminal-repeat (LTR) retrotransposons should be divided into three major groups (or families), namely the Ty1-copia, gypsy-Ty3, and Pao-like groups.

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.

Nested retrotransposons on the W chromosome of the wild silkworm Bombyx mandarina.

The W chromosome of the silkworms Bombyx mori or B. mandarina is recombinationally isolated from the Z chromosome and the autosomes. We previously characterized a female‐specific randomly amplified polymorphic DNA (RAPD), designated W‐Yamato, derived from the W chromosome of the wild silkworm Bombyx mandarina. To further analyse the W chromosome of B. mandarina, we obtained a lambda phage clone that contains the W‐Yamato RAPD sequence and sequenced the 16.7 kb DNA insert. We found that this DNA comprises a nested structure of at least seven elements: six retrotransposons and one transposable element‐like sequence. The transposable element‐like sequence is inserted into a micropia‐like retrotransposon (Karate). The Karate and the non‐long terminal repeat (non‐LTR) retrotransposon BMC1 are inserted into a 412‐like retrotransposon (Judo). Furthermore, this Judo, and two non‐LTR retrotransposons (Kurosawa and Kendo) are inserted into a Pao‐like retrotransposon (Yamato). These results indicate that the retrotransposons inserted into the W chromosome are not efficiently removed but accumulate gradually as strata without recombination.

The female-killing chromosome of the silkworm, Bombyx mori, was generated by translocation between the Z and W chromosomes

Bombyx mori is a female-heterogametic organism (female, ZW; male, ZZ) that appears to have a putative feminizing gene (Fem) on the W chromosome. The paternally transmitted mutant W chromosome, Df(pSa + pW + od)Fem, derived from the translocation-carrying W chromosome (pSa + pW + od), is inert as femaleness determinant. Moreover, this Df(pSa + pW + od)Fem chromosome has been thought to have a female-killing factor because no female larvae having the Df(pSa + pW + od)Fem chromosome are produced. Initially, to investigate whether the Df(pSa + pW + od)Fem chromosome contains any region of the W chromosome or not, we analyzed the presence or absence of 12 W-specific RAPD markers. The Df(pSa + pW + od)Fem chromosome contained 3 of 12 W-specific RAPD markers. These results strongly indicate that the Df(pSa + pW + od)Fem chromosome contains the region of the W chromosome. Moreover, by using phenotypic and molecular markers, we confirmed that the Df(pSa + pW + od)Fem chromosome is connected with a partially deleted Z chromosome and that this fused chromosome behaves as a Z chromosome during male meiosis. Furthermore, we demonstrated that the ZZW-type triploid female having the Df(pSa + pW + od)Fem chromosome is viable. Therefore, we concluded that the Df(pSa + pW + od)Fem chromosome does not have a female-killing factor but that partial deletion of the Z chromosome causes the death of the ZW-type diploid female having the Df(pSa + pW + od)Fem chromosome. Additionally, our results of detailed genetic analyses strongly indicate that the female-killing chromosome composed of the Df(pSa + pW + od)Fem chromosome and deleted Z chromosome was generated by translocation between the Z chromosome and the translocation-carrying W chromosome, pSa + pW + od.