Toshikazu Oshiki

Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori

The sex chromosomes of the silkworm, Bombyxmori, are designated ZW(XY) for females and ZZ(XX) for males. The W chromosome of B. mori does not recombine with the Z chromosome and autosomes and no genes for morphological characters have been mapped to the W chromosome as yet. Furthermore, femaleness is determined by the presence of a single W chromosome, regardless of the number of autosomes or Z chromosomes. To understand these interesting features of the W chromosome, it is necessary to analyze the W chromosome at the molecular biology level. Initially to isolate DNA sequences specific for the W chromosome as randomly amplified polymorphic DNA (RAPD) markers, we compared the genomic DNAs between males and females by PCR with arbitrary 10-mer primers. To the present, we have identified 12 W-specific RAPD markers, and with the exception of one RAPD marker, all of the deduced amino acid sequences of these W-specific RAPD markers show similarity to previously reported amino acid sequences of retrotransposable elements from various organisms. After constructing a genomic DNA lambda phage library of B. mori we obtained two lambda phage clones, one containing the W-Kabuki RAPD sequence and one containing the W-Samurai RAPD sequence and found that these DNA sequences comprised nested structures of many retrotransposable elements. To further analyze the W chromosome, we obtained 14 W-specific bacterial artificial chromosome (BAC) clones from three BAC libraries and subjected these clones to shotgun sequencing. The resulting assembly of sequences did not produce a single contiguous sequence due to the presence of many retrotransposable elements. Therefore, we coupled PCR with shotgun sequencing. Through these analyses, we found that many long terminal repeat (LTR) and non-LTR retrotransposons, retroposons, DNA transposons and their derivatives, have accumulated on the W chromosome as strata. These results strongly indicate that retrotransposable elements are the main structural component of the W chromosome.

W-derived BAC probes as a new tool for identification of the W chromosome and its aberrations in Bombyx mori

We isolated four W chromosome-derived bacterial artificial chromosome (W-BAC) clones from Bombyx mori BAC libraries by the polymerase chain reaction and used them as probes for fluorescence in situ hybridization (FISH) on chromosome preparations from B. mori females. All four W-BAC probes surprisingly highlighted the whole wild-type W sex chromosome and also identified the entire original W-chromosomal region in W chromosome-autosome translocation mutants. This is the first successful identification of a single chromosome by means of BAC-FISH in species with holokinetic chromosomes. Genomic in situ hybridization (GISH) by using female-derived genomic probes highlighted the W chromosome in a similar chromosome-painting manner. Besides the W, hybridization signals of W-BAC probes also occurred in telomeric and/or subtelomeric regions of the autosomes. These signals coincided well with those of female genomic probes except one additional GISH signal that was observed in a large heterochromatin block of one autosome pair. Our results support the opinion that the B. mori W chromosome accumulated transposable elements and other repetitive sequences that also occur, but scattered, elsewhere in the respective genome.

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.

Identification of the female-determining region of the W chromosome in Bombyx mori

The W chromosome of the silkworm Bombyxmori is devoid of functional genes, except for the putative female-determining gene (Fem). To localize Fem, we investigated the presence of W-specific DNA markers on strains in which an autosomal fragment containing dominant marker genes was attached to the W chromosome. We produced new W-chromosomal fragments from the existing Zebra-W strain (T(W;3)Ze chromosome) by X-irradiation, and then carried out deletion mapping of these and sex-limited yellow cocoon strains (T(W;2)Y-Chu, -Abe and -Ban types) from different Japanese stock centers. Of 12 RAPD markers identified in the normal W chromosomes of most silkworm strains in Japan, the newly irradiated W(B-YL-YS)Ze chromosome contained three, the T(W;2)Y-Chu chromosome contained six, and the T(W;2)Y-Abe and -Ban chromosomes contained only one (W-Rikishi). To investigate the ability of the reduced W-chromosome translocation fragments to form heterochromatin bodies, which are found in nuclei of normal adult female sucking stomachs, we examined cells of the normal type p50 strain and the T(W;2)Y-Chu and -Abe strains. A single sex heterochromatin body was found in nuclei of p50 females, whereas we detected only small sex heterochromatin bodies in the T(W;2)Y-Chu strain and no sex heterochromatin body in the T(W;2)Y-Abe strain. Since adult females of all strains were normal and fertile, we conclude that only extremely limited region, containing the W-Rikishi RAPD sequence of the W chromosome, is required to determine femaleness. Based on a comparison of the normal W-chromosome and 7 translocation and W-deletion strains we present a map of Fem relative to the 12 W-specific RAPD markers.

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.

Effect of the brain on the suboesophageal ganglion and determination of voltinism in Bombyx mori

Abstract It is known that voltinism in the silkworm is affected by inhibitory and promoting functions of the brain through the nerve commissures on the suboesophageal ganglion or the corpora allata. It was demonstrated that the brain has only an accelerative function through the nerve commissures on the secretion of the suboesophageal ganglion and has no inhibitory function. Voltinism in the silkworm is determined by the amount of the suboesophageal ganglion hormone. When silkworms have a large quantity of this hormone, they lay diapause egg batches, and when they have a small quantity, they lay non-diapause egg batches. The quantity of its hormone is modified by two factors; one is the brain-suboesophageal ganglion system (diapause direction) and the other is the brain-corpora allata system (non-diapause direction).

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.

Impaired yolk protein uptake by oocytes of a Bombyx mori mutant

Abstract A spontaneous mutant of silkworms which produces small eggs is designated sm . To clarify the function of the sm gene, we examined the biosynthesis and distribution of three major yolk proteins, vitellogenin, 30K protein and egg specific protein. The concentration of protein in hemolymph in sm female pupae was higher than normal in the last half of the pupal stage. The 30K protein concentration in the hemolymph of the sm strain was above normal throughout the pupal stage. Vitellogenin in hemolymph was similarly elevated above normal, especially in the last half of the normal stage. However, sm ovaries, beginning with the fourth day of the pupal stage had lower than normal concentrations of these two proteins together with egg specific protein. These results suggest that while sm fat bodies produced and secreted vitellogenin and 30K protein, the sm ovaries had an impaired uptake of proteins. This abnormality might be caused by gaps between follicle cells, impaired protein receptors, or some other abnormality of follicle cells. In the sm mutant, the egg specific protein gene which is expressed only in follicle cells, was transcribed and translated normally in the follicle cells, but did not attain normal concentrations in the oocytes. The in situ hybridization experiments showed that mRNA of egg specific protein was dispersed throughout sm follicle cells, while in normal cells the signals were located on the side of the follicle cells facing the oocyte. The oocytes of the sm strain have impaired uptake of proteins from both hemolymph and follicle cells.