Summary of Research Project Results under JSPS FY2003
"Research for the Future Program"

1.Research Institution The University of Tokyo
2.Research Area Life Sciences
3.Research Field Mechanisms of the Advanced Function in Insects and Proposal of New Insectech
4.Term of Project FY 1999 - FY 2003
5.Project Number 99L01201
6.Title of Project Genetic Network for the Sex Determination in Insects

7.Project Leader
Name Institution,Department Title of Position
Toru,Shimada The University of Tokyo; Graduate School of Agricultural and Life Science Associate Professor

8.Core Members
Names Institution,Department Title of Position

9.Summary of Research Results
  1. Genome analyses of the silkworm, Bombyx mori, for analysis of sex determination:

  2. We performed genome analysis of Bombyx mori, especially the EST analysis, microarray analysis, and BAC-based of the sex chromosomes as a collaboration with other projects for the silkworm genome.
  3. Structural, expressional and functional analyses of Bmdsx, Bombyx mori ortholog of Drosophila doublesex:

  4. Bmdsx, an ortholog of doublesex, the most downstream gene in the sex determination cascade of Drosophila, was found in the EST database of Bombyx. We revealed the gene structure of Bmdsx, and that it is transcribed into sex-specific mRNA isoforms. The mechanism of the sex-specific splicing was different from that in dsx in Drosophila. It depends on male-specific repression of splicing. To understand the function of Bmdsx, we introduced female- and male-type genes into the germ line of Bombyx. Ectopic expression of female Bmdsx mRNA induced vitellogenin mRNA in males. We demonstrated that BmDSX proteins bound to the promoter of the vitellogenin gene by gel retardation assay. We also established a transgenic line that expresses male-type Bmdsx mRNA produced male-like abnormal structures in genitalia, pheromone gland, and internal sexual organs.
  5. Other genes homologous to Drosophila sex-determining genes.

  6. Bmfru, a homolog of Drosophila fruitless gene was found in the Bombyx EST database. The analysis of its genomic and mRNA structures revealed many isoforms encoding different zinc-finger motifs. We also found the orthologs of Drosophila sex determining genes, transformer-2, sans fille, intersex, ovo, ovarian tumor, etc.
  7. Structure and function of the W chromosome.

  8. Twelve female-specific RAPD markers that enable us to screen BACs, were developed. The nucleotide sequences of W-chromorome-derived BACs showed complicated structure of nested retrotransposons and transposons, and extremely low abundance of genes; this situation was specific to the W chromosome. The W chromosome of a sex-limited strain contained only one marker “Rikishi” out of the 12 ones, indicating this region can solely determine femaleness. Genomic subtraction experiment between the female and male obtained only one DNA fragment, which was close to Rikishi on the W chromosome. The sequence of the BAC contig around Rikishi contained several functional genes.
  9. Differential screening of female-specific mRNAs in embryos.

  10. We compared mRNA in early embryos between the female and male in the sex-limited black egg strain, and found 6 female-specific mRNAs.
  11. Structure and function of the Z chromosome.

  12. We analyzed 4 BAC contigs for the Z chromosome, and found that the Z chromosome mainly contains differentiated cell-specific genes, and their expression does not show gene dosage compensation. There was only a weak synteny between the Bombyx Z chromosome and Drosophila chromosomes.

10.Key Words
(1)sex determination (2)sex chromosome (3)doublesex gene
(4)sex-specific splicing (5)transgenic silkworm (6)morphogenesis of sexual organs
(7)sex-limited silkworm strains (8)vitellogenin (9)gene dosage compensation