Masataka G. Suzuki

The construction of an EST database for Bombyx mori and its application

To build a foundation for the complete genome analysis of Bombyx mori, we have constructed an EST database. Because gene expression patterns deeply depend on tissues as well as developmental stages, we analyzed many cDNA libraries prepared from various tissues and different developmental stages to cover the entire set of Bombyx genes. So far, the Bombyx EST database contains 35,000 ESTs from 36 cDNA libraries, which are grouped into ≈11,000 nonredundant ESTs with the average length of 1.25 kb. The comparison with FlyBase suggests that the present EST database, SilkBase, covers >55% of all genes of Bombyx. The fraction of library-specific ESTs in each cDNA library indicates that we have not yet reached saturation, showing the validity of our strategy for constructing an EST database to cover all genes. To tackle the coming saturation problem, we have checked two methods, subtraction and normalization, to increase coverage and decrease the number of housekeeping genes, resulting in a 5–11% increase of library-specific ESTs. The identification of a number of genes and comprehensive cloning of gene families have already emerged from the SilkBase search. Direct links of SilkBase with FlyBase and WormBase provide ready identification of candidate Lepidoptera-specific genes.

A single female-specific piRNA is the primary determiner of sex in the silkworm

The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

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.

Role of the male BmDSX protein in the sexual differentiation of Bombyx mori

Summary The sex determination pathway is different between Drosophila melanogaster and Bombyx mori in the initial signal. Here we show evidence that the sex determination pathway in B. mori is similar to that of D. melanogaster at the level of the terminal regulator, doublesex (dsx), which is essential for the proper differentiation of the sexually dimorphic somatic features of D. melanogaster. In B. mori, a homolog of dsx (Bmdsx) is expressed in various tissues, and its primary transcript is alternatively spliced in males and females to yield sex‐specific mRNAs that encode male‐specific (BmDSXM) and female‐specific (BmDSXF) polypeptides. In the studies reported here, transgenic silkworms carrying a construct with a Bmdsx male cDNA placed under the control of either an hsp70 promoter or a Bombyx actin3 promoter were generated by piggyBac‐mediated germline transformation. Ectopic expression of the male cDNA in females resulted in abnormal differentiation of certain female‐specific genital organs and caused partial male differentiation in female genitalia. Transgenic analysis also revealed that the expression of BmDSXM in females caused repression of the female‐specifically expressed gene, the vitellogenin gene, and also resulted in activation of the pheromone‐binding protein gene that is dominantly expressed in males. These results provide evidence that the role of BmDSXM includes the activation of some aspects of male differentiation as well as the repression of female differentiation. Taken together with our previous data on the function of BmDSXF, we can conclude that Bmdsx is a double‐switch gene at the final step in the sex‐determination cascade of B. mori.

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.

Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin

ABSTRACT The herpes simplex virus (HSV)-based amplicon vector, a bacterial-viral-mammalian cell shuttle system, holds promise as a versatile gene delivery vehicle because of its large transgene capacity. However, amplicon-mediated transgene expression is often transient. We hypothesized that the presence of prokaryotic DNA sequences within the packaged vector genome can trigger transcriptional silencing of the entire vector sequence. To test this, we constructed a novel amplicon vector devoid of bacterial sequences (minicircle [MC] amplicon). Although the same dose of the minicircle amplicon vector in normal human fibroblasts resulted in an expression of luciferase approximately 20 times higher than that caused by the conventional amplicon vector, no significant difference was observed in copy numbers of luciferase DNA between MC amplicon- and control-transduced cells. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. Chromatin immunoprecipitation PCR analyses of several regions of vector genomes revealed that the bacterial sequences found in the conventional amplicon DNA were associated with an inactive form of chromatin immediately after infection. The presence of bacterial sequences also affected the remaining vector sequences in the conventional amplicon vector. Finally, nude mice injected with the MC amplicon exhibited higher and more sustained expression of luciferase than those injected with the conventional amplicon, confirming the usefulness of the MC amplicon devoid of bacterial sequences. Although additional improvements are absolutely required, these findings are a significant first step toward developing a novel HSV amplicon vector that can achieve enhanced long-term transgene expression.

Large Scale Full-Length cDNA Sequencing Reveals a Unique Genomic Landscape in a Lepidopteran Model Insect, Bombyx mori

The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

Establishment of a Novel In Vivo Sex-Specific Splicing Assay System To Identify a trans-Acting Factor That Negatively Regulates Splicing of Bombyx mori dsx Female Exons

ABSTRACT The Bombyx mori homolog of doublesex, Bmdsx, plays an essential role in silkworm sexual development. Exons 3 and 4 of Bmdsx pre-mRNA are specifically excluded in males. To explore how this occurs, we developed a novel in vivo sex-specific splicing assay system using sexually differentiated cultured cells. A series of mutation analyses using a Bmdsx minigene with this in vivo splicing assay system identified three distinct sequences (CE1, CE2, and CE3) positioned in exon 4 as exonic splicing silencers responsible for male-specific splicing. Gel shift analysis showed that CE1 binds to a nuclear protein from male cells but not that from female cells. Mutation of UAA repeats within CE1 inhibited the binding of the nuclear protein to the RNA and caused female-specific splicing in male cells. We have identified BmPSI, a Bombyx homolog of P-element somatic inhibitor (PSI), as the nuclear factor that specifically binds CE1. Down-regulation of endogenous BmPSI by RNA interference significantly increased female-specific splicing in male cells. This is the first report of a PSI homolog implicated in the regulated sex-specific splicing of dsx pre-mRNA.

The first murine zygotic transcription is promiscuous and uncoupled from splicing and 3′ processing

Initiation of zygotic transcription in mammals is poorly understood. In mice, zygotic transcription is first detected shortly after pronucleus formation in 1‐cell embryos, but the identity of the transcribed loci and mechanisms regulating their expression are not known. Using total RNA‐Seq, we have found that transcription in 1‐cell embryos is highly promiscuous, such that intergenic regions are extensively expressed and thousands of genes are transcribed at comparably low levels. Striking is that transcription can occur in the absence of defined core‐promoter elements. Furthermore, accumulation of translatable zygotic mRNAs is minimal in 1‐cell embryos because of inefficient splicing and 3′ processing of nascent transcripts. These findings provide novel insights into regulation of gene expression in 1‐cell mouse embryos that may confer a protective mechanism against precocious gene expression that is the product of a relaxed chromatin structure present in 1‐cell embryos. The results also suggest that the first zygotic transcription itself is an active component of chromatin remodeling in 1‐cell embryos.

Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII

Neonatal gene therapy is a promising strategy for treating a number of congenital diseases diagnosed shortly after birth as expression of therapeutic proteins during postnatal life may limit the pathologic consequences and result in a potential “cure.” Hemophilia A is often complicated by the development of antibodies to recombinant protein resulting in treatment failure. Neonatal administration of vectors may avoid inhibitory antibody formation to factor VIII (FVIII) by taking advantage of immune immaturity. A helper-dependent adenoviral vector expressing human factor VIII was administered i.v. to neonatal hemophilia A knockout mice. Three days later, mice produced high levels of FVIII. Levels declined rapidly with animal growth to 5 wk of age with stable factor VIII expression thereafter to >1 y of age. Decline in factor VIII expression was not related to cell-mediated or humoral responses with lack of development of antibodies to capsid or human factor VIII proteins. Subsequent readministration and augmentation of expression was possible as operational tolerance was established to factor VIII without development of inhibitors; however, protective immunity to adenovirus remained.