A two-element Activator/Dissociation (Ac/Ds) gene trap system was successfully established in rice (Oryza sativa ssp. japonica cv. Nipponbare) to generate a collection of stable, unlinked and single-copy Ds transposants. The germinal transposition frequency of Ds was estimated as an average of 51% by analyzing 4413 families. Study of Ds transposition pattern in siblings revealed that 79% had at least two different insertions, suggesting late transposition during rice development. Analysis of 2057 Ds¯anking sequences showed that 88% of them were unique, whereas the rest within T-DNA. The insertions were distributed randomly throughout the genome; however, there was a bias toward chromosomes 4 and 7, which had two times as many insertions as that expected. A hot spot for Ds insertions was identi®ed on chromosome 7 within a 40-kbp region. One-third of Ds¯anking sequences was homologous to either proteins or rice expressed sequence tags (ESTs), con®rming a preference for Ds transposition into coding regions. Analysis of 200 Ds lines on chromosome 1 revealed that 72% insertions were found in genic region. Anchoring of more than 800 insertions to yeast arti®cial chromosome (YAC)-based EST map showed that Ds transposes preferentially into regions rich in expressed sequences. High germinal transposition frequency and independent transpositions among siblings show that the ef®ciency of this system is suitable for large-scale transposon mutagenesis in rice.

BackgroundOur knowledge on zebrafish reproduction is very limited. We generated a gonad-derived cDNA microarray from zebrafish and used it to analyze large-scale gene expression profiles in adult gonads and other organs.Methodology/Principal FindingsWe have identified 116638 gonad-derived zebrafish expressed sequence tags (ESTs), 21% of which were isolated in our lab. Following in silico normalization, we constructed a gonad-derived microarray comprising 6370 unique, full-length cDNAs from differentiating and adult gonads. Labeled targets from adult gonad, brain, kidney and ‘rest-of-body’ from both sexes were hybridized onto the microarray. Our analyses revealed 1366, 881 and 656 differentially expressed transcripts (34.7% novel) that showed highest expression in ovary, testis and both gonads respectively. Hierarchical clustering showed correlation of the two gonadal transcriptomes and their similarities to those of the brains. In addition, we have identified 276 genes showing sexually dimorphic expression both between the brains and between the gonads. By in situ hybridization, we showed that the gonadal transcripts with the strongest array signal intensities were germline-expressed. We found that five members of the GTP-binding septin gene family, from which only one member (septin 4) has previously been implicated in reproduction in mice, were all strongly expressed in the gonads.Conclusions/SignificanceWe have generated a gonad-derived zebrafish cDNA microarray and demonstrated its usefulness in identifying genes with sexually dimorphic co-expression in both the gonads and the brains. We have also provided the first evidence of large-scale differential gene expression between female and male brains of a teleost. Our microarray would be useful for studying gonad development, differentiation and function not only in zebrafish but also in related teleosts via cross-species hybridizations. Since several genes have been shown to play similar roles in gonadogenesis in zebrafish and other vertebrates, our array may even provide information on genetic disorders affecting gonadal phenotypes and fertility in mammals.

Myosins are actin-based motor proteins responsible for various motility and signal transduction. Only a small set of myosin classes is present inplants, and little is known about their functions. Here we showed how a rice myosin gene controlled pollen development by sensing changed environmental factors. The analysis is based on a gene-trapped Ds insertion mutant Oryza sativa myosin XI B (osmyoXIB). This mutant showed male sterility under short day length (SD) conditions and fertility under long day length (LD) conditions. Under both SD and LD conditions, the OSMYOXIB transcript was detected in whole anthers. However, under SD conditions, the OSMYOXIB-GUS fusion protein was localized only in the epidermal layer of anthers due to the lack of 3'-untranslated region (3'-UTR) and to dilute (DIL) domain sequences following the Ds insertion. As a result, mutant pollen development was affected, leading to male sterility. By contrast, under LD conditions, the fusion protein was localized normally in anthers. Despite normal localization, the protein was only partially functional due to the lack of DIL domain sequences, resulting in limited recovery of pollen fertility. This study also provides a case for a novel molecular aspect of gene expression, i.e., cell layer-specific translation in anthers.

Phenotype screens of Ds insertional lines identified a male sterile Orysa sativa no pollen (Osnop) mutant with a pollen-less phenotype at the flowering stage. The mutant phenotype showed linkage to Ds insertion into Osnop gene region. This mutant contained a deletion of 65 kb chromosomal region at the site of Ds insertion containing 14 predicted genes. Out of these deleted genes, Delegen 5-7, 9-10 were redundant, as two or three copies were present with 100% homology in other regions of rice genome. RT-PCR analysis showed that Delegen 5-7 were expressed not only in wild type plants but also in the mutant plants. In addition to this, Delegen 8-10 transcripts could not be detected under normal growth conditions, and Delegen 12 was expressed only in roots, thus deletion of these genes may not affect the pollen development. Our data and analysis also ruled out the possibility of delegen 1-4, 11, and 13 as candidates contributing to the pollen-less phenotype. Further investigation showed that the delegen 14 was expressed only in late stage of pollen development with the highest expression at the stage of pollen release and germination by RT-PCR, Northern blotting, in situ hybridization, and promoter-GUS transgenic plants. Thus, the delegen 14 gene is the best candidate for Osnop, corresponding to the pollen-less phenotype in the mutant. Our data suggest that delegen 14 may play an important role during late stage of pollen development and its germination. Since the delegen 14 gene has both C(2) and GRAM domains, it can be assumed that this gene cross-links both calcium and phosphoinositide signaling pathways. This is the first report to suggest possible functions for this gene in plant development.

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKC␣ is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ϳ60% of the catalytic activity of the mutant PKC␣, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKC␣ in immune complex kinase assays. The PKC␣ C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKC␣ immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKC␣ immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKC␣ is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKC␣ function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.