Eiji Nitasaka

NBRP databases: databases of biological resources in Japan.

The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

The FEATHERED gene is required for polarity establishment in lateral organs especially flowers of the Japanese morning glory (I pomoea nil ).

Most strains harboring the feathered (fe) mutation in the Japanese morning glory (Ipomoea nil or Pharbitis nil) show deformed phenotypes such as upcurled leaves and separated or tubular petals. These phenotypes seem to be caused by loss of abaxial identity in lateral organs. The FE gene was isolated using the inserted transposon as a tag. An En/Spm-related transposable element, Tpn102, inserted in the fourth intron of the FE gene, was responsible for the fe mutation. FE encodes a GARP transcription factor closely related to ArabidopsisKANADI1 (KAN1), which promotes an abaxial cell fate. Genetic analyses and molecular studies, which showed that all fe mutant strains have the same fe allele despite their phenotypic differences, revealed that fe strains with strong phenotypes have additional mutations enhancing the fe phenotype. These findings and historical records of fe phenotypes suggest that these enhancer mutations were accumulated in the fe background during selection for strong phenotypes. The mutant phenotypes and molecular analysis of fe strains suggest that FE regulates the abaxial identity of lateral organs redundantly with modifier genes, as KAN1 does in Arabidopsis. FE, however, affects flower phenotype even in the single mutant unlike KAN1, moreover, modifier mutations affect flower phenotype only in the fe mutant background, suggesting that FE may play a more crucial role in promotion of abaxial cell fate in flowers of the Japanese morning glory.

The Gravity-Regulated Growth of Axillary Buds is Mediated by a Mechanism Different from Decapitation-Induced Release

When the upper part of the main shoot of the Japanese morning glory (Pharbitis nil or Ipomoea nil) is bent down, the axillary bud situated on the uppermost node of the bending region is released from apical dominance and elongates. Here, we demonstrate that this release of axillary buds from apical dominance is gravity regulated. We utilized two agravitropic mutants of morning glory defective in gravisensing cell differentiation, weeping (we) and weeping2 (we2). Bending the main shoots of either we or we2 plants resulted in minimal elongation of their axillary buds. This aberration was genetically linked to the agravitropism phenotype of the mutants, which implied that shoot bending-induced release from apical dominance required gravisensing cells. Previous studies have shown that basipetal translocation of auxin from the apical bud inhibits axillary bud growth, whereas cytokinin promotes axillary bud outgrowth. We therefore compared the roles of auxin and cytokinin in bending- or decapitation-induced axillary bud growth. In the wild-type and we plants, decapitation increased cytokinin levels and reduced auxin response. In contrast, shoot bending did not cause significant changes in either cytokinin level or auxin response, suggesting that the mechanisms underlying gravity- and decapitation-regulated release from apical dominance are distinct and unique.

Geko, a novel gene involved in olfaction in Drosophila melanogaster

To characterize genes involved in olfactory responses to chemical attractants, we screened 3000 P-element-tagged lines for their attraction to ethanol. Ten lines showed reduced levels of response, and revertants of these lines were obtained by excising the inserted P-element. The olfactory response of one line reverted to wild-type behavior compared to the original mutant line. The gene affected by this P-lacW insertion was named geko (gk). A 1.3-kb transcript was found to emanate from close to the P-insertion site, and the 5″ upstream region was interrupted by the P-element. The amount of mRNA of gk gene in the P-lacW inserted line was about half that of the control strain. The response to ethanol of the gk1 mutant was restored by transforming the genomic region containing this transcription unit. lacZ expression (stemming from this reporter-genes presence in the transposon) was observed in the antenna and the antennal-maxillary complex (the olfactory organ of adults and larvae, respectively). gk mRNA was detected at the antenna and from other parts of the body. The deduced gk product showed no overall similarity to any reported amino-acid sequences.

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.

The relationship between DNA structural variation and activities of P elements in P and Q strains of Drosophila melanogaster

To characterize the relationship between P element activities and their structures, we cloned P elements from genomic libraries of three isogenic P and Q strains derived from natural populations in Japan. These P elements were mapped with BamHI, AvaII and PstI and were classified by their size. The majority of P elements cloned were classified as either complete or relatively small P elements rather than medium size. The numbers of full length (2.9 kb) P elements per haploid genome of NP280 (P), AK194 (weak P) and WY113 (Q) were at least four, five and one, respectively. However, the 2.9 kb P element of WY113 was thought to be defective since this strain has no transposase activity. In our previous work, we demonstrated that the ORF 3-deleted P element is essential for P cytotype determination in WY113. A similar P element also exists in NP280, and this may have an important role for P cytotype determination in this strain. Two and one copies of the KP element, a deletion derivative of the P element, were found in NP280 and AK194, respectively. One of four complete P elements in NP280 was fully sequenced, and the base sequence was completely identical to that of pπ25.1 originally derived from the U.S.A. This result is consistent with the notion that these P elements have a relatively recent origin in Drosophila melanogaster.

THE MOLECULAR ANALYSIS OF BROWN EYE COLOR MUTATIONS ISOLATED FROM GEOGRAPHICALLY DISCRETE POPULATIONS OF DROSOPHILA MELANOGASTER

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.

Development of EST-SSR markers of Ipomoea nil

Although Japanese morning glory (Ipomoea nil (L.) Roth.) has been used intensively for genetic studies, DNA markers have not been developed in Ipomoea nil sufficient to cover all chromosomes. Therefore, we conducted microsatellite (simple sequence repeats, SSR) marker development in I. nil for future genetic studies. From 92,662 expressed sequence tag (EST) sequences, 514 unique microsatellite-containing ESTs were identified. Primer pairs were designed automatically in 326 SSRs. Of 150 SSRs examined, 75 showed polymorphisms among strains. A phenogram based on the SSR genotypes revealed the genetic relation among seven Japanese morning glories from five different regions of the world and an ivyleaf morning glory (I. hederacea Jacq.). The developed SSR markers might be applicable for genetic studies of morning glories and their relatives.

A novel microsatellite locus isolated from an AFLP fragment in the mangrove species Kandelia obovata (Rhizophoraceae)

The study of AFLP analysis in Kandelia obovata, one of the major mangrove species in Japan, revealed the existence of a unique fragment showing stuttered peaks. We cloned this fragment and found a novel microsatellite locus. We report the method used for isolation and the polymorphic nature of this locus among the populations on Iriomote Island.