NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF GENE EXPRESSION AND REGULATION I

Professor:
Shigeru Iida
Associate Professor (adjunct):
Yoshihiro Ozeki
Research Associate:
Rie Terada
Yoshiki Habu
Yoshishige Inagaki
Atsushi Hoshino
Postdoctoral Fellow:
Yasuyo Johzuka-Hisatomi2
Hong-Qing Li2
Graduate Student:
Naoko Ishikawa
Takashi Kagami
Toshio Yamaguchi
Technical Staff:
Sachiko Tanaka-Fukada
Tomoko Mori
Yoshiko Inoue
Visiting Fellows:
Yasumasa Morita
Fumiyoshi Myoga


The main interest of the group is in understanding the biology of the dynamic genome, namely, genome organization and reorganization and its impact on gene expression and regulation. Although there are many elements affecting organization and reorganization of the genomes, we are currently focused on mobile genetic elements in general and plant transposable controlling elements in particular.


I. Identification and characterization of mutable alleles in the Japanese morning glory

The Japanese morning glory (Pharbitis nil or Ipomoea nil) is a traditional horticultural plant in Japan, and extensive physiological and genetical studies on the plant have been conducted. A number of mutants related to the color and shape of its flowers and leaves have been isolated since the 17th century, and more than 200 genetic loci including about 20 mutable loci have been assigned to one of the 10 linkage groups. We have identified that the mutable flecked allele bearing white flowers with colored flecks is the DFR-B gene having the En/Spm-related transposable element Tpn1 inserted into its second intron. The non-autonomous Tpn1 element carries a genomic DNA segment containing at least four exon sequences encoding part of a HMG-box sequence. Spliced hybrid transcripts containing the DFR-B exon(s) and the HMG exons from Tpn1 were detected in the flower buds of the flecked line, and they were polyadenylated within Tpn1. Thus Tpn1 can be regarded as a specialized transducing transposon carrying a part of the genomic sequence for a HMG-box (Takahashi et al. (1999) Mol. Gen. Genet. in press).

The plants with the recessive mutable speckled allele, in the presence of the dominant Speckled-activator, produce colorless flowers with fine and round colored spots distributed over the corolla, while plants carrying the speckled allele without active Speckled-activator bear pale yellow flowers. Currently, we are analyzing the CHI gene containing another En/Spm-related element Tpn2 in the mutable speckled lines. We are also characterizing not only other mutable alleles such as purple-mutable but also stable alleles displaying white flowers.

Fig. 1
The mutable lines of the common morning glory: heavily (A) and lightly (B) variegated lines; white-flower lines (C and D); structures of the CHS-D genes in the lines A, B and C (E) and the line D (F).


II. Identification of the mutable alleles in the common morning glory

The mutable aflaked line of the common morning glory (Pharbitis purpurea or Ipomoea purpurea) also bears white flowers with colored flakes and sectors (Fig. 1). The mutable aflaked allele is known to exhibit incomplete dominance. Interestingly, not only intensely colored flakes but also white spots and sectors were often observed in lightly colored flowers of the morning glory with the heterozygous state A/aflaked. We showed that the mutable aflaked allele is caused by insertion of a new transposable element, Tip100, into the CHS-D gene intron. Tip100 is 3.9 kb long and belongs to the Ac/Ds family. Although the timing and frequency of the flower variegation vary in different lines (Fig. 1A and B), they carry the identical mutable allele (Fig. 1E).

It has been postulated that the timing and the frequency of the variegation are determined by the active state of another genetic element modulator. It was further postulated that some lines bearing white flowers must be a modulator inactive line whereas other white-flower lines must be deficient at the A locus, (Fig. 1C and D), because in the presence of active modulator the latter can produce variegated flowers while the former still display white flowers. Indeed, the structure of the CHS-D gene in one of the former lines we analyzed was found to be indistinguishable from that in the mutable aflaked lines (Fig. 1E) whereas a white-flower line deficient at the A locus carries double insertions of Tip100 (Fig. 1F). Presumably, excision of one of the two copies of Tip100 from the CHS-D gene in the latter line appears to be insufficient to restore the CHS-D function and probably both elements are rarely excised in the same tissue.


III. Characterization of the genes for anthocyanin pigmentation in morning glories.
As an initial step to characterize the genes for anthocyanin pigmentation in morning glories, we sequenced segments of about 17 kb genomic DNA containing three DFR genes in the Japanese and common morning glories. The three DFR genes in both plants are arranged in tandem array, and all of them comprise six exons with identical intron positions. Their DFR-B genes, which carry longer introns than the DFR-A and DFR-C genes, are responsible for pigmentation in flowers, stems and coloring leaves. Indeed, the DFR-B gene of the common morning glory is expressed extensively in the young buds of pigmented flowers, considerably in stems, and moderately in sepals and leaves, whereas the DFR-A and DFR-C genes were expressed scarcely but significantly in the young flower buds and stems. Several novel mobile element-like sequences of around 200 bp were found in the genomic DFR regions.

A phylogenetic tree indicated that each DFR gene in the Japanese morning glory is most closely related to the corresponding DFR gene in the common morning glory and that the DFR-B gene is the most diversified gene among the three DFR genes. At least two gene duplication processes must be involved in generating three tandem copies from a single copy, and significant sequence divergence in both exons and introns of the duplicated DFR genes might occur during evolution of the genomes of these morning glories. Since homologies between the corresponding DFR genes extend beyond the exon sequences to introns and intergenic sequences, the results can be interpreted to mean that duplications and major divergence occurred prior to speciation of the Japanese and common morning glories (Inagaki et al. (1999) Gene in press).


Publication List:
Habu, Y., Hisatomi Y. and Iida, S. (1998) Molecular characterization of the mutable flaked allele for flower variegation in the common morning glory. Plant J. 16, 371-376.
Habu Y. and Iida, S. (1998) AFLP (amplified restriction fragment length polymorphism)-based mRNA fingerprinting. Plant Biotechnology 15, 249-251.
Hasebe, A., Tsushima S. and Iida, S. (1998) Isolation and characterization of IS1416 from Pseudomonas glumae, a new member of the IS3 family. Plasmid 39, 196-204.
Iida, S., Hiestand-Nauer, R., Sandmeier, H., Lehnherr H. and Arber, W. (1998) Accessory genes in the darA operon of bacteriophage P1 affect anti-restriction function, generalized transduction, head morphogenesis and host cell lysis. Virology 251, 49-58.
Nakai, K., Inagaki, Y., Nagata, H., Miyazaki C. and Iida, S. (1998) Molecular characterization of the gene for dihydroflavonol 4-reductase of Japonica rice varieties. Plant Biotechnology 15, 221-225.
Saito, N., Tatsuzawa, F., Kasahara, K., Iida S. and Honda, T. (1998) Acylated cyanidin 3-sophorosides in the brownish-red flowers of Ipomoea purpurea. Phytochemistry 49, 875-880.


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Last Modified: 12:00, May 28, 1999