NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

OFFICE OF DIRECTOR

Director-General:
Hideo Mohri
Associate Professors:
Shigeru Itoh
Ryuji Kodama
Kohji Ueno
Research Associate:
Akinao Nose (- May 31, 1998)
Masuo Goto (on leave)


Evolution of photosynthesis

Shigeru Itoh

We study the evolution of photosynthesis in molecular level. Oxygen-evolving photosynthesis of cyanobacteria seems to be evolved from the anoxygenic bacterial photosynthesis at 2.7-3.5 billion years ago just after the evolution of life. Symbiosis of cyanobacteria inside the eukariotic cells, then, produced the first plant 2 billion years ago. We recently focus our target of research on the evolutionary process from anoxygenic to oxygenic photosynthesis.

(1) Hunting for missing links of evolution "New photosynthesis".

A newly discovered bacterium Acidiphilium rubrum isolated from acidic mine drainage, was shown to use Zn-bacteriochlorophyll in its anoxygenic photosynthesis by us. This was the first case of photosynthesis based on pigments other than Mg-chlorophyllıs that are ubiquitously used in the ever-known oxygenic and anoxygenic photosynthesis.

Acaryochloris marina, a newly discovered oxygen-evolving cyanobacteria-like organism, was shown to use a far-red absorbing new pigment chlorophyll-d in its photosystem I reaction center pigment protein complex in collaboration with a Marine Biotechnology Institute Kamaishi. Although chlorophyll a that absorbs visible red light has been known to be indispensable for the oxygenic photosynthesis of plants and cyanobacteria, this organism efficiently undergoes oxygenic photosynthesis even with far-red light that has lower quantum energy. We named the special pair chlorophyll-d of the newly identified photosystem I reaction center P740 (Fig. 1). The organism can be a missing link between the anoxygenic photosynthesis that can use far-red light and the oxygenic photosynthesis that uses only visible-light. Molecular mechanisms and structures of the new photosynthetic systems are now being studied.

(2) Survey through optimization mechanism of photosynthesis.

We compared the structure-function relationships of the photosynthetic reaction center complexes of plants, green sulfur bacteria, Heliobacteria and newly found organisms. We replaced quinone cofactor molecules by artificial compounds inside the proteins and studied their localization by the ps-ns absorption, fluorescence and spin-echo ESR spectroscopy at 4-280 K. Comparison of structure-function correlation in various photosynthetic systems also revealed unique cytochrome reactions in Green-sulfur bacteria. Molecular architecture of plant and bacterial photosynthetic proteins are shown to be highly optimized but in different directions. This gave us a hint for the design of ancestral photosynthetic apparatus.

Fig. 1
Absorption spectrum (A) and light-induced difference spectrum of reaction center chlorophyll-d P740(B) of newly identified photosystem I (PS I) complex of Acaryochloris marina. Broken lines in (A) and (B) represent absorption and difference spectra (P700) in spinach PS I reaction center containing chlorophyll-a, respectively. Molecular structures of chlorophylls are also shown.


Mechanisms determining the outline shape of the adult lepidopteran wings

Ryuji Kodama

Wings of the lepidopteran insects (butterflies and moths) develop from the wing imaginal disc, which is a hollow sac made of simple epithelium. When the pupariation is completed, the wing, which was hidden inside the body wall of the larvae, is exposed on the surface of the pupa, which gradually turns into the adult wing. The outline shape of the adult wing is often different from that of the pupal wing. This difference is brought about by the programmed cell death of the marginal area of the pupal wing, while the internal area develops as adult wing blade. The marginal dying area is called the degeneration region and the internal area is called the differentiation region, hereafter.

The cell deaths in the degeneration region proceeds very rapidly and completes in a half to one day period in Pieris rapae or several other species examined. It was shown that the dying cells in the regeneration region have two characteristics common with the apoptotic cell death in mammalian cells. These are i) the presence of apoptotic bodies, which are heavily condensed cells or their fragments engulfed by other cells or macrophages, shown by transmission electron microscopy and ii) the presence of conspicuous accumulation of fragmented DNA evidenced by the TUNEL histological staining (Kodama, R. et al., Rouxıs Arch. Dev. Biol. 204, 418-426, 1995).

The cells in the degeneration region are actively engulfed by the macrophages in the cavity beneath the wing epithelium. Moreover, the macrophages seem to be concentrated beneath the degeneration region by the strong adhesion between basal surfaces of the dorsal and ventral epithelium in the differentiation region. By injecting the india ink or ferritin solution to the body cavity of the pupa, we have confirmed that this adhesion is tight enough to exclude the macrophages from the differentiation region, because the injected probes was found mostly concentrated in the degeneration region when observed several minutes later (Yoshida, A. (Biohistory Research Hall) and Kodama, R., unpublished).

Studies using another lepidopteran species, Orgyia recens approximans, provided by Drs. Y. Arita and K. Yamada (Meijo University) is underway. In this species, the wing is normally formed until the beginning of the pupal period, but becomes conspicuously degenerated only in the female adult. In our preliminary study, it was shown that the pupal wing is normally formed both in male and female pupa, but after about two days, female pupal wing starts degeneration on its margin, as if the degeneration region is continuously formed deep into the center of the wing (Kodama, R. et al., unpublished). It is thus suggested that the control mechanism which demarcates the region to be degenerated is defective in the female in this species. Further investigation using this species might give important insight into such mechanisms.

Another collaborative work with the laboratory of Dr. K. Watanabe (Hiroshima University) concerns mostly on the development of trachea and tracheole pattern in the swallow tail butterfly. According to their observations, the pattern formation of wing epithelium is often dependent on tracheal and tracheole patterns. Basic research on the development of tracheal pattern formation is being done including SEM observation (Fig.2) and histological observations to provide the basis of the morphogenetic analysis of wing epithelium as a whole.

Fig. 2
The inside of the larval wing disc of a swallow tail butterfly. The dorsal and the ventral epithelium of the disc were peeled apart with adhesive tape after critical-point-drying. The upper panel shows a low magnification view, whose lower right corner is shown in high magnification in the lower panel. Thick and smooth-surfaced tubes in the upper panel are trachea. A bundle of much thinner tubes are tracheoles, whose details are shown in the lower panel.


Protein palmitoylation and cell development at embryogenesis

Kohji Ueno

Thus far I have studied the molecular mechanisms of abdominal leg development in the silkworm Bombyx mori. From these studies, I have elucidated that abdominal leg development is regulated by a homeotic gene which specifies the identities of the abdominal segments. I have found that a high molecular weight protein, p260/270, is expressed in abdominal leg cells during early embryonic stages. The purified p260/270 was found to transfer palmitate to the cysteine residues of synthetic peptides in vitro. Most small, GTP-binding, heterotrimeric G, and G-protein-linked receptor proteins are known to be modified with palmitate through thioester linkages. Thus, these dynamic modifications may be important in the regulation of signal transduction. Therefore, I speculated that p260/270 may be involved in protein palmitoylation and may function in abdominal leg development (Ueno, K. and Suzuki, Y. J. Biol. Chem. 272: 13519-13526, 1997).

To understand the molecular mechanisms of how protein modification by palmitoylation regulates the development of cells and organs, I have attempted to find a vertebrate homologue of Bombyx p260/270. In order to accomplish this, I have searched an EST (Expressed Sequence Tag) data base for cDNAs encoding amino acid sequence residues corresponding to p260 and p270. From these searches, I found that several mouse embryonic cDNA clones were highly homologous to the amino acid sequences of p260 and p270. This suggested that a homologue of p260/270 was expressed in mouse embryos. One of these mouse p260/270 cDNAs was therefore used for in situ hybridization analyses of mouse embryos. Transcripts homologous to p260/270 were detected mainly in the central and peripheral nervous systems of mouse embryos from embryonic day 10 (E10). These transcripts were detected in the regions of the forebrain, midbrain, hindbrain and spinal cord of the central nervous system as well as in the cranial ganglia and dorsal root ganglia of the peripheral nervous system. The result of in situ hybridization on one mouse embryo section is shown in Fig.3. From these results, I speculate that a homologue of Bombyx p260/270 regulates development in the central and peripheral nervous system of mouse embryos. Further study is necessary to identify the specific cells which express these transcripts and understand the mechanisms of how this homologue regulates cell development.

Fig. 3
Localization of transcripts of a mouse homologue of p260/270 by in situ hybridization of a sagittal section of mouse E11.5 embryo. The transcripts are stained purple and nuclei are stained with methyl green. FB, forebrain; MB, midbrain; CG cranial ganglia.


Akinao Nose

How individual nerve cells find and recognize their targets during development is one of the central issues in modern biology. Our aim is to elucidate the molecular mechanism of axon guidance and target recognition by using the simple and highly accessible neuromuscular system of Drosophila.

The musculature of Drosophila embryos consists of 30 identifiable muscle fibers per hemisegment. Each muscle fiber is innervated by a few motoneurons in a highly stereotypic manner. The high degree of precision and previous cellular manipulations of neuromuscular connectivity suggest the presence of recognition molecules on the surface of specific muscle fibers which guide the growth cones of motoneurons. We have previously isolated several enhancer trap lines that express the reporter gene b-galactosidase (b-gal) in small subsets of muscle fibers prior to innervation. By molecularly characterizing these lines, we identified two genes, connectin and capricious that play roles neuromuscular connectivity.

I. connectin

Connectin is expressed on a subset of muscle fibers (primarily lateral muscles) and on the axons, growth cones of the motoneurons which innervate these muscles (primarily SNa motoneurons) and on several associated glial cells. When coupled with its ability to mediate homophilic cell adhesion in vitro, these results led to the suggestion that Connectin functions as an attractive signal for SNa pathfinding and targeting.

To study the role of connectin in vivo, we ectopically expressed Connectin on all muscles by using MHC (myosin heavy chain) promoter (MHC-connectin) in the P-element mediated transformants. In MHC-connectin, SNa nerves were observed to send extra axon branches that form ectopic nerve endings on muscles 12, muscles they would never innervate in wild type. Furthermore, the ectopic innervation on muscle 12 was dependent on the connectin expression on SNa. These results showed that connectin functions as an attractive and homophilic guidance molecule for SNa in vivo.

II. capricious

capricious (caps) is expressed in subsets of neurons and muscles, including the RP5 motoneuron and its target, muscle 12. The cDNA cloning and sequencing revealed that caps encodes a novel transmembrane protein that like connectin belongs to LRR family. Within the LRR family, Caps protein was found to be most related to the product of the Drosophila tartan gene that have been implicated in neural and muscular development. We found that in the loss-of-function mutants of caps, the synaptic arborization pattern on muscle 12, a caps-positive muscle, was abnormal. The nerve terminal failed to stabilize on muscle 12, and instead extended to and arborized on the adjacent muscle, muscle 13. Ectopic expression of caps in all muscles by GAL4-UAS system also resulted in aberrant synapse formation on muscle 12. Like in the loss-of-function alleles, the muscle 12 terminal axon branch often formed collaterals that turned back and innervated muscle 13. These results suggest that Caps is involved in neuromuscular target recognition and/or stabilizaion of the synapses.


Analysis of meiosis

Masuo Goto

The major goal of our research is to elucidate regulatory mechanisms of meiosis. Meiosis is a crucial step in gamete formation and is essential for sexual reproduction. Meiotic steps are highly conserved among eukaryotic species. We have isolated and analyzed a number of cloned mouse genes which may be relevant to spermatogenesis. In addition, efforts have been also paid to elucidate the regulatory mechanisms of meiosis in fission yeast in more detail. Some examples of the analysis are presented below.

I. Regulators of meiosis II in fission yeast.

We analyzed the mechanisms of fission yeast meiosis using a meiosis II deficient mutant mes1. Even though the mes1+ gene is essential to progress to meiosis II, its function has not yet been defined. To better understand the downstream pathway of mes1 we decided to isolate a suppressor. We isolated our suppressor, mes1 suppressor, from a S. pombe genomic library. The mes1 mutation was suppressed by overexpression of the slp1 gene. The slp1 gene is relevant to spindle checkpoint in mitosis and radiation sensitivity. This gene is homologous to S. cerevisiae CDC20, but lacks the corresponding 35 amino acids C-terminal. The authentic slp1+ gene encodes 488 amino acids, and its carboxyl region has significant homology to WD-repeat protein family. The C-terminal 35 amino acid portion includes a part of the seventh WD-repeat. In our experiments, full-length Slp1p could not suppress the mes1 defect. In addition, under nitrogen starvation, the slp1 mutant cells produce mature asci containing one or two diploid spores. This indicates that the slp1+ gene is essential in both meiosis I and meiosis II.

We determined that the physical interaction between Mes1p and Slp1p depends on the Slp1p C-terminal 35 amino acids. This physical interaction was prevented when an amino acid substitution occurred at aspartate-457 or tryptophan-463 in the seventh WD-repeat. The mutant Slp1p could suppress the mes1 defect similarly to the truncated 35 C-terminal amino acids form. Therefore, the seventh WD-repeat of Slp1p is a key region for mes1 suppression and binding to Mes1p. New insight into the crystal structure of G-protein heterotrimer and the G b g dimer revealed that G b is shaped like a seven-bladed propeller (seven WD-repeats) with a central shaft tunnel connecting the two faces. The seven WD-repeats of Slp1p could form a similar structure. Mutations in the seventh WD-repeat might destruct the propeller form since both the aspartate and the tryptophan residues are well conserved between WD-repeat protein families. This result indicates that the Slp1p may not be active in the seven propeller structure at the onset of meiosis II. In wild-type cells, the physical interaction between Mes1p and Slp1p is a critical step in activating Slp1p and overcoming the meiosis II checkpoint. This study implies that there are different cell cycle checkpoint mechanisms between mitosis and meiosis even though the same cell cycle regulators act at the appropriate steps.

II. Molecular cloning and characterization of mouse testis poly(A) binding protein II.

A cDNA clone from mouse testis cDNA library was isolated by a transcomplementation method using a S. pombe meiotic mutant (sme2) that arrests in the first meiotic division. The cDNA clone isolated has an open reading frame encoding 302 amino acids and has a strong similarity to mouse poly(A) binding protein II (mPABII) and bovine poly(A) binding protein II (PABII). PABII is known to bind the growing poly(A) tail. Northern blot analysis of the cDNA clone identified by mPABII revealed a single transcript of 1.2kb, detectable exclusively in adult testis. Immunohistochemical analysis using the polyclonal antibody demonstrated that mPABII protein was expressed in the nucleus at the specific stages from late pachytene spermatocytes to round spermatids. Genetic mapping showed the Pabp3 gene encoding mPABII located near at position 19.5 on mouse chromosome 14. Although mPABII function is not clearly linked to spermatogenesis, these results suggest that mPABII might be involved in specific spermatogenetic cell differentiation.


Publication List:
Hara, H., Tang, J., Kawamori, A., Iwaki, M. and Itoh, S. (1998) Anomalous pulse-angle and phase dependence of Hahnıs electron spin echo and multiple-quantum echoes of the spin correlated radical pair P700+A1- in Photosystem I. Applied Magnetic Resonance 14, 367-379.
Itoh, S., Iwaki, M., Wakao, N., Aoki, A. and Tazaki, K. (1998) Accumulation of Fe, Cr and Ni metals inside cells of acidophilic bacterium Acidiphilium rubrum that produced Zn containing bacteriochlorophyll a. Plant Cell Physiol. 39, 740-744.
Iwaki, M., Itoh, S., Hara, H., and Kawamori, A. (1998) Spin-polarized radical pair in Photosystem I reaction center that contains different quinones and fluorenones as the secondary electron acceptor. J. Phys. Chem. 102, 10440-10445.
Kagami, O., Gotoh, M., Makino, Y., Mohri, H., Kamiya, R. and Ogawa, K. (1998) A dynein light chain of sea urchin sperm flagella is a homolog of mouse Tctex 1, which is encoded by a gene of the t complex sterility locus. Gene, 211, 383-386.
Kosaka, M., Kodama, R. and Eguchi, G. (1998) In vitro culture system for iris-pigmented epithelial cells for molecular analysis of transdifferentiation. Exp. Cell Res. 245, 245-251.
Kubo-Irie, M., Irie M., Nakazawa, T. and Mohri, H. (1998) Morphological changes in eupyrene and apyrene spermatozoa in the reproductive tract of the male butterfly Atrophaneura alcinous Klug. Invertebrate Reprod. Dev. 34, 259-268.
Nose, A., Isshiki, T. and Takeichi, M. (1998). Regional specification of muscle progenitors in Drosophila: the role of the msh homeobox gene. Development 125, 215-223.
Ohoka, H., Iwaki, M. and Itoh, S. (1998) Membrane-bound cytochrome cz couples quinol oxidoreductase to the P840 reaction center complex in isolated membranes of the green sulfur bacterium Chlorobium tepidum. Biochemistry 37, 12293-12300.
Qiang, H., Miyashita, H., Iwasaki, I., Kurano, N., Miyachi, S., Iwaki, M. and Itoh, S. (1998) A photosystem I reaction center driven by chlorophyll d in oxygenic photosynthesis. Proc. Natl. Acad. Sci. USA. 95, 13319-13323.
Shishido, E., Takeichi, M. and Nose, A. (1998). Drosophila synapse formation: regulation by transmembrane protein with Leu-rich repeats, Capricious. Science 280, 2118-2121.
Yoshida, A., Arita, Y., Sakamaki, Y., Watanabe, K. and Kodama, R. (1998) Transformation from the pupal to adult wing in Oidaematophorus hirosakianus (Lepidoptera: Pterophoridae). Ann. Entomol. Soc. Am. 91, 892-857.

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