NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

RADIOISOTOPE FACILITY

(Managed by NIBB)

Head:
Masaharu Noda
Associate Professor:
Kazuo Ogawa
Technical Staffs:
Kazuhiko Furukawa (Radiation
Protection Supervisor),
Yosuke Kato (Radiation Protection Supervisor),
Yoshimi Matsuda (Radiation Protection Supervisor)
Supporting Staff:
Takayo Ito, Risako Shirai


This Facility consists of a central experimental station which is placed in the Common Building I and three smaller substations which are placed in National Institute for Basic Biology (NIBB) Building, Laboratories of Gene Expression and Regulation (LGER) Building which opened in May 1, 1997, and National Institute for Physiological Sciences (NIPS) Building. These spaces are controlled areas and only the persons who are permitted by the Radiation Protection Supervisor can enter. The members of the Radioisotope Facility have a variety of businesses such as monitoring and recording the going in and out the controlled areas of the persons, purchasing the radioisotopes for the users from the Japan Radioisotope Association (JRA), and passing the radioisotope wastes to the JRA. They also serve as lecturers of the training course required for the registration of beginners and for annual registrations of users. At every opportunity, the Radiation Protection Supervisors are providing them appropriate guidance for radioisotope handling and the law of interest.

The total number of users in and out the controlled areas from April to December, 1997 was 8,922. As shown in Figure 1A, the number of users in the LGER substation was comparable to that in the central station. The total radioisotopes purchased in this period for each controlled area and nuclide are shown in Figure 1. Although a variety of nuclides can be used in the central station, only 125I, 35S, 32P, 14C, 33P, and 3H were purchased within this period. From April, 1997, we have been allowed to use 33P in all the controlled areas.


The teaching staff of the Radioisotope Facility is also engaged in their own research. They are interested in clarifying the structure and function of dynein motor protein. Dyneins are a group of microtubule-activated ATPases that serve to convert chemical energy into mechanical energy. They have been divided into two large subgroups, namely, the axonemal and cytoplasmic dyneins. Figure 2 shows the localization of two dyneins in the outer arms (Ogawa et al., 1977) and the mitotic apparatus (Mohri et al., 1976) that have been visualized by the same antibodies directed against the motor domain of axonemal dynein (fragment A).

Fig.2.


The native dyneins are very large. They range in molecular mass up to 1 to 2 mega daltons and they are complex proteins as shown in Figure 2. Each dynein contains two or three heavy chains (HCs) with ATPase activity, which range in molecular mass up to 500 kDa. The motor activity of dynein is associated with these chains. Some functional differences have been reported between HCs of outer arm dynein. Sea urchin outer arm dynein is a heterodimer of HCs (alpha and beta) and at least the beta-HC is able to induce gliding of microtubules in vitro. The alpha-HC might amplify the function of beta-HC and it has been reported to have no motile activity. After the first cloning of beta-HC from sea urchin ciliary axonemes (Gibbons et al., 1991; Ogawa, 1991), the sequences of HCs of axonemal and cytoplasmic dyneins from a variety of organisms were determined in their entirety. Without exception, all the HCs cloned to date contain four P-loop (ATP-binding) sequences in the midregion of the molecule. Thus, they can be classified as a four P-loop family.

The outer arm dyneins contain two or three proteins that range in molecular mass from 70 to 120 kDa and copurify with HCs. ICs of sea urchin outer-arm dynein are abbreviated as IC1, IC2, and IC3. Those of Chlamydomonas are called IC78 and IC69, and ICs of cytoplasmic dynein are called IC74. Chlamydomonas IC78 and IC69 were cloned by Wilkerson et al. (1995) and Mitchell and Kang (1991), respectively. The sequences of sea urchin IC2 and IC3 were determined by Ogawa et al. (1995). Finally, the sequence of IC1 was determined this year by Ogawa et al. (1996). Thus, all the ICs found in the axonemal and cytoplasmic dyneins of the model organisms used for studies of dynein function have been completely sequenced. Comparison of amino acid sequences of IC2 and IC3 with those of IC78 and IC69 and with that of IC74 showed that, although all five ICs are homologous, IC2 is much more closely related to IC78, and IC3 is much more closely related to IC69, than either sea urchin chain or either Chlamydomonas chain is related to each other. Regions of similarity between all five ICs are limited to the carboxy-terminal halves of the molecules. Similarity are due primarily to conservation of the WD repeats in all of these chains. The WD repeats are involved in protein-protein interactions in a large family of regulatory molecules (Neer et al., 1994). A parsimony tree for these chains (Ogawa et al., 1995) shows that, although the carboxy-terminal halves of all of these chains contain WD repeats, the chains can be divided into three distinct subclasses (IC3 plus IC69, IC2 plus IC78, and IC78).

Fig . 3.
Substructures of outer arm dyneins from sea urchin sperm flagella and Chlamydomonas flagella.

By contrast, sea urchin IC1 is not a member of the WD family. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. Then, a question arises as whether Chlamydomonas outer arm dynein contains IC1. The answer is "no". Because it consists of just two intermediate chains. Alternative answer is "yes". Because the sequences of two light chains (LC16 and LC14) from Chlamydomonas outer arm dynein shows that they are members of novel family of thioredoxin (Patel-King et al. 1996). The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin.

Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. They propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence.

They are now isolating cDNA clones that encode LCs of sea urchin outer arm dynein as part of an effort to understand why dynein is so very large and complex. The outer arm dynein of sea urchin sperm flagella contains six light chains with molecular masses of 23.2, 20.8, 12.3, 11.5, 10.4, and 9.3 kDa, respectively. They have cloned the cDNA for the 23.2 kDa (LC1) and the 12.3 kDa (LC3) polypeptides and found that they are highly homologous to mouse Tctex2 and Tctex1, respectively. These mouse proteins are encoded by the t complex region that is involved in transmission ratio distortion (TRD), male sterility and the development of the germ cells. Their finding raises the possibility that axonemal dyneins are involved in this phenomenon. TRD may be caused by the dysfunction of multiple axonemal dyneins.


Publication List:
Kazuo Ogawa, and Hideo Mohri (1997) Establishment of a dynein motor superfamily. In Recent Advances in Marine Biotechnology. Vol I. Endocrinology and Reproduction (Edited by M. Fingerman, R. Nagabhushanam, and M.-F. Thompson), pp 249-281. Oxford & IBH Publishing Co. New Delhi.

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Last Modified: 12:00, May 28, 1998