The laboratory consists of four regular divisions and conducts research into regulatory mechanisms of gene expression in higher plants and animals.
Closed during 1995 and will be reinitiated in 1996 on new projects.
Ito, T., Hirano, Y., Shimura, Y. and Okada, K. (1995) Two touch-inducible calmodulin and calmodulin-related genes tandemly located on chromosome of Arabidopsis thaliana. Plant Cell Physiol. 36, 1369-1373.
Mikami, K., Katsura, M., Okada, K., Shimura, Y. and Iwabuchi, M. (1995) Developmental and tissue-specific regulation of the gene for the wheat basic/leucine zipper protein HBP-1a(17) in transgenic Arabidopsis plants. Mol. Gen. Genet. 248, 573-582.
Okada, K., Ito, T., Sawa, S., Yano, A., Ishiguro, S. and Shimura, Y. (1995) Mutational analysis of flower development in Arabidopsis thaliana. In "Modification of gene expression and non-Mendelian inheritance" (eds. Oono, K. and Takaiwa, H.) NIAR Japan, pp. 145-156.
Professor: Takashi Horiuchi
Research Associate: Masumi Hidaka, Takehiko Kobayashi, Ken-ichi Kodama
Institute Research Fellow: Katsuki Johzuka
Graduate Student: Katsufumi Ohsumi, Keiko Taki
Technical Staff: Kohji Hayashi, Yasushi Takeuchi
Homologous recombination may occur in all organisms. While related functions apparently involve exchange between two parent-derived chromatids and repair of DNA damage incurred by physical and chemical reagents, many questions remain unanswered. As deduced from our analyses of recombinational hotspots of E. coli & S. cerevisiae, in particular the activity related to DNA replication fork blocking events, the physiological function of homologous recombination, especially in normally growing cells is better understood.
HOT1 is a mitotic recombinational hotspot in the yeast S. cerevisiae
and was first identified by Keil and Roeder. HOT1 stimulates both
intra- and inter-chromosomal recombination, and for a precise
analysis enhancement of excisional recombination between directly
repeated DNAs at its nearby site was investigated. HOT1 was originally
cloned on a 4.6 kb BglII fragment which locates in rRNA repeated
genes (about 140 copies) on chromosome XII. A single rRNA unit
consists of two transcribed 35S and 5S rRNA genes and two non-transcribed
regions, NTS1 and NTS2, the former is between 3'-ends of 35S and
5S rRNA genes, and the latter is 5' ends of these two genes. The
HOT1 DNA fragment contains the NTS1, 5SRNA gene and NTS2 region
but it was later found to be composed of two non-contiguous cis-elements,
E and I, located in NTS1 and NTS2, respectively. Because E and
I positionally and functionally overlapped with the enhancer and
initiator of the 35S rRNA transcription, respectively, Roeder's
group suggested that transcription by RNA polymerase I, initiated
at the 35S rRNA promoter site may stimulate recombination of the
downstream region, thereby revealing Hot1 activity.
The NTS1 has a site at which the replication fork is blocked and we termed this site SOG, but later called RFB (replication fork block). By assaying Rfb activity for various DNA fragments derived from the NTS1 and cloned on plasmids, we determined the minimal region, about 100 bp long, located near the enhancer region of the 35S rRNA transcription and essential for blocking replication fork advancing in a direction opposite that for transcription. The RFB sequence has no homology to any other known sequence and has no characteristic structure such 2-fold symmetry, repeated structure, etc.; hence, trans-factor(s) may have a role in blocking the fork. Interestingly, this region is included in one of two cis-elements required for a recombinational hotspot, Hot1, activity.
To investigate functional relationships between the fork blocking activity in RFB and the hotspot activity in HOT1, we first isolated mutants defective in Hot1 activity and examined whether these mutations would also affect Rfb activity. Using a colony color sectoring assay method, we isolated 23 Hot defective mutants from approximately 40,000 mutagenized colonies. Among these Hot- mutants, one proved to be a rad52 mutant; the other 4 mutants lose fork blocking activity (Fig. 1).
Genetic analysis of these mutants revealed that all four were
recessive for the Rfb phenotype and defined one complementation
group. This mutation was designated fob1 ( fork
blocking function) and one of the fob1 mutants,
fob1-4, was further analyzed. First, from yeast cDNA bank,
we cloned FOB1 gene by selecting a DNA fragment which had
suppressive activity for Hot deficiency of the mutant. The minimal
FOB1 plasmid was shown to complement both the Hot-
and Rfb- phenotype of the fob1-4 mutant, suggesting
that both phenotypes are caused by a mutation in the FOB1
gene (Fig. 1). DNA sequencing of the FOB1 gene revealed
that the putative Fob1 protein consists of 566 amino acids and
has a molecular mass of 65,000 daltons. We have overproduced and
purified a protein with this molecular weight and the expected
amino acid sequence of N-terminus of Fob1 protein. We are testing
whether the Fob1 protein has binding activity to RFB sequence
or not. We found the same sequence as that of the FOB1
gene in sequence of the chromosome IV cosmid 9727. Thus, the FOB1
gene is apparently not linked with the rRNA gene cluster present
in chromosome XII. Sequencing of the fob1-4 mutant gene
revealed two mutational changes in the open reading frame, one
is non-sense (amber) and other is a miss-sense mutation. The amber
mutation may account for the two defective phenotypes of the fob1-4
mutant and why it is non-leakyness.
Other workers showed that transcription of the rRNA gene is involved in the Hot1 activity, while the transcription appears not to be responsible for the Rfb activity. Our finding indicates that fork blocking event is required for Hot1 activity and seems not to be responsible for rRNA transcription. Thus, in yeast, both two independent events, one is fork blocking at RFB site and other is transcription of the rRNA gene, are required for activation of Hot1.
In E. coli RNase H defective (rnh-) mutants,
we found specific DNA fragments, termed Hot DNA, when DNA in the
ccc form is integrated into the E. coli genome by homologous
recombination to form a directly repeated structure, a strikingly
enhanced excisional recombination between the repeats occurs.
We obtained 8 groups (HotA-H) of such Hot DNA, 7 of which (HotA-G)
were clustered in a narrow region, called replication terminus
region (about 280 kb) on the circular E. coli genome. Analysis
of the HotA, B and C revealed that blocking of replication fork
at the Ter, replication terminus, sites is responsible
for these Hot activities. Further analysis of these Hot led to
design of a putative model, in which the ds(double stranded)-break
occurs at the fork arrested at the DNA replication fork blocking
(Ter) site, through which the RecBCD recombinational enzyme
enters the ds-DNA molecule and enhances recombination between
directly repeated Hot DNA, when the enzyme meets an appropriately
oriented Chi sequence.
To know how to activate other termination event-independent Hots, especially HotG, D and H, DNA sequences of these Hot fragments were determined and found that all Hot DNAs contained Chi sequence, thereby suggesting that RecBCD enzyme may be involved in these Hot activation such as in HotA. We now examine what event occurs near the Hot DNA sites, which probably provides an entrance site for RecBCD enzyme.
Horiuchi, T. and Fujimura, Y. (1995) Recombinational rescue of the stalled DNA replication fork: a model based on analysis of an Escherichia coli strain with a chromosome region difficult to replicate. J. Bacteriol. 177, 783-791.
Horiuchi, T., Nishitani, H. and Kobayashi, T. (1995) A new type of E. coli recombinational hotspot which requires for activity both DNA replication termination events and the Chi sequence. In "Molecular Mechanism of Genetic Recombination". Advance Biophysics vol. 31, 133-147.
Professor: Tetsuo Yamamori
Research Associates: Satoshi Koike, Yuriko Komine
Technical Staffs: Hideko Utsumi
Our research goal is to understand mechanisms underlying evolution of the nervous system. In order to approach this question, we are currently focusing on two systems.
Recently, it has been recognized that cytokines, defined as intercellular
mediators in the immune system, have a variety of roles in the
nervous system as well. One such a factor, LIF (leukemia inhibitory
factor) known also as CDF (Cholinergic Differentiation Factor),
is a pleiotropic factor which shows a remarkable repertoire of
activities from embryonic stem cells to neurons (Yamamori, T.
In Chemical Factors in Neuronal Growth, Degeneration and Regeneration
(Ed. by C. Bell), Elsevier, in press). Recent study have revealed
that CDF/LIF and its receptors belong to the IL-6 family and the
Based on Bazan'S model which predicted the cytokine receptor family as a member of immunoglobulin super gene family (1990) and the model of the interaction among the members of the IL-6 family (ligand) and the IL-6 receptor family (Taga and Kishimoto, 1992; Stahl and Yancopoulos, 1993), we proposed that the evolution of the IL-6/class IB receptor family may have occurred in at least two major steps (Yamamori and Sarai, 1994). Firstly, binding subunits of an IL-6 receptor and for a CDF/LIF receptor evolved and secondly, a third binding subunits of a CNTF receptor evolved. Our evolutional consideration predicts that the binding subunits generally determine the specificity of the receptors and it is possible that novel members of the cytokine family and their receptors exist in the nervous system.
In order to know roles of the genes involved in long-term memory, we choose the cerebellum as a model system. In the cerebellum the conjunctive stimuli of parallel fibers and a climbing fiber to a Purkinje cell induce prolonged reduction of a synaptic efficacy between the paralleled fiber to the Purkinje cell (LTD; long-term depression, Ito et al., 1982).
Previously, we examined the expression of 10 immediate early genes
(IEGs) including all the known Fos and Jun family in cerebellar
slices under the pharmacological condition that cause long-term
desensitization of the Purkinje cell to AMPA (a glutamate analogue).
Among the IEGs examined, Fos and Jun-B were predominantly induced
under the conjunctive condition (Nakazawa et al., 1993).
Recently, we have examined Jun-B expression in vivo under a conjunctive protocol of AMPA, a pharmacological substitute for parallel fiber stimulation, and climbing fiber stimulation via electric stimulation of Inferior Olive. June-B are predominantly induced around the local area where the AMPA and climbing fiber stimulation were conjunct. These results suggest that the coincidence mechanism may exist at gene expression level and lead to a cerebellar long-term plasticity.
We are currently working to identify the molecules that are induced after Jun-B induction and playing roles in cerebellar long-term plasticity.
Yamamori, T. Mikawa, S. and Kado, R. (1995) Jun-B expression in Purkinje cells by conjunctive stimulation of climbing fibre and AMPA. NeuroReport 6, 793-796.
The Division will be initiated in 1996.