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Reseach
Our research group is studying the molecular structures and functions of
nuclei and chromosomes, mainly in plants. Our recent goal is to construct
plant artificial chromosomes by analyzing chromosome functional elements;
centromeres, telomeres and replication origins.
1. T-DNA-insertion-induced centromere
breakage in Arabidopsis thaliana
Two novel minichromosomes (α
and δ) in addition to two other aberrant chromosomes (β and γ) were found in a
transgenic Arabidopsis plant produced by an in planta vacuum infiltration
technique. Fluorescence in situ hybridization (FISH) indicated that both
of the two minichromosomes originated from the short arm of chromosome 2. The
mini α chromosome contained the whole short arm 2S and a truncated centromere
(180-bp repeat cluster), whereas mini δ lacked the terminal region including
telomere repeats. Pachytene FISH clearly revealed that mini δ comprised a ring
chromosome carrying two copies of the region from the 180-bp repeat cluster to
BAC-F3C11. Both of the 180-bp clusters (each ca. 500 kb in length) were thought
to possess normal centromere functions since the centromere-specific histone H3
variant (HTR12) was detected on both clusters. Notwithstanding this dicentric
and ring form, mini δ was stably transmitted to the next generations perhaps due
to its compact size (<4 Mb). Chromosome β also comprised a dicentric-like
structure, with one of the two 180-bp repeat sites derived from chromosome 1 and
the other from chromosome 2. However, the latter was quite small and failed to
bind HTR12. The data obtained in this study indicated that 500 kb of the 180-bp
array of the chromosome 2 centromere is sufficient to form a functional
domain.
2. Analysis of centromere-specific DNA sequence in
tobacco
Centromeres have an important role to segregate chromatids
into daughter cells at mitosis and meiosis. Though the centromere function have
been conserved among all eukaryotes including yeasts, animals and plants,
centromeric DNA sequences involved in the centromere function are diverged among
closely related species. Since long DNA is able to be transform into tobacco,
tobacco has a potential to be a model plant for artificial chromosomes
construction. However, centromeric DNA and proteins these are necessary to
construct and characterize artificial chromosomes had not been investigated in
tobacco. Hence, we started investigations of centromeric components in tobacco,
and isolated and characterized centromere specific histone H3 variants in
tobacco (NtCENH3) last year. In this year, we isolated a centromeric DNA
sequence (Nt2-7) from tobacco by a chromatin immunoprecipitation using an
antibody against the NtCENH3, and localization of the Nt2-7 was investigated by
fluorescence in situ hybridization. Hybridization signals of the Nt2-7 were
observed on all tobacco centromeres. These results suggesting the Nt2-7 is a
functional centromeric DNA in tobacco.
 
3. Molecular characterization of Luzula
holocentric chromosomes
The centromere is an essential region for
distributing chromosomes into daughter cells, and only a centromere exists in a
chromosome at a primary constriction. However, as exceptional cases,
‘holocentric chromosomes’ do not have any constriction-like structures, and
their entire regions look as if they act as a centromere. Luzula species
have been known to possess holocentric chromosomes for a long time, but no
critical studies of their kinetochore proteins have been made. In this study, we
successfully isolated a cDNA encoding a putative centromere-specific histone H3
(LnCENH3) by RT-PCR and RACE-PCR. The deduced amino acid sequence was then used
to raise an anti-LnCENH3 antibody. Immunostaining clearly revealed the diffuse
centromere-like structure that appears in the linear shape at prophase to
telophase. Furthermore, it was shown that the amount of LnCENH3 decreased
significantly at interphase. The polar side positioning on each chromatid at
metaphase to anaphase also confirmed that LnCENH3 represents one of the
centromere-specific proteins in L. nivea.
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Institute of Plant Science and Resources, Okayama University
2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan
Tel: +81-86-434-XXXX
Fax: +81-86-434-XXXX
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