75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

313 Basis for CACTA Transposon Immobilization by DNA Methylation
Asuka Miura, Miyuki Nakamura, Masaomi Kato, Testuji Kakutani
Department of Integrated Genetics, National Institute of Genetics, Mishima, Japan
Transposon and their derivatives comprise the major components of heterochromatin, and they are often highly
methylated. Our previous studies have shown that endogenous transposon CACTA1 is mobilized in the DNA
hypomethylation mutant ddm1 (Decrease in DNA Methylation) or in the mutant of DNA methylatransferase genes,
suggesting that DNA methylation is necessary for the immobilization of this transposon in wild type (Miura et at. 2001,
Nature 411, 212-214; Kato et al. 2003, Curr. Biol. 13, 421-426). The immobilization effects by DNA methylation can
be trans-acting (such as transcriptional repression of transposase gene) or cis-limited (such as inhibition of transposase
access by heterochromatin formation).
In order to distinguish the possible cis-limited and trans-acting components, we used autonomously mobile CACTA1
and non-autonomous CACTA2. CACTA2 has internal deletion and did not transpose in the absence of the autonomous
CACTA1. CACTA1 transposes by itself when activated by the ddm1 mutation. CACTA2 transposed only when both
CACTA1 and CACTA2 were derived from ddm1. Notably, CACTA2 derived from wild type did not transpose even in
the presence the ddm1-derived CACTA1. Moreover, we examined the transposition of CACTA element in transgenic
lines with 35S-CACTA-GUS construct. Mobility of the non-autonomous CACTA element inserted between 35S and GUS
reporter was monitored in the presence of the ddm1-activated CACTA1. When the mobility was examined for various
35S-CACTA-GUS lines in the background of ddm1(-/-) and ddm1(-/+), they displayed variable GUS staining patterns
depending on their insert locus. These results suggest that the immobilization by DNA methylation is mediated not
only through trans-acting but also through cis-limited effects. In order to identify the target sequences of the cis-limited
immobilizing effects of DNA methylation, we are currently investigating the immobilizing effect of RNA-induced DNA
methylation (RdDM) in various parts of CACTA1 and flanking regions.
314 Silencing Of A Basic Chitinase Gene In Arabidopsis Thaliana Using Short Hairpin-
Polymerase III Vector Based System
Ewelina Rodakowska, Marta Derba, Przemyslaw Wojtaszek
Department of Molecular and Cellular Biology, Adam Mickiewicz University, Miedzychodzka 5, 60-371
Poznan, Poland
Chitinases (EC 3.2.1.14) are ubiquitous enzymes hydrolyzing ß-1,4-glycosidic linkages between adjacent N-acetyl-
D-glucosamines. There are 25 genes coding for chitinases and chitinase-like proteins in Arabidopsis and they are grouped
in the families 18and 19 of glycosyl hydrolases. Plant chitinases are usually considered as pathogenesis-related proteins,
although their involvement in stress responses is also well known. However, growing evidence indicates that at least
some representatives participatein the regulation of plant growth and development; they are involved in nodulation, PCD
and somatic embryogenesis; in the last case probably through interactions with wall-associated arabinogalactan proteins.
We are interested firstly in the function of chitinases in plant morphology and development and secondly in short hairpin
(shRNA) methodology - a new approach in plants reverse genetics. For theanalysis, a basic chitinase gene (At3g12500)
has been chosen. It is thought to be a pathogenesis-related protein, since its expression level increases upon pathogen
attack. On the other hand, its expression in rootsis constitutive during whole plants life. Transgenic plants with decreased
expression of chitinase mRNA have been obtained using short hairpin approach. In this communication, the generation
of transgenic plants with silenced chitinase gene, and preliminary phenotype analysis will be presented. This work is
funded by the Ministry of Education and Science grant PBZ-KBN-089/P06/2003 to P.W.