Download Methylation changes in specific sequences in

This article was downloaded by: [Universita' Milano Bicocca]
On: 26 November 2012, At: 03:20
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Plant Biosystems - An International Journal Dealing
with all Aspects of Plant Biology: Official Journal of
the Societa Botanica Italiana
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tplb20
Methylation changes in specific sequences in
response to water deficit
c
a
a
Massimo Labra , Candida Vannini , Marcella Bracale & Francesco Sala
b
a
Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi dell'Insubria,
Via J.H. Dunant 3, Varese, Italy
b
Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria 26, Milano, Italy
c
Dipartimento di Scienze dell'Ambiente e del Territorio, Piazza della Scienza 1,
Universitd degli Studi di Milano, Bicocca, 20126, Milano, Italy
Version of record first published: 05 Aug 2006.
To cite this article: Massimo Labra, Candida Vannini, Marcella Bracale & Francesco Sala (2002): Methylation changes in
specific sequences in response to water deficit, Plant Biosystems - An International Journal Dealing with all Aspects of
Plant Biology: Official Journal of the Societa Botanica Italiana, 136:3, 269-275
To link to this article: http://dx.doi.org/10.1080/11263500212331351179
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to
anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contents
will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should
be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,
proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in
connection with or arising out of the use of this material.
PLANTBIOSYSTEMS,136 (3) 269-276, 2002
Methylation changes in specific sequences in response
water deficit
to
Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012
M. LABRA,C. VANNINI,E SALAand M. BRACALE
received 20 June 2001; revised version accepted 18 February 2002
ABSTRACT - Conditions of environmental stress may lead to epigenetic
variability in plants. In particular, variation in genome methylation may be a
mechanism of plant adaptation to abiotic stresses, We have investigated the
relationship between water deficit and DNA changes in specific sequences by
using the CRED-RA (Coupled Restriction Enzyme Digestion and Random
Amplification) approach. Three polymorphic bands have been detected in
dehydrated and rehydrated pea root tips. One of these bands is a zinc-finger
sequence with three non canonical motifs, that could be involved in signal
transduction pathways.
KEY WORDS - Pisum sativum L., Pea, CRED-RA, Zinc finger, DNA
methylation,RAPD
An increasing body of experimental evidence indicates
that conditions of environmental stress may lead to
genotypic variability in plants. Heritable changes
induced by the environment have been shown in flax
(CULLIS, 1986), in Mesembryanthemum crystallinum L.
(BOHNERT et aI., 1995), in Brassica nigra L. (WATERS&
SCHAaL, 1996), and in Ustilago rnaydis L. (CULL~S,
1990). Moreover, genomic changes have been observed
frequently in plants regenerated from tissue or cell cultures (KAEPPLER& PHILLIPS, 1993). Recently, epigenetic
changes have also been associated to abiotic stress.
Hypermethylation of heterochromatic loci has been
reported in tobacco, either in response to osmotic stress
(KOVARIKet al., 1997) or in silenced genes in transgenic
plants (MEYERet al., 1992; MEYERet al., 1994). On the
contrary, hypomethylation has been documented in
chicory root tips (DEMEULEMEESTERet al., 1999) and in
Arabidopsis lhaliana L. (FINNEGAN et al, 1998) when
exposed to low temperature. Epigenetic changes have
been observed in tissue cultures, while methylation polymorphisms have been frequently observed during the
propagation of tissue cultures at the level of repeated
sequences (SMULDERSet al., 1995) and may contribute to
somaclonal variation (KAEPPLERet al., 2000).
All these variations in genome methylation might be part
of the plant's adaptation mechanisms to abiotic stresses
(KOVARIKet al., 1997; MARTINESSEN& R~CHARDS,1995).
However, more work is needed in order to clarify the
metabolic processes, and the role of genes involved in
these types of stress response.
Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012
270 M. LABRa et al.
In the present study we faced the problem of understanding the correlation between environmental stress
and DNA methylation. Using the CRED-RA (Coupled
Restriction Enzyme Digestion and Random
Amplification) technique (CaI et al., 1996) we wanted to
identify the genes involved in DNA methytation change
in response to water stress. The CRED-RA technique is
based upon the coupled restriction enzyme digestion
and random amplification of DNA. It uses restriction
endonucleases sensitive and insensitive to cytosine
methylation in their targeting sites, so that DNA digestion is methylation-dependent (CAt et al., 1996). Results
reveal the presence of three polymorphic bands, out of
a total of 222 bands detected, one of which is a zinc-finger-like sequence induced in response to water stress.
MATERIALS AND METHODS
Elmer Gene Amp PCR System 9600. RAPD analysis
requires only the presence of a single randomly chosen
oligonucleotide (KARP et al., 1998). The PCR reaction
was performed in 20 Ill reaction mixture containing 2 ml
10x-Dynazyme buffer (Celbio), 200 I~M of each dNTP,
20 ng DNA primer, 0.5 U Dynazyme II (Celbio) and 20
ng template DNA. An annealing step (1 rain at 36°C for
10-bp primers) or at 45°C (for 20-bp primers) and an
extension step (72°C for 1 rain) followed an initial
denaturation step (1 rain at 94°C). Primers, listed in
Table 1, were obtained from Life Technologies.
Amplification products were separated by electrophoresis on 2% ultrapure agarose gel, stained with ethidium
bromide and visualised under UV light (Gel Doc 2000
Biorad). All bands detected were reproducible as
assessed by three independent repetitions of the analysis.
Plants and DNA extraction
PoIymorphic bands analysis
Pea (Pisum sativum L. cv. Lincoln) seeds were surface- Polymorphic bands were excised and gd-purified using
sterilised in 10% NaC10 and germinated in moistened the QIAEX II agarose gd extraction kit (Qiagen) as
agriperlite at 25°C in the dark. After 72 h, the seedlings described by the supplier. The purified fragments were
were transferred to glass dissectors on top of 25 % glyc- cloned in the pUC57 plasmid by the T-doning kit (MBI
erol (approx. 90% relative humidity) for 3 days in the Fermentas) and sequenced (MWG). Sequence analysis
dark. In these conditions, the water content is reduced was performed by using the 2.0 version of the
up to 82 %. These seedlings represented the dehydrated Washington University BLAST (http://www.ncbi.nlm.
sample (D). Previous data showed that this treatment nih.gov/BLAST) software for gene and protein identifiinduces a drought stress able to promote a significant cation through sensitive, selective and rapid similarity
cellular response, and that primary root tips always sur- search of protein and nucleotide sequence database.
vive and resume growth upon rewatering (BRACALEet Sequence analysis was also performed by MOTIF dataal., 1997). To obtain rehydrated samples (R), some of the base (http://motiLgenome.AD.JP).
dehydrated seedlings were transferred in water for 5 h
and subsequently in moistened agriperlite at 25 °C in the Northern analysis
dark. Control seedlings (C) were grown in agriperlite in Total RNA was extracted by TRIZOL (GibcoBRL) from
the dark at 25°C for the same period of time.
whole pea seedlings. Samples containing 10 lag of total
Twenty-five root tips (2 mm in length) were excised RNA were electrophoresed on formaldehyde 1.2%
from C, D and R seedlings and were ground to a fine (w/v) agarose gels and blotted onto Hybond-N+ mempowder in liquid nitrogen with a mortar and pestle. brane (Amersham). Filter hybridisation was performed
Total genomic DNA was extracted and purified by using with the probes of the three clones of interest, labelled
the DNeasy Plant mini kit (Qiagen).
by using the Rediprime kit (Amersham), under high
stringency wash conditions. Filters were finally exposed
Genomic DNA digestion and CRED-RA
to Kodak X-Omat AR X-ray film.
The methylation status of the genome was analysed
using the restriction enzymes MspI and HpaII. One pg Southern blotting
of genomic DNA from each sample was digested with Ten pg of DNA were digested with Hind III and HhaI
15 U of each enzyme. Both enzymes recognise the restriction enzymes and separated on 0.8% (w/v)
CCGG sequence, but HpaII is active only if both agarose gel in 1% (v/v) TBE buffer. The DNA was
cytosines are unmethylated (CCGG), whereas MspI transferred by capillary blot onto Hybond-N+ memdoes not cleave the DNA when the external cytosine is branes (Amersham) according to the manufacturer's
methylated (5=CCGG) (KovaRZKet al., 1997).
instructions. Prehybridisation and hybridisation were
Restricted DNA was amplified by the RAPD (Random performed at 65°C with constant agitation. Probes were
Amplified Polymorphic DNA) method using a Perkin- generated from pUC57-CHL24-3 (accession number
Methylation changes and water deficit 271
EMBLY14837, MARKAUSAKAS& DREGUNIENE, unpublished) plasmid by double digestion with EcoRI and
BamHI. The insert was then labelled by using the Gene
Images Alkphos direct labelling and detection system
(Amersham). Autoradiography was performed at room
temperature with Kodak X-Omat AR X-ray film and an
intensifying screen.
MCDR
RESULTS
Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012
D N A polymorphism detection by the C R E D - R A
technique
To determine the sequences involved in methylation
change by water stress, DNA from 72-h dehydrated (D),
rehydrated (R) and control (C) samples was fractionated with HpalI or MspI restriction enzymes and analysed
3500bp-.~
lO00bp-~
TABLE1
DNA primers for RM~D analysis
Name
FS-5
FS-6
FS-7
FS-8
FS-9
FS-10
FS-11
FS-I2
FS-13
FS-I4
FS-15
FS-16
FS-25
FS-26
FS-27
FR-27
FS-28
CHL9
CHL12
CHL16
CHL17
CHL18
CHL19
CHL20
CHL21
CHL22
CHL24
CHL26
CHL30
CHL34
CHL35
CHL24-3
CHL24,2
CHL24-I
DNA sequence
5'-GGGATCCGGC-3'
5'-GCTTCGATAC -3'
5'-CCGAATTCGC-3'
5'-TGAGTCACCG-3'
5'-GGAGAGACTC-3'
5'.ACCAGGTTGG-3 '
5'-GACGCCACAC-3'
5'-TGTAGCTGGG-3'
5'-GGAAGTCGCC-3'
5'-GTGGCAAGCC-3'
5'-ATCGGCTGGG-3'
5'-CTCCCGGTAC-3'
5'-CTTGCCCACG-3'
5'-ATCGGCTGGG-3'
5'-AGCCGGCCTT-3 '
5'-ACGCGCGGGA-3 '
5'-TGGCCCCGGT-3'
5'-GGCATGTCAAGCCCTGGTAA -3'
5'- CGGAAGCAATTTGCTTGGCT-3'
5'-CTCCCTCATGATTCTTGGGA -3'
5'-AAACTCAAGGCCACCCTTCC -3'
5'- CCTTTGCCTTAGGATTCGGT-3'
5'- GAGGATATGTTCGCCGTCCT-3'
5'- ACTAATGGCAACCCTTCGAG-3'
5'- AGAAGCATCCCAAAAGCGTC-3'
5'- TTGTTCCTGACCCTGGTTCA-3'
5 '-CATCCGCCAATATKFTCCCG-3 '
5'-CAATTCGAGGATCCAGAGAC4'
5'-CAGGCAATCCTAAACTCTCT-3'
5'-ATCGGGAAAGACTCCGCAAA-3'
5'-GAGTACAGGTATGACAGGCA-3'
FIGURE1 - CRED-RAanalysisperformedby usingCHL24 primer on
DNA from control (C), dehydrated (D) and rehydrated (R) root tips.
CHL24-1, CHL24-2 and CHL24-3 indicate bands isolated from the
gd and sequenced.
by CRED-RA. Since HpaII is active only if both
cytosines are unmethylated (CCGG), whereas MspI
cleaves the DNA when only the second cytosine is
methylated (CsnlCGG), polymorphic bands detected
were originated from a different methylation status in
CCGG sequences localised in the amplification region.
When cytosines in the CCGG sequence are methylated,
HpaII and MspI are inactive, and consequently the
amplification product can be obtained. On the contrary;
in demethylation status, enzymes cut the DNA in a specific CCGG sequence, resulting in the absence of amplification fragments.
Different RAPD primers were tested to detect scorable
bands on the analysing gel. Bands on the gels were consistent and reproducible. The 32 primers listed in Table
1 produced a total of 222 bands, three of which were
polymorphic. Polymorphic bands were obtained by
0
o
0
d')
0
9
N
0
o~
!
it-
Io
ffl
IC~
I1i~-
0
H
I-I
t~
~
0
Inii_il o
r0t
i~i.~
H
r~
ffl
di~.
t-I
q
r',t
t~
o
a"
P_,
0
rl.
":::1
b,l
ff
o
n,
0
u~
H
H
Q
q
Q
;~
Q
~
q
©
q~
~
Q
H
~
~
H
H
~
Q
C~
~
H
H
Q
~
H
q
q~
Q
Q
Q
Q
Q
d~
Q
q~
H
H
Q
©
Q
q
H
Q
H
~
~
~
H
~
H
Q
H
~
~
H
H
H
Q
H
Q
~
H
Q
Q
q
q
H
~
~
Q
©
H
~
q
~
Q
Q
~
~
©
Q
~
~
~
~
0
H
q
Q
Q
~
Q
H
q
~
q
~
~
Q
Q
~
~
0
Q
Q
H
~
H
Q
~
q
~
H
~
H
Q
O
~
H
H
q~
Q
H
~
Q
©
q
H
Q
0
~
Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012
Q
H
H
©
~
~
q
q~
Q
~
~
©
~'
Q
q
H
d~
~
Q
d~
~
q
Q
~
q
Q
Q
Q
H
H
Q
Q
Q
H
~
©
©
Q
©
q
q
H
H
H
q
q
Q
H
q
q
~
~
Q
Q
Q
Q
~
Q
~
q
©
q
Q
H
~
q
Q
~
q~
H
Q
Q
H
Q
H
q
Q
Q
Q
MethyIation changes and water deficit 273
amplifying HpaII digested DNA with the CHL24
primer. Electrophoresis of the amplification products
showed two bands (indicated as CHL24-1 and CIcLL243) present only in D and R samples, and one band (indicated as CHL24-2) detected only in C sample (Figure 1).
D
R
C
CHCL 24-1
Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012
Sequences analysis
CHL24d and CHL24-3 (from D and R samples) and
CHL24-2 (from C sample) bands were excised from the
gel, cloned in the pUC57 plasmid and sequenced.
Sequences of CHL24-1 and CHL24-3 are identical in
bands purified from D and R samples.
The sequences were submitted to the EMBL database
and have received the following accession numbers:
AJ311167 for CHL24-1, AJ312062 for CHL24-2 and
AJ312063 for CHL24-3. The HpaII target sequence was
detected in all three fragments by restriction analysis
conducted using the Restriction Enzymes Map
Database. This confirmed that polymorphic bands can
be ascribed to methylation changes in CCGG
sequences.
CHL24-1 is 719-bp long and does not reveal significant
similarity with sequences present in GenBank or EMBL
database, while CHL24-2 (744 bp) shows a high
sequence similarity (97% overlap) with mitochondrial
DNA of Arabidopsis thaliana L.
CHL24-3 is 807-bp long with an ORF of 612 bp (Figure
2A), and does not show similarity with other genes or
sequences present in GenBank or EMBL. MOTIF
analysis inside this clone reveals three non canonical
zinc-finger motifs, two of the C-Xs-C-X;C-X;H type
and a third of the C-XT-C-Xs-C-X;H type (Figure 2B).
These data may suggest a relationship between CHL243 and a transcription factor containing the zinc-finger
motif. Further analysis is needed in order to ascertain
whether or not CHL24-3 is a putative zinc-finger transcription factor involved in methylation metabolisms.
To establish a relationship between CHL24-3 and other
zinc-finger sequences, a MOTIF - Genome Net database was used. The results shown in figure 2B describe
the zinc-finger motif and the similarity between the
zinc-finger motif of Arabidopsis thaliana (Athb-1) and
our sequence. Athb-1 recognised a 9-bp symmetric
sequence of DNA, and specific analysis showed that this
gene could be involved in the signal transduction pathway necessary for plant growth and development (SESSA
et al., t993).
,Northern and Southern blot analysis"
As shown in Figure 3, northern analysis on the total
RNA from control and treated seedlings confirmed' the
results obtained using the CRED-RA technique.
D
R
C
CHCL 24-2
D
R
C
CHCL 24-3
FIGURE 3 - Expression of CHL24-1, CHL24-2 and CHL24-3
mRNAs.Eachlanewas loadedwith 10 pg total t~NApreparedfrom
C (control),D (dehydrated)and R (rehydrated)pea seedlings. Gel
stainingwas used to verifythat equal amountsof RNA werepresent
in eachlane.
Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012
274 M. LaBr,a et al.
Drought stress induces the appearance of CHCL24-1
and CHCL24-3 clones: their mRNA, absent in control
conditions, accumulated upon drought treatment and
slightly decreased after rehydration. On the contrary,
CHCL24-2 mRNA levels remained high as long as the
seedlings were kept under control conditions, and
rapidly declined after drought treatment.
To characterise the putative zinc-finger transcription
factor, Southern blot analysis was performed by using as
probe the CHL24-3 sequence. The analysis was performed on genomic DNA extracted from C, D and R
samples digested with HindIII and HpaII. CHL24-3
sequence did not show a target sequence for Hind III,
but the target sequence of HpaII was present in position
286 of the CtlL24-3 fragment. HpaII recognises the
GCGC sequence, but this enzyme is active only if both
cytosines are unmethylated. Figure 4 shows the results
of the Southern blot. Using CHL24-3 as a probe, a
1200-bp fragment was detected in D and R samples,
while it was absent in control sample.
M
CDR
",ql"--800bp
FIGURE4 - Southern blot analysis of genomic DNA isolated from pea
DISCUSSION
In the present study, a relationship is established
between water deficit and DNA methylation changes in
specific sequences. Indeed, on the basis of the CREDRA approach, three polymorphic bands have been
detected. This means that these sequences are regulated
by specific DNA changes in response to water deficit.
On the basis of these results, we may consider epigenetic change as an important mechanism in response to abiotic stress in plants. This consideration is in agreement
with several papers describing a relationship between
methylation changes and environmental stress (SABBAtI
et al., 1995; KOVAPdKet J., 1997; FINNEGANet al., 1998;
DEMEULEMEESTZR,1999). To define the role of the three
isolated sequences, a BLAST analysis was performed.
Analysis of the CHL24-3 sequence revealed three zincfinger domains. This type of zinc-finger domain is found
in a number of proteins in a wide range of organisms; it
is present in many regulatory proteins, including viral
transcription factors and oncoproteins, and in plants it
is involved in DNA repair and recombination
(FREEMONTet aL, 1991).
Much work has been done on the protein-DNA interaction of the zinc-finger protein. Crystallographic
(PAVLETICH& PABO, 1991) and nuclear magnetic resonance studies (OMICHINSKI et al., 1993) have revealed
DNA-protein interaction between a single zinc-finger
motif and its target DNA sequence. The amino acids
responsible for base recognition have been found by statistical sequence analysis. By domain swapping studies,
root.
Ten l~g of DNA were digested with HindIII and HpaII restriction
enzymes, electrophoresed on a 0.8% agarose gel, transferred to nylon
membranes and probed with CHL24-3. (C) Control root tips; (D)
Dehydrated root tips; (R) Rehydrated root tips;
(M) Molecular marker.
it was shown that each zinc finger in the clustered zincfinger motifs spans a unit of three bases in the target
sequences (NARDELLIet al., 1991).
CHL24-3 is a zinc-finger sequence with three non
canonical motifs. The repetition for three times of this
motif, inside a sequence of only 807 bp, suggests that
these amino acid combinations are not casual, but result
from the translation of nucleotidic sequences related to
specific zinc-finger functional domains.
On the basis of our results and from database analysis,
it is difficult to define a functional role for CHL24-3
zinc-finger sequences; however, we have shown a relationship between water stress, DNA methylation
change and zinc-finger sequences. Further investigation
will be necessary to characterise this sequence and to
describe its metabolic rote in response to water stress.
Finally, on the basis of our data, we consider CRED-RA
to be a powerful tool in understanding whether methylation changes are directed to specific sequences or are
a random response to stress found equally in all
sequences. In addition, CRED-RA can be used to isolate
genes or sequences involved in plant response to abiotic stress.
Methylation changesand waterdeficit 275
Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012
ACKNOWLEDGEMENTS
KOVaRIK A., KOUKALOVAB., BEZDEK M., OPKINNY Z., i997 -
Hypermethylation of tobacco heterochromatictoci in response to
osmotic stress. Theor. Appl. Genet. 95: 301-306.
W e would like to t h a n k Cristian Savini a n d Valentina
MaRTINESSENR.A. & R~CHaRDSE.J., 1995 - DNA methylation in
H a u p t for technical assistance.
eukaryotes. Curr. Op. Genetics Development. 5: 234-242.
MEYER
P., LINN F., HEIDMANNI., MEYERZ A.H., NIEDENHOFI.,
REFERENCES
SAEDLER H., 1992 - Endogenous and environmental factors
influence 35S promoter methylation of a maize A1 gene
BONHERT HJ., NELSON D., JENSEN RG., 1995 - Adaptation to
construct in transgenicpetunia and its co[our phenotype. Mol.
environmental stresses. Plant Cell 7: 1099-1111.
Gen. Genet. 231: 345-352.
BRACALE M., LEVI M., SAVlNI C., DICORATO W., GALLI M.G.,
MEYER P., NIEDENHOFI., LOHUIS M., 1994 - Evidencefor cytosine
1997 - Water deficit in pea root tips: effects on the cell cycle and
methytation of non-symmetrical sequences in transgenic
on the production of dehydrin-Iike proteins. Ann. Bot. 97: 593Petunia hybrida. European Molecular Biology Organization
600.
Journal 13: 2084-2088.
CAI Q., GuY C.L., MooK~ G.A., 1996 - Detection of cytosine
NARDELLIJ., GILSON T.J., VESQUE C., CAARNAYR, 1991- Base
methylation and mapping of a gene influencing cytosine
sequence discrimination by zinc finger DNA binding domains.
methylation in the genome of Citrus. Genome 39: 235-242.
Nature 349: 175-178.
CULLIS C.A., 1986 - Phenotypic consequences of environmentally
OMICHINSKI J.G., CLOm¢ G.M., SCHK~ O., FELSENFELD G.,
induced changes in plant DNA. Trends in Genetics 2: 307-309.
TRAINOR C., APPELLA E., STAHL S.J., GRONENBORN A.M.,
CULLISC.A., 1990 In: SCANDALIOSJ.G. (ed), DNA rearrangements
1993 - NMR structure of a specific DNA complex of Znin response to environmental stress, 73-97. Academic, New
containing DNA binding domain GATA-1. Science 261: 438York.
446.
DEMEULEMEESTER M.A.C., VAN STALLEN N., DE PROFT M.E,
PAVLETICHN. & PABO C., 1991 - Zinc finger-DNA recognition:
1999 - Degree of DNA methylation in chico~7 (Cichorium
Crystal structure of a Zzf268-DNA complex at 2.1. Science 252:
intybus L.): influence of plant age and vernalization. Plant
809-817.
Science. 142: 101-108.
SABBAttS., RAISEM., TM~ M., 1995-Methylation of DNA in NaClFINNEGANEJ., GENGERR.K., PEACOCKW.J., DENNIS E.S., 1998
adapted cells of potato. Plant Cell Rep. 14 (7): 467-470.
- DNA methylation in plants. Ann. Rev. Plant Physiol. Mol.
SESSAG., MOm~LLIG., RUBEgrI I., 1993 - TheAthb-1 and-2 HDBiol. 49: 223-247.
Zip domains homodimerize forming complexes of different
FREEMONT RS., HANSON I.M., TROWSDALEJ., 1991 ~ A novel
DNA binding specificities. European Molecular Biology
cysteine-rich sequence motif Cell 64: 483-484.
Organization Journal 12: 3507-3517.
KAEPPLER S.M. & PHILLIPS R.L., 1993 - Tissue culture-induced SMULDERSM.J.M., Rus-KORTEKAASW., VOSMANB., 1995 - Tissue
DNA methylation variation in maize. Proc. Natl. Acad. 8d.,
culture-induced DNA methytation polymorphism in repetitive
USA. 90: 8773-8776.
DNA of tomato catli and regenerated plants. Theor. Applied
KaEPPLER S.M., KAEPPLER H.E, RttEE Y., 2000 - Epigenetic
Genet. 91:1257-1264.
aspects of somadonal variation in plants. Plant Mol. Biol. 43:
WATERSE.R. & SC14AALB.A., 1996 - Heat shock induces a loss of
179-188.
rRNA-encoding DNA repeats in Brassica nigra. Proc. Nail
KA~ A., ISAACRG., INGRAMD.S. (Eds), 1999 - Molecular tools
Acad. Sci., USA. 93: 1449-1452.
for screening biodiversity. Chapman and Hall, London.
AUTHORS
Massimo Labra (corresponding author), Dipartimento di Scienze dell'Ambiente e del Territorio, Piazza della Scienza 1, Universitd degh"
Studi di Milano - Bicocca, 20126 Milano, Italy
Candida Vannini, MarcellaBraeale, Dipartimento di Biologia Strutturale e Funzionale, Via J.H.Dunant 3, Universit~ degli Studi
delHnsubria, Varese, Italy
Francesco SaIa, Dipartimento di Biologia, Via Celoria 26, Universitd degli Studi di Milano, Milano, Italy