presentación de powerpoint · molecular markers (physiological, genetic or epigenetic hallmarks)...
TRANSCRIPT
AGEING, A BARRIER IN FORESTRY PRODUCTION
↓ ↓ ↓ CLONAL MULTIPLICATION CAPACITY
COMPLEX ARCHITECTURE↓ ROOTING↓ MORPHOGENIC ABILITY↓ IN VITRO ESTABLISHMENT
REPRODUCTIVE GROWTH
↑↑ ↑ CLONAL MULTIPLICATION CAPACITY
SIMPLE ARCHITECTURE↑ ROOTING
↑MORPHOGENIC ABILITY↑↑ IN VITRO ESTABLISHMENT
VEGETATIVE GROWTH
Characterization Of Developmental PhasesCharacterization Of Developmental Phases
NEW STRATEGIES FOR IMPROVING HIGH-QUALITY PLANT PRODUCTION FROM MATURE
SELECTED TREES
REPRODUCTIVE MATURE
PHASE-CHANGE
VEGETATIVE MATURE
JUVENILE
EMBRYOGENIC
REINVIGORATION
MOLECULAR MARKERS (Physiological, Genetic Or Epigenetic Hallmarks) OF AGEING AND REINVIGORATION
UNDERSTANDING OF THE MOLECULAR BASIS OF AGEING AND REINVIGORATION
OPTIMIZATION OF BREEDING PROGRAMS
GENE REGULATION, EPIGENETICS
GENOMIC MAPINGGENE SEQUENCIATION
QTLs
GENE EXPRESSION
PROFILESPHYSIOLOGY
POLYAMINES AS PHYSIOLOGICAL MARKERS
Fraga M.F., Berdasco, M., Diego, L.B., Rodríguez, R yCañal, M.J. 2004. Changes of polyamine content associated with Pinus radiata D.Don and Prunus persica [L.]Bascth“Hakuho” ageing. Tree Physiology, en prensa.
Fraga M.F, Rodríguez, R. y Cañal M.J. 2003. Reinvigoration of Pinus radiata is associated with partial recovery of juvenile-like polyamine concentrations. Tree Physiology 23: 205-209.
Fraga M.F., Cañal, M.J. y Rodríguez, R. 2002. Phase-change related epigenetic and physiological changes in Pinus radiataD. Don. Planta 215: 672-678.
Fraga M.F., Cañal, M.J. y Rodríguez, R. 2002. In vitromorphogenic potential of differently aged Pinus Radiata D. Don. trees correlates with polyamines and DNA methylation levels. Plant Cell, Tissue and Organ Culture 70: 139-145.
AGEING
REINVIGORATION
PHASE-CHANGE
IN VITROBEHAVIOUR
EPIPHYSAGE Group
EPIGENETIC MARKERS, DNA METHYLATION
Fraga, M.F., Uriol, E., Diego L. B., Berdasco, M., Esteller, M., Cañal, M. J. y Rodriguez, R. 2002. High performance capillary electrophoretic method for the quantification of 5-methyl 2’-deoxycytidine in genomic DNA: Application to plant, animal and human cancer tissues. Electrophoresis 23: 1677-1681.
Fraga, M.F., Rodriguez, R. y Cañal, M. J. 2000. Rapid quantification of DNA methylation by high performance capillary electrophoresis. Electrophoresis 54 :2990-2994.
Fraga M.F, Rodríguez, R. y Cañal M.J. 2002. Genomic DNA methylation-demethylation during aging and reinvigoration of Pinus radiata. Tree Physiology 22: 813-816.
Fraga M.F., Cañal, M.J. y Rodríguez, R. 2002. Phase-change related epigenetic and physiological changes in Pinus radiata D. Don. Planta 215: 672-678.
QUANTIFICATION METHOD
QUANTIFICATION METHOD
AGEINGREINVIGORATION
PHASE-CHANGE
GENETIC MARKERS, RPT5 ATPase
Diego, L.B., Berdasco, M., Fraga M.F., Cañal, M.J., Rodríguez, R. y Castresana, C. 2004. A Pinus radiata AAA-ATPase, the expression of which increases with tree maduration. Journal of Experimental Botany 55 (402):1597-1599.
AGEING REINVIGORATION
EPIPHYSAGE Group
J APolyacrilamide
Gel
ds-cDNA
TTTTTTTTTTT-3´5´3´ 5´P PRIMER
5´5´3´
3´T PRIMER
AAAAAAAAAA-3´5´TTTTTTTTTTT-3´3´
RNA-DNAhybrid
First strand synthesis(MMLV retrotranscriptase)
Reamplification of cDNAS (PCR)
AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´AAAAAAAAAA-3´5´
AAAAAAAAAA-3´5´
Juvenile Sample Adult SampleRNA isolation
(Dong & Dunstan, 1996)
DNAase I treatment
3 low stringency cycles (T. Anneal. = 40°C)
25 high stringency cycles(T. Anneal. = 60°C)
PCR
J APolyacrilamide
Gel
J APolyacrilamide
Gel
ds-cDNAds-cDNA
TTTTTTTTTTT-3´5´3´ 5´P PRIMER
TTTTTTTTTTT-3´5´3´ 5´
TTTTTTTTTTT-3´5´3´ 5´P PRIMER
5´5´3´
3´T PRIMER
5´5´3´
3´T PRIMER
5´5´3´
3´T PRIMER
AAAAAAAAAA-3´5´TTTTTTTTTTT-3´3´
RNA-DNAhybrid
First strand synthesis(MMLV retrotranscriptase)
AAAAAAAAAA-3´5´ AAAAAAAAAA-3´AAAAAAAAAA-3´5´TTTTTTTTTTT-3´3´ TTTTTTTTTTT-3´3´
RNA-DNAhybrid
First strand synthesis(MMLV retrotranscriptase)
Reamplification of cDNAS (PCR)
Reamplification of cDNAS (PCR)
AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´AAAAAAAAAA-3´5´
AAAAAAAAAA-3´5´
Juvenile Sample Adult SampleRNA isolation
(Dong & Dunstan, 1996)AAAAAAAAAA-3´5´ AAAAAAAAAA-3´AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´ AAAAAAAAAA-3´AAAAAAAAAA-3´5´AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´
AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´
Juvenile Sample Adult SampleRNA isolation
(Dong & Dunstan, 1996)
DNAase I treatmentDNAase I treatment
3 low stringency cycles (T. Anneal. = 40°C)
25 high stringency cycles(T. Anneal. = 60°C)
PCR
3 low stringency cycles (T. Anneal. = 40°C)
25 high stringency cycles(T. Anneal. = 60°C)
PCR
DIFFERENTIAL DISPLAY-RTPCR
F1 oripGEM®-T Easy Vector
(3015 pb) Apa IAat IISph IBstZ INco IBstZ INot ISac IIEcoR ISpe IEcoR INot IBstZ IPst ISal INde ISac IBstX INsi I
T7
SP6
+ cDNApGEM®-T
Easy
LIGATION
E.coli DH5competentes
TRANSFORMATION
LB-Amp
X-galSELECTIONMULTIPLICATIONMINIPREPS
AUTOMATICSEQUENCING
F1 oripGEM®-T Easy Vector
(3015 pb)
F1 oripGEM®-T Easy Vector
(3015 pb)pGEM®-T Easy Vector
(3015 pb) Apa IAat IISph IBstZ INco IBstZ INot ISac IIEcoR ISpe IEcoR INot IBstZ IPst ISal INde ISac IBstX INsi I
T7
SP6
Apa IAat IISph IBstZ INco IBstZ INot ISac IIEcoR ISpe IEcoR INot IBstZ IPst ISal INde ISac IBstX INsi I
T7T7
SP6SP6
+ cDNApGEM®-T
Easy
LIGATION+ cDNA
pGEM®-T Easy
LIGATION
E.coli DH5competentes
TRANSFORMATION
E.coli DH5competentes
TRANSFORMATION
LB-Amp
X-galSELECTION
LB-Amp
X-galSELECTIONMULTIPLICATIONMULTIPLICATIONMINIPREPSMINIPREPS
AUTOMATICSEQUENCINGAUTOMATICSEQUENCING
cDNA CLONING AND SEQUENCING
GENE EXPRESSION PROFILES IN
JUVENILE AND ADULT P. radiata
NEEDLES
PLANT MATERIAL
CANDIDATE GENES
pGEMT
cDNAs
cDNA INFERRED PROTEIN BEST ALINEATION
Pr β284 A1 Apoprotein, Photosystem I 93% Pinus thumbergii
Pr β286 P Glycoprotein 69% Arabidopsis thaliana
Pr β288 ATPase RPT5, 26S proteasome 95% Arabidopsis thaliana
Pr β381 Enoyl-ACP reductase 73% Nicotiana tabacum
Pr β382 Disulfide isomerase 75% Prunus avium
A P. radiata AAA-ATPase WHICH EXPRESSIONINCREASES WITH TREE MATURATION
Viveros Oihamberri, Vitoria (Spain).
Juvenile Trees SystemOpen pollinization
Selected Clones
Adult Trees SystemOpen pollinization
Selected Clones
PHASE-CHANGE VALIDATION
MACROGRAFTTEMPORALEVOLUTION
(1 month – 3 years)
CONSECUTIVEGRAFT
CONSECUTIVEGRAFT
NORMAL DEVELNON FLOWERING,CLONAL
MULTIPLICATION,VERY LOW RPT5 EXPRESSION
JUVENILE-LIKE
FLOWERING,LOW MORPHOGENIC
POTENTIALHIGH RPT5 EXPRESSION
ADULT FloweringBranches (A1)
FloweringBranches (A1)
EpicormicShoot (ES)
EpicormicShoot (ES)
Non FloweringBranches (A2)
Non FloweringBranches (A2)
REINVIGOR.
Juvenile Trees SystemOpen pollinization
Selected Clones
Adult Trees SystemOpen pollinization
Selected Clones
PHASE-CHANGE VALIDATION
Juvenile Trees SystemOpen pollinization
Selected Clones
Adult Trees SystemOpen pollinization
Selected Clones
PHASE-CHANGE VALIDATION
MACROGRAFTTEMPORALEVOLUTION
(1 month – 3 years)
CONSECUTIVEGRAFT
CONSECUTIVEGRAFT
NORMAL DEVELNORMAL DEVELNON FLOWERING,CLONAL
MULTIPLICATION,VERY LOW RPT5 EXPRESSION
JUVENILE-LIKE
FLOWERING,LOW MORPHOGENIC
POTENTIALHIGH RPT5 EXPRESSION
ADULT FloweringBranches (A1)
FloweringBranches (A1)
EpicormicShoot (ES)
EpicormicShoot (ES)
Non FloweringBranches (A2)
Non FloweringBranches (A2)
NON FLOWERING,CLONAL
MULTIPLICATION,VERY LOW RPT5 EXPRESSION
JUVENILE-LIKE
FLOWERING,LOW MORPHOGENIC
POTENTIALHIGH RPT5 EXPRESSION
ADULT
NON FLOWERING,CLONAL
MULTIPLICATION,VERY LOW RPT5 EXPRESSION
JUVENILE-LIKE
NON FLOWERING,CLONAL
MULTIPLICATION,VERY LOW RPT5 EXPRESSION
JUVENILE-LIKE
FLOWERING,LOW MORPHOGENIC
POTENTIALHIGH RPT5 EXPRESSION
ADULT
FLOWERING,LOW MORPHOGENIC
POTENTIALHIGH RPT5 EXPRESSION
ADULT FloweringBranches (A1)
FloweringBranches (A1)
EpicormicShoot (ES)
EpicormicShoot (ES)
Non FloweringBranches (A2)
Non FloweringBranches (A2)
REINVIGOR.
Diego et al. (2004) Journal of Experimental Botany 55(402):1597-1599
Cytokinin ResponsesCytokinin Responses
CRE1CK
ARR5
CYDC3
NIA1
Activator
GROWTH RESPONSES
RPN12a
Stress ResponsesStress Responses
REGULATORYPROTEOLYSISREGULATORYPROTEOLYSIS
Cell Cycle ControlCell Cycle Control
SEXUAL MATURATION(Physcomitrella,, Lilium)
SEXUAL MATURATION(Physcomitrella,, Lilium)
TISSUEDIFFERENTIATION
(Zynnia)
TISSUEDIFFERENTIATION
(Zynnia)
Auxin ResponsesAuxin Responses
REPRESOR DEGRADATIONGENE EXPRESSION CONTROLREPRESOR DEGRADATION
GENE EXPRESSION CONTROL
Degradationof IAA /AUX
proteins
MORPHOGENIC ABILITIES
Cytokinin ResponsesCytokinin Responses
CRE1CK
ARR5
CYDC3
NIA1
Activator
GROWTH RESPONSES
RPN12a
Cytokinin ResponsesCytokinin Responses
CRE1CK
CRE1CK
ARR5
CYDC3
NIA1
Activator
GROWTH RESPONSES
RPN12aRPN12aRPN12a
Stress ResponsesStress Responses
REGULATORYPROTEOLYSISREGULATORYPROTEOLYSIS
Cell Cycle ControlCell Cycle Control
Stress ResponsesStress Responses
REGULATORYPROTEOLYSISREGULATORYPROTEOLYSIS
Cell Cycle ControlCell Cycle Control
SEXUAL MATURATION(Physcomitrella,, Lilium)
SEXUAL MATURATION(Physcomitrella,, Lilium)
TISSUEDIFFERENTIATION
(Zynnia)
TISSUEDIFFERENTIATION
(Zynnia)
SEXUAL MATURATION(Physcomitrella,, Lilium)
SEXUAL MATURATION(Physcomitrella,, Lilium)
TISSUEDIFFERENTIATION
(Zynnia)
TISSUEDIFFERENTIATION
(Zynnia)
Auxin ResponsesAuxin Responses
REPRESOR DEGRADATIONGENE EXPRESSION CONTROLREPRESOR DEGRADATION
GENE EXPRESSION CONTROL
Degradationof IAA /AUX
proteins
MORPHOGENIC ABILITIES
Auxin ResponsesAuxin Responses
REPRESOR DEGRADATIONGENE EXPRESSION CONTROLREPRESOR DEGRADATION
GENE EXPRESSION CONTROL
Degradationof IAA /AUX
proteins
MORPHOGENIC ABILITIES
26S PROTEASOME FUNCTIONS IN PLANTS
Proteasomicproteolysis
Auxin reception
IAA/AUXProteins
Messengers
Efectors
Primary Response
SHORT-TERM EFFECTS
SHORT-TERM EFFECTS
MIDDLE- AND LONG-TERM EFFECTS
MIDDLE- AND LONG-TERM EFFECTS
RPT5
ES A1 A2
PROTEOLYTICCOMPETENCE
MODULATION?
PROTEOLYTICCOMPETENCE
MODULATION? Proteasomicproteolysis
Proteasomicproteolysis
Proteasomicproteolysis
Auxin reception
Auxin reception
IAA/AUXProteins
IAA/AUXProteins
IAA/AUXProteins
MessengersMessengers
Efectors
Primary Response
SHORT-TERM EFFECTS
SHORT-TERM EFFECTS
MIDDLE- AND LONG-TERM EFFECTS
MIDDLE- AND LONG-TERM EFFECTS
EfectorsEfectorsEfectors
Primary ResponsePrimary ResponsePrimary ResponsePrimary Response
SHORT-TERM EFFECTS
SHORT-TERM EFFECTS
MIDDLE- AND LONG-TERM EFFECTS
MIDDLE- AND LONG-TERM EFFECTS
MIDDLE- AND LONG-TERM EFFECTS
MIDDLE- AND LONG-TERM EFFECTS
RPT5
ES A1 A2
PROTEOLYTICCOMPETENCE
MODULATION?
PROTEOLYTICCOMPETENCE
MODULATION? RPT5
ES A1 A2
PROTEOLYTICCOMPETENCE
MODULATION?
PROTEOLYTICCOMPETENCE
MODULATION? RPT5
ES A1 A2
RPT5RPT5RPT5
ES A1 A2ES A1 A2
PROTEOLYTICCOMPETENCE
MODULATION?
PROTEOLYTICCOMPETENCE
MODULATION?
IS PROTEASOME MODULATING
CELLULAR COMPETENCE?
EXPRESSION LEVELS OF RPT5 homologue in Pinus radiata
Molecular marker of ageing and reinvigoration
Does it perform a role in competence modulation auxin-
dependent?
EPIGENETIC FACTORS
DNADNAiRNAs
5-
PROTEINSPROTEINSNON- HISTONES ALTERATIONS
HISTONES MODIFICATIONS:
- ACETYLATION
- METHYLATION
- PHOSPHORYLATION
-
DNA 5-
PROTEINS
-
-
-
-
-
WHICH INFLUENCES:TRANSCRIPTION,
REPLICATION,REPAIR
AND RECOMBINATION PROCESSES
METHYLCYTOSINE
UBIQUITINATION, ...
EPIGENETIC FACTORS
DNA iRNAs
PROTEINSNON HISTONES ALTERATIONS
HISTONES MODIFICATIONS:
ACETYLATION
METHYLATION
PHOSPHORYLATION
CONFORMATIONCHROMATIN
EPICORMICSHOOT
MATURE VEGETATIVE
MATURE REPRODUCTIVE
1-MONTH-OLD
NEEDLES (b1)
<1-MONTH-
OLDNEEDLES
(b0)
APICAL
BASAL
APICAL
BASALM
AT
UR
AT
ION
MA
TU
RA
TIO
N
MA
TU
RA
TIO
N
ONTOGENIC DEVELOPMENT
ONTOGENIC DEVELOPMENT
ONTOGENIC DEVELOPMENT
ONTOGENIC DEVELOPMENT
ARE HISTONE ACETYLATION LEVELS STABLE DURING AGEING AND REINVIGORATION? (If so, can AcH4 act as a molecular marker?)
MOLECULAR BASIS OF MATURATION AND DIFFERENTIATION
H4
HISTONE H4 TAIL MODIFICATIONS
HISTONE ACETYLATION DURING AGEING AND CHANGES DURING REINVIGORATION
DAPI AcH4
DAPI AcH4
ACETYLATED HISTONE H4
INMUNOLOCATION IN MATURE
VEGETATIVE (top) AND MATURE
REPRODUCTIVE (bottom) SHOOTS
DAPI + AcH4
DAPI
AcH4
ACETYLATED HYSTONE H4 INMUNOLOCATION IN
EPICORMIC SHOOT
LEVELS OF ACETYLATED H4 IN Pinus radiata NEEDLES ARE AGEING-DEPENDENT
GLOBAL HISTONE H4 ACETYLATION STRONGLY DECREASES WITH ONTOGENIC AGE.
REINVIGORATION COURSES WITH INCREASES IN ACETYLATED H4 LEVELS.
INMUNOLOCATION* Results are given as acetylation index (%).
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
APICAL BASAL APICAL BASAL
48
93 40 39
60 50 28 19
100
8 25 15
* Results are given as band intensity
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
34
34
34
34
34
34
* Results are given as acetylation index (%).
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
APICAL BASAL APICAL BASAL
48
93 40 39
60 50 28 19
100
8 25 15
* Results are given as band intensity
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
34
34
34
34
34
34
* Results are given as acetylation index (%).
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
APICAL BASAL APICAL BASAL
48
93 40 39
60 50 28 19
100
8 25 15
* Results are given as band intensity
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
34
34
34
34
34
34
* Results are given as acetylation index (%).
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
APICAL BASAL APICAL BASAL
48
93 40 39
60 50 28 19
100
8 25 15
* Results are given as acetylation index (%).
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
APICAL BASAL APICAL BASAL
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
APICAL BASAL APICAL BASAL
48
93 40 39
60 50 28 19
100
8 25 1548
93 40 39
60 50 28 19
100
8 25 15
* Results are given as band intensity
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
34
34
34
34
34
34
* Results are given as band intensity
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
INMUNOBLOTTING* Results are given as band intensity
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
1-MONTH-OLDNEEDLES (b1)
< 1-MONTH-OLDNEEDLES (b0)
1
0,77
0,34
0,63
0,30
0,23
106 kD80 kD
49,5 kD
32,5 Kd
27,5 kD18,5 kD
AMID
OBLACKST
ANDARS
EPICORM
IC SH
OOT
106 kD80 kD
49,5 kD
32,5 Kd
27,5 kD18,5 kD
AMID
OBLACKST
ANDARS
EPICORM
IC SH
OOT
MOLECULAR MARKER OF
AGEING
ESMVMR
STRUCTURAL ANALYSIS OF
BASAL PORTIONS OF 1-MONTH-OLD
NEEDLES
Top : MATURE VEGETATIVE
Bottom : MATURE REPRODUCTIVE
MERISTEM (HIPODERMIS)
MERISTEM (EPIDERMIS)
MESOPHYLL OF PARENCHYMA CELLS
MERISTEM (ENDODERMIS)
PERICYCLE
FLOEM
NOMARSKY
NOMARSKY
NOMARSKY
SIEVE PORES (CALLOSE
DEPOSITION)
REPLICATION
GLOBAL ACETYLATED H4 IS PREFERENTELLY CORRELATED WITH REPLICATION RATHER THAN WITH TRANSCRIPTION.
Jasencakova et al. (2000),The Plant Cell 12:2087-2100.
IN DIFFERENTIATED CELLS MOST OF THE GENOME CONSISTS OF HYPOACETYLATED INACTIVE CHROMATIN, WHICH MAY BE CONSIDERED THE “GROUND”STATE.
NOMARSKY
OPTIC MICROSCOPY
MERISTEM
EPICORMIC SHOOT
STRUCTURE
MERISTEMATIC CELLS IN EPICORMIC SHOOTS
CELL DIVISION
CELL MATURATION IN MATURE NEEDLES
GLOBAL H4 HYPOACETYLATION
GLOBAL ACETYLATED HISTONE H4 LEVELS
Molecular marker of ageing and reinvigoration
Molecular basis of organ maturation/
differentiation
HOW DOES DNA METHYLATION ACT IN THESE PROCESSES? DOES SPECIFIC METHYLATION MODULATE AGE-, PHASE-CHANGE- AND REINVIGORATION-RELATED EVENTS?
GLOBAL DNA METHYLATION (HPCE)MOLECULAR MARKER of:
-Ageing
- Reinvigoration
-Phase-change
BUT...
TFPROMOTER CODING
SEQUENCENON CODING
SEQUENCECODING
SEQUENCENON CODING
SEQUENCE MICROSATELLITES
CH3 CH3
TF
TFTFPROMOTER CODING
SEQUENCENON CODING
SEQUENCECODING
SEQUENCENON CODING
SEQUENCE MICROSATELLITESPROMOTER CODINGSEQUENCE
NON CODINGSEQUENCE
CODING SEQUENCE
NON CODINGSEQUENCE MICROSATELLITES
CH3CH3 CH3CH3
TFTFTF
TFPROMOTER CODING
SEQUENCENON CODING
SEQUENCECODING
SEQUENCENON CODING
SEQUENCE MICROSATELLITES
CH3 CH3
TF
TFTFPROMOTER CODING
SEQUENCENON CODING
SEQUENCECODING
SEQUENCENON CODING
SEQUENCE MICROSATELLITESPROMOTER CODINGSEQUENCE
NON CODINGSEQUENCE
CODING SEQUENCE
NON CODINGSEQUENCE MICROSATELLITES
CH3CH3 CH3CH3
TFTFTF SITE-SPECIFIC METHYLATION
WHAT KIND OF CELL ARE PREFERENTLY METHYLATED? DOES METHYLATION AFFECT MERISTEM AND/OR DIFFERENTIATED CELLS?
INMUNOLOCATION OF 5-mC
INMUNOLOCATION OF 5-METHYLCYTOSINE IN BASAL PORTIONS OF 1-MONTH-OLD P. radiata NEEDLES
Top : MATURE VEGETATIVE
Bottom : MATURE REPRODUCTIVE
DAPI
DAPI
5-mC
5-mC
OPTIMIZATION OF BISULFITE GENOMIC SEQUENCING IN PLANTS
-ALKALINE DENATURATION
- DEAMINATION
-DESULFONATION
-NEUTRALIZATION AND DESALTING
-AMPLIFICATION (PCR)
-CLONING AND SEQUENCING
DNAMethyl-easy
Mario F.FragaCecilia FerreroManel Esteller
DNAMethyl-easy
Mario F.FragaCecilia FerreroManel Esteller
CpG ISLAND
DO PROMOTER OR FIRST INTRON SEQUENCES CONTAIN
ANY METHYLATION-SUSCEPTIBLE ISLAND?
IS POLYAMINE BIOSYNTHESIS REGULATED BY SPECIFIC- METHYLATION?
ADC2
ARGININE DECARBOXYLASE EXPRESSION IN Arabidopsis thaliana
IS NOT REGULATED BY PROMOTER METHYLATION
ADC1POLYAMINE BIOSYNTHETIC PATHWAYS
OIHANBERRI S.A.
Ignacio GarcíaAitor Telleria
UNIVERSITAT AUTÒNOMA DE BARCELONA
Antonio TiburcioTeresa Altabella
UNIVERSIDAD DE OVIEDOEPIPHYSAGE GROUP (www.uniovi.es)
Roberto RodríguezMª Jesús CañalMaría BerdascoL. Borja DiegoRodrigo HasbunLuis ValledorCarlos Noceda
NEIKER
Enrique RitterSantiago EspinelAna Aragonés
CENTRO DE INVESTIGACIONES BIOLOGICAS.-C.S.I.C
Mª Carmen RisueñoPilar TestillanoPablo González- Melendi
CNIOMario F. FragaManel Esteller