hc70al final presentation
DESCRIPTION
HC70AL Final Presentation. Chris McQuilkin June 4 th , 2009. Gene One: AT4G36540. A transcription factor involved in DNA binding A bHLH Gene “basic Helix-Loop-Helix” refers to the structure of the protein bHLH genes are found in Eukaryotes and are highly conserved across species. - PowerPoint PPT PresentationTRANSCRIPT
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HC70ALFinal Presentation
Chris McQuilkinJune 4th, 2009
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Gene One: AT4G36540
• A transcription factor involved in DNA binding
• A bHLH Gene – “basic Helix-Loop-Helix” refers to the
structure of the protein– bHLH genes are found in Eukaryotes and
are highly conserved across species
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AT4G36540What is the Structure of the Gene?
116 288 85 180 92 207 91 134 91 93 331
Exon 1 Exon 5 Exon 4Exon 3 Exon 2
Translational Start Codon
Translational Stop Codon
Intron 1 Intron 2 Intron 3 Intron 4
Total length= 1,714 base pairs
T-DNA Insertion Site
Forward Primer
Reverse Primer
LB
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Where is the Gene Active?
CM Arabidopsis RT-PCR Gene: AT4G36540 (Leaf and Silique cDNA)
ScD
NA
GD
NALcD
NA
10
0 bp
•Why is there no band in the positive control?
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RT-PCR Forward Primer
Forward Primer
Intron
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Where is the Gene Active?mRNA accumulation
0
500
1000
1500
2000
2500
Chala
zal S
eed
Coat/G
lobu
lar S
tage
Chala
zal S
eed
Coat/P
regl
obul
ar S
tage
Flor
al B
ud/R
epro
duct
ive
Leaf
/Veg
etat
ive
Ovule
/Rep
rodu
ctive
Seed
/24-
Hr Pos
t-Fer
tiliza
tion
Seed
ling/
3 Day
s Afte
r Im
bibi
tion
(3DAI)
Stem
/Veg
etat
ive
Seed
/Pre
glob
ular
Sta
ge
Plant region/ stage of development
mR
NA
Acc
um
ula
tio
n
Series1
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Where is the Gene Active?Promoter Cloning
Strategy of Promoter ActivityStrategy of Promoter ActivityAnalysisAnalysis
Arabidopsis Genomic DNA
•PCR amplification of upstream region
•With Gene-specific Primers
•And High Fidelity DNA Polymerase
PCR Product pENTR/D-TOPO vector
Ligation: Population of Recombinant Plasmid(vector+PCR product) and NON-recombinant plasmid(vectory only)
Transformation of competent E.coli cells
Screening for E.coli cells harboring recombinantplasmid
Confirmed Recombinant plasmid DNA: Verifying the authenticity ofrecombinant plasmid DNA by Restriction Enzyme Digestion
DNA sequences: verification of the clonedPromoter Region by Sequencing Analysis.
Sequence Analysis and confirmed identity of thecloned upstream region
Recombinant Plasmid DNA + Beta-Gluronidase (GUS) gene carrying T-DNAVector
Krista Templeton and Auni Hovanesian, HC70AL 2008
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Promoter Cloning*Two fragments were amplified by PCR
PCR of AT4G3540 Promoter Region
1 hr 120 volts
1 k
b La
dd
er
iPro
of
Poly
mera
se
Positiv
e C
on
trol
~1.7 kb= Expected Size of PCR-amplified promoter~0.8 kb= Unexpected PCR product
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Expected Plasmid alone= 2.5 kb
Expected Plasmid + Promoter= 4.2kb
Observed= 2.5kb and 3.5 kb
Promoter Cloning
AscI-Digested pENTR Plasmid DNA From Six E. coli Colonies
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Gene one: Genotyping
1 hr, 120 volts
Wild-type Control
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04/16/2009
1 hr, 120 volts
Gene one: Genotyping
Hom
ozy
go
us
Hem
izygous
Hem
izygou
s Hem
izygous
What is the expected size of the T-DNA band?
887 Bases
What is the expected size of the Wild-Type Band?
~250 bases
~250 base pairs
~900 base pairs
~1000 base pairsWhy are there two bands in the Lanes 1 and 3?
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Concatamers
119 288 85 180 92 207 91 134 91 93 334
Exon 1 Exon 5 Exon 4Exon 3 Exon 2
Translational Start Codon
Translational Start Codon
Intron 1 Intron 2 Intron 3 Intron 4
116 288 85 180 92 207 91 134 91 93 331
Exon 1 Exon 5 Exon 4Exon 3 Exon 2
Translational Start Codon
Translational Start Codon
Intron 1 Intron 2 Intron 3 Intron 4
T-DNA Insertion Site
Forward Primer
Reverse Primer
LB LB
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Nomarski Observation
Mutant Embryo and wild-type embryo show no phenotypic differences
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Nomarski Observation
Mutant seed coat and wild- type seed coat show no phenotypic differences
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Results
• Three hemizygous and one homozygous T-DNA plants were identified– Two bands were observed in each of the
lanes containing DNA with a T-DNA insert
• Gene AT4G36540 mRNA accumulation was observed in both the leaf and silique of Arabidopsis
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• The promoter did not insert into any of the E. coli plasmids that were screened.– Four of the colonies contained an unidentified
insert about 1 kb long
• No phenotypic changes were observed in either the hemizygous or homozygous T-DNA Arabidopsis plants using Nomarski Observation
Results
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Conclusion
• AT4G36540 is not lethal, or there may be another DNA sequence that codes for the same protein
• Although no phenotypic differences were observed in the seed coat or embryo using Nomarski, there could be differences that are more subtle or appear at different stages of development
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What next?
• Look further for phenotypic changes in Arabidopsis mutants—examine different tissues and more stages of development
• Sequence the unidentified DNA fragment taken up by the E. coli C600 plasmids
• Repeat PCR of the promoter– Excise the band containing recombined plasmid DNA, and
repeat transformation– If a promoter is identified, use GUS to study where the gene is
expressed• Grow more plants from the identified mutant lines• Repeat sequencing reaction of region between LB and
T-DNA primers to confirm location of the T-DNA insert
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Gene Two: AT1G75240
• A homeobox gene
• Homeobox genes regulate development and cell differentiation– A homeobox is a sequence
• They are found in animals, plants, and fungi
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AT1G75240What Is The Structure of the Gene?
330 77 97 927 254
Translational Start Codon
Translational Stop Codon
Forward Primer Reverse Primer
Total Length: 1,685 Base Pairs
Predicted T-DNA Insert
LB
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Where is the Gene Active?
1 hr 120 volts
AT1G75240 mRNA in Arabidopsis Leaf and Silique
Leaf c
DN
A
Siliq
ue c
DN
A
Gene is active in the silique but not the leaf
Tubulin mRNA AT1G7524
0 mRNA
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Where is the Gene Active?
mRNA Accumulation of AT1G75240
0500
100015002000250030003500
Em
bryo
Pro
per/
Hea
rt S
tage
Em
bryo
Pro
per/
Mat
ure
Ovu
le/R
epro
duct
ive
See
d/C
otyl
edon
Sta
ge (
7-8D
AP
)
See
d/M
atur
e G
reen
Sta
ge
See
d/P
ost-
Mat
ure
Gre
en S
tage
(18
-
Developmental Stages
Ave
rag
e m
RN
A A
ccu
mu
lati
on
Series1
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Genotyping
Arab
ido
psis 1
Arab
ido
psis 8
Arab
ido
psis 2
Arab
ido
psis 7
Arab
ido
psis 6
Arab
ido
psis 5
Arab
ido
psis 4
Arab
ido
psis 3
Arab
ido
psis 9
Arab
ido
psis 10
Arab
ido
psis 11
Arab
ido
psis 12
WT
Co
ntro
l
100 bp
100 bp
LB
b1 C
on
trol
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Results
• All the plants screened were wild-type
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Conclusion
• The chance of getting all wild-type plants is extremely low, so it is possible that the Salk Institute sent the wrong kind of plant
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Thank you!
• Anhthu Bui• Brandon Chen• Bob Goldberg • Daisy Robinton• Ingrid Nelson• Kristin Gill• Min Chen
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SRB Contig Sequencing
Contig Length: 50.7 KB
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Approach
• Contig entered into three online databases that look for predicted genes: FGENESH, GENSCAN, and GeneMark
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396-507 999-10921324-1428
-2086 2126-2351 2990
1901-
281- 427 459- 572
1 2,000
2,001 4,000
628- 729 759-846 999-1097 1134-1642 1843-
2894 3046
-2550
240 396 - 507 1134 - 1442 1901 -
-2550 3272
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Approach
• Predicted Genes were entered into a BLAST search to see if the predicted gene matched Expressed Sequence Tags (ESTs) found in other species
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• Whole Contig was searched for DNA repeats
Approach
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Results: GENSCANPredicted Gene (BLASTN)
Accession Number E-value Description
1 BP033723.1 1e-110 Lotus Japonicus
2 FN014331.1 1e-52 Petunia axillaris subsp. axillaris pool of root and petal tissue
3 GO259467 3e-26 Tissue from peanut Arachis hypogaea
4 EX522455.1 0.0 Trichome from stem of Medicago sativa
5 FK023593.1 8e-77 cDNA from Glycine max (soy bean)
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Results: GENSCANPredicted Protein (tBLASTn) Accession Number E-value Description
1 Y18169 5e-70 Pisum sativum mitochondrial ccb248 gene and partial rps7 gene.
2 FM179380 2e-10 Vitis vinifera complete mitochondrial genome, cultivar Pinot noir clone ENTAV115
3 L40816 3e-35
4 DQ008791 5e-60 Thottea tomentosa large subunit ribosomal RNA gene, partial sequence; mitochondrial
5 AC144406 4e-14 Medicago truncatula clone mth2-5h18, complete sequence.
Predicted Protein (BLASTp)
4 YP_173352 2e-41 hypothetical protein NitaMp002 [Nicotiana tabacum].
5 No significant match
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Results: FGENESHPredicted Gene (BLASTn)
Accession Number
E-value Description
1 EX527626 0.0 MTGland_A031_2007-05-22/MTGlandA031_B09_039_1 Medicago truncatula A17 glandular trichome Medicago truncatula cDNA, mRNA sequence
2 CD721224 2e-25 Chardonnay Vitis vinifera
3 GD544655 1e-40 Scarlet Runner Bean globular-stage suspensor region
4
5 FN014331 9e-88 Pool of root and petal tissue of Petunia axillaris
6 FF554629.1 9e-143 Vigna Unguiculata
Predicted Protein (tBLASTn)
1 Y18169 18e-118 Pisum sativum mitochondrial ccb248 gene and partial rps7 gene.
6 AC144406 8e-18 Medicago truncatula clone mth2-5h18, complete sequence
Predicted Protein (BLASTp)
1 CAB43024 2e-118 cytochrome c biogenesis protein [Pisum sativum]
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Results: GeneMarkPredicted Protein (tBLASTn)
Accession Number (of Lowest E-Value)
E-value Description
1 AC192958 1e-13 Medicago truncatula chromosome 2 BAC clone mte1-45m19, complete sequence
2 AC147537 1e-23 Medicago truncatula clone mth2-133k2, complete sequence.
3 EX527626 3e-113 Glandular trichome Medicago truncatula
13 BA000042 1e-18 Nicotiana tabacum mitochondrial DNA, complete genomeLength=430597
15 L40816 5e-25 Glycine max mitochondrion polymorphic marker DNA sequence.
16 L40816 7e-46 Glycine max mitochondrion polymorphic marker DNA sequence.
17 AC145156 1e-50 Medicago truncatula clone mth2-7h6, complete sequence.
18 DQ647831 1e-108 Chlorokybus atmophyticus large subunit ribosomal RNA gene, partial
sequence; mitochondrial.
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Results: Gene Mark19 BA000042 1e-41 Nicotiana tabacum
mitochondrial DNA, complete genome.
20 AP004975 1e-26 Lotus japonicus genomic DNA, chromosome 5, clone: LjT21J12, TM0158, complete sequence.
21 AP004975 7e-17 Lotus japonicus genomic DNA, chromosome 5, clone: LjT21J12, TM0158, complete sequence.
23 XM_002336124 2e-12 Populus trichocarpa predicted protein, mRNA.
32 FM179380 5e-29 Vitis vinifera complete mitochondrial genome, cultivar Pinot noir clone ENTAV115.
33 AC144406 7e-29 Medicago truncatula clone mth2-5h18, complete sequence.