probing plant gene function with transposons july 12th, 2000
TRANSCRIPT
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Probing Plant Gene Function With Transposons
July 12th, 2000
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Genome sequencing assignments
CSHSC
ESSA
KazusaGenoscope
TIGR
SPP
I II III IV VKazusa
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• Only 1 in 20 genes have an assigned genetic role
• Systematic determination of gene expression and genetic
function is a major goal of the Arabidopsis Genome Project
Functional Genomics
Zn-finger
kinase
CLL E(z)
hexose transporter
prolifera
AtKAP Zn-fingerMutL
SAR1CTP
synthase
= known gene
= cDNA/EST
= hypothetical protein
= pseudogene
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Genetraps Gene traps are reporter genes that integrate
at random in the plant genome
Gene traps simultaneously monitor gene expression and gene function
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Genetrap transposons
reporter gene
intron
positivemarker
plant gene fusion protein
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Random Insertions
negative marker
positive marker
reporter gene
selection
transposition
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Epidermal cells
Reporter gene expression mimics expression of the plant gene•Guard cells•Unbranched trichomes
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Meristem cells
Root meristem (columella initials)
Shoot meristem (rib zone)
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Thermal Assymetric Interlaced (TAIL) PCR
nested anchorsarbitrary degenerate primer
TAIL product
Reference:Liu et al., 1995 Plant J. 8, 457
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Genetrap database
1. Sequence insertion sites
2. Stain for reporter expression
3. Functional screens
GeneTrap database
GeneTrapTransposants
Genome sequence
(map position)
Arabidopsis ESTs
Crop ESTs
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Profile for ET 79Parent GenotypeSequenceCharacterizationExpression Data
ET 79Date Sequenced: 4/14/1996Primer: DS5-4NLength: 137
Orientation of Insertion
Upstream of gene(5')
Annotation
gb|AC002291|AC002291 Arabidopsis thaliana chromosome I BAC F22K20 genomic sequence, Score = 83.8 bits (42), Expect = 4e-15 Identities = 45/46 (97%), Positives = 45/46 (97%)
1.7kb upstream of F22K20.10 (annotated "unknown")GRR1 homolog with LRR repeatscdk2 associated p45/skp2 protein homolog
(in 6kb gap between genes)
Sequence
TAATAAAACNGTTATACGATAACGGTCGGTACGGGATTTCCCATCCTACTTTCATCCCTGCTCATTAGCCCCCAATGTGCATTTGTGTTATCTTTACCAGTTGCGTGTAANCTTCTGTGTATTNCTNCTTCTCACTC
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Summary of Genetrap tags
3726 insertion sites sequenced• 3292 annotated gene hits• 74 transcription factors• 211 kinases (114 receptors)• 53 disease resistance genes• 80 retrotransposons
www.cshl.org/genetrap
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Genetraps in genome biology
• Mapping insertions on chromosomes
• Targets for regulatory genes
• Secretion traps
• Lethal genes
• Redundant genes
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Cytogenetic map of chromosome 4S
NOR
knob
cen
3Mb
2Mb
0.5Mb
2Mb
0.5Mb
Paul Fransz
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Transposon distribution
3Mb Bac contig 1874 genetrap insertions106 on chromosome 4S
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250500
7501000
12301500
17502080
0
10
20
30
40
50
60
DsE/G
transcribed genes
total genes
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Dandelion alters dorsoventral patterning
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cycloidea-like
ET 3964
ET3964ET3964
ET3964 is expressed dorsally
500bp
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ET3964 is over expressed in dnd1-2 plants
AGO CYC-like dorso/ventral patterning??
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GUS Inhibition by Glycosylation
35S
SignalSequence
Alpha amylase GUS
Asn-358
No treatment 20uM Tunicamycin
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Secretion Trap
- Tunicamycin + Tunicamycin
Cytosolic Protein
- Tunicamycin + Tunicamycin
Secreted or Plasma Membrane-Spanning Protein
SignalSequence
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Minus Tunicamycin Plus Tunicamycin
GT7487 secreted hypothetical
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GT6249 hypothetical
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Genes Tagged by Secretion Trap Screen
GT6666 Glutathione conjugate ABC transporter GT7137 Beta-gylcosidase
GT5376 Seven-pass receptorGT6114 Receptor kinase GT6224 Receptor kinase
GT7059 Predicted transmembrane protein with VWF domainGT7134 Hypothetical transmembrane protein with armadillo repeats
GT6943 Similar to aminopeptidase P GT7106 Serine protease with predicted secretion signal
GT5211 Unknown protein with predicted secretion signalGT7243 Unknown protein with predicted secretion signalGT7487 Unknown protein with predicted secretion signal GT5397 “Between genes”
2059 Gene traps screened464 Stain (23%)33 Secreted (7% of stain)
GT5397GT5397
GT6224GT6224
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PROLIFERA is expressed in dividing cells
In heterozygote plants, the PROLIFERA gene is expressed in dividing cells throughout the plant - not just in embryos
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Essential Genes• 2000 Arabidopsis genes are essential for cellular function
• Early lethality obscures role later in development
• gene expression patterns reveal function
SCN1
BRCA2
tRNA
ribosome protein S14
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PROLIFERA is an essential geneGenetrap insertions in the PROLIFERA gene are lethal in homozygous embryos, and haploid megagametophytes
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PROLIFERA encodes an MCM7 protein
PROLIFERA encodes a homolog of the DNA replication licensing factor CDC47 (MCM7)
Gene fusions with the Green Fluorescent Protein (GFP) are localized in the nucleus during the cell cycle
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PROLIFERA encodes an MCM7 protein
PROLIFERA encodes a homolog of the DNA replication licensing factor CDC47 (MCM7)
Gene fusions with the Green Fluorescent Protein (GFP) are localized in the nucleus during the cell cycle
QuickTime™ and aQuickDraw decompressor
are needed to see this picture.
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Reporter gene expression is found in the carpel wall and inflorescence meristem
AGL8 ET3214 interrupts the untranslated leader of the MADS box gene AGL8
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AGL8 Gene FunctionLoss of AGL8 function affects•carpel wall differentiation
The AGL8 gene was renamed FRUITFULL (ful1)
•fruit elongation•fruit shattering
+
+
ful
ful
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FRUITFULL-related genesFRUITFULL is expressed in the inflorescence meristem, but the meristem is normal in fruitfull mutants
AGAMOUS
AGL1
AGL5 AGL7 (APETALA1)
AGL8 (FRUITFULL)
AGL10 (CAULIFLOWER)
APETALA1 and CAULIFLOWER are closely related to FRUITFULL , and also expressed in the floral meristem
Are these three genes redundant with respect to meristem function?
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Genetic Redundancy
apetala1 cauliflower double mutants have proliferating floral meristems ressembling cauliflowers
ap1 cal ful triple mutants have flowers replaced by shoots
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Genetic Redundancy
• Many genes in Arabidopsis have one or more close homologs in Arabidopsis
• Disruption of most individual genes in Arabidopsis has no obvious effect on growth and development
• Gene redundancy may be the primary cause
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CreditsCatherine KidnerMary ByrneAndrew GrooverBruce MayCristy YordanJoe SimorowskiAnna-Marie ArroyoRulan ShenJohn Healy
Thanks to:
Shiv GrewalCaroline DeanScott PoethigKathy BartonSarah HakeMarty YanofskyMarja TimmermansAndrew HudsonDick McCombiePaul Fransz
Former lab members:
Patty SpringerQing GuMark CurtisMaitreya DunhamBen Lehner