Download Probing Plant Gene Function With Transposons.

Probing Plant Gene Function
With Transposons
July 12th, 2000
Genome sequencing assignments
I
II
III
IV
Kazusa
CSHSC
V
TIGR
SPP
ESSA
Genoscope
Kazusa
Functional Genomics
• 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
Zn-finger CLL E(z)
kinase
prolifera
hexose
MutL
transporter
SAR1
CTP
synthase
AtKAP
Zn-finger
= cDNA/EST
= hypothetical protein
= pseudogene
= known gene
Genetraps

Gene traps are reporter genes that integrate
at random in the plant genome

Gene traps simultaneously monitor gene
expression and gene function
Genetrap transposons
reporter gene
intron
plant gene fusion protein
positive
marker
Random Insertions
transposition
selection
reporter gene
negative marker
positive marker
Epidermal cells
Reporter gene expression
mimics expression of the
plant gene
•Guard cells
•Unbranched trichomes
Meristem cells
Shoot meristem (rib zone)
Root meristem (columella initials)
Thermal Assymetric Interlaced
(TAIL) PCR
TAIL product
Reference:
Liu et al., 1995
Plant J. 8, 457
arbitrary
degenerate
primer
nested anchors
Genetrap database
Crop
ESTs
GeneTrap
Transposants
Arabidopsis
ESTs
Genome
sequence
(map
position)
GeneTrap
database
1. Sequence
insertion sites
2. Stain for
reporter
expression
3. Functional
screens
Profile for ET 79
Parent Genotype
Sequence
Characterization
Expression Data
ET 79
Date Sequenced: 4/14/1996
Primer: DS5-4N
Length: 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 repeats
cdk2 associated p45/skp2 protein homolog
(in 6kb gap between genes)
Sequence
TAATAAAACNGTTATACGATAACGGTCGGTACGGGATTTCCCATCCTACTTTCATCCCTGC
TCATTAGCCCCCAATGTGCATTTGTGTTATCTTTACCAGTTGCGTGTAANCTTCTGTGTATT
NCTNCTTCTCACTC
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
Genetraps in genome biology
•
•
•
•
•
Mapping insertions on chromosomes
Targets for regulatory genes
Secretion traps
Lethal genes
Redundant genes
Cytogenetic map of chromosome 4S
3Mb
NOR
2Mb
0.5Mb
0.5Mb
knob
2Mb
cen
Paul Fransz
Transposon distribution
3Mb Bac contig
1874 genetrap insertions
106 on chromosome 4S
60
50
40
30
DsE/G
transcribed genes
20
total genes
10
0
250
500
750
1000
1230
1500
1750
2080
Dandelion alters dorsoventral patterning
ET3964 is expressed dorsally
ET3964
ET 3964
500bp
cycloidea-like
ET3964
ET3964 is over expressed in dnd1-2 plants
AGO
CYC-like
dorso/ventral patterning??
GUS Inhibition by
Glycosylation
Signal
Sequence
35S
Alpha amylase
No treatment
Asn-358
GUS
20uM Tunicamycin
Secretion Trap
Secreted or Plasma MembraneSpanning Protein
Cytosolic Protein
Signal
Sequence
- Tunicamycin
+ Tunicamycin
- Tunicamycin
+ Tunicamycin
GT7487 secreted hypothetical
Minus Tunicamycin
Plus Tunicamycin
GT6249
hypothetical
Genes Tagged by Secretion Trap
Screen
GT6666
GT7137
Glutathione conjugate ABC transporter
Beta-gylcosidase
GT5376
GT6114
GT6224
Seven-pass receptor
Receptor kinase
Receptor kinase
GT7059 Predicted transmembrane protein with VWF
domain
GT7134 Hypothetical transmembrane protein with
armadillo repeats
GT6943
GT7106
Similar to aminopeptidase P
Serine protease with predicted secretion signal
GT5211
GT7243
GT7487
GT5397
Unknown protein with predicted secretion signal
Unknown protein with predicted secretion signal
Unknown protein with predicted secretion signal
“Between genes”
2059 Gene traps screened
464 Stain (23%)
33 Secreted (7% of stain)
GT6224
GT5397
PROLIFERA is expressed in
dividing cells
In heterozygote plants, the PROLIFERA gene is expressed in
dividing cells throughout the plant - not just in embryos
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
PROLIFERA is an essential gene
Genetrap insertions in the PROLIFERA gene
are lethal in homozygous embryos, and haploid megagametophytes
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
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 a
QuickDraw decompressor
are needed to see this picture.
AGL8
ET3214 interrupts the
untranslated leader of the
MADS box gene AGL8
Reporter gene
expression is
found in the
carpel wall and
inflorescence
meristem
AGL8 Gene Function
Loss of AGL8 function affects
•carpel wall differentiation
•fruit elongation
•fruit shattering
+
ful
+
ful
The AGL8 gene was
renamed FRUITFULL (ful1)
FRUITFULL-related genes
FRUITFULL is expressed in the inflorescence meristem, but the meristem is normal in
fruitfull mutants
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?
AGAMOUS
AGL1
AGL5
AGL7
(APETALA1)
AGL8
(FRUITFULL)
AGL10
(CAULIFLOWER)
Genetic Redundancy
ap1 cal ful triple mutants have
flowers replaced by shoots
apetala1 cauliflower double
mutants have proliferating
floral meristems ressembling
cauliflowers
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
Catherine Kidner
Mary Byrne
Andrew Groover
Bruce May
Cristy Yordan
Joe Simorowski
Anna-Marie Arroyo
Rulan Shen
John Healy
Former lab members:
Patty Springer
Qing Gu
Mark Curtis
Maitreya Dunham
Ben Lehner
Credits
Thanks to:
Shiv Grewal
Caroline Dean
Scott Poethig
Kathy Barton
Sarah Hake
Marty Yanofsky
Marja Timmermans
Andrew Hudson
Dick McCombie
Paul Fransz