Probing Plant Gene Function With Transposons

July 12th, 2000

Genome sequencing assignments

CSHSC

ESSA

KazusaGenoscope

TIGR

SPP

I II III IV VKazusa

• 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

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

positivemarker

plant gene fusion protein

Random Insertions

negative marker

positive marker

reporter gene

selection

transposition

Epidermal cells

Reporter gene expression mimics expression of the plant gene•Guard cells•Unbranched trichomes

Meristem cells

Root meristem (columella initials)

Shoot meristem (rib zone)

Thermal Assymetric Interlaced (TAIL) PCR

nested anchorsarbitrary degenerate primer

TAIL product

Reference:Liu et al., 1995 Plant J. 8, 457

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

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

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

NOR

knob

cen

3Mb

2Mb

0.5Mb

2Mb

0.5Mb

Paul Fransz

Transposon distribution

3Mb Bac contig 1874 genetrap insertions106 on chromosome 4S

250500

7501000

12301500

17502080

0

10

20

30

40

50

60

DsE/G

transcribed genes

total genes

Dandelion alters dorsoventral patterning

cycloidea-like

ET 3964

ET3964ET3964

ET3964 is expressed dorsally

500bp

ET3964 is over expressed in dnd1-2 plants

AGO CYC-like dorso/ventral patterning??

GUS Inhibition by Glycosylation

35S

SignalSequence

Alpha amylase GUS

Asn-358

No treatment 20uM Tunicamycin

Secretion Trap

- Tunicamycin + Tunicamycin

Cytosolic Protein

- Tunicamycin + Tunicamycin

Secreted or Plasma Membrane-Spanning Protein

SignalSequence

Minus Tunicamycin Plus Tunicamycin

GT7487 secreted hypothetical

GT6249 hypothetical

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

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 geneGenetrap 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 aQuickDraw decompressor

are needed to see this picture.

Reporter gene expression is found in the carpel wall and inflorescence meristem

AGL8 ET3214 interrupts the untranslated leader of the MADS box gene AGL8

AGL8 Gene FunctionLoss of AGL8 function affects•carpel wall differentiation

The AGL8 gene was renamed FRUITFULL (ful1)

•fruit elongation•fruit shattering

+

+

ful

ful

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?

Genetic Redundancy

apetala1 cauliflower double mutants have proliferating floral meristems ressembling cauliflowers

ap1 cal ful triple mutants have flowers replaced by shoots

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

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