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Probing Plant Gene Function With Transposons

Probing Plant Gene Function With Transposons. July 12th, 2000. Genome sequencing assignments. II. III. IV. V. I. CSHSC. Kazusa. TIGR. SPP. ESSA. Genoscope. Kazusa. Functional Genomics. Only 1 in 20 genes have an assigned genetic role

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Probing Plant Gene Function With Transposons

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  1. Probing Plant Gene Function With Transposons July 12th, 2000

  2. Genome sequencing assignments II III IV V I CSHSC Kazusa TIGR SPP ESSA Genoscope Kazusa

  3. Functional Genomics • Only 1 in 20 genes have an assigned genetic role • Systematic determination of gene expression and genetic functionis a major goal of the Arabidopsis Genome Project CTP synthase CLL E(z) Zn-finger prolifera SAR1 hexose transporter kinase MutL AtKAP Zn-finger = cDNA/EST = hypothetical protein = pseudogene = known gene

  4. Genetraps • Gene traps are reportergenes that integrate at random in the plant genome • Gene traps simultaneously monitor gene expressionand gene function

  5. Genetrap transposons positive marker reporter gene intron plant gene fusion protein

  6. transposition selection reporter gene negative marker positive marker Random Insertions

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

  8. Meristem cells Shoot meristem (rib zone) Root meristem (columella initials)

  9. Thermal Assymetric Interlaced (TAIL) PCR TAIL product arbitrary degenerate primer nested anchors Reference: Liu et al., 1995 Plant J. 8, 457

  10. Crop ESTs 1. Sequence insertion sites Arabidopsis ESTs 2. Stain for reporter expression Genome sequence (map position) GeneTrap database 3. Functional screens Genetrap database GeneTrap Transposants

  11. 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 TAATAAAACNGTTATACGATAACGGTCGGTACGGGATTTCCCATCCTACTTTCATCCCTGCTCATTAGCCCCCAATGTGCATTTGTGTTATCTTTACCAGTTGCGTGTAANCTTCTGTGTATTNCTNCTTCTCACTC

  12. 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

  13. Genetraps in genome biology • Mapping insertions on chromosomes • Targets for regulatory genes • Secretion traps • Lethal genes • Redundant genes

  14. NOR 3Mb 2Mb knob 0.5Mb 0.5Mb cen 2Mb Cytogenetic map of chromosome 4S Paul Fransz

  15. Transposon distribution 3Mb Bac contig 1874 genetrap insertions 106 on chromosome 4S

  16. Dandelion alters dorsoventral patterning

  17. ET 3964 cycloidea-like ET3964 is expressed dorsally ET3964 ET3964 500bp

  18. ET3964 is over expressed in dnd1-2 plants AGO CYC-like dorso/ventral patterning??

  19. Signal Sequence Asn-358 35S Alpha amylase GUS GUS Inhibition by Glycosylation No treatment 20uM Tunicamycin

  20. Secreted or Plasma Membrane- Spanning Protein Signal Sequence Secretion Trap Cytosolic Protein - Tunicamycin + Tunicamycin - Tunicamycin + Tunicamycin

  21. GT7487 secreted hypothetical Minus Tunicamycin Plus Tunicamycin

  22. GT6249 hypothetical

  23. Genes Tagged by Secretion Trap Screen 2059 Gene traps screened 464 Stain (23%) 33 Secreted (7% of stain) GT6666 Glutathione conjugate ABC transporter GT7137 Beta-gylcosidase GT5376 Seven-pass receptor GT6114 Receptor kinase GT6224 Receptor kinase GT7059 Predicted transmembrane protein with VWF domain GT7134 Hypothetical transmembrane protein with armadillo repeats GT6943 Similar to aminopeptidase P GT7106 Serine protease with predicted secretion signal GT5211 Unknown protein with predicted secretion signal GT7243 Unknown protein with predicted secretion signal GT7487 Unknown protein with predicted secretion signal GT5397 “Between genes” GT6224 GT5397

  24. PROLIFERA is expressed in dividing cells In heterozygote plants, the PROLIFERA gene is expressed in dividing cells throughout the plant - not just in embryos

  25. 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

  26. PROLIFERA is an essential gene and haploid megagametophytes • Genetrap insertions in the PROLIFERA gene are lethal in homozygous embryos,

  27. 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

  28. 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

  29. AGL8 ET3214 interrupts the untranslated leader of the MADS box gene AGL8 • Reporter gene expression is found in the carpel wall and inflorescence meristem

  30. fruit elongation • fruit shattering + ful + The AGL8 gene was renamed FRUITFULL (ful1) ful AGL8 Gene Function • Loss of AGL8 function affects • carpel wall differentiation

  31. AGAMOUS AGL1 AGL5 AGL7 (APETALA1) AGL8 (FRUITFULL) AGL10 (CAULIFLOWER) FRUITFULL-related genes APETALA1 and CAULIFLOWER are closely related to FRUITFULL , and also expressed in the floral meristem • FRUITFULL is expressed in the inflorescence meristem, but the meristem is normal in fruitfull mutants Are these three genes redundant with respect to meristem function?

  32. Genetic Redundancy ap1 cal ful triple mutants have flowers replaced by shoots • apetala1 cauliflower double mutants have proliferating floral meristems ressembling cauliflowers

  33. 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

  34. Credits Catherine Kidner Mary Byrne Andrew Groover Bruce May Cristy Yordan Joe Simorowski Anna-Marie Arroyo Rulan Shen John Healy Thanks to: Shiv Grewal Caroline Dean Scott Poethig Kathy Barton Sarah Hake Marty Yanofsky Marja Timmermans Andrew Hudson Dick McCombie Paul Fransz Former lab members: Patty Springer Qing Gu Mark Curtis Maitreya Dunham Ben Lehner

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