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Lecture 9-10. Plant genomics II
Functional genomics: Identify the function of each and every gene in the genome. Since the characterization of the function of a protein domain in one organism generally provides hint to its function in another organism, the first goal of functional genomics is to identify as many genes as possible in major model organisms
Basic ApproachesA. Forward genetics: Random mutagenesis, screen for traits of interests
Chromosome walking or transposon-tagging
B. Reverse genetics: disrupt a particular gene or set of genes with known seq.
C. Fine structure geneticsD. Gene expression profile (analyses of transcriptome)
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B. High-throughput reverse genetics
1. PCR-based screen for T-DNA or transposon insertion mutations in specific genes---Wisconsin knockout facility
2. Database searches--salk institute lines
3. TILLING
4. RNAi (RNA interference)
5. Gain-of-function (activation-tagging) mutagenesis
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Gene X
NPTII
FR
LB
RB
PCR products:
1. Screen for T-DNA (or Ds) insertion in specific genes
Screening pools (p1-p5)
p1 p2 p3 p4 p5 1kb ladder p1 p2 p p4 p5
LB/F RB/R
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2. Data-base searches for T-DNA insertions in the genes of interests
Gene X
LB
RB
Enzyme digestion
Ligase
PCR Sequence database
Salk Institute Genomic Group (http://signal.salk.edu/cgi-bin/tdnaexpress)
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3. TILLING (Targeting Induced Local Lesions IN Genomes)Arabidopsis Tilling website:http://tilling.fhcrc.org:9366/
EMSIsolated DNA
poolGene-specific
primersPCRheat & coolCEL I
Denature
1 2 3 4 5 6 7 8 9 10
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4. RNA interference (RNAi)
loss of gene activity
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Gene X
NPTII
35S enhancer
5. Gain-of-function (Activation tagging) mutagenesis
Over-express Gene XLeading to gain-of-function phenotype
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C. Fine Structure Genetics
1. Modifier screens: enhancer and suppressor screens, synthetic lethal
2. Enhancer-trap (using GFP or GUS)
3. GAL4-mediated over-expression
4. Yeast Two-hybrid screen (Y2H)
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ap1-1 ap1-1 cal-1
ap1-1 cal-1
1. Enhancer or suppressor mutations
cal-1: wild-type lookingap1-1: flower mutantap1-1 cal-1: cauliflower
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Gene X
GUS
Minimal promoter
Enhancer of gene X
T-DNA
2. Enhancer-trap
Dr. Tom Jack’s websitehttp://www.dartmouth.edu/~tjack/
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GAL4
Minimal promoter
Tissue-specific Enhancer
3. GAL4-mediated over expression
X
5X UASGene Z
www.plantsci.cam.ac.uk/Haseloff/gene_expression/geneExpFrameset.html
5X UASGFP
and/or
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4.Yeast Two Hybrid (Y2H) Assay to test interaction between two proteins
GAL4DB
GAL4AD
X Y
HIS3 or lacZ5XUAS
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LUFS
LUFS+Q-rich
Q-rich+WD
Positive Control
LUFS+Q-rich+WD
Yeast Two -Hybrid Assay for Interaction Between LUG and SEU
SEU-GAL4-ADVS.
Q-rich (89-184, 449-470)
7 WDLUFS Q-rich (89-184, 449-470)
7 WDQ-rich (89-184, 449-470)
LUFS
LUFS
LUG-GAL4-DB constructs
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D. Analyses of the transcriptomeDocumenting gene expression on a genome wide scale
Transcriptome: complete set of transcripts and their relative expression levels in a particular cell or tissue under defined conditions
I. cDNA microarrays
II. Oligonucleotide arrays
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I. cDNA microarrays
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II. Oligonucleotide microarrays (Affymetrix GeneChip)
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mask
Light deprotection
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Sporulation gene expression profile in budding yeastChu et al., (1998) Science 282, 699-705
Several classes of sporulation gene expression after transfer to sporulation media
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Survey of 1116 genes during sporulation in budding yeastChu et al., (1998) Science 282, 699-705