Functional Genomics
Functional Genomics
Functional Genomics
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<strong>Functional</strong> <strong>Genomics</strong> <br />
Frans Tax!<br />
Associate Professor!<br />
Department of Molecular!<br />
and Cellular Biology,!<br />
School Plant Sciences!<br />
!<br />
Other relevant courses:!<br />
MCB 546-genetic and molecular networks!<br />
!<br />
IGERT students:!<br />
!<br />
Michael Nodine-GMI-Vienna!<br />
Amanda Durbak-UM-Columbia-postdoc!<br />
Adriana Racolta-PhD Spring 2013!<br />
Ivan Dimitrov
Todays outline <br />
• Introduction- !<br />
• Function?!<br />
• How can you test function?!<br />
– Reverse genetics!<br />
• Mutation-several more steps!<br />
• Gene families-SHP paper!<br />
• Forward genetics
What is functional genomics? <br />
• What does one mean by gene function?<br />
(What do gene products do?)
What is functional genomics? <br />
• What does one mean by gene function?<br />
– Role in the organism (in vivo) <br />
– Role under environmental conditions <br />
– Biochemical activity (in vitro) <br />
– Role in the cell <br />
– Interaction with other genes <br />
• (Which is most important?)
Genetics vs. Biochemistry <br />
• See<br />
http://bio.research.ucsc.edu/people/sullivan/<br />
savedoug.html <br />
• And <br />
• http://bio.research.ucsc.edu/people/kellogg/<br />
contents/Demise%20of%20Bill.html
What is functional genomics? <br />
• How does one find gene function? <br />
– Say you find a DNA sequence, you think it<br />
encodes a gene….
What is functional genomics? <br />
• How does one find gene function? <br />
– BLAST! Homology <br />
– Transcriptional response <br />
– Proteomic response <br />
– Make a mutant<br />
• homologous recombination-knockout <br />
• Identify an allele or polymorphism <br />
• RNAi <br />
• Design a blocking antibody <br />
• Existing mutant already known
What is the geneticists or<br />
genomicists most powerful functional<br />
tool?
What is the geneticists or<br />
genomicists most powerful functional<br />
tool? <br />
EMS mutagenesis <br />
(Forward functional genomics)
How to find mutants <br />
• What is the spontaneous rate of mutations<br />
per gene?
How to find mutants <br />
• What is the spontaneous rate of mutations<br />
per gene? <br />
• Looking at a single gene in mice,<br />
11/1,000,000 gametes have a mutation <br />
• We use mutagens to increase that 1000 fold.
Variation in strains is useful <br />
• Natural variation can be used as a source of<br />
allelic variation <br />
• Used commonly in agriculture, human<br />
genetics, population genetics
Common mutagens
Common mutagens <br />
• EMS/MMS/NSG <br />
• Transposons/T-DNA <br />
• Ionizing radiation <br />
• UV <br />
• Spontaneous mutations <br />
• DEB/Psoralen/ENU <br />
• New Jersey <br />
How do these affect DNA?
Common mutagens <br />
• EMS/MMS/NSG <br />
G to A transitions <br />
• Transposons/T-DNA insert into gene <br />
• Ionizing radiation <br />
breaks in DNA <br />
• UV <br />
thymidine dimers <br />
• Spontaneous mutations <br />
• DEB/Psoralen gene-sized<br />
deletions<br />
of DNA <br />
• ENU G to A, C to T, deletions
Kinds of mutations based on<br />
effects on DNA sequence: <br />
• DNA sequence to RNA-CCX for proline
Kinds of mutations based on<br />
effects on DNA sequence: <br />
• DNA sequence to RNA-CCX for proline <br />
• Synonomous <br />
• Non-synonomous <br />
• nonsense
What can you get from 1 basepair<br />
mutation in CCX?
Does every mutation result in a<br />
change in amino acid sequence?
Does every mutation result in a<br />
change in amino acid sequence? <br />
• No <br />
– Synonymous changes <br />
• 3rd base wobble in codons <br />
• Some amino acids are specified by 6 triplets
Does every change in an amino<br />
acid kill the protein?<br />
Serine: UCX <br />
Threonine-ACX
Does every change in an amino<br />
acid kill the protein?<br />
• No, single base pair changes often lead to a<br />
change in a similar amino acid <br />
• Only a few changes <br />
can give you stop codons
The genetic code protects against<br />
• Agree? <br />
mutation
What kinds of mutations do you<br />
want? <br />
• Lets say you are studying a receptor kinase <br />
• What kinds of mutations can you get?
What kinds of mutations do you<br />
want? <br />
• Lets say you are studying a receptor kinase <br />
• What kinds of mutations can you get? <br />
No receptor"<br />
Less active receptor"<br />
Overly active receptor"<br />
Interfering interaction with co-receptor"
What kinds of mutations do you<br />
• Nulls <br />
want? <br />
• A variety of missense changes that might<br />
tell you about the roles of domains within<br />
that protein, or tell you about roles in<br />
specific cell types.
Nomenclature <br />
• Nonsense <br />
• Missense <br />
• Frameshift <br />
• Knockout <br />
• Splice site changes <br />
– Which can be nulls? <br />
– Which do you want?
EMS-mechanism
What can you get from 1 change<br />
in CCX?
Even with a mutagen that<br />
changes 50% of bases, the effects<br />
are limited
The genetic code protects against<br />
mutation-implications for genetic<br />
screens <br />
• Its a numbers game. Be ambitious.
How do you know if you have a<br />
null?
How do you know if you have a<br />
null? <br />
• No protein <br />
• No mRNA <br />
• No DNA for gene <br />
• Genetic inference-null=null
How do frameshift mutations<br />
create nulls?
How do nonsense mutations<br />
create nulls? <br />
• Nonsense-mediated decay
• 5 <br />
How do splice site mutations<br />
create nulls? <br />
• 3
Arabidopsis as a model system <br />
• Brassica!<br />
• 5-6 week generation time!<br />
• Small!<br />
• Diploid!<br />
• Transformable!<br />
• Genome sequenced, can go!<br />
• from mutant to gene quickly!<br />
• Lots of tools-genome sequence, !<br />
!insertions, microarrays, enhancer traps,<br />
activation tagging lines !
The Arabidopsis genome<br />
sequence<br />
• 27,739 protein coding genes, 1312 nc RNAs,<br />
4827 pseudogenes!<br />
• >humans, 23,000, >fruit fly 19,000,!<br />
!>nematode 14,000!<br />
!<br />
30% of genes dont match animal or fungal!<br />
!genes!<br />
• 17% of genes are tandem!<br />
• Larger gene families (40% of genes are in<br />
families with 5 or more members, 12% for<br />
yeast, 20% for C. elegans)!<br />
– (1 gene in flies, 2-3 in mammals)
The rice genome sequence <br />
• More genes than non-vertebrate model systems;<br />
37,500!<br />
• 30% of genes dont match animal or fungal genes !<br />
• Larger gene families!<br />
• Lots of tandem genes (almost 34%)!
<strong>Functional</strong> genomics <br />
• What is the function in vivo of a gene or<br />
group of genes?
How do you determine the<br />
function of a gene in plants?<br />
(Arabidopsis)
How do you determine the<br />
function of a gene in plants?<br />
(Arabidopsis) <br />
Mutants-forward or reverse genetics <br />
Expression analyses <br />
Cell and tissue specific <br />
induction based on environmental conditions <br />
Overexpression <br />
Biochemical tests of function <br />
In silico-deductions based on predicted amino acid sequence
Ways to inactivate genes
Ways to inactivate genes <br />
• EMS or other mutagens (TILLING) <br />
• Insertions <br />
• RNAi
The TILLING Method <br />
• Seeds are mutagenized using EMS to cause<br />
point mutations.<br />
• DNA is collected from the M2 generation<br />
and is used for screening.<br />
• The DNA is pooled and arrayed on microtiter<br />
plates.<br />
• Using gene-specific primers already<br />
designed, PCR is performed. The heating and<br />
cooling of the PCR products forms<br />
heteroduplexes.<br />
.<br />
• CelI is used to cut mismatch sites.<br />
• Cleaved PCR products are then run out on<br />
polyacrylamide gels in order to isolate<br />
mutants.<br />
• DNA is sequenced and the specific point<br />
mutation . is identified.<br />
Image taken from: Henikoff, Steven and Comai, Luca. 2003. Single-Nucleotide<br />
Mutations for Plant <strong>Functional</strong> <strong>Genomics</strong>. Annual Review of Plant Biology. 54.<br />
375-401.<br />
• M3 seeds are sent to the investigator.
RNAi to knockdown genes <br />
Introduce a T-DNA with an<br />
inverted repeat sequence <br />
or hairpin <br />
http://www.rnaiweb.com/RNAi/What_is_RNAi/index.html
Insertional mutagenesis <br />
Valentine, 2003 Plant Phys 143:948
T-DNA mutagenesis <br />
Insertion is essentially random throughout the genome <br />
T-DNA ends are mostly LB or RB
SAIL database
Identification of shp1 mutant by T-DNA insertion mutagenesis
SHATTERPROOF MADS-box genes control seed<br />
dispersal in Arabidopsis<br />
Liljegren SJ, Ditta GS, Eshed HY, et al.<br />
Nature 404, 766-770 (2000)<br />
Flower formation of wild-type<br />
Sepals<br />
Petals<br />
stamens<br />
Capels
ABC Model of Floral Development<br />
C gene expression<br />
C C C <br />
B gene expression<br />
B B B B <br />
A gene expression<br />
A A A A <br />
A group<br />
B group<br />
C group<br />
APETALA1 (AP1) and<br />
APETALA2 (AP2)<br />
APETALA3 (AP3) and<br />
PISTILLATA (PI)<br />
AGAMOUS (AG)<br />
Sepals Petals Carpels stamens<br />
Flower formation of AGAMOUS mutants
MADS-box genes<br />
Family of transcription<br />
regulator genes<br />
Conserved in yeast, plants,<br />
human<br />
17 genes in plants-floral<br />
architecture<br />
The MADS box gene<br />
involved in diverse aspects<br />
of plant development,<br />
extensive redundancy<br />
between family members.<br />
Phylogenetic Tree for Arabidopsis and Antirrhinum MADS Box Genes<br />
Rounsley et al. Plant Cell (1995)
Expression tools <br />
Image from Arabidopsis eFP Browser: http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi
How were shp2-1 and shp1-1 <br />
isolated?
Identification of shp1 mutant by T-DNA insertion mutagenesis
Homologous recombination disrupted SHP2 (AGL5) gene in Arabidopsis<br />
Kempin et al. Nature (1997)
Why did they have to isolate <br />
more shp1 and shp2 alleles?
How did they isolate <br />
more shp1 and shp2 alleles?
Practical aspects of forward<br />
genetic screens <br />
1-3 germ!<br />
cells in!<br />
the seed-!<br />
homozygous!<br />
recessive!<br />
mutants in!<br />
the next!<br />
Generation!<br />
!<br />
Each M1=!<br />
2 -6 !<br />
chromosomes!<br />
In screen!<br />
1 hit per gene/!<br />
Per 1000-3000!<br />
chromosomes!
What kinds of mutations did they<br />
get from EMS mutagenesis?
The Arabidopsis genome sequence<br />
(true for other plants so far too) <br />
• More genes than non-vertebrate model systems <br />
• 30% of genes dont match animal or fungal genes<br />
• Larger gene families<br />
• Lots of tandem genes (almost 20%) <br />
What would be the consequence of having larger gene families <br />
for geneticists?
What would be the consequences of this for geneticists? <br />
• Forward genetics screens not as efficient <br />
• Genetic redundancy is a problem when you<br />
want to find out what a gene does <br />
• Estimates in Arabidopsis for embryo<br />
essential genes are around 1000 (4%) <br />
– Male and female gametophyte?
Reverse genetics is an answer <br />
Plant Physiology, 138, 558-9
Interesting unresolved issues in<br />
plant functional genomics <br />
• Why are gene families bigger? <br />
• Why are duplicate genes maintained? <br />
• Why are there tandem genes?
Most duplicates are thought to become pseudogenes!<br />
quickly; simple redundancy is not stable."<br />
Moore and Purruganan, 2005
Potential questions: <br />
I may ask you something about how to determine <br />
the function of a gene or gene family. <br />
I may ask you about gene duplication and what can <br />
happen after two or more duplicate copies are still around- <br />
see the figure in the Moore and Purugganan paper. Based <br />
on the shp story and its close relatives (AG and AG11), <br />
how are these genes maintained in the Arabidopsis genome?
Plant gene families <br />
Members of a gene family are defined as<br />
having a BlastP value of E < 10 -20 over 80%<br />
of the length of the protein
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