1. Chapter 11: Functional genomics
Finding genes and their function
Structural genome analysis: find genes
Functional genome analysis: find function

2. As we will see in chapter 12, most transgenic plants that are in the fields now have been made with bacterial genes (easier to find and analyse)
For the newer transgenic plants, plant genes have been introduced.
How can these interesting plant genes be found?

3. Structural genome analysis
Eukaryotic genomes contain much DNA outside the genes
Human
Yeast
Z. mays
E. coli
Genomes 2
T.A. Brown, Bios

4. Structural genome analysis
Eukaryotic genomes contain much DNA outside the genes
How to find these genes that are scattered on the genome?
When the genome is sequenced, there are several techniques to find the genes in the sequence:
look for coding regions (open reading frames from ATG to stopcodon), not so easy in eukaryotes because the open reading frame is interrupted by introns.
compare DNA with mRNA that is transcribed from the genes (RNA can be extracted and analysed)
Structural characteristics (such as GC%)
Compare with information from other organisms
Map based cloning: for genes for which a mutant phenotype is known

5. Map based cloning
Map based cloning: for genes for which a mutant phenotype is known
For this technique, physical and genetic (chapter 9) maps are needed.
A physical map is based on a collection of clones that are overlapping each other and preferentially the corresponding sequence.
Such a physical map can be correlated with a genetic map by comparing the sequence of a genetic marker with the sequence of the physical map.
If the mutant phenotype is genetically mapped, than the gene can be cloned on the basis of the correlated physical map = map based cloning

6. Physical map Overlapping clones DNA sequence
Genetics, analysis of genes and genomes, Hartl and Jones (Jones and Bartlett publishers)

7. Integration of physical and genetic maps

8. Functional genome analysis
To find the function of the genes
search for homology with known genes (chapter 3: BLAST)
Expression analysis, for example a gene that is expressed only in the flower must have a function in the flower
By mutant analysis
By making transgenic plants with higher or lower expression of the gene

9. Functional genome analysis
By making transgenic plants with much higher or lower expression of the gene
Higher expression: by introducing construct with strong promoter- gene fusion
Lower expression: with RNAi or antisense technology
Antisense produces the reverse of the normal mRNA  hybridisation with mRNA prevents translation and makes mRNA unstable
RNAi uses double stranded RNA and has a similar effect (complex mechanism)

10. Functional genome analysis
By making transgenic plants with much higher or lower expression of the gene, for example a gene that makes plant grow taller
Higher expression: transgenic plant will be larger than normal
Lower expression: transgenic plant will be smaller than normal

11. ANTISENSE STRATEGY messenger RNA ANTISENSE protein coding sequence
promoter
promoter
DNA
messenger RNA
ANTISENSE
protein