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

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?

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

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

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

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

Integration of physical and genetic maps

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

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)

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

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