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Overview Genomics Martin Polz [email protected] I. Genome sizes II. How to sequence entire genomes III. Genome evolution 1. Point mutations 2. Regional changes 3. Global changes IV. Environmental genomics I. Genome sizes Bacterial genome: 6×105 to more than 107 Smallest known: Mycoplasma genitalium (480 protein coding genes, 3 rRNA genes, 33 tRNA genes) Prokaryotes genome sizes are roughly proportional to gene numbers. Eukayotic genome: 8.8×106~ 6.9×1011 Smallest known include: Saccharomyces cerevisiae and other fungi Eukaryotes genome sizes are NOT proportional to gene numbers or anatomical complexity Genome size (bp) Chromosome number (n) Amoeba dubia 670,000,000,000 12 Trumpet lily 90,000,000,000 20 Human 3,454,200,000 23 Chicken 1,200,000,000 39 Housefly 900,000,000 6 Tomato 655,000,000 12 1 Chapter 16 II. How can we sequence entire genomes? BACTERIA ARCHAEA EUKARYA ia er ria ct m te iu s ba ac ria a s a ns b e er cu bus s e eri v r id ia c o t i ct s ria a t u ad st lex ts s id g si ld lf bac ct cte ba oco ogl u o u a o t o an on pla mp por mo ae ls x b a s le b n an ea o b pl m o fe mo -p l o s a n i t o e oeb ma o o o m ee rp an h th ch d ngi lo ne c f v t r i i l m n p qu her ra e u a i e i r P i p y Fl ic Sl Am An A M Ar D G K G A Su M C La Fu T M Figure 16.3 upper Figure 16.3 middle SEQUENCING A GENOME Genomic DNA 1. Cut DNA into fragments of ~160 kb, using different restrictions enzymes. 4. Cut each 160 kb fragment into 1 kb fragments. “Shotgun clones” BAC library 2. Insert fragments into bacterial artificial chromosomes, grow in E. coli cells. 3. Analyze fragments, locate each on map of genome. 5. Insert 1 kb fragments into plasmids, grow in E.coli cells. Shotgun sequence ...ATTTAGACTCGATAA TAGACTCGATAAGGATGC... 6. Sequence each fragment (note that ends of fragments overlap). 2 Figure 16.3 lower ...ATTTAGACTCGATAAGGATGC... Figure 16.4 7. Assemble 1 kb fragments from within each 160 kb fragment by matching overlapping ends. Open reading frame (ORF) Start Stop 5’ …C T C A A T G G G T A C G T A G G AT C G G G A A T C G T A C A G G A A C G T T T G A A A T C G... 3’ … G A G T T A C C C A T G C A T C C T A G C CC T T A G C A T G T C C T T G C A A A C T T T A G C... Draft sequence Genome map 8. Assemble fragments from different BACs by matching overlapping ends. III. Genome evolution 1. Point mutations - measure evolutionary relationships (phylogeny) --> see last lecture 2. “Regional” change - duplication - deletion - mobile elements - chromosome rearrangements 3. “Global” change - genome duplication --> not covered 3 Mechanisms of gene deletion or duplication replication errors unequal crossing over Example: evolution of antifreeze gene in arctic fish 5’ 1 2 3 4 5 3’ 6 Ancestral trypsinogen gene Deletion 1 5’ 6’ 3’ Thr Ala Ala Gly 4 fold duplication + addition of spacer sequence 1 5’ 6’ Internal duplications + addition of intron sequence Consequences of gene duplication 1 5’ 1 - part of a gene: if functional domain is duplicated --> functional enhancement -complete gene gene dosage enhanced New function can evolve due to relaxed selection on one copy 2 3 4 5 6 3’ Spacer: Gly 7 … 37 38 39 40 41 6’ 3’ Antifreeze glycoprotein gene Mobile Elements (selfish DNA) Human genome: gene duplications 5% of the human genome is found to be recently-duplicated large segments (>500bp, identity>95%). [JA Bailey, Science, 2002] The duplicated regions create mosaic structure. Some of the duplicated segments contain new genes. Insertion sequences Only encode for transposase, no exogenous genes. Transposons Carry both transposase and exogenous genes (e.g. drug resistant genes). Non-autonomous transposable elements Do not carry transposase itself, depend on other autonomous transposable elements. Retroelements Carry reverse transcriptase. -Retrovirus -Retroposons -Retrotrnasposons 4 Example: transposons Chromosome rearrangements Transposons disrupt genes but can also carry genes around chromosome and among different cells. Figure 16.6 Example: comparison of bacterial genomes Lateral gene transfer - very important mechanisms in prokaryotes! ARCHAEA BACTERIA ia er ia ct ter ba ac ia a ia b e er ri iv r ga it lfu ct te ter s to os su ba ac ac x fi e rmu rmo m-p en ple nob ob r a av qu e e a e A Th Th Gr Gr Pu Cy Fl m iu s s us us ter us cu bu c c r te m cc co glo us iu us pro opy oba o t c no eo occ ic lob o n an 7 o 8 a a d a 1 c m h h P7 L ro lfo erm eth eth er et rc alo pJ pS Py Su Th M M Th M A H When genes are transferred laterally, they move between species that are not necessarily closely related 5 Recognizing laterally transferred DNA: - different GC content - “new” DNA in related organisms - phylogenetic differences Mechanisms of lateral gene transfer Example: Pathogenicity islands - many pathogens acquire genes from other bacteria 2 1 3 1 Transformation: uptake of DNA from environment 2 Transduction: DNA transfer by viruses 3 Conjugation: plasmid transfer between bacterial cells Environmental Genomics - applying genomic sequencing to DNA directly obtained from the environment BAC clone obtained directly from environment 16 S rRNA gene Proteorhodopsin: Proteorhodopsin: A new way to convert solar energy to chemical energy in the ocean Protein coding gene revealing function of organism in environment 6 7