1. BIOL 2416 Genetics Chapter 9: Functional and Comparative genomics

2. Let’s say we get some genomic sequence… cgctggggga cagcctgcag gcttcaggag gggacacaag catggagcgg ctttggggtc tattccagag agcgcaacaa ctgtccccaa gatcctctca gaccgtctac cagcgtgtgg aaggcccccg gaaagggcac ctggaggagg aagaggaaga cggggaggag ggggcggaga cattggccca cttctgcccc atggagctga ggggccctga gcccctgggc tctagaccca ggcagccaaa cctcattccc tgggcggcag caggacggag ggctgccccc tacctggtcc tgacggccct gctgatcttc actggggcct tcctactggg ctacgtcgcc ttccgagggt cctgccaggc gtgcggagac tctgtgttgg tggtcagtga ggatgtcaac tatgagcctg acctggattt ccaccagggc agactctact ggagcgacct ccaggccatg ttcctgcagt tcctggggga ggggcgcctg gaggacacca tcaggcaaac cagccttcgg gaacgggtgg caggctcggc cgggatggcc gctctgactc aggacattcg cgcggcgctc tcccgccaga agctggacca cgtgtggacc gacacgcact acgtggggct gcaattcccg gatccggctc accccaacac cctgcactgg gtcgatgagg ccgggaaggt cggagagcag ctgccgctgg aggaccctga cgtctactgc ccctacagcg ccatcggcaa cgtcacggga gagctggtgt acgcccacta cgggcggccc gaagacctgc aggacctgcg ggccaggggc gtggatccag (ETC!)

3. Recall genomic DNA sequence can lead to structural (“used”) gene annotation – how? Look for ORFs –Open Reading Frame = potential structural gene (ATG…TGA) –May use species-specific codon bias patterns to find genuine ORFs Use ESTs –Expressed Sequence Tag –Unique sequence produced from 5’/3’ ends of cDNA clones –May be used as a probe to find/map complementary mRNA transcripts –If found, means genomic DNA is expressed - bingo! “used” gene! –Can also use entire cDNA instead of ESTs Look for Conserved Sequences –More likely to be important/expressed (regulatory or structural) –Use Bioinformatics tools, e.g. BLASTn and BLASTp –Basic Local Alignment Search Tools = computer algorithms that align and compare a query sequence against all sequences in a database to find a match –BLAST Tutorial: http://www.DigitalWorldBiology.com/dwb/Tutorials/Entries/2009/1/26_BLAST_for_Be ginners.html http://www.DigitalWorldBiology.com/dwb/Tutorials/Entries/2009/1/26_BLAST_for_Be ginners.html

4. Structural gene annotation leads to reverse genetics Forward genetics starts with a phenotype and attempts to discover and isolate the gene that is responsible Reverse genetics starts with a candidate structural gene and attempts to figure out the associated phenotype –Involves lots of bioinformatics = fusion of math, computer science and life science

5. Functional Genomics Starts with an annotated genomic DNA sequence Seeks to describes gene function, gene expression patterns, and control of gene expression of all genes in a genome Individual gene function may be assigned experimentally by –Gene knockouts –Gene knockdowns

6. Gene Knockouts: Knockout Mice Involves DNA mutation Knockout mice have BOTH normal copies of a gene (AA) replaced by two mutant copies (aa) The mutant copies cannot produce the normal protein (like taking away the brakes on a car!) Knockout mice are commercially available for different disease models –What happens with no brakes? –How can we fix it?

7. Optional Info: How to Make a Knockout Mouse Knockout mice produced by: –Using PCR to make a nonfunctional, mutant copy of a gene (a) –The mutant gene is introduced into mouse stem cells that have two normal gene copies (AA) –In some of the embryonic stem cells, one of the two normal gene copies is swapped out for a mutant copy by natural homologous recombination; these stem cells are now Aa –The Aa embryonic stem cells are added to a regular AA embryo –The chimeric (mixed) embryo is implanted into a surrogate mom –Hopefully some of the Aa stem cells of the embryo become germ line cells in the chimeric baby mice Germ line cell are used to make egg or sperm cells An Aa germ line means = half of the egg or sperm cells will be A, and the other half will be a –Allow the chimeric baby mice to grow up and breed with a regular AA mouse Each grandbaby mouse will get an A gamete from the regular parent If the mutated gene copy (a) made it into the germ line, one or more of the grandbabies may also inherit an a from the chimeric parent –Some of the grandbabies should be Aa in ALL of their cells, including THEIR germ line cells –Breed two Aa grandbabies 25% of the great-grandbabies will be aa knockout mice See slide show: http://genome.wellcome.ac.uk/doc_WTD021038.htmlhttp://genome.wellcome.ac.uk/doc_WTD021038.html

8. PCR: Polymerase Chain Reaction Invented by Kary Mullis (Nobel Prize) Amplification of target DNA in a test tube –IMPORTANT, cell-free alternative to DNA amplification by cloning Exponential process In a thermal cycler Uses Taq Polymerase –Heat-stable –Cannot proofread… 3 steps:

9. PCR Animations Narrated animation: http://www.youtube.com/watch?v=_YgXcJ4n-kQ http://www.youtube.com/watch?v=_YgXcJ4n-kQ DNAi.org animation (no sound): http://www.dnai.org/text/mediashowcase/index2.html ?id=582 http://www.dnai.org/text/mediashowcase/index2.html ?id=582 PCR rap: http://www.youtube.com/watch?v=oCRJ4r0RDC4 http://www.youtube.com/watch?v=oCRJ4r0RDC4 BioRad’s PCR song: http://www.youtube.com/watch?v=x5yPkxCLads http://www.youtube.com/watch?v=x5yPkxCLads

10. http://www.pbs.org/wgbh/nova/sciencenow 3210/02.html Gene Knockdowns: RNAi Method

11. RNAi: RNA interference Production of small dsRNA molecules that function in knock out or knock down gene expression from a specific gene Does NOT involve DNA mutation! Found in all eukaryotes except yeast RNAi is a cellular defense mechanism RNAi mechanism is triggered by –Exogenous molecules: Viral DNA or dsRNA coming in from the outside –Endogenous molecules (from the inside): aberrant transcripts from transposons or repetitive regions Pre-miRNA molecules transcribed form e.g. introns Small dsRNAs have been implicated in every major disease Tremendous potential in medicine and research

12. RNAi: RNA Interference RNAi gene silencing can happen at any of three levels: –Cleavage of mRNAs Dicer chops up longer dsRNAs into small siRNAs (silencing RNAs) One strand of siRNA becomes part of RISC (RNA-Induced Silencing Complex) in cytosol; siRNA finds and binds specific mRNA in a complementary way Slicer part (“Argonaute”) of RISC cleaves/destroys the mRNA –Translational repression Longer dsRNAs chopped up into miRNAs (micro RNAs) by Dicer miRNAs become part of RISC Bind at 3’ UTR of mRNA in a partially complementary way Slicer part of RISC not activated, but RISC stuck on target mRNA and translation inhibited –Transcriptional repression Affects chromatin structure/DNA packaging, making genes more or less accessible for transcription (eu = more accessible, hetero = less accessible) Least well understood Involves siRNA-dependent initiation of heterochromatic silencing by RITS complex (RNA-induced Initiation of Transcriptional gene Silencing)

13. http://en.wikipedia.org/wiki/File:RNAi-simplified.png

14. http://www.silencinggenomes.org

15. Comparative Genomics Seeks to compare all or part of the genomes of two or more strains or species Allows researchers to hone in on crucial genetic differences between strains/species/cell types. For example: –Using computer analysis: May discover which gene(s) cause one virus to be more virulent than another Could rapidly identify ideal candidate gene products of pathogens for extra-quick vaccine production (“reverse vaccinology”) In humans and other non-microbial species, may be able to associate certain haplotype blocks with certain disease resistance, or other desirable features for further study –Using molecular analysis: Diagnose e.g. viral infections by using patient cDNA to probe a “Virochip” with 20,000 diagnostic viral genes Or use tumor cell cDNA to probe a DNA microchip with gene sequences known to play a role in cancer to maximize treatments (pharmacogenomics):

16. 1 2 3 4 http://www.dnai.org

17. Metagenomics Seeks to compare entire microbial (bacteria, viruses or fungi) communities Identify community members by the presence of diagnostic gene sequences in a DNA sample taken from the community (DNA sample is cloned and shotgun-sequenced; computers sort out who is who) A.k.a. environmental genomics Useful for e.g. finding new antibiotics by screening lots of community members simultaneously, allowing identification of genes with new antibiotic function

18. What other “omes” can we study? Transcriptome –All mRNA transcripts Proteome –All proteins Interactome –All interactions between proteins Epigenome –Epi = above –How DNA is packaged –Affects expression