Download BIOL 2416 Genetics

BIOL 2416 Genetics
Chapter 9: Functional and
Comparative genomics
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!)
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
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–
–
–
–
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
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
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
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?
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.html
PCR:
3
steps:
• 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…
PCR Animations
• Narrated animation:
http://www.youtube.com/watch?v=_YgXcJ4n-kQ
• DNAi.org animation (no sound):
http://www.dnai.org/text/mediashowcase/index2.html
?id=582
• PCR rap:
http://www.youtube.com/watch?v=oCRJ4r0RDC4
• BioRad’s PCR song:
http://www.youtube.com/watch?v=x5yPkxCLads
Gene Knockdowns: RNAi Method
http://www.pbs.org/wgbh/nova/sciencenow 3210/02.html
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
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)
http://en.wikipedia.org/wiki/File:RNAi-simplified.png
http://www.silencinggenomes.org
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):
http://www.dnai.org
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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
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