L16: Comparative Genomics Flashcards
What is Francis Crick central dogma of molecular biology?
DNA is converted to RNA which produces protein
How big is the human genome?
3x10^9 base pairs
What is the mutation rate of human genome?
~10^(-8) base pairs / generation
10-100 new mutations/generation
Which selection increases fitness?
Positive selection causes mutation to spread through the population
Which selection reduces fitness?
negative selection will tend to remove the mutaiton form population
What is the most recent hypothesis that makes us human?
Human DNA has udnergone accelerated evolution since divergence from common ancestor with our closest primate relative (the chimpanzee)
What is the proof that humans underwent accelerated human evolution?
- Rats and mice diverged 3x as long ago as humans and chimps
- arguably rats and mice are far more similar than humans and chimps
What is comparative genomics?
comparison between mouse, chimpanzee, and human genome DNA sequence allows us to identify sequences that have undergone accelerated evolution in the human lineage
Which parts of comparative anatomy shows evolution of human skeleton comapred to chimp?
- limb evolution
- brain evolution
how much of the genomic DNA sequence do humans and chimps share?
- Humans and chimpanzees share ~99% genomic DNA sequence conservation
- 1% makes around 30 million bases
- some of these will likely be functionally important in driving human/chimp divergence
What are Human Accelerated Regions of DNA sequence?
HARs are DNA sequence conserved throughout mammal evolution then rapidly changed when humans evolved
What are the possible DNA sequencing techniques?
- Sanger DNA sequencing
- Next generation sequencing (NGS)
How are HARs identified?
for example:
- mouse, chimp and human genomic DNA is acquired through NGS
- comparative genomics studies identifying HARs, three main types of sequences identified:
- conserved between human and non-humans - likely to be functionally important and subject to positive selection but not to account for human specific phenotype
- conserved in non-human mammals and changed in humans –> HAR - strong candidates for being functionally important AND altered function positively selected during human evolution
- not conserved between human and non-humans - more likely to be silent mutations
What are the DNA functions?
- DNA sequence regulating gene expression: enhancers and promoters regulate gene transcritpion to messenger RNA (mRNA); tend to be located in non-coding DNA (ie introns or intergenic DNA)
- DNA sequence transcribed into RNA: protein coding messenger RNA [mRNA] exons translated to PROTEIN; non protein coding RNA (micro RNA [miRNA], long non-coding RNA [IncRNA], pseudogene RNA)
How do HARs map within genomic location?
vast majority of HARs:
- do NOT map to protein coding regions (mRNA/exons)
- do NOT map to non-coding RNA
- DO map to intergenic/intron regions where Promoters/enhancers tend to be found
What is the hypothesis of HARs function and how to test it?
- most HARs map outside protein coding DNA
- one hypothesis is that HARs regulate gene expression
- can be tested using LacZ reporters to investigate DNA promoter and enhancer function
What is the function of DNA enhancers?
- DNA sequence that regulate the transcription of protein coding genes
- enhancers control the time and place of gene expression (which cells express a gene and when)
- proteins bind to enhancer DNA sequences and direct gene transcription from promoters
What is LacZ?
- Metabolic enzyme found in bacteria (a beta-galactosidase)
- mammals have no LacZ gene so no beta-galactosidase activity
- in nature LacZ catalyses the breakdown of disaccharide sugars
- LacZ also catalyses the breakdown of the colourless synthetic chemical Xgal to give a bright blue insoluble product
What is the example of LacZ exprssion to compare HARs?
- transgene construct DNA injected into mouse embryos
- embryos stained with Xgal to identify where the putative enhancer drives LacZ expression
experiment:
- compare HAR (putative enhancer) DNA fragment from human and corresponding ‘ancestral’ DNA fragment from chimp to drive LacZ reporter expression in transgenic mice
Example 1: different human vs chimpanzee drive LacZ reporter expression in developing mouse brain - this human HAR sequence drives weaker expression in developing brain
Example 2: different human vs chimpanzee driven LacZ reporter expression in developing mouse limb - this human sequence drives weaker expression in developing limb
What is the next step after LacZ expression for comparative genomics?
study genes whose expression these enhancers rgulate to specify human limb and brain adaptations
HARs can function as brain enhancers, well and good, BUT what is the evidence that HARs actually contributed to human brain evolution?
arguably it is our complex social and cognitive behaviour that most distinguishes us from the chimps and makes us human
- there are number of neurodevelopmental conditions in which social and cognitive behaviour are affected
- autism spectrum conditions (ASC) describe several symptoms and behaviours which affect the way in which a group of people understand and react to the world around them
What is the hypothesis of HAR and ASCs?
- HAR mutations cause ASC
- if this hypotheses is supported would provide functional evidence that HARs make us human from a genetic perspective because they influence social and cognitive behaviours
What does HAR426 cause? How was it tested?
mutation (G>A) in HAR426 is a genetic risk factor for ASC - people harbouring a homozygous HAR426 G>A mutation are more likely to have ASC than other people, including their relatives, who don’t
HAR426 is near to the CUX1 gene - HAR426 could physically interact with the CUX1 gene promoter to regulate CUX1 expression
How is CUX1 gene important?
- vertebrate homologue of Drosophila Cut
- DNA binding protein regulating gene expression (transcription factor)
- loss or gain of function affect neuronal development (‘dosage sensor’)
What is the hypothesis of HAR426? What is the experiment done?
Hypothesis:
- HAR426 is an enhancer that regulates expression of CUX1
- G>A mutation to HAR426 alters the expression of CUX1 and affects brain development
- HAR426 ‘G’ allele not linked to ASC
- HAR426 ‘A’ allele linked to ASC
Experiment:
- use transgenes in which reporter expression dirven by these two HAR426 allels
Prediction:
- G>A mutation affects enhancer function so alters gene expression
How is Luciferase assay used in HAR426 experiment? What is the result
- HAR426 putative enhancer coupled to a CUX1 luciferase reporter (chemiluminsecent assay) a cell line
- luciferase activity gives a readout of enhancer activity in vitro
result:
- the ‘G’form does enhance CUX1 promoter activity but mutating to the ‘A’ form enhances it still further
- HAR426 functions as an enhancer
- mutation to HAR426 results in overexpression of CUX1
Luciferase assay in a cell line, does this hypothesis hold in an actual brain under physiological conditions (in vivo)? What is the further experiment? What is the result?
GFP reporter assay
- HAR426 enhancer coupled to a CUX1-GFP reporter in transgenic mouse
- GFP level gives a readout of enhancer activity in vivo
Result: HAR426 ‘A’ form linked to ASC drives higher levels of GFP expression in developing brain
- in vivo assay agrees with in vitro assay
- suggests HAR426 has evolved to drive a particular level of CUX1 and that mutations alter CUX1 expression
- CUX1 itself regulates gene expression so increasing CUX1 levels may affect expression of many other genes - collateral effects
- hypothesis that evolution of HAR426 has contributed to human brain evolution supported by these experiments
