W1 L2 negative selection W

Question 1

Distribution of constraint in mammals: Human vs. chimp divergence

Answer 1

Human vs. chimp divergence: sequence conservation reflects function
- pseudo-gene evolve at the highest rate
- 4 fold degenerate site: evolve like pseudo gene due to codon bias
-non-synonymous site evolve at a lower rate (deleterious)
Much more sequence conservation in the sites that encode for amino acids than other sites.

Question 2

Purifying selection/negative constraint/negative selection: Divergence between D. melanogaster & D. simulans

Answer 2

• Very closely related species: Pseudogenes evolve fastest, synonymous sites (4-fold degenerate sites) slightly less.
• CDS evolves with constraint (expected) & UTRs showed high constraint (mRNA half-life, miRNAs).
• AA encoding regions accumulate many deleterious mutations => purged from population via negative selection.

Question 3

Footprinting DNase1

Answer 3

-using dnase 1, it will bind to area of the gene where protein normally bind to and cut it

Question 4

Phylogenetic Footprinting

Answer 4

To identify regulatory elements.
-Closer related species may not have had enough time to diverge
-Not enough divergence between human & chimp to distinguish, & cannot vs. mice (do not have Apo gene). Get divergence by (not pair-wise comparisons) with ApoA.
sequencing lots of closely related species : Conserved & variable regions added in front of a reporter => Distinct difference. time for divergence to occur.
* Further related species that still retain a similar ‘peak’ (i.e. nt) are of interest to determine function.

Question 5

ApoA

Answer 5

High plasma levels are a risk indicator for cardiovascular disease
* But the gene is only found in Old world monkeys and Hominids
Get divergence by sequencing lots of closely related species > “phylogenetic shadowing”

Question 6

Phylogenic shadowing with ApoAo

Answer 6

Not enough divergence between human & chimp to distinguish, & cannot vs. mice (do not have Apo gene). Get divergence by (not pair-wise comparisons) with ApoA.sequencing lots of closely related species
: Conserved & variable regions added in front of a reporter => Distinct difference.

Question 7

Extreme levels of conservation

Answer 7

Ultra-Conserved Sequences:Berjano et al. (2004) Science
Human/rat/mouse
481 segments of 200bp with 100% identity
* Conserved non-coding elements Woolfe et al. (2005) PloS
Human/Fugu
1373 non-coding of 200bp with 80% identity
* Conserved Non-Genic Sequences
Dermitzakis et al (2003) Science
Human/mouse
327,000 segments of 100bp with 70% identity

Question 8

CNGs coding non genic region

Answer 8

Not transcribed (no Expressed Sequence Tags (ESTs)/cDNAs)
* Don’t show substitution patterns of coding or noncoding RNA sequences
-Higher level of purifying selection acting on it than coding sequences

Question 9

Ultra Conserved Sequences

Answer 9

• Many of them, but each is unique
• Often Clustered
• Often in gene deserts
• Many are near genes that are involved in regulation of transcription or development
• Some overlap with exonic and these are highly enriched for genes involved in RNA binding and splicing regulation

Question 10

Reporter genes and CNS enhancer

Answer 10

Using reporter gene to test the fuction of CNS
* Reporter constructs (transgenic mice) recapitulate a component of the expression pattern.
* Add CNS upstream of a reporter & transient transfection in Zebrafish to detect presence of nearby credible gene.
* Pax6 (eye development): Test 7 CNS to detect if it drives GFP (reporter) expression in Zebrafish.
* Some CNGs express exclusively in eye: suggests conserved region 19 involved in driving express of adjacent gene (pax6)
* Here, ultra-conserved regions are regulatory elements/modules far away from gene itself (true for some genes).

Question 11

Conservation of function rather than sequence: Even-skipped (eve) counter-example:

Answer 11

Stripe 2 enhancer in early body plan: a regulatory module composed of multiple TF binding sites.
Sequence region has a conserved function but the sequence itself has changed a lot over evolutionary time.
Expect body plan gene conserved over Drosophila or more but binding site variation analyses show slight variation in D. yakuba & D. erecta, but D. pseudoobscura had hardly any conservation.
: D. p s2 enhancer expresses same as D. m s2 enhancer, even though it is not conserved at all: .
Not only does the D. pseudoobscura enhancer express at correct place (in D. m), but it rescues to the same extent. Chimeric (half-half) enhancers don’t recapitulate pattern (stripe shifted). Wrong no. binding sites & alters express.
; enhancer is defined by TF binding sites. ; moved with respect to each other.

Question 12

what is the phylogeny process in sequences comparison

Answer 12

• Phylogeny: Compare genome & identify slow-evolving regions to find AA encoding seq (=> function & role)
  Harder to identify e.g. promoter 5’ region with a regulatory role.