1. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) DNA content of organisms appears to increase with complexity ORGANISMDNA CONTENT (bp) Mycoplasma10 6 Bacteria5 x 10 6 Algae 5 x 10 7 Molluscs 2 x 10 9 Insects 3 x 10 9 Plants5 x 10 10

2. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) There are problems with the assumption that as organisms grow larger and more complex, they need more DNA. DNA content = 10 9 bp DNA content is 10 11 bp “C” value paradox – organisms which are similar have widely different DNA contents.

3. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) The major fraction of eucaryotic DNA consists of repetitive, non-coding sequences This was discovered using a DNA/DNA hybridisation technique called a C o t curve The C o t curve measures the time taken for single stranded DNA molecules to reanneal to each other, and is dependent on how long it takes for each fragment to find its complement. C o (mol conc of DNA) X t (Time in secs)

4. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) To do a C o t determination on an eucaryotic genome, 1) Mechanically shear DNA into fragments of approx 1000 bp 2) Denature fragments by heating to approx 95 o C 3) Lower temperature so that hybrids form 4) Measure the amount of SS and DS DNA present at unit time after the temperature has been lowered

5. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) 5) To measure the amount of SS / DS DNA at unit time, the DNA solution is passed through a hydroxyapatite column. 6) SS DNA passes through the column, DS DNA binds. 7) All you are doing is measuring the time taken for similar sequences to anneal to each other i.e. the amt of SS DNA passing through at unit time 8) If the genome is repetitive, repetitive fragments will find their complements, bind to HA and very little SS DNA will pass through the column

6. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1)

7. OrganismSpeciesGenomeSc DNA% Rep BacteriumE. coli4.2 x 10 6 0 *** YeastS.cerevisiae1.3 x 10 7 0 *** WormC. elegans8.0 x 10 7 6.7 x 10 7 17 *** Fruit flyD. melano1.4 x 10 8 1.0 x 10 8 30 *** Sea urchinS.purpuratus8.6 x 10 8 4.3 x 10 8 50 MouseM. musculus2.7 x 10 9 1.5 x 10 9 42 ToadX. laevis3.1 x 10 9 1.7 x 10 9 46 PlantN. tabacum4.8 x 10 9 5.0 x 10 8 67 (***) NewtT. cristatus2.2 x 10 10 4.7 x 10 9 53

8. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) 70% 30%

9. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) In the human genome, genes and gene related sequences account for 30% of the total DNA. However, only 3% of the genome is coding, the rest is Non-coding (27%) and Repetitive ( 70%) Repetitive DNA is organised into two classes: Tandemly repeated DNA and Interspersed genome wide repeats Tandemly repeated DNA = Satellite, Mini satellite and Micro satellite DNA.

10. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) Satellite DNA can be isolated using Caesium chloride density centrifugation Main band = single copy DNA 40.3% GC content Satellite bands have different GC contents and band above or below the main band

11. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) Satellite DNA consists of long tandem arrays of short Or long ( 200 bp) repeated sequences: Satellite band tandem repeats can be 100 kb in length

12. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) Mini and Micro satellites are other types of satellite DNA Satellite DNA – tandemly repeated in clusters up to 100kb Mini satellite DNA – clusters up to 20 kb in length Micro satellites – clusters up to 150 bp in length. Microsatellites are variable – number of repeats of a particular sequence varies between individuals This variability is the basis of DNA fingerprinting

13. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) Suspect 1 has a DNA profile which matches the sperm sample Suspect 2 and “boyfriend” have profiles which are completely different. Remember, different sized bands have different NUMBERS of repeats

14. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) Tandemly repeated DNA is frequently found at chromosomal centromeres (specialised regions holding sister chromatids)

15. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) Repetitive DNA is organised into two classes: Tandemly repeated DNA and Interspersed genome wide repeats Interspersed – scattered throughout the genome, NOT in repeats Mechanism of movement Is TRANSPOSITION

16. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1) Two examples of Interspersed Repeats are: S hort I nterspersed N uclear E lements S 300bp, 10 6 copies (TRANSPOSE) L ong I nterspersed N uclear E lements S 6.0 kb, 4,000 copies

17. BSL 2016 – Lecture 3 – Genome evolution and repetitive DNA (1)

19. Recent genome sequencing programs have found that have large lengths of identical intergenic DNA, despite separating 80m years ago (function?) Q – What happens when these identical regions are deleted? Knock out 3MB conserved region Insert altered genome into mouse ES cells Introduce ES cells into denucleated eggs Phenotypic analysis