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

DNA content of organisms appears to increase with complexity

ORGANISM DNA CONTENT (bp)

Mycoplasma 10 6

Bacteria 5 x 10 6

Algae 5 x 10 7

Molluscs 2 x 10 9

Insects 3 x 10 9

Plants 5 x 10 10

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

There are problems with the assumption that as organismsgrow 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 widelydifferent DNA contents.

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 Cot curve

The Cot curve measures the time taken for single stranded DNA molecules to reanneal to each other, and is dependenton how long it takes for each fragment to find its complement.

Co (mol conc of DNA) X t (Time in secs)

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

To do a Cot determination on an eucaryotic genome,

1) Mechanically shear DNA into fragments of approx 1000 bp

2) Denature fragments by heating to approx 95oC

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

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

5) To measure the amount of SS / DS DNA at unit time, theDNA 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 takenfor 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 findtheir complements, bind to HA and very little SS DNA willpass through the column

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

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

Organism Species Genome Sc DNA % Rep

Bacterium E. coli 4.2 x 10 6 4.2 x 10 6 0 ***

Yeast S.cerevisiae 1.3 x 10 7 1.3 x 10 7 0 ***

Worm C. elegans 8.0 x 10 7 6.7 x 10 7 17 ***

Fruit fly D. melano 1.4 x 10 8 1.0 x 10 8 30 ***

Sea urchin S.purpuratus 8.6 x 10 8 4.3 x 10 8 50

Mouse M. musculus 2.7 x 10 9 1.5 x 10 9 42

Toad X. laevis 3.1 x 10 9 1.7 x 10 9 46

Plant N. tabacum 4.8 x 10 9 5.0 x 10 8 67 (***)

Newt T. cristatus 2.2 x 10 10 4.7 x 10 9 53

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

70%30%

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

In the human genome, genes and gene related sequencesaccount 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 andMicro satellite DNA.

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

Satellite DNA can be isolated using Caesium chloridedensity centrifugation

Main band = single copyDNA 40.3% GC content

Satellite bands have differentGC contents and band aboveor below the main band

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

Satellite DNA consists of long tandem arrays of shortOr long (<5 to>200 bp) repeated sequences:

Satellite band tandem repeatscan be 100 kb in length

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 100kbMini satellite DNA – clusters up to 20 kb in lengthMicro 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

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 completelydifferent.

Remember, different sized bandshave different NUMBERS ofrepeats

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

Tandemly repeated DNA is frequently found at chromosomalcentromeres (specialised regions holding sister chromatids)

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 movementIs TRANSPOSITION

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

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

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

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

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