1. Repetitive elements Evolutionary ‘signposts’
Significance
Evolutionary ‘signposts’
Passive markers for mutation assays
Actively reorganise gene organisation by creating, shuffling or modifying existing genes
Chromosome structure and dynamics
Provide tools for medical, forensic, genetic analysis

2. Repetitive sequences AAA, ATATATAT, CGTCGTCGT etc.. 5 main classes
Tandem repeats
Transposon-derived repeats
Segmental duplications
Processed pseudogenes

3. 1) Tandem repeats Blocks of tandem repeats at subtelomeres
pericentromeres
Short arms of acrocentric chromosomes
Ribosomal gene clusters

4. Tandem / clustered repeats
Broadly divided into 4 types based on size
class
Size of repeat
Repeat block
Major chromosomal location
Satellite
5-171 bp
> 100kb
centromeric
heterochromatin
minisatellite
9-64 bp
0.1 – 20kb
Telomeres
microsatellites
1-13 bp
< 150 bp
Dispersed
HMG3 by Strachan and Read pp

5. Satellites Large arrays of repeats Some examples Satellite 1,2 & 3
- found in all chromosomes
a (Alphoid DNA)
b satellite
HMG3 by Strachan and Read pp

6. Minisatellites Moderate sized arrays of repeats Some examples
Hypervariable minisatellite DNA
- core of GGGCAGGAXG
- found in telomeric regions
- used in original DNA fingerprinting technique by Alec Jeffreys
HMG3 by Strachan and Read pp

7. Microsatellites 1-13 bp repeats e.g. (A)n ; (AC)n Individual genotype
VNTRs - variable number of tandem repeats, SSR - simple sequence repeats
1-13 bp repeats e.g. (A)n ; (AC)n
2% of genome (dinucleotides - 0.5%)
Used as genetic markers (especially for disease mapping)
A father might have a genotype of 12 repeats and 19 repeats, a mother might have 18 repeats and 15 repeats while their first born might have repeats of 12 and 15.
Individual genotype
HMG3 by Strachan and Read pp

8. Microsatellite genotyping
The most common way to detect microsatellites is to design PCR primers that are unique to one locus in the genome and that base pair on either side of the repeated portion
Therefore, a single pair of PCR primers will work for every individual in the species and produce different sized products for each of the different length microsatellites
Fig 7.7 HMG3 by Strachan and Read pp 190

9. Microsatellite genotyping.

10. CA repeat genotyping Marker D17S800 Allele types A (3,6) B (1,5).
Marker D17S800 A B C D E
Allele types
A (3,6)
B (1,5)
C (3,5)
D (2,5)
E (3,6)
N.B. ‘stutters’ or shadow bands
Caused by strand slippage
On rare occasions, microsatellites can cause the DNA polymerase to make an extra copy of CA similar to the way we find it difficult to say toy boat several times in a row with consistent accuracy. If an individual¹s DNA polymerase adds to the repeated sequence, then this slightly larger version can be passed on to offspring who will usually replicate it accurately. Over time, as animals in a population breed, they will recombine their microsatellites during sexual reproduction and the population will maintain a variety of microsatellites that is characteristic for that population and distinct from other populations which do not interbreed
Fig 7.8 HMG3

11. strand slippage during replication
Fig 11.5 HMG3 by Strachan and Read pp 330

12. strand slippage during replication
Fig 11.5 HMG3 by Strachan and Read pp 330

13. 2) Transposon-derived repeats
Repetitive elements…
2) Transposon-derived repeats
A.k.a. interspersed repeats
45% of genome
Arise mainly as a result of
transposition either through
a DNA or a RNA intermediate
4 main types
LINES, SINES, LTRs and DNA transposons

14. LINEs (long interspersed elements)
Transposon-derived repeats…
LINEs (long interspersed elements)
Most ancient of eukaryotic genomes
Autonomous transposition (reverse trancriptase)
~6-8kb long
Internal polymerase II promoter and 2 ORFs
3 related LINE families in humans
– LINE-1, LINE-2, LINE-3.
Believed to be responsible for retrotransposition of SINEs and creation of processed pseudogenes
Nature (2001) pp
HMG3 by Strachan & Read pp

15. SINEs (short interspersed elements)
Transposon-derived repeats…
SINEs (short interspersed elements)
Non-autonomous (successful freeloaders! ‘borrow’ RT from other sources such as LINEs)
~ bp long
Internal polymerase III promoter
No proteins
Share 3’ ends with LINEs
3 related SINE families in humans
– active Alu, inactive MIR and Ther2/MIR3.
Nature (2001) pp
HMG3 by Strachan & Read pp

16. LINES and SINEs have preferred insertion sites
In this example, yellow represents the distribution of mys (a type of LINE) over a mouse genome where chromosomes are orange. There are more mys inserted in the sex (X) chromosomes.

17. Try the link below to do an online experiment which shows how an Alu insertion polymorphism has been used as a tool to reconstruct the human lineage

18. Long Terminal Repeats (LTR)
Transposon-derived repeats…
Long Terminal Repeats (LTR)
Repeats on the same orientation on both sides of element
e.g. ATATATNNNNNNNATATAT
Autonomous or non-autonomous
Autonomous retroposons encode gag, pol genes which encode the protease, reverse transcriptase, RNAseH and integrase
Nature (2001) pp
HMG3 by Strachan & Read pp

19. DNA transposons (lateral transfer?)
Transposon-derived repeats…
DNA transposons (lateral transfer?)
DNA transposons
Inverted repeats on both sides of element
e.g. ATGCNNNNNNNNNNNCGTA
From
GenesVII by Levin
Nature (2001) pp

20. Transposon derived repeats
major types
class
family
size
Copies*
% genome*
LINE
LINE-1 (Kpn family)
~6.4kb
0.8x106
15.4
SINE
Alu
~0.3kb
1.3x106
10.7
LTR
e.g.HERV
~1.3kb
0.7x106
7.9
DNA
transposon
mariner
~0.25kb
0.4x106
2.7
* Updated from HGP publications
HMG3 by Strachan & Read pp

21. 3) Segmental duplications
Closely related sequence blocks at different genomic loci
Transfer of 1-200kb blocks of genomic sequence
Segmental duplications can occur on homologous chromosomes (intrachromosomal) or non homologous chromosomes (interchromosomal)
Not always tandemly arranged
Relatively recent

22. Segmental duplications
Interchromosomal segments duplicated among non-homologous chromosomes
Intrachromosomal duplications occur within a chromosome / arm
Nature Reviews Genetics 2, (2001);

23. Segmental duplications
Segmental duplications in chromosome 22
Segmental duplications

24. Segmental duplications - chromosome 7.

25. Nature Reviews Genetics 2, 791-800 (2001)

26. 4) Pseudogenes - processed

27. Repetitive sequences AAA, ATATATAT, CGTCGTCGT etc.. 5 main classes
Tandem repeats
Transposon-derived repeats
Segmental duplications
Processed pseudogenes

28. Insights from the HGP………
7) Repeat content
Age distribution
Comparison with other genomes
Variation in distribution of repeats
Distribution by GC content
Y chromosome
Nature (2001) 409: pp

29. Repeat content…….
a) Age distribution
Most interspersed repeats predate eutherian radiation (confirms the slow rate of clearance of nonfunctional sequence from vertebrate genomes)
LINEs and SINEs have extremely long lives
2 major peaks of transposon activity
No DNA transposition in the past 50MYr
LTR retroposons teetering on the brink of extinction
Most IR predate eutherian radiation (confirms the slow rate of clearance of nonfunctional sequence from vertebrate genomes)
LINEs and SINE have extremely long lives
2 major peaks of transposon activity
No DNA transposition in the past 50MYr
LTR teetering on the brink of extinction

30. a) Age distribution
overall decline in interspersed repeat activity in hominid lineage in the past 35-40MYr
compared to mouse genome, which shows a younger and more dynamic genome
Most IR predate eutherian radiation (confirms the slow rate of clearance of nonfunctional sequence from vertebrate genomes)
LINEs and SINE have extremely long lives
2 major peaks of transposon activity
No DNA transposition in the past 50MYr
LTR teetering on the brink of extinction

31. b) Comparison with other genomes
Higher density of transposable elements in euchromatic portion of genome
Higher abundance of ancient transposons
60% of IR made up of LINE1 and Alu repeats
whereas DNA transposons represent only 6%
(a few human genes appear likely to have resulted from horizontal transfer from bacteria!!)

32. c) Variation in distribution of repeats
Some regions show either
High repeat density
e.g. chromosome Xp11 – a 525kb region shows 89% repeat density
Low repeat density
e.g. HOX homeobox gene cluster (<2% repeats)
(indicative of regulatory elements which have low tolerance for insertions)

33. d) Distribution by GC content
High GC – gene rich ; High AT – gene poor
LINEs abundant in AT-rich regions
SINEs lower in AT-rich regions
Alu repeats in particular retained in actively transcribed GC rich regions E.g. chromosme 19 has 5% Alus compared to Y chromosome

34. Repeat content…….
e) The Y chromosome !
Unusually young genome (high tolerance to gaining insertions)
Mutation rate is 2.1X higher in male germline
Possibly due to cell division rates or different repair mechanisms

35. Working draft published – Feb 2001
Finished sequence – April 2003
Annotation of genes going on

36. References
Text:
1) Human Molecular Genetics 3 by Strachan and Read – Chapter 9 pp
Optional Reading
Batzer MA, Deininger PL Alu repeats and human genomic diversity Nature Rev Genet 3 (5): May 2002
BS Emanuel & TH Shaikh Segmental duplications: an 'expanding' role in genomic instability and disease Nature Reviews Genetics 2, (2001)
Nature (2001) 409: pp