repetitive elements significance evolutionary signposts passive markers for mutation assays actively...

Repetitive elements

Significance

Evolutionary ‘signposts’ Passive markers for mutation assays Actively reorganise gene organisation by

creating, shuffling or modifying existing genes

Chromosome structure and dynamicsProvide tools for medical, forensic,

genetic analysis

Repetitive sequencesAAA, ATATATAT, CGTCGTCGT etc..

5 main classes

1) Tandem repeats2) Transposon-derived

repeats3) Segmental duplications4) Processed pseudogenes

1) Tandem repeats

Blocks of tandem repeats at subtelomeres pericentromeres Short arms of acrocentric

chromosomes Ribosomal gene clusters

Tandem / clustered

repeats

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 265-268

Broadly divided into 4 types based on size

SatellitesLarge arrays of repeats

Some examplesSatellite 1,2 & 3

- found in all chromosomes

Alphoid DNA) satellite

HMG3 by Strachan and Read pp 265-268

MinisatellitesModerate sized arrays of repeats

Some examplesHypervariable minisatellite DNA

- core of GGGCAGGAXG- found in telomeric regions- used in original DNA fingerprinting technique by Alec Jeffreys

HMG3 by Strachan and Read pp 265-268

MicrosatellitesVNTRs - variable number of tandem repeats, SSR - simple sequence repeats

1-13 bp repeats e.g. (A)n ; (AC)n

HMG3 by Strachan and Read pp 265-268

2% of genome (dinucleotides - 0.5%)Used as genetic markers (especially for disease mapping)

Individual genotype

Microsatellite genotyping

Fig 7.7 HMG3 by Strachan and Read pp 190

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

Microsatellite genotyping.

CA repeat genotyping.

Fig 7.8 HMG3

A B C D E Allele typesA (3,6)B (1,5)C (3,5)D (2,5)E (3,6)

Marker D17S800

N.B. ‘stutters’ or shadow bands

Caused by strand slippage

strand slippage during replication

Fig 11.5 HMG3 by Strachan and Read pp 330

Fig 11.5 HMG3 by Strachan and Read pp 330

strand slippage during replication

2) Transposon-derived repeats

A.k.a. interspersed repeats45% of genomeArise mainly as a result of transposition either through a DNA or a RNA intermediate

4 main typesLINES, SINES, LTRs and DNA transposons

Repetitive 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

LINEs (long interspersed elements)

Transposon-derived repeats…

Nature (2001) pp879-880 HMG3 by Strachan & Read pp268-272

Non-autonomous (successful freeloaders! ‘borrow’ RT from other sources such as LINEs)

~100-300bp 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.

HMG3 by Strachan & Read pp268-272

SINEs (short interspersed elements)

Transposon-derived repeats…

Nature (2001) pp879-880

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.

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

http://www.geneticorigins.org/geneticorigins/pv92/intro.html

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

Long Terminal Repeats (LTR)Transposon-derived repeats…

Nature (2001) pp879-880 HMG3 by Strachan & Read pp268-272

DNA transposons Inverted repeats on both sides of elemente.g. ATGCNNNNNNNNNNNCGTA

DNA transposons (lateral transfer?)

Transposon-derived repeats…

Nature (2001) pp879-880

FromGenesVII by Levin

Transposon derived repeats

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

major types

* Updated from HGP publications HMG3 by Strachan & Read pp268-272

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

Segmental duplicationsInterchromosomal segments duplicated

among non-homologous

chromosomes

Intrachromosomal duplications occur within a

chromosome / arm

Nature Reviews Genetics 2, 791-800 (2001);

Segmental duplicationsSegmental duplications in

chromosome 22

Segmental duplications - chromosome 7.

Nature Reviews Genetics 2, 791-800 (2001)

4) Pseudogenes - processed

Repetitive sequencesAAA, ATATATAT, CGTCGTCGT etc..

5 main classes1) Tandem repeats2) Transposon-derived repeats3) Segmental duplications4) Processed pseudogenes

7) Repeat content

a) Age distribution

b) Comparison with other genomes

c) Variation in distribution of repeats

d) Distribution by GC content

e) Y chromosome

Nature (2001) 409: pp 879-891

Insights from the HGP………

Repeat content…….

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

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

a) Age distribution

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!!)

c) Variation in distribution of repeatsSome regions show eitherHigh 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)

High GC – gene rich ; High AT – gene poor

LINEs abundant in AT-rich regionsSINEs 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

d) Distribution by GC content

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

e) The Y chromosome !

Repeat content…….

• Working draft published – Feb 2001

• Finished sequence – April 2003

• Annotation of genes going on

ReferencesText: 1) Human Molecular Genetics 3 by Strachan

and Read – Chapter 9 pp 265-268

Optional Reading

1) Batzer MA, Deininger PL Alu repeats and human genomic diversity Nature Rev Genet 3 (5): 370-379 May 2002

2) BS Emanuel & TH Shaikh Segmental duplications: an 'expanding' role in genomic instability and disease Nature Reviews Genetics 2, 791-800 (2001)

3) Nature (2001) 409: pp 879-891