Lecture 2 – Repeat elements BB30055: Genes and genomes Genomes - Dr. MV Hejmadi (bssmvh@bath.ac.uk) Lecture 2 – Repeat elements

Lecture 2 Repeat elements What are repeat elements? How did they originate? Why are they important?

Repetitive DNA Main classes based on origin Tandem repeats Interspersed repeats Segmental duplications

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

Tandem / clustered repeats Broadly divided into 3 types based on size and location 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 Which of the classes of tandem repeats are likely to be found in a mRNA and which are unlikely? HMG3 – Chap 9 pp 265-270

Satellites Large arrays of repeats Some examples Satellite 1,2 & 3 Sat a (Alphoid DNA) - found in all chromosomes Sat b satellite Coutesy: Evani Viegas-Pequignot Alphoid contains centromere binding protein CENPB ICF syndrome (Immunodeficiency- Centromeric instability-Facial anomalies syndrome) Caused by hypomethylation of Sata in chromosomes 1,9,16

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 Similar to ecoli chi sequence – hotspot for recombination

Microsatellites VNTRs - Variable Number of Tandem Repeats, SSR - Simple Sequence Repeats /STR – short tandem 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.

Microsatellite genotyping design PCR primers unique to one locus in the genome a single pair of PCR primers will produce different sized products for each of the different length microsatellites .

Genotype test in a large family using (CA)/(TG) marker D17S800 1 (3,6) 2 (1,5) 3 (3,5) 4 (2,5) 5 (3,6) etc Alleles Samples What is the genotype of sample 10, 13, 16 ? Why do you get the shadow bands? Suggested to be due to replication slippage

strand slippage during replication How are tandem repeats generated in the genome? strand slippage during replication Fig 11.5 HMG3 by Strachan and Read pp 330

strand slippage during replication

2) Interspersed repeats A.k.a. Transposon-derived repeats ~ 45% of genome Arise mainly as a result of transposition either through DNA or RNA retrotransposons (retroposons) ‘copy and paste’ DNA transposons (‘cut & paste’) See lecture 3 for transposition

Classes of transposable elements Classes of mobile elements. DNA transposons, e.g., Tc-1/mariner, have inverted terminal inverted repeats (ITRs) and a single open reading frame (ORF) that encodes a transposase. They are flanked by short direct repeats (DRs). Retrotransposons are divided into autonomous and nonautonomous classes depending on whether they have ORFs that encode proteins required for retrotransposition. Common autonomous retrotransposons are (i) LTRs or (ii) non-LTRs (see text for a discussion of other retrotransposons that do not fall into either class). Examples of LTR retrotransposons are human endogenous retroviruses (HERV) (shown) and various Ty elements of S. cerevisiae (not shown). These elements have terminal LTRs and slightly overlapping ORFs for their group-specific antigen (gag), protease (prt), polymerase (pol), and envelope (env) genes. They produce target site duplications (TSDs) upon insertion. Also shown are the reverse transcriptase (RT) and endonuclease (EN) domains. Other LTR retrotransposons that are responsible for most mobile-element insertions in mice are the intracisternal A-particles (IAPs), early transposons (Etns), and mammalian LTR-retrotransposons (MaLRs). These elements are not present in humans, and essentially all are defective, so the source of their RT in trans remains unknown. L1 is an example of a non-LTR retrotransposon. L1s consist of a 5'-untranslated region (5'UTR) containing an internal promoter, two ORFs, a 3'UTR, and a poly(A) signal followed by a poly(A) tail (An). L1s are usually flanked by 7- to 20-bp target site duplications (TSDs). The RT, EN, and a conserved cysteine-rich domain (C) are shown. An Alu element is an example of a nonautonomous retrotransposon. Alus contain two similar monomers, the left (L) and the right (R), and end in a poly(A) tail. Approximate full-length element sizes are given in parentheses Science 12 March 2004: Vol. 303. no. 5664, pp. 1626 - 1632

Interspersed repeats (transposon-derived) major types class family size Copy number % genome* LINE L1 (Kpn family) L2 ~6.4kb 0.5x106 0.3 x 106 16.9 3.2 SINE Alu ~0.3kb 1.1x106 10.6 LTR e.g.HERV ~1.3kb 0.3x106 8.3 DNA transposon mariner ~0.25kb 1-2x104 2.8 * Updated from HGP publications HMG3 by Strachan & Read pp268-272

Repeat elements in the human genome

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

Segmental duplications Segmental duplications in chromosome 22

Repeat elements How did they originate? Tandem repeats – replication slippage etc Interspersed repeats – transposition events Segmental duplications – strand exchange, recombination events

Repeat elements Why are they important? Evolutionary ‘signposts’ Passive markers for mutation assays Actively reorganise gene organisation by creating, shuffling or modifying existing genes Provides information on chromosome structure and dynamics Provides tools for medical, forensic, genetic analysis

Pathogenic potential of Short Tandem Repeats (STR) Reduction or expansion of STR can be pathogenic 1) Unstable expansion of short tandem repeats Characterised by anticipation Large expansions outside coding sequences Modest expansions within coding sequences FRAXA, FRAX E Huntington disease (HD) Myotonic dystrophy (DM1) SCA 1,2,3,6,7, 17 Friedrich ataxia (FA) Kennedy disease Spinocerebellar ataxia 8,11 Anticipation – lower age of onset and/or severity is worse over subsequent generations FA – Freidrich’s ataxia HD 10-30 CAG repeats – non pathogenic 40-200 repeats – unstable repeats cause huntingtin protein to aggregate and kill cells Pg 477 HMG3 HMG 3 Chapter 11 pp 337 - 344

2) Unstable deletions of STRs STRs tend to be deletion hotspots Pg 358-HMG3

Interspersed repeats are susceptible to deletions/duplications E.g. Kearns-Sayre syndrome- encephalomyopathy External opthalmoplegia Ptosis Ataxia Cataract Common 4977bp deletion in mt DNA External opthalmoplegia Ptosis Ataxia Cataract Mt is recombination deficient therefore such deletions could arise due to replication slippage just like the STR

Pathogenic potential of segmental duplications Nature Reviews Genetics 2, 791-800 (2001)

STRs are…  A) microsatellite sequences. B) generally highly polymorphic. C) Useful markers along the chromosome. D) a and b b and c a and c

Which of the following is not one of the four classes of transposon-derived repeat sequences?  A. LINES B. SINES C. DNA transposons D. Long terminal repeat transposons E. pseudogenes

When repeats of a region lie adjacent to each other they are called ___________ duplications ________________ gene transfer is the direct transfer of genes from one species in the germ line of another species.

True or False All Repetitive DNA elements in the human genome arose through a common mechanism

Match the columns Recombination events Tandem repeats Replication slippage Transposition events Tandem repeats Interspersed repeats Segmental duplications

Reading Chapter 9: HMG 3 by Strachan & Read Repeat elements and disease Nature Reviews Genetics 2, 791-800 (2001)

CpG islands show no methylation TpG Methyl CpG Deamination methylated at C CpG islands show no methylation Significance of CpG islands Non-methylated CpG islands associated with the 5’ ends of genes Usually overlap the promoter region Aberrant methylation of CpG islands linked to pathologies like cancer or epigenetic diseases like Rhett’s syndrome http://www.sanger.ac.uk/HGP/cgi.shtml

Inheritance of CpG methylation