Alu Human Polymorphism

1. Alu Human Polymorphism (Differences between individuals at chromosomal regions) Polymorphic means “many forms” Many polymorphisms are located in the non-coding DNA.

2. Human Genome • How many chromosomes does each human cell have? • 22 pairs of autosomal chromosome and 1 pair of sex chromosomes • What is the size of the human genome? • The human genome is made up of 3 billion base pairs • How many genes does the Human Genome code for?

3. Genome Comparison

4. Human Genome • What is a gene? • A gene is segment of a genome that encodes a single specific end product (a protein or RNA) or a functional unit or information • How much of the genome are “genes”? • ~1.5% of the genome • What accounts for the rest of the DNA in the genome?

6. Human Genome • Other items in genome • Introns • Repeated sequences (telomeres, centromeres) • Non-coding RNAs • Transposons (SINES and LINES) (Short Interspersed Elements: less than 500 bp long and present 500,000 or more in the genome and Long Interspersed Elements) • Psuedogenes (genes that no longer produce functional products)

7. Transposons – Transposable Element • Alu is a transposable element. • A member of SINEs. 300 nucleotides. • Name comes from a endonuclease AluI that is found in the middle. • Sometimes called “jumping genes” but not really a jumping gene because it doesn’t code for a protein. Retrotransposon L1 (a LINE) supplies the rt (reverse transcriptase) needed to jump.

8. Alu elements • http://www.geneticorigins.org/pv92/aluframeset.htm • Under media/animations • Illustrations about the Alu element listed as How Alu jumps • Pedigree on Alu inheritance listed as PV92 inheritance • Theory: PV92 locus on Chromosome 16; • Homozygous ++; Heterozygous + -; Homozygous – (inherited in Mendelian fashion) • 731 bp with; 416 bp without • Readily separated by gel electrophoresis

9. Alu elements • Alu is a “defective” transposon of the SINE (Short INterspersed Element) family • Alu is about 300 nucleotides (base pairs) in length • Human chromosomes have more than 1 million Alu copies (10% of the genome by mass) • There is no evidence that an Alu can be lost from a site – it can only be inserted

10. Alu elements • Alu has the necessary sequences to be transcribed by RNA Pol III • The LINE (Long INterspersed Element) L1 supplies the reverse transcriptase needed for Alu to jump • L1 is a defective retrovirus • It nicks the DNA close to the sequence AATTTT • The AAAA of Alu transcript binds to the TTTT of the nicked DNA – the acts as a primer for the L1 to bind to

11. Alu Elements • What are the consequences and benefits of having an Alu element? • Alu inserting into exons has been implicated in neurofibromatosis, cancer and heart attacks • They can provide alternate splicing in 5% of genes which leads to genetic diversity • Can provide information on migration and evolution

12. Alu elements • Alu elements are only found in the primate branch • Each Alu insertion is a unique event and is inherited from each parent • Most occurred millions of years ago and are often on both pairs of chromosomes • There are Alu elements that have occurred since humans branched from other primates • This gives rise to dimorphic Alus from the last hundreds of thousands of years • Estimated to jump 1:200 live human births

13. Alu Elements • The Alu element that we will be looking at in on chromosome 16 • There is no phenotypic data associated with this locus (i.e. does not associate with disease, trait, or sex determination)

14. Human Genome Online resources for mapping genetic diseases • http://gslc.genetics.utah.edu/units/disorders/sloozeworm/scenario.cfm • http://gslc.genetics.utah.edu/units/disorders/pedigree/mapgene.cfm

15. Alu Lab • What is the benefit of saline? Saline is a very reproducible way of harvesting cells – it loosens a large number of single cheek cells • What about food contamination? • Don’t eat before lab; Brush your teeth, etc. after you • Have breakfast. If molecules of food get in, it’s ok. • No effect because the element is only found in primates • Why are we boiling the samples? • To lyse open the cells and release the DNA • What is Chelex? • Chelex is a metal binding resin • It is used to scavenge metal that can contaminate PCR reactions

16. Alu Lab • Where do the primers amplify? • Primers amplify region on chromosome 16 – outside of the Alu region – the primers do not amplify Alu itself • What will the products (+/+, +/- and -/-) look like? • -/- has one smaller band around 400bp, +/+ has one larger band around 700bp; +/- has two bands one at 400 and one at 700 • There is a picture of a control gel on: http://www.geneticorigins.org/pv92/aluframeset.htmunder the theory section • Why is there no control for the PCR reaction? • There is no need for a control because all samples will produce a band therefore it will tell us if the DNA was good without a need for an additional control

17. The primer/loading dye is sucrose and cresol red. Allows it to be loaded in the gel wells and stay there and see where it is. • The Ready-to-Go PCR bead contains reagents: Taq DNA polymerase, Tris-HCl (ph 9.0); KCl; MgCl2; and dNTPs • Plasmid pBR322 is digested with restriction endonuclease BstNI – produces markers. (This will be the ladder). It produces fragments at 1857 bp; 1058 bp; 929 bp; 383 bp; and 121 bp.

18. Alu Lab Possible problems • Lose cell pellet at the beginning • No amplification • Maybe didn’t get complete lysis

19. Alu within the Alu Alu paper is on the wiki listed under Human DNA Polymorphism: Recent Insertion of an Alu element within a Alu insertion Online alignment tool • http://www.ch.embnet.org/software/LALIGN_form.html • To use Alignment tool copy the mutated large Alu sequence into the box labeled 1st query sequence • Make sure to name the sequence in box labeled First sequence title (I named this Mut) • Paste Alu element (non-mutated) sequence into box labeled 2nd sequence query • Make sure to name the sequence in box labeled Second sequence title (I named this Alu) • Click Run Align at the bottom of the page • This will produce the alignment with the details about what bases were aligned and the % of identity • This will give you three alignments with the Alu element

20. Alu within the Alu Mutated larger Alu sequence (from GenBank extension number AF302689): aactgggaaa atttgaagag aaagtcacac agatacattt cagtaaggtt gtctctgtta 61 cttgaggctt acaagaagga aagaaggccg ggcgcggtgg ctcacgcctg taatcccagc 121 actttgggag gccgaggcgg gcggatcacg aggtcaggag atcgagacca tcccggctaa 181 aacgctgaaa cctcgtctct actaaaaata caaaaaattg ccgggcgcgg tggctcacgc 241 ctgtaatccc agcactttgg gaggccgagg cgggtggatc atgaggtcag gagatcgaga 301 ccatcctggc taacaaggtg aaaccccgtc tctactaaaa atacaaaaaa ttagccgggc 361 gcggtggcgg ggcgcctgta gtcccagcta ctcgggaggc tgaggcagga gaatggcgtg 421 aacccgggaa gcggagcttg cagtgagccg agattgcgcc actgcagtcc gcagtccggc 481 ctgggcgaca gagcgagact ccgtctcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 541 aaaaaaaaaa aaaataccaa aaattagccg ggcgtagtgg cgggcgcctg tagtcccagc 601 tacttgggag gctgaggcag gagaatggcg tgaacccggg aggcggagct tgcagtgagc 661 cgagatcctg ccactgcact ccagcgtggg cgacagagcg agactccgtc tcaaaaaaaa 721 aaaaaaaaaa aaaaaaagaa agaattccct ctctaaacac actctaacac acaggagttg 781 agaactca

21. Alu within the Alu Alu sequence (non-mutated): ggccgggcgcggtggctcacgcctgtaatcccagcactttgggaggccgaggcgggcggatcacgaggtcaggagat gagaccatcccggctaaaacgctgaaacctcgtctctactaaaaatacaaaaaattagccgggcgtagtggcgggcgctgtagtcccagctacttgggaggctgaggcaggagaatggcgtgaacccgggaggcggagcttgcagtgagccgagatcctgccactgcactccagcctgggcgacagagcgagactccgtctcaaaaaaaaaaaaaaaaaaaaaaaaaaa

22. Alu within the Alu

24. Alu within the Alu: sequence results ½ Alu sequence Full Alu sequence ½ Alu sequence

25. Website National Geographic articles on using the Y-chromosome to trace migration out of Africa http://news.nationalgeographic.com/news/2002/12/1212_021213_journeyofman.html DNA interactive website: links to Human Identification, Recovering the Romanovs, Human Origins and Genes and Medicine http://www.dnai.org/d/index.html