Brain Journal , 2004, Everett & Wood, pp. 2385-2405

1. (Huntington’s Disease) Brain Journal, 2004, Everett & Wood, pp. 2385-2405

2. Brain Journal, 2004, Everett & Wood, pp. 2385-2405

3. Different regions of brain affected by Triple Repeat Diseases (Page 413, book)

4. EcoR1 restriction site EcoR1 restriction site Isolation Of The Gene Implicated In Spinocerebellar Ataxia Type-1 From Five Primate Species S.J. Richards, E.B. Whitledge, J.M. Lau, and D.L. Robinson Department of Biology, Bellarmine University, 2001 Newburg Rd, Louisville, KY 40205 Introduction CAG Repeats In The SCA-1 Geneof Healthy Animals Initial SCA-1 PCR Second SCA-1 PCR • Spinocerebellar Ataxia Type-1 (SCA-1) is a rare, dominantly-inherited, neurodegenerative disease that results from a tri-nucleotide (CAG) expansion. Six to thirty-nine CAG repeats occur in the Ataxin-1 gene of healthy people.3 • What makes this neurodegenerative disease unique is ‘anticipation’. As the mutant gene is passed from generation to generation the age of onset of symptoms decreases as a result of enlargement of the poly-Q (polyglutamine) region.2 • The expansion of the poly-Q repeat region occurs during DNA replication, and can reach as high as 81 CAG repeats in the Ataxin-1 gene of people with SCA-1. • As a result, the protein Ataxin-1 gains a toxic function that results in the eventual death of the Purkinje cells of the cerebellum and spinal cord. • The length of the poly-Q region is negatively correlated with the age of onset. 1 2 3 4 • Lane: • 100 bp MW ladder • Human DNA • Lemur DNA • Macaque DNA • Lane: • 100 bp MW ladder • Macaque • Orangutan • Vervet • Wooly Monkey • 100 bp MW ladder 400 bp 300 bp 200 bp 100 bp Transformation • After PCR amplification, UA cloning was used to ligate the PCR product into a plasmid vector (Qiagen). • Heat-shock transformation was then used to incorporate the plasmid vector into bacterial cells. • Transformants were selected using blue-white screening. • Vector plasmid was purified, quantified, and a sample was digested using EcoRI to remove the insert from the plasmid. • Multiple sequence alignments of the CAG repeat region from 14 species were analyzed using the ClustalW program (Vector NTI). • This program allows for analysis of this region in an evolutionary context. ABNORMAL Purkinje Cell NORMAL Purkinje Cell Projected Phylogenic Relationship Objectives • Isolate and sequence the CAG repeat region of the Ataxin-1 gene from five primate species: • Macaca assamensis (Assamese Macaque) • Cercopithecus aethiops (Vervet Monkey) • Eulemur macaco (Black Lemur) • Pongo pygmaeus (Orangutan) • Lagothrix lagothrica (Woolly Monkey) • Search various DNA databases for other SCA-1 sequences, and use that information to examine the evolution of the CAG repeat region. Lemur SCA-1 EcoRI Digestion Lane: (1) 100 bp MW Ladder (2) Lemur SCA-1 [3] (3) Lemur SCA-1 [4] (4) Lemur SCA-1 [7] (5) Lemur SCA-1 [8] (6) 100 bp MW Ladder 1 2 3 4 5 6 300 bp 200 bp Conclusions • We have successfully isolated the CAG repeat region of SCA-1 from 5 primates. • The number of CAG repeats in healthy primates ranged from 2 to 29. • ClustalW analysis of the CAG repeat region showed fairly predictable phylogenetic relationships between the species examined. • The closer the evolutionary relationship a species has to humans, the more CAG repeats appear in the SCA-1 gene. • It appears that a greater number of CAG repeats begin to appear in primates as the development fine motor skills and dexterity become refined. Methods • Primer design and optimization. • PCR amplification and purification. • Ligation to a plasmid vector. • Heat-shock transformation into competent cells. • Plasmid prep and purification. • Sequencing. 1 2 3 4 5 DNA Sequencing • Transformed plasmid samples were sent to the University of Louisville for sequencing. • Sequences were analyzed using Vector NTI (Version 10.0). PCR References • PCR primers were designed using published DNA sequences for the Ataxin-1 gene in both Chimpanzee and Human (NCBI GenBank). • Forward 5’-ACCTATGCCAGCTTCATCCCATC-3’; TM: 59.0˚ C • Reverse 5’-GTCATGCAGGTGTAAAGGTCAAGA-3’; TM: 56.8˚ C • DNA was extracted from blood or muscle tissue from healthy animals. Five ng of DNA template was used for PCR. • PCR conditions: initial denaturing at 95 C for 5 min, followed by 35 cycles at 95 for 1:20 min, 52 for 1:43 min, 73 for 1:20 min, and 6 min extension at 73. Vector DNA Acknowledgements 1 Banfi, S., A. Servadio, M.Y. Chung, T.J. Kwiatowski, Jr., A.E. McCall, L.A. Duvick, Y. Shen, E.J. Roth, H.T. Orr and H.Y. Zoghbi. 1994. Identification and characterization of the gene causing type 1 spinocerebellar ataxia. Nature 7: 513-520. 2 Orr, H.T., M.Y. Chung, S. Banfi, T.J. Kwiatowski, Jr., A. Servadio, A.L. Beaudet, A.E. McCall, L.A. Duvick, L.P.W. Ranum and H.Y. Zoghbi. 1993. Expansion of an unstable CAG repeat in spinocerebellar ataxia type 1. Nature 4: 221-226. 3 Everett C.M., N.W Wood. 2004. Trinucleotide Repeats and Neurodegenerative Diseases. Brain. 127: 2385-2405. • We would like to thank Dr. Roy Burns and the Louisville Zoo for their help. • This project was partially supported by NIH Grant Number P20 RR16481 from the BRIN Program of the National Center for Research Resources. • We would also like to thank Dr. Ric Devor, Integrated DNA Technologies INC., Coralville, IA. • We would also like to thank Dr. Steven Wilt, Bellarmine University.

6. 1966 Arlo Woody Guthrie (died at 55, in 1967) Sarah

7. Huntington's disease is… • …a genetic disease of the central nervous system that produces • speech slurring, involuntary movements, & progressive dementia. • It usually starts between the ages of 30 and 50, and causes death • after about 20 years (usually of pneumonia, choking, or heart failure). • Suicide is common. • Between 100,000-250,000 Americans have it • (or will when they get older). • …one birth in every 10,000 has the disease • It is a dominant mutation which is easily passed on • because people don’t know they have it until later in life. • There is no known cure.

8. This disease is named after Dr. Huntington (Long Island) who first diagnosed himself with the disease in 1872. His father and grandfather both died of the disease. His distant relative (who first came to America in the 1630’s) did so after being persecuted in Europe for consorting with the “devil” and for practicing witchcraft. It was probably the Huntington’s Disease that caused people to conclude he was “possessed”. (At first, people thought Woody Guthrie was an alcoholic…. then schizophrenic….)

9. ? ? The story of Nancy Wexler

10. Nancy Wexler (Ph.D. in psychology) Her mother died of Huntington’s so she may have the disease herself. In 1979, she saw a film about a Venezuelan village where an excessive number of people had the disease…... She got federal grant to visit the village and interview the people.

11. Once they found out she might have the disease too, they eventually grew to trust her (and answered her prying questions)

12. She built a pedigree chart of 15,000 Venezuelans and collected blood from 3,500 of them. This took 13 years….

13. How did this disease originate in this little village in Venezuela? In the 1800’s a Portuguese sailor come to the village. Some rumored he was a drunkard because he always walked as if he was intoxicated. Eventually, he married a local woman and had numerous children. Later, he died of unknown causes...... But his gene for Huntington’s Disease still survives in this village today (seven generations later)…. Of his 5,000 direct relatives, 250 of them have Huntington’s Disease (that is 1 out of every 20).

14. 1966 Arlo Woody Guthrie (died 1967) Sarah

15. The gene was isolated in 1993. Chromosome 4 Dominant. The CAG repeats occur in the first exon. Normal = 6 - 35 repeats Diseased = 40 – 121 repeats

16. What is affected by the mutated Huntington Protein? Granular and filamentous Brain Journal, 2004, Everett & Wood, pp. 2385-2405

17. And that’s why Positional Cloning is important, honey!

22. Some Human Disease Genes identified by Positional Cloning: 1986 = Duchennes Muscular Dystrophy 1989 = Cystic Firbrosis 1990 = 4 more 1991 = Fragile X Syndrome & 3 others 1992 = Lowe Syndrome & 2 others 1993 = Huntingtons Disease & 11 more 1994 = BRCAI (Breast Cancer), Dwarfism & 11 others 1995 = Alzheimers II, BRCAII & 9 others 1996 = X-linked Myotubular Myopathy & 15 others 1997 = Deafness (DFNAI), Tuberous Sclerosis, Juvenile Glaucoma & 13 others 1998 = Congenital Night Blindness, Juvenile Parnkinsons Disease & 10 others

23. How does the Huntington’s Disease gene actually cause disease? A “degenerative disease”.. ..it is 10-20 years before becoming fatal.

24. Apparently, it is a mutation that causes the repetition of the sequence “CAG”. Whereas a healthy person has 20 or so repeats (CAGCAGCAGCAGCAG...) people who have this disease have from 39 to 125 CAG repeats in a row. The more CAG repeats they have, the earlier the disease shows up.

25. # of CAG Repeats Median Age at Onset * 39 66 years 40 59 41 54 42 49 43 44 44 42 45 37 46 36 47 33 48 32 49 28 50 27 *Age by which 50% of individuals will be affected Why are these repeats so harmful?

26. CAG is the code for the amino acid Glutamine

28. This CAG gets repeated up to 125x Loci is on Chromosome 4 “HAP-1” is a protein that occurs in brain cells. Its normal function (whatever that is) might be blocked by the Glutamines.

29. This is why the mutation is Dominant. Even if an individual is Hh, they will have this faulty protein in their brain. It doesn’t matter if the other allele is normal. It took another 10 years to discover all of this.. So, maybe extra HAP-1 protein could be delivered to patients brains, or some other molecule could be added to stop the Glutamines from attacking it….there is alot of research going on right now.

30. Basically, there are three molecular approaches that can be taken once a genetic disease is described at the biochemical level: 1) develop pharmaceuticals 2) gene therapy 3) early diagnosis What are the advantages of early diagnosis?

31. There is a genetic test for Huntington’s Disease… It costs $1300. Only 3% of the people in the U.S. who are “at risk” actually take the test…. Why so few do you think?

32. There are other “Triple Repeat” diseases…… These genes are sometimes called “Stuttering” genes…they usually affect the neurological system Mental Retardation …X Chromosome A type of Muscular Dystrophy.. …Chromosome 19

33. What does all this have to do with DNA Replication? Stuttering genes, and other regions in the genome that have repeats, likely became that way because of mistakes during DNA replication

34. DNA Replication A very quick look..

35. What does the “S” stand for ??

37. The “Klenow Fragment” of DNA Polymerase Hyperlink to molecular movies... (1&2 are best)

38. See places where replication is taking place?

40. The building block of a new DNA strand QUESTION: What is the function of the three phosphate groups? Energy....the last two phosphates break away when the phosphodiester bond is formed to the 3’ end of the adjacent nucleotide.

41. QUESTION: If another nucleotide base was going to be added to this molecule, where would it be added?

43. DNA Polymerase is a “stupid” enzyme…. It has to be told when & where to start doing “its thing”... “Primers” tell the enzyme where to begin DNA replication. “priming” before you paint “priming” a water pump

44. ?

45. The two new strands grow in the opposite directions RNA Primase

46. DNA (and RNA) bases are always added at the 3’ end of the nucleic acid chain. Click Here for an animation

48. Proteins involved in eukaryotic DNA replication: 1. Origin Replication Complex (ORC) = binds to DNA sequences that represent “initiation sites”....eukaryotes have lots of these initiation sites available to start replication. They allow the next two enzymes to do their thing. 2. Helicase = unwinds DNA where the ORC is, separating the two strands 3. Topoisomerase = prevents original DNA from getting tangled 4. RNA Primase = adds 11 RNA bases near the initiation site ...this tell DNA Polymerase where to start...

49. Proteins involved in eukaryotic DNA replication (continued): • DNA Polymerase = a large complex of proteins that grab • the appropriate nucleotide triphosphate (one • that is complementary to the DNA strand and • adds it to the 3’ end of the new strand. 6. RNAase = an enzyme that removes RNA primers after replication. When that’s been done DNA Polymerase fills in these gaps with the appropriate DNA sequence. 7. DNA Ligase = forms phosphodiester bonds between the DNA pieces that are not yet connected (called “nicks”). A nick is when the phosphodiester bond is broken on one strand but not the other.

50. Do cells ever make mistakes in copying DNA? Absolutely. Remember that cells can replicate all of their DNA in hours or even minutes....so there are bound to be errors.