Biology 423 Research Paper: Genetics behind cloning of a human gene:

1. Biology 423 Research Paper: Genetics behind cloning of a human gene: Topics due next week 1 page outlines due Oct. 29

2. Writing a scientific paper: Choose a topic: Pick a genetic disease for which the responsible gene has been cloned. You can find lists of these diseases at the following Yahoo site: http://dir.yahoo.com/Health/Diseases_and_Conditions/Genetic_Disorders/ The OMIM database is also a useful place to get information to start. Please do not choose Huntingtons syndrome or Fibrodysplasia ossificans progressiva. Collect papers Define the problem you want to present

3. Make a title – active statements work well • eg. Grant’s disease is caused by a defective potassium pump • Make an outline: • Define a title for each section – even each paragraph. • Use active statements again. • Eg. Grant’s disease is a genetic disease that affects breathing • The Grant’s disease mutation is on Chromosome 7 • A mutation in a potassium pump is linked to Grant’s disease • (The two above will be the main sections) • Expression of wild type potassium pump reverts Grant’s disease • effects in cultured cells • Grant’s potassium pump message is expressed in lung cells • Treatment of patients with potassium pump blockers has no • effect on progress of disease. • Microarray analysis suggests potential treatment. • These can be used as section titles or as topic sentences (see below)

4. Choose figures and make them. • You can copy figures from some of the papers you read. • If so cite them properly eg (from Smith et al., 1987). • The figure with its figure legend should be comprehensible • without reading the text. • Write paragraphs • First sentence is a topic sentence • Last sentence is a concluding sentence • active voice • don’t try to sound academic • define your terms • if referring to a figure, define the figure but do not duplicate the figure legend. • Maintain the same tense, either past or present. • Make and abstract to put at the beginning of the paper • summarizing the experiments you will describe, • the results and the conclusions.

5. Citations: When you discuss published work, cite the paper. Do the citation in the first sentence in which the study is mentioned. Eg. Seven large families with a high incidence of cystic fibrosis were surveyed for DNA markers linked to the disease (Smith et al., 1987). References: at the end of the section: in alphabetical order Smith J, Jones, P.A. and White, K. 1987 Family studies map cystic fibrosis to Chromosome 7 Genetics 130: 147-156. Use the journal “Cell” as an example of how to format the paper, the citations and the references. There are several nice reference managers available. We use EndNote for making bibliographies and storing references.

6. Mapping genes by recombination frequency Test cross to monitor recombination between different genes Frequency of recombination is directly related to distance between genes (loci) on chromosome Three point cross

7. Drosophila, a model organism for genetics

8. Traits for our three point cross Body color; yellow vs wild type Bristles: forked vs straight (wild type) Crossveins: crossveinless vs wild type

9. Fig. 5.12

10. Test cross vg b pr / vg+ b+ r+X vg b pr / vg b pr Punnet square: Male Female vg+b+pr+vg b+pr+ vg+b pr+ vg b pr+ vg+b+ pr vg b+pr vg+b pr vg b pr Vg b pr vg+b+pr+vg b+pr+ vg+b pr+ vg b pr+ vg+b+ pr vg b+pr vg+b pr vg b pr 1:1:1:1:1:1:1:1 ratio of phenotypes if genes are not linked If genes are linked, parental combinations of alleles are overrepresented in progeny

11. Fig. 5.12

12. 3 genes, which is in the middle?

13. Fig. 5.13

14. Calculate distance between pairs: vg to pr: add up all classes with a recombination event between vg+ and pr or vg and pr+ 252 + 241 + 13 + 9 = 525 Divide by total number of chromosomes scored: 525/4197 X 100 cM = 12.5

15. Calculate distance between pairs: pr to b: add up all classes with a recombination event between pr+ and b or pr and b+ 131 + 118 + 13 + 9 = 271 Divide by total number of chromosomes scored: 271/4197 X 100 cM = 6.4 The distance between vg and b is the sum of the distance between vg-pr and pr-b 12.3 + 6.4 = 17.7

16. Fig. 5.12

17. Fig. 5.15

18. How do we map genes in humans? Relative association of markers: Allelic variants will co-segregate if the genes are closely linked on a chromosome. Map distances depend on frequency of recombination

19. To map a human genetic trait: Look for association between mapped markers and a trait of interest Markers can be traits, proteins or DNA sequences Anything that is polymorphic can be mapped We can translate map position into DNA sequence by determining the linkage between DNA-based markers and traits.

20. Pedigree Analysis: symbols

21. Screen family members for RFLP markers linked to trait

22. RFLP polymorphisms reveal genetic differences 2. Separate DNA fragments by size on an agarose gel 3. Hybridize to single copy radioactive probe- Southern Blot 1. Cut genomic DNA with Restriction enzymes

23. Test degree of linkage: odds of linkage Data looks like M1 is linked to SF. Mother has two M1 alleles. Her chromosome is uninformative, like a test-cross. Father has two different M alleles. Recombination of his alleles can be seen in this pedigree. In this family, there are 8 informative chromosomes. 1 has a recombination event. Therefore, we estimate map distance as 1/8X 100 cM. 12.5%

24. Odds of Linkage is (Probability gene and marker are linked at a certain map distance) divided by (Probability they are unlinked). In our case: L = 6.1 Log of L or LOD = 0.8 Maximum likelihood odds of linkage; Change estimated linkage Distance p(.1) to get the best LOD score for the data.

25. Calculate Lod score Odds of linkage Probability gene and marker are linked at a certain map distance / Probability they are unlinked. Calculate maximum odds for data. Predicted linkage distance gives best odds Add up log of odds for many families to get more data

26. To achieve significant LOD score: Combine odds of linkage for many families: p1(L)/p1(NL) x p2(L)/p2(NL) xp3(L)/p3(NL) In practice we combine the log of odds: LOD1 + LOD2 + LOD3. Continue until LOD > 3.0 before linkage is accepted Linkage distance is based on the linkage distance that gives the maximum value for the data.

27. If genes and markers are unlinked the p(L)/p(NL) will be <1.0 in some families and the LOD will be Negative. Therefore, as you add more families the LOD will only increase if the data of the majority of families supports linkage.

28. Huntington’s Disease A neurodegenerative condition that results in degeneration of basal ganglia late in life. Autosomal Dominant Fully penetrant Affects 1 in 10,000 individuals One of the first genes identified by map based cloning

29. Gusella et al 1984. Science 225, 1320-1326

30. 100’s of DNA markers mapped onto each chromosome – high density linkage map. the relative location of 100s of polymorphic DNA markers on chromosomes can be mapped using mapping panels. First step. Find approximate chromosomal location of gene using few large families or many small families. Use LOD score to determine if markers are linked to gene in human families. The LOD score allows you to compare families in which marker and gene are either in repulsion or in coupling.

31. Gusella et al 1984. Science 225, 1320-1326

32. DNA was collected from family members Many sequences known to be polymorphic in humans Were tested to find sequences that are linked to disease. i.e. find polymorphic alleles that cosegregate with disease allele. RFLPs were tested. Restriction Fragment Length Polymorphisms One locus with a HindIII polymorphism was linked in two large families tested.

33. HindIII polymorphism is closely linked to disease Marker G8 from a randomly chosen phage clone with a 17.6 kb human DNA insert.

34. Allele C is always found in affected individuals with one exception Marker G8 is linked to Huntingtons disease at a distance of 2 cM With a LOD score of 12.1 Some unaffected individuals also have allele C but in these cases it is from an unaffected parent

35. Physical map of region that contains Huntingtons gene Huntington Disease Collaborative Research Group 1993 75 families from all over the world tested for closer markers 500 kb region between D4S180 and D4S182 – no recombinations

36. Once a small interval has been defined, how to find the correct gene? List of genes predicted from sequence. Examine mRNA expression patterns to focus on genes expressed in tissues affected by disease. Compare alleles of candidate genes between healthy and affected individuals.

37. Exon trap used to identify expressed genes in Huntingtons interval Bucker et al. 1991 PNAS 88, 4005-4009

38. Screened sequences from clones representing contig, 4 transcripts found.

39. IT15 encoded a gene that was different in healthy family members from affected family members. Expressed in many tissues including brain

40. Structure of Huntingtin protein Polyglutamine repeats (CAG codon) are expanded in diseased individuals Sawa 2001 J. Mol. Med. 79, 375-381.

41. Once the gene is available, new studies can be initiated to understand disease Mouse model: transgenic mouse with expanded polyglutamine repeats. Heterozygote has neurodegenerative disease similar to Huntington’s. Develop abnormal deposits of huntingtin fragments in inclusion bodies of neurons. Express huntingtin protein in cultured nerve cells Mutant fragments accumulate in clumps and cells die Normal protein also fragmented but clumps do not develop. May be clumps are toxic or Maybe fragments with longer polyglutamine repeats are toxic.

42. Possible toxic effects of huntingtin leading to nerve cell death Huntingtin binds some transcription factors. Mutant Htt also binds May take transcription factors out of action. Mutant but not normal Htt binds p53 transcription factor, p53 is a regulator of cell death As a result,Ca2+ flow altered in mitochondria, Cell death. Mutant also binds a histone deacetlyase. Modification of gene expression.

43. Normal Huntingtin may protect nerve cells from death by performing essential functions. Normal Htt is part of a complex that transports a hormone, Brain derived neurotrophic factor along axons of medium spiny neurons. These are first to degenerate in Huntingtons Disease. Mutant Htt cannot perform transport function. But HD is dominant . It may be toxic or heterozygotes may have insufficient functional protein for normal development. Haploinsufficient.

44. Possible functions of Huntingtin leading to disease

45. Although the function of Huntingtin is still not known, treatments are being tested to reduce the symptoms Caspase blockers Anti-apoptosis