Mutants and Disease

1. Mutants and Disease MUPGRET Workshop

2. Mutation • Heritable change in the DNA sequence. • Naturally occurring • Induced

3. Types of mutations • Chromosomal • Point • Insertion/Deletion • DNA repair

4. Mutagens • Environmental • Chemical

5. Mutations as a tool • Associating a phenotype with a gene. • Understanding gene function. • Studying protein interactions. • Understanding cell lineage and organ development.

6. Associating a phenotype with a gene • Changes in the DNA sequence that non-functional or reduced function proteins often cause a visible change in the appearance of the organism. • Some changes do not give visible phenotypes. • Often identified as an “off-type” in plant species.

7. Cell may not be able to follow damaged instruction OR Damaged protein is made OR Spelling error may be harmless X X Damaged protein may or may not be able to function in the cell. Cell does not make the protein Functional protein made by the cell Misspelled Genes: 3 Possible Outcomes DNA A misspelled gene

8. Dwarfing • Gibberellic acid (GA) is a plant hormone. • GA levels influence growth. • Mutants in genes for GA synthesis, reduce plant height.

9. Associating a phenotype #2 • This is often the first step towards understanding the function of a gene or to dissecting a biochemical pathway. • The mutation can be either a naturally occurring one or an induced one. • Can be targeted or random.

10. Understanding gene function • “You don’t know how something really works until you have to fix it.” • Disruptions of the gene can be either non-functional or “leaky”. • Often the “leaky” phenotypes will really help you understand how to gene works.

11. Understanding gene function • In the case of targeted mutagenesis where you know what the other genes in that would/could be co-regulated with the mutant are you can understand the pathway better by looking at expression of the co-regulated genes.

12. Understanding gene function • In the case of site directed mutagenesis where you can target particular sequences, you can dissect the part of the protein that is important for function. • Can help to ID the catalytic site or a site involved in protein-protein interactions or a site involved in transport, etc.

13. Protein Explorer • Protein Explorer http://molvis.sdsc.edu/protexpl/frntdoor.htm • Also available at Biology Workbenchhttp://workbench.sdsc.edu/ • Tutorials at http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/pdb.shtml • Troubleshooting http://molvis.sdsc.edu/protexpl/troubles.htm

14. Studying protein-protein interaction • Often use a series of alleles that have defects in different parts of the gene to identify the site that is required for protein-protein interaction. • The series can be insertions, deletions, or point mutations and may come from nature or be induced or a combination of the two.

15. Interaction Maps • Molecular Interaction Maps http://discover.nci.nih.gov/mim/index.jsp

16. Understanding cell lineage • Usually used with transposon mutagenesis. • Transposons are mobile pieces of DNA that can insert into a gene and disrupt its function. • Insertion can happen throughout development and can be used to track where cells came from with visible marker.

17. Ac/Ds in Maize

18. Corn example of cell lineage

19. Methods for detection mutations • Alteration in electrophoretic mobility • Sequencing • Protein trunctation test

20. Blazing a Genetic Trail • It tells the story of how mutations are involved in several different diseases. • http://www.hhmi.org/genetictrail/

21. Association Genetics • Usually used for medical genetics. • Recently applied to plant genetics. • Which genes were involved in domestication? • Is this gene responsible for part of the difference we see in a particular trait such as plant height?

22. Dwarf 8 • Mutagenesis and trait analysis suggested that d8 might influence flowering time and plant height.

23. D8 study • Sequenced D8 in many ~100 maize lines. • Measured flowering time and plant height in the same material. • Compare DNA sequence to flowering time and plant height.

24. D8 summary • Found several polymorphisms that are associated with changes in flowering time. • Data also indicate that D8 has undergone selection. • Compare synonymous vs. nonsynonymous substitutions.

25. Plants as a Model for Disease • Sometimes mutations in the same gene in different organisms have similar phenotype. • This allows researchers to choose the organism with the best genetic resources to study the normal function of that gene. • This also allows researchers to identify prospective genes for a phenotype in one species, based on another.

26. Xeroderma pigmentosa • Autosomal recessive. • UV exposure damages DNA. • Defect in DNA damage repair. • Risks include cancer, telangiectasia, disfigurement. • Can be diagnosed before birth. • Take total protection measures from sun/fluorescent light.

27. Xeroderma pigmentosa

28. UV damages tissue that contains molecules that can absorb light.

29. Mechanisms of UV damage • Low penetration into tissues. • Molecular fragmentation—proteins, enzymes, and nucleic acids contain double bonds that can be ruptured by UV. • Free radical generation—molecules of susceptible tissues absorb UV and eject an electron, which is taken up by oxygen, then termed superoxide, a free radical.

30. Free radicals • Are scavenged by superoxide dismutase, vitamin C, vitamin E, glutathione peroxidase, carotene.

31. Lesion mutant in maize

33. Prions and Disease • Proteins that can change shape. • And make other proteins change their shape! • As number of changed proteins increases a phenotype is observed. • Causal agent of mad cow disease, scrapie in sheep and Creutzfeldt-Jakob disease in humans.

34. Prions II • Previously thought only nucleic acid encoded changes caused disease. • Stanley Prusiner discovered prion’s ability to change other protein’s structure and won the Nobel Prize. • Sup35 is a prion-like protein in yeast.

35. Sup35 • Translation termination factor • Carboxyl end binds to the ribosomal complex to terminate translation. • If Sup35 is converted to an alternate conformation (infectious prion conformation) the shape change spreads throughout the cell and is passed to daughter cells.

36. Sup35 • In prion conformation causes ribosomes to read through stop codons altering shape and function of proteins. • Not adaptively advantageous so why is it maintained?

37. Why? • True et al. 2000. Nature 407: 477-483. • Reduced translation fidelity, extends proteins. • Some of these are antibiotic resistant. • Could lead to stabilization of new phenotype under correct environment.