Your view on genetics

1. Your view on genetics A: I am pretty good at it. B: I am not too familiar with genetics, but I am eager to learn more. C: I am not too familiar with genetics and I do not like it much.

2. Just for fun Which statement is closer to your belief? A: Biochemistry and Genetics are two distinct research fields. Every graduate student needs to choose between them for learning and research. B: Biochemistry and Genetics interact closely in today’s research, but each lab should stay with one discipline and just collaborate with others. C: Genetics and Biochemistry are two different research approaches that are no longer clearly separated. If needed, today’s students should use both to tackle biological problems.

3. Using genetic alterations to dissect functions of gene products - Development of one gene one enzyme concept. - molecular lesions, biochemical defects and genetic natures - The nature of mutations ("morphs"). - Deficiencies and duplications - Genetic mapping.

4. Development of one gene one enzyme concept 1900, Archibold Garrod recognized that absence of a functional enzyme causes certain inherited disorders in humans. 1911. Bateson recognized the link between genes and enzymes. Genes are inherited and enzyme is for phenotypes 1941. George Beadle and Edwin Tatun: one gene one polypeptide - set up by earlier work in Drosophila work on eye colors in 1935 at Caltech and Europe. - Genetic control of biochemical reactions in Neurospora. (PNAS 27: 499-506). Work done at Stanford. - Nobel Prize in 1958. Credit to Garrod

5. Edward Lawrie Tatum Born in Boulder George Wells Beadle

6. arg 2-3 - + + - arg 4-7 - + + + Grwoth medium Minmal + Arginine + Minmal +citulline - Minmal +Ornithine - Mutant arg 1 Minimal - arg 1 arg 4-7 arg 2-3 ornithine citulline arginine C B A Srb and Horowitz, 1944

7. Gene is a stretch of DNA 1926 Fredric Griffith showed that hereditary substance could be transferred from dead bacteria to living bacteria 1944. Oswald Avery et al. show that the Griffith’s substance is DNA 1953. DNA structure. Watson and Crick deduced the structure 1953. S. Benzer demonstrated intragenic recombination in phage: gene is a segment of DNA. 1960s. Charles Yanofsky: linear relationship between mutations in the NT sequence and changes in AA sequence of the protein

8. Using genetic alterations to dissect the functions of gene products - Development of one gene one enzyme concept. - molecular lesions, biochemical defects and genetic natures - The nature of mutations ("morphs"). - Deficiencies and duplications - Genetic mapping.

9. Mutation: heritable change in the nucleotide sequence of a cell’s DNA Spontaneous mutation Depurination Depyrimidination cytosine deamination mutation Induced mutation (by mutagen) radiation (ionizing, nonionizing) chemicals (Base analogs, intercalating agents)

10. Same sense missense nonsense Substitution Point mutation Deletion insertion frameshift inframe Mutation Transposable elements P element, TC, sleeping beauty etc Deletion Duplication Inversions Translocation Fission and fusion Chromosome change

11. Using genetic alterations to dissect functions of gene products - Development of one gene one enzyme concept. - molecular lesions, biochemical defects and genetic natures - The nature of mutations ("morphs"). - Deficiencies and duplications - Genetic mapping.

12. The nature of mutations ("morphs") - loss-of-function mutations - hyperactive mutations - dominant negative mutations - Change-of-function (neomorphic) mutations. - phenotypes created by over-or misexpression

13. Before talking about morphs Let us first make sure we understand: Recessive mutations m/m with phenotype Dominant mutations m/+ with phenotype Statement 1: most human diseases are recessive. A: yes. B: no. C: not sure. Statement 2: Most of oncogenes contain dominant mutations. A: yes. B: no. C: not sure.

14. Nonsense missense deletion insertion chromosomal rearrangement Null, KO, amorph Loss-of-function lof or lf Reduction-of-function, KD or partial loss-of-function = hypomorph • - Recessive ? • What situation is dominant? • What is hyploid-insufficiency? • - Caused by what type lesions?

15. Broader definition that fits the meaning of the word: Hyperactive = hypermorph Misexpression = neomorph/hypermorph Gain-of-function dominant negative < antimorph Neomorphic Narrow definition and often used: Gain-of-function mutation = hyperactive mutation

16. Hyperactive mutations = hypermorph, let us call it gf - Protein (enzyme) is more active than wt - Protein activity can no longer be turned off - Protein was expressed at a higher level transcriptional control translational control RNA or protein stability Genotype Phenotype gf/gf mutant gf/+ may be mutant gf/null ? gf/df ? gf/gf/+ gf/+/+

17. Exercise 1. Compare the phenotype severity between gf/null mutants and gf/gf mutations. gf/gf is more severe. A: yes, B: no. 2. Compare gf/null with gf/+ A: gf/null is less severe than gf/+ B: gf/null is more severe than gf/+

18. dn/dn > dn/null > +/dn > +/null ~ +/+ having dn is worse than having null Dominant negative, antimorphic The mutant gene has a negative effect in the same direction as loss-of-function mutations. Its product is toxic to the wild-type protein in a dn/+ heterozygote. It competes with wild type. Null/+ wild type phenotype Dn/+ mutant phenotype Mechanisms: 1. Competes with wt for another positive factor - common Forms a non-functional multimers with wt. Please read Herskowitz’s review in 1987. He made the proposal without experiments

19. Key: adding normal gene copy neither enhance its phenotype nor reduce its phenotype. neo/+ = neo/+/+ (regarding the new phenotype) Neomorphic: the mutant gene generate a new function that is different from its normal role. Protein changed its activity to do something different Protein binds to another protein that the wt does not bind

20. Exercise Gene A is normally expressed only in muscle cells. Gene A(lf) cause muscle reduction. Gene A (gf) causes over production of muscle. A mutation in gene A’s promoter, cause it to be expressed at a high level in skin and abnormal skin development. Is this mutation a gf allele, or Neo allele? How do we determine that? Do we need to?

21. Using genetic alterations to dissect functions of gene products - Development of one gene one enzyme concept. - molecular lesions, biochemical defects and genetic natures - The nature of mutations ("morphs"). - Deficiencies and duplications - Genetic mapping.

22. Deficiency (Df) = deletion of a segment of chromosome Key: Df reduces the dosage of many genes Df/+ is not exactly the same as null/+ because dosage effects of other genes in Df likely exist. Duplication (Dp) = duplication of a segment of chromsome 1. Free duplication = small extra chromosome 2. Attached duplication, more stable. Regarding a particular gene in dp, dp is not same as adding a copy of the gene. However, the side effect is smaller than df.

23. Ras biochemistry • GTPase : cycle betwen GTP and GDP • functional switch • - Activator SOS for the exchange reaction • - Negative: GAP • - Effector region GDP GTP SOS target RAS GDP RAS GTP GAP Active Inactive Pi

24. Three-d structure

26. Ras oncogene always on. Lack of GTPase v12 E13 E61 Not dependent on Sos, GAP has no role on it. Still binds to target Still binds to GAP GDP GTP SOS target RAS GDP RAS GTP X GAP Active Inactive Pi Question: are Ras oncogenes gf alleles?

28. Figure 8.2. Life cycle of C. elegans Fertilized egg egg laid embryogenesis ~14 hrs Hatching/L1 larva ~11.5 hrs starvation L2 larva ~7hrs dauer larva gonadogenesis (many months) L3 larva ~7.5hrs food L4 larva Spermtoagenesis ~9.5hrs adult oogenesis eggs

29. Figure.8.3. The sexes of self-fertilized and cross-fertilized C. elegans progeny. Hermaphrodite Male XX XO meiosis Sperm Oocyte Sperm X gametes X O X 100% 50% 50% 100% fertilization XX XX XO 100% 50% 50% Self-progeny cross-progeny

31. AC WT 3° 3° 2° 1° 2° 3° X - AC 3° 3° 3° 3° 3° 3° Indicating: - AC is required for vulval induction - AC may send a signal to induce vulval cells

32. signal pathway function

33. E E E E E E ras(lf)/ras(lf) ras(dn)/+ Vulvaless 0% V V V V V V Multivulva 200% ras(gf)/ras(gf) anchor cell inductive signal Ras function signal E E V V V E Wild type 100% induction

34. Let us work on things 1. Isolated a Vulvaless mutant, called mutant sy94 sy94/+ Vulvaless sy94/sy94 more severe, die early Question: lf (A) gf (B) Dn (C) or Neo (D) ? Df/+ is wild type, so it is not haploid insufficient (lf). What is the key to make the distinction? What do we do?

35. 1. Isolated a lf mutant. Revertant screen dn/+ becomes null/+ 2. Determined the phenotype for lf alleles Making the null/sy94 strain If sy94 is a gf, is sy94/null more severe than sy94/+ ? A: yes, B: no. If sy94 is a dn, is sy94/null more severe than sy94/+ ? A: yes, B: no. 3. Df and Dp test.

36. Dosage analysis of dominant mutations in the ras gene in C. elegans. Under induction ras genotyhpe dn/dn dn/Df dn/dn/Dp dn/+ dn/+/dp Lethal Lethal 96% 59% 0% Gene activity/dosage Phenotype severity

37. Mechanism of dn of Ras

38. gf story: 1. Determine that it is in the same gene as that in dn 2. Determine that it is gf, not dn, not neomorphic 3. Oncogene connection. ras genotyhpe Multivulva ras genotyhpe Under induction gf/gf/Dp gf/gf gf/+/Dp gf/+ gf/Df dn/dn dn/Df dn/dn/Dp dn/+ dn/+/dp Lethal Lethal 96% 59% 0% 100% 93% 53% 23% 8% Phenotype severity Phenotype severity Gene activity/dosage Gene activity/dosage

39. Question John is studying the nature of a mutation in gene A in the fly. He found that m/m has a severe mutant phenotype. m/+ has a very weak phenotype. He introduced an additional copy of the wild type gene (using transposible element) into the m/m mutant and found the m/m/+ animals are significantly less severe in the phenotype. A: m is dn mutation B: m is gf allele C: m is a lf allele

40. question Regarding the mutant a phenotype caused by a gf mutation A: gf/gf/+ is always less severe than gf/gf B: gf/gf/+ is always more severe than gf/gf C: can be either