2. 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.

3. 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.

4. 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.

5. 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

6. George Wells Beadle Edward Lawrie Tatum Born in Boulder

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

8. 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

9. 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.

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

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

12. 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.

13. 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

14. 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.

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

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

17. 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/gfmutant gf/+ may be mutant gf/null? gf/df ? gf/gf/+ gf/+/+

18. 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/+

19. 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. dn/dn > dn/null > +/dn > +/null ~ +/+ having dn is worse than having null Mechanisms: 1. Competes with wt for another positive factor - common 2.Forms a non-functional multimers with wt. Please read Herskowitz’s review in 1987. He made the proposal without experiments Null/+ wild type phenotype Dn/+ mutant phenotype

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

21. 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?

22. 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.

23. Deficiency (Df) = deletion of a segment of chromosome 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. 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.

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

25. Three-d structure

27. 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 RAS GDP RAS GTP Pi GTPGDP target Active Inactive SOS GAP X Question: are Ras oncogenes gf alleles?

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

30. Figure.8.3. The sexes of self-fertilized and cross-fertilized C. elegans progeny. Sperm OocyteSperm gametes X X OX 50% 100% XX 100% XX 50% XO 50% Self-progeny cross-progeny XX XO meiosis HermaphroditeMale fertilization

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

33. signalpathwayfunction

34. anchor cell inductive signal EEVVVE Wild type 100% induction signal Rasfunction EEE EEE Vulvaless 0% ras(lf)/ras(lf) ras(dn)/+ Multivulva200% VV V VVV ras(gf)/ras(gf)

35. 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?

36. 1. Isolated a lf mutant. Revertant screen dn/+ becomes null/+ 2. Determined the phenotype for lf alleles Making the null/sy94 strain 3. Df and Dp test. 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.

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

38. Mechanism of dn of Ras

39. 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 genotyhpeMultivulva gf/gf/Dp gf/gf gf/+/Dp gf/+ gf/Df ras genotyhpeUnder induction dn/dn dn/Df dn/dn/Dp dn/+ dn/+/dp Lethal 96% 59% 0% 100% 93% 53% 23% 8% Gene activity/dosage Phenotype severity Gene activity/dosage

40. 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

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