Lecture 2

1. Lecture 2 • Lab2 • Allele classification • Genetic screens • Epistasis

2. Groups Group 1-1 Josh Farhi Tyler Madden Group 1-2 Youssef Neema Christine Schmidt Group 2-1 Caitlin Carlisle Kourtney Gordon Nina Nissan Group 2-2 Nicole Stabler Rachel Edgar Group 2-3 Kathleen Shah Camille Fong Chih Kai

3. Sunday heat shock times

4. Monday heat shock times

5. Where do we do the heat shocks? Room 361 of the Western Science Center. How do I get there on Sunday if the front doors of WSC are locked? Tunnels. A) Middlesex College front door turn left down to the staircase in the middle of the hallway. Go down to tunnel that leads to the WSC. B) Natural Science Center to Physics and find a tunnel leading to WSC. C) Natural Science Center to B&G third floor to WSC

6. Allele classification Functional allele wild-type allele Active gene product

7. Loss-of-function allele (lf) Generally recessive Two classes important for this course

8. Loss-of-function allele (lf) Null alleles-amorphic alleles Completely inactive gene product

9. Loss-of-function allele (lf) Null alleles-amorphic alleles No gene product Regulatory mutant resulting in no expression

10. Loss-of-function allele (lf) weak alleles-hypomorphic alleles Partially inactive gene product

11. Loss-of-function allele (lf) weak alleles-hypomorphic alleles Partially inactive gene product: multifunctional protein with only 1 of 2 functions affected by the change.

12. Loss-of-function allele (lf) weak alleles-hypomorphic alleles Partial expression of a gene product Regulatory mutant resulting in partial expression

13. Hypomorphic regulatory mutant Gut enhancer ORF gene something Brain Gut embryo of something

14. Hypomorphic regulatory mutant Gut enhancer ORF gene something Brain Brain Gut Gut mutant Wild-type

15. Gain-of-function alleles (gf) Generally associated with misregulation of a gene product’s activity, and are generally dominant. Three examples

17. Many developmentally important genes are expressed in a spatially restricted pattern. The pattern of the expression is important for the phenotype of the organism. Non- or mis-expression of the gene can result in a phenotype. Antennapedia is expressed in the second thoracic segment where the second leg will form. Leg to antenna transformation.

18. Antennapedia can be misexpressed either by spontaneous mutation or by genetic engineering. Antp ry+ hsp Antenna to second leg transformation

19. Summary of experiments on Antennapedia Leg primordia Antenna primordia antenna leg wild type antenna antenna Antplf leg leg Antpgf

20. Dominant negative (dn) antimorph Criteria The gene product works in a complex, either with itself or another protein, and the activity of all the proteins in the complex is essential for the total activity of the complex.

21. heterodimer homodimer X X Complex inactive Complex inactive X Mutation must not affect the ability of the complex to form.

22. A dominant negative allele reduces activity to a greater extent than a null allele when heterozygous. dn X wt wt x x x x 1 : 2 : 1 1/2 wild type activity 1/4 wild type activity

23. Multiple copies of the dn allele or overexpression will strongly inhibit wild type activity. dn x x X Only rarely will a complex of two wild type proteins form. X x x X x x X x x x wt

24. Genetic screens Genetic analysis requires genetic variants.

25. Saturation screens An attempt to identify as many genes whose products contribute to the process that you are studying as is statistically and technically possible.

26. Genetic screen for leucine auxotrophic yeast Yeast cells mutagenesis Replica plate to minimal media +leu -leu Random pool of DNA sequence changes

27. Complementation analysis leu1 X leu2 leu1 leu1 leu+ X X X X leu2 leu+ leu2 leu- leu+ Non-complementation in same complementation group =same gene. Complementation

28. Example of 100 mutants and 1,000 genes required for leucine biosynthesis. 901 900 Poisson distribution zero group=e-m # Complementation groups (genes) 98 100 1 0 1 2 3 #hits (numbers of independent allele/ complementation group (gene))

29. Example of 100 mutants and 10 genes required for leucine biosynthesis. e-10=4.5X10-5 3 # Complementation groups (genes) 2 1 0 10 20 #hits (numbers of independent allele/ complementation group (gene))

30. Real example

31. A Biochemical Pathway A B C D E

32. Epistasis Epistatic interactions are assayed by comparing the phenotype of a double mutant organism with that of the singly mutant organisms.

33. Epistasis: Criteria for the two mutations A. Have related phenotypes growth control sex determination dorsal ventral axis determination B. Work on a pathway that makes a distinct decision growth/nongrowth male/female expression/nonexpression C. The two mutations have distinct/opposite phenotypes all males versus all females expression always ON versus always OFF Ventralized versus Dorsalized

34. Control of sporulation by sporulation inducing factor (sif) in a hypothetical fungus No sporulation Sporulation

35. The pathway that controls sporulation Receptor protein kinase Inhibitor of sporulation Inducer of sporulation Kinase Spo genes -sif No expression OFF Active ON Inactive OFF Inactive OFF Inactive OFF +sif Active ON Inactive OFF Active ON Active ON Expression ON

37. Epistasis example Drosophila embryos have a dorsal and ventral side. Mutations exist where the mother lays eggs where the embryo develops with only the dorsal side lacking The ventral side (dorsalized). Mutations exist also that ventralize the embryo.

38. Loss-of-function alleles phenotype spz dorsalized Toll dorsalized pelle dorsalized tube dorsalized dl dorsalized cact ventralized Double mutants spz cact ventralized Toll cact ventralized pelle cact ventralized tube cact ventralized dl cact dorsalized What does the above data tell you about the order of function? Draw out the pathway indicating positive and negative interactions.

39. Tollgf ventralized Tollgf spz ventralized pelle Tollgf dorsalized tube Tollgf dorsalized dl Tollgf dorsalized What does this additional information tell you about the order of function?