1. Gene Interaction

2. Modifier: a second mutation that influences the phenotype of another mutation. Modifiers may make a mutant phenotype more severe (=enhancers) or less severe (=suppressors). The modifier interactions may either be recessive (requiring homozygosity at the modifier locus to modify the original phenotype) or dominant (requiring only heterozygosity at the modifier locus to modify the original phenotype).

3. Suppression: a form of epistasis whereby the expression of one mutation (the “suppressor” mutation) normalizes the phenotype of another mutation (the “suppressed” mutation). The suppressor mutation may display no other phenotype.
Intragenic suppression: “pseudo-reversion”; can be same codon or different/interacting region of gene.

4. Extragenic suppression: suppressor mutation is in
different gene than suppressed mutation
Classic example: tRNA anticodon mutations that suppress nonsense/frameshift
mutations in other genes
Classic example: eye color suppression in Drosophila

5. A molecular mechanism for suppression
Figure 6-22

6. Ommatidium: 8 photoreceptors (R1-8)
12 accessory cells
sev mutations remove R7 photoreceptor
(cell develops as non-neuronal cone cell)
*non-lethal: functions only in precursor
of R7 photoreceptor!
sev encodes transmembrane protein tyrosine kinase
closely related to mammalian c-ros
Created sev ts mutation based on known v-src ts
mutants, using in vitro mutagenesis

7. RTK Signal Transduction Pathway

8. Screened ~30,000 F1 animals for absence of R7, using an microscopic optical projection method (reduced corneal pseudopupil effect)
Identified 20 mutations: Enhancers of sevenless, (E)sev
Detail histological examinations….

10. Mitotic recombination generates homozygous clones within heterozygous flies
Normal
X-rays
E(sev)+
E(sev)3D
w E(sev)
w+ E(sev)+
E(sev)1A
E(sev)2A
w E(sev)
w+ E(sev)+
Rare, small clones
w E(sev)
w+ E(sev)+
white clone red clone
lacks pigment granules pigment granules
E(sev)2B
E(sev)3C

11. Over expression of actin and many actin-associated proteins is cell lethal,
implying that stoichiometry of cytoskeletal components is critical
Fimbrin (actin-associated protein) encoded by SAC6 in yeast
Overexpression of SAC6 from GAL1 promoter inhibits cell growth
Screen for “spontaneous” mutations in which pGAL1-SAC6 plasmid-bearing
cells survive on galactose-medium (suppression of SAC6-overexpression lethality)
1326 suppressor strains
numerous possible explanations for gain-of-function and, therefore, suppression
of gain-of-function mutations

12. Potential classes of “suppressor” mutations:
Plasmid mutations of pGAL1 or SAC6
Plasmid loss due to marker “events”
(e.g., conversion of genomic lys2 with plasmid LYS2)
Genomic mutations in genes that regulate pGAL1
Genomic suppressors of SAC6 product (fimbrin)
Used dominance test in diploids to distinguish plasmid from genomic events

13. Gal toxicity is dominant
mutation is in the LYS2 plasmid

14. “recessive genomic”: heterozygote grows on neither –Lys or –Leu (unable to survive with a GAL1-SAC6 plasmid)
“dominant genomic”: heterozygote grows with either plasmid (on –Lys or –Leu)
Tested 126 genomic suppressors by crossing to pGAL-lacZ reporter.
For only 2 strains was reporter expression normal (heavily induced on GAL).
Both are mutations in ACT1 gene: act1-20, act1-21

15.
ACT1
act1-20
act1-21

16. + pGAL1-SAC6 plasmid
ACT1
act1-20
act1-21
Larger, rounder, delocalized actin
(actin cross-linked?)
Therefore, suppressor mutations normalize actin dynamics in presence of SAC6 overexpression

18. Highly specific screen for physically interacting proteins
Mutant actins polymerize ~normally
Mutant actins are reduced in Sac6p binding
Mutant actin filaments show reduced cross-linking (bundling)
Highly specific screen for physically interacting proteins
Mutations define the site(s) of interaction on the protein

19. A model for synthetic lethality
Figure 6-23

20. Transgenesis

21. Methods of introducing a transgene
Figure 20-22

22. Two results of transformation by simple yeast vectors
Figure 20-23

23. Creation of Caenorhabditis elegans transgenes
Figure 20-28

24. Creation of Mus musculus transgenes
Figure 20-30

25. Producing cells containing a targeted gene knockout
Figure 20-31a

26. Producing cells containing a targeted gene knockout
Figure 20-31c

27. Producing a mouse containing the targeted gene knockout
Figure 20-32a

28. Creation of Drosophila melanogaster transgenes
Figure 20-29