Gene Interaction

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).

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.

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

A molecular mechanism for suppression Figure 6-22

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

RTK Signal Transduction Pathway

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….

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

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

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

Gal toxicity is dominant mutation is in the LYS2 plasmid

“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

+ - + - ACT1 act1-20 act1-21

+ 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

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

A model for synthetic lethality Figure 6-23

Transgenesis

Methods of introducing a transgene Figure 20-22

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

Creation of Caenorhabditis elegans transgenes Figure 20-28

Creation of Mus musculus transgenes Figure 20-30

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

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

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

Creation of Drosophila melanogaster transgenes Figure 20-29