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Flies Mega-Review Basics 4 chromosomes o Chromosome I is the sex chromosome Females: XX Males: XY (XO is sterile male) No meiotic recombination in males (doesn’t matter which chromosome) Y has few genes (heterochromatic) so generally, if you put a transgene on the 1st chromosome it would be on the X chromosome o Chromosomes II, III are large autosomes o Chromosome IV is a tiny autosome – mostly heterochromatin o 99% of genes are on chromosomes I, II, and III. So, we will ignore chromosome IV. Some code: o Separate chromosomes with semicolons Remember, we can ignore the 4th chromosome I ; II ; III If a genotype is always +/+, you can ignore it Instead of +/+ ; UAS-GFP/+ ; Ubi-Gal4/+ You can write UAS-GFP/+ ; Ubi-Gal4/+ o Separate (trans)genes on the same chromosome with commas UAS-GFP,Ubi-Gal4/+ ; Gal80ts/+ o Single male: Usually used in a cross to isolate a certain genotype (in screens, to isolate different mutations in mutagenized males) o Multiple males: Usually used for a cross if you’ve already isolated your genotype/mutant allele o Single female: Use with caution (might already be inseminated by males from their stock bottle!) o Multiple females: Use with caution for same reason as above o Single virgin female: Use if trying to isolate genotype on the female side – for purposes of Pat’s lectures this isn’t usually done since mutagenesis is done in males o Multiple virgin females: For purposes of the screens described in Pat’s lectures, you almost always use multiple virgin females 1 Balancers Important Characteristics: o Suppresses recombination o Includes dominant marker o Homozygous lethal How to write out balancers: “BALANCER, Dominant marker” o Ex: Second chromosome balancer with a Curly marker: SM5, Cyo st o 1 chromosome balancers: FM1, FM2, FM3…etc o 2nd chromosome balancers: SM1, SM2, SM3…etc o 3rd chromosome balancers: TM1, TM2, TM3…etc o Or, for purposes of the final, you can make up something: “I am using a first chromosome balancer named Purp that gives flies purple eyes.” You can also use double balancers; for example, TM1,Sb/TM2,Ser THIS IS VIABLE If a mutatation is homozygous lethal, you can use balancers to keep a stock: lethal = homozygous lethal mutation lethal/TM3,Sb X lethal/TM3,Sb lethal/lethal (dead) TM3,Sb/TM3,Sb (dead) Lethal/TM3,Sb (alive, stubble) The Gal4 System Uses: to control transgene expression (reporter, RNAi, dominant negative) in a spatially and/or temporally specific way Components: o Driver-Gal4: you can control where Gal4 protein is expressed using a tissue-specific driver o UAS-Transgene: Gal4 binds to the UAS promoter region to activate transgene expression. Therefore, the transgene is expressed wherever Gal4 expression is driven. You can also use this to drive any transgene (RNAi to knock down gene expression, dominant negative) in certain tissues/at certain times o Gal80: For optional temporal control (protein inhibits Gal4 activation of UAS) tub-Gal80 - tubulin driver constitutively expresses Gal80 tub-Gal80ts (Gal80 protein active at 18 deg Celsius, inactive at 29 deg Celsius) Flp/frt system Uses: to induce clonal populations of negatively-marked cells (that can also be homozygous mutant for a gene that is homozygous lethal to the organism when mutant) 2 Components: o Frt sites: inserted at the base of the chromosome arm at which you want flippaseinduced recombination to occur o Flippase (flp): driven by heat-shock (hs-flp) or a Gal4/UAS system (UAS-flp) o Ubi-Reporter (or UAS-Reporter if you want clonal induction to be tissue-specific) o Driver-Gal4 (if you want clonal induction to be tissue-specific) o Recessive mutant allele: if you want your negatively marked cell to be mutant - put on the arm at which you want flippase-induced recombination to occur MARCM system Uses: to induce clonal populations of positively-marked cells (that can also be homozygous mutant for a gene that is homozygous lethal to the organism when mutant) Components o UAS/Gal4/Gal80 system o Frt sites o hs-flp o Recessive mutant allele: if you want your positively-marked cell to be mutant - put on the arm at which you want flippase-induced recombination to occur 3 Flp-out system Uses: o Caveat for using flp/frt or MARCM system: only half of the cells are marked/become homozygous mutant because mitotic recombination needed to induce clones Flp-out system doesn’t rely on mitotic recombination so has better chance of marking all/most cells (greater clonal induction) Components: o Frt sites Flanking a STOP codon that disrupts expression of a transgene so that when flipped out, transgene will be expressed Flanking a Gal80 construct that is inhibiting a Gal4 system, so that when Gal80 is flipped out, Gal4-induced expression will begin o flippase: (either hs or UAS-induced) Practice problem: Mosaic analysis and crossing scheme to get desired genotypes 4