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Tetrad Genetics Drosophila melanogaster Todd Nystul, Ph.D. UCSF Depts. of Anatomy & OBGYN-RS Center for Reproductive Sciences Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research Gene function is conserved from Drosophila to mammals Expression of the master regulator, eyeless, causes ectopic eyes to form Expression of mouse eyeless (called Pax6) produces a Drosophila eye! Halder, Callaerts and Gehring, Science 1995 The life cycle The Life Cycle Embryogenesis Larval development Model for studying: Model for studying: • The cell cycle • Growth control • Morphogen signaling • Signal transduction • Embryogenesis • Developmental neurobiology • Epithelial morphogenesis • Hematopoiesis • Epithelial polarity The Life Cycle Model for studying: Model for studying: •Organogenesis • Stem cell biology •Hormonal cues • Homeostasis and aging •Circadian rhythms • Nutrition and fat storage • Behavior and neurobiology • Cancer biology The Drosophila karyotype 4 pairs of chromosomes X Chromosome is telocentric; 2 and 3 are metacentric; 4 is small and mostly heterochromatic The equal left and right arms are called 2L and 2R, and 3L and 3R Each arm carries ~20% of the gene of the fly Y is heterochromatic – few genes, fertility factors XO is a viable sterile male Sex is determined by the X:autosome ratio (not the presence of a Y, as in humans) There is recombination in females, but NOT in males Means that genes on the same chromosome behave as if they are 100% linked in males. The illustrious history of Drosophila genetics 1910: T. H. Morgan isolates a spontaneous mutant with white eyes that is sex-linked. Why was the first mutation he isolated sex-linked? In class Q1: Red-eyed male: w+/Y x White-eyed female: w-/w- ☿ w- w- w+ w+/wRed w+/wRed Y w-/Y White w-/Y White ♂ The illustrious history of Drosophila genetics 1910: T. H. Morgan isolates a spontaneous mutant with white eyes that is sex-linked. Why was the first mutation he isolated sex-linked? In class Q2: wts1; ry506/+ male x wildtype female: wts1/+; ry506/+ or wts1/+; ry+/+ The illustrious history of Drosophila genetics 1910: T. H. Morgan isolates a spontaneous mutant with white eyes that is sex-linked. Why was the first mutation he isolated sex-linked? 1913: Sturtevant constructed the first genetic map. 1914-1916: Bridges discovers non-disjunction in XXY females, providing first proof that chromosomes must contain genes. Chromosome theory of heredity (1933 Nobel Prize) w+/Y x w-/wX X non-disjunction All females have red (w+) eyes X(w-) X(w-) Y are white-eyed females The illustrious history of Drosophila genetics 1910: T. H. Morgan isolates a spontaneous mutant with white eyes that is sex-linked. Why was the first mutation he isolated sex-linked? 1913: Sturtevant constructed the first genetic map. 1914-1916: Bridges discovers non-disjunction in XXY females, providing first proof that chromosomes must contain genes. Chromosome theory of heredity (1933 Nobel Prize) The illustrious history of Drosophila genetics 1927: Muller showed that x-ray irradiation causes gene mutation, including chromosomal rearrangements (1946 Nobel Prize). 1935-38: Bridges published polytene physical maps of such accuracy that they are still used today. 1978: E. B. Lewis’s characterization of the bithorax complex (a Hox gene cluster) provides foundation for understanding genetic regulatory elements. (1995 Nobel Prize) 1980: Nusslein-Volhard and Wieschaus complete a systematic genomewide mutational screen to attempt to identify all genes involved in embryonic axial patterning (1995 Nobel Prize). 1981: Rubin and Spradling make transgenic flies with the use of transposable element vectors. 1993: Brand and Perrimon create a two-component transgenic system for controlling ectopic gene expression. 2000: Drosophila genome sequenced (www.flybase.org). Nomenclature Genes are traditionally named for phenotype and given 1-4 letter abbreviations. Gene name is italic and protein name is roman and capitalized (e.g. hedgehog encodes for Hedgehog First letter is lowercase for recessive alleles, uppercase for dominant alleles. Allele name is superscript (eag1 or eagEY00714). Wild-type is a plus sign (+) Homozygote: single allele written by itself implies homozygosity: eag101 Heterozygote: alleles are above and below a line or separated by a slash (/): eag101/eagEY00714 or eag101/+ Hemizygote: For X-linked in males, single allele is written over Y: w/Y Multiple alleles: Alleles on the same chromosome are separated by a comma Alleles on different chromosomes are separated by a semi-colon: y, w, eag1; shgR21; ry506/TM6 Chromosomes listed in order (X, II, III) and anything unlisted is assumed to be wildtype: f ; cn bw ; TM2, Ubx130 / tra Balancers A balancer is a chromosome that has been massively reorganized (by inducing many translocations) to prevent recombination. It is one of the two homologs, not an extra chromosome. Balancer chromosomes provide two main advantages: 1.They preserve disadvantageous alleles in a stock automatically! 2.They make it easier to trace alleles of interest through multiple generations Preserving disadvantageous alleles: 1.Autosomal balancers carry recessive lethal mutations A stock in which the lethal allele, EGFRco is balanced by the balancer CyO: ♂ ☿ EGFRco CyO EGFRco EGFRco/EGFRco (dead) EGFRco/CyO CyO EGFRco/CyO CyO/CyO (dead) parental genotype is maintained Balancers Balancer chromosomes provide two main advantages: 1.They preserve disadvantageous alleles in a stock automatically! 2.They make it easier to trace alleles of interest through multiple generations Preserving disadvantageous alleles: 1.Autosomal balancers carry recessive lethal mutations. 2.X chromosome balancers must be viable as hemizygotes but usually carry a recessive female-specific sterile mutation. Why? ♂ ☿ dlg1 FM7 Y dlg1/Y (dead) FM7/Y FM7 dlg1/FM7 FM7/FM7 (sterile) parental genotype is maintained Balancers Balancer chromosomes provide two main advantages: 1.They preserve disadvantageous alleles in a stock 2.They make it easier to trace alleles of interest through multiple generations 1. Preserving disadvantageous alleles: A.Autosomal balancers carry recessive lethal mutations B.X chromosome balancers must be viable as hemizygotes but usually carry a recessive female-specific sterile mutation. A stock in which the lethal allele, EGFRco is balanced by the balancer CyO: ♂ ☿ EGFRco CyO ♂ EGFRco EGFRco/EGFRco (dead) EGFRco/CyO CyO EGFRco/CyO CyO/CyO (dead) parental genotype is maintained ☿ Lgl1 EGFRco Lgl1 Lgl1/Lgl1 (dead) Lgl1/EGFRco EGFRco Lgl1/EGFRco EGFRco/EGFRco (dead) parental genotype is maintained? Balancers 1. Preserving disadvantageous alleles: A.Autosomal balancers carry recessive lethal mutations. B.X chromosome balancers must be viable as hemizygotes but usually carry a recessive female-specific sterile mutation. 1 co Lgl Meiosis EGFR Lgl1 X EGFRco A.Balancers have chromosomal inversions that suppress meiotic recombination a b c d e f g h a b c d e f g h a b c d e f g h b a d c f e h g balancer chromosome wild type a b c d e f g h a b c d e f g h selection pressure for wild type chromosomes cause loss of lethal mutation acentric and dicentric chromosomes cause lethality of gametes Balancers 2. Tracing alleles of interest through multiple generations X: FM7 (dominant marker: Bar eyes) II: CyO (dominant marker: Curly wings) III: TM3: (dominant marker: Stubbly back hairs) IV: virtually no meiotic recombination so balancer is unnecessary. Sensitized backgrounds to identify genetic interactions • A sensitized background is a genotype that works with other genetically related mutationis to produce a phenotype or enhance a phenotype. • The alleles that interact in a sensitized context may or may not produce a phenotype on their own Erika A. Bach et al. Genetics 2003;165:1149-1166 The P-element revolution 1977: P-M Hybrid Dysgenesis (Kidwell, Kidwell and Sven) wild “P” ♂ x lab “M” ☿ sterile progeny lab “M” ♂ x wild “P” ☿ fertile progeny 1982: “P-elements”: Rubin, Kidwell, and Bingham demonstrate that the “P” cytotype is due to transposable elements. But, why are wild females protected? Hmmm... (notice how the use of italics creates suspense) 1982: Spradling and Rubin clone the P-element and demonstrate that it can be used to generate transgenics. 1988: Cooley and Spradling publish a method for efficient generation and screening of insertional mutants. 1993- present: Starting with Brand and Perrimon’s two-component expression system, P-elements become the basis for many genetic tools. Useful features of P-elements Natural P-element Transposition and copy number can be controlled: P-elements used in stable lines lack transposase, so they cannot hop. P{ry+ Δ2-3} has transposase that can only be translated in the germline but lacks the 31 bp repeats at each end that are essential for transposition. Why do they lack the 31 bp repeats? To mobilize, cross a P-element source to Δ2-3 and then cross Δ2-3 away again. Score for presence/absence of P-element. Lab strains lack endogenous P-elements so copy number can be tightly controlled. Enhancer traps P-elements have a strong bias for inserting near the 5’ end of genes, but otherwise transposition is somewhat random. Enhancer traps are generated by P-elements carrying a reporter gene with a minimal promoter must land within the regulatory region of a gene usually an approximation of the cellular expression pattern of the gene, but not the subcellular localization of the protein Typical enhancer trap construct LacZ LacZ Protein traps Protein traps are generated by P-elements carrying a reporter gene that is flanked by splice acceptors and donors are hopped around the genome. Must land within the transcriptional unit of a gene to be expressed Must be in frame to form fusion protein (otherwise, it disrupts normal protein translation Fusion proteins are accurate reports of both cellular transcription pattern and subcellular protein localization patterns. Typical protein trap construct Gal4/uas transcription system Gal4 is a transcription factor that activates the UAS promoter. Gal4 P-element can use endogenous enhancers (enhancer trap) or can include a promoter. Gal4 can be driven by a ubiquitous promoter (e.g. Tub-Gal4 or Ubi-Gal4) a tissue-specific promoter (e.g. elav-Gal4 is expressed in all neurons and MHC-Gal4 is expressed in all muscle cells) Variations to provide temporal control: Gal80 inhibits Gal4; Gal80ts is only active at permissive temp. (18° - 22°C) Gal4ER is only active in the presence of an estrogen analog In class Q1 ϕC-31 integrase attB insertion sites in the original collection In the TRiP RNAi collection, RNAi constructs are inserted using ϕC-31 technology In class Q2 MiMIC lines Inserted DNA can: 1. Manipulate gene expression 2. Tag the protein (protein trap) 3. Utilize the enhancers of the gene (enhancer trap) 4. Mutate (or reverse a mutation of) the gene Flp/FRT recombination Flipase (FLP) is an enzyme that catalyzes recombination between two FRT sites. FRT sites can be on the same chromosome (e.g. tub-FRT-STOP-FRT-Gal4) or homologous chromosomes. Flp can be controlled by a cell-type specific promoter (e.g. eyeless), or it can be inducible (e.g. heat-shock) The result is a genetically heritable rearrangement that positively or negatively labels cells. When labeled cells divide they form a “clone” that is distinguishable from surrounding unlabeled cells. Cells in clone can become homozygous mutant for, or specifically overexpress, a gene of interest. Flp/frt recombination Positive marking, all cells are wildtype Negative marking, labeled cells are mutant Tub FRT FRT LacZ hs-Flp GFP hs-Flp FRT Tub FRT FRT FRT LacZ GFP * GFP+ wildtype cell lethal mutation FRT GFP+ heterzygous cell FRT * GFP- homozygous mutant Planar cell polarity Cell autonomous: only cells within the clone are affected. Cell non-autonomous: cells within the clone affect cells outside the clone (or visa-versa) Tumor suppressor screens 1967: Gateff and Schneiderman identify lethal giant larvae, the first in vivo example of a tumor suppressor. 1995: Xu et al., identify the first component of the hippo pathway by generating clones in larval imaginal discs and screening adults for mutations. Dual clone technologies Dual-marked clones Tracking multiple lineages simultaneously Differentially labeling single cells FSCs FSCs DNA GFP GFP B-gal DAPI B-gal Stem cell niche cells