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Download P elements Problem Set 4 for this and the last lecture. Quiz coming
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P elements P elements Problem Set 4 for this and the last lecture. Quiz coming up next week: covers material up to today--7 lectures (includes lecture Chromosomes 4 from Sept. 12) Hybrid Dysgenesis Two Strains Laboratory strain: M cytotype Strains from the wild: P cytotype 1. Heat sensitive sterility 2. Increased rates of mutagenesis-unstable alleles Mf x Pm Pf x Pm Pf x Mm 3. Increased rates of chromosomal rearrangements 4. All effects in the germline, not in somatic tissues Mf x Mm HD No HD M strains have no P elements. P strains have many P elements. P elements produce an inhibitor of transposition, which is inherited maternally in germline. Therefore, inhibition of transposition a maternal effect. All somatic cells produce an inhibitor. No P elements Mf x Mm Repressor provided maternally No HD No repressor provided maternally Pf x Pm Pf x Mm Mf x Pm No HD HD 1 Rubin and colleagues showed that HD caused by a transposable element. Rationale: white gene had been cloned, so induced HD white mutations and looked for insertions in white gene. Mf x Pm 1. All seven white mutants spontaneously reverted to red. 2. All seven contained insertions that the wild-type white gene lacked. HD m x Basc/Basc f contains M X chromosome no P elements Isolated seven new white alleles Basc-balancer chromosome containing an inversion, Bar and a w allele. Screened for white-eyed flies w*/Basc, where w* is new HD allele. Does the 2.9 kb element contain transposase activity? 3. All of the insertions were related (Ac like). 4. Many elements present in P strains, but none in the M strains. 5. The largest element in the genome was 2.9 kbp. Singed bristle assay: snw is a weak allele of singed. snw has two P elements in the singed promoter. When one is excised, get a more extreme phenotype (sne), When the other is excised, get wild-type (sn+). Inject plasmid containing 2.9 kb P element Injected snw/Y m x sn-/sn- f snw/Y snw/sn- females Also get wild type (sn+/sn-) and more extreme phenotype (sne/sn-) 2.9 kb element has transposase activity. Can use P elements to integrate genes into the genome Uses of P elements 1. New alleles of gene 2. Insert genes into genome e.g., test whether cloned correct gene by mutant rescue. 3. Genetic mapping 4. Reverse genetics 5. Enhancer trapping 6. Missexpression of genes. ry-/ry - ry+ maps to different loci in different lines. Therefore, integration at different sites. 2 The bipartite GAL4-UAS system Uses of P elements 1. New alleles of gene 2. Insert genes into genome e.g., test whether cloned correct gene by mutant rescue. 3. Genetic mapping 4. Reverse genetics 5. Enhancer trapping 6. Missexpression of genes. • GAL4 is a transcriptional activator from yeast that recognizes a DNA sequence called the UAS (upstream activating sequence) • We can use this to control expression of YFG in a tissue specific manner by using enhancer elements specific for the tissue we are interested in Mix-N-Match! Gal4 has no effect; Can’t bind DNA GAL 4 Eye enhancer Eye enhancer UAS GFP No expression w/o GAL4 GAL 4 CNS enhancer UAS GFP UAS GAL 4 GFP Posterior wing enhancer UAS GAL 4 GFP brain enhancer UAS GAL 4 GFP GFP is a ‘reporter’ gene GAL 4 antennal enhancer UAS eyeless Halder, Callaerts, and Gehring, Science, 1995 3