Transcript Lecture 4
Lecture 4 • Mosaic analysis • Maternal/zygotic screens • Non-complementation screen • Cytogenomics to genomics • Designer deletions • Gene disruption in Drosophila • Systematic RNAi screens Lab 4 schedule • MONDAY SECTION: Sunday 9:30-11:30 AM come in to start your in situ. WSC 339 • TUESDAY SECTION: Monday 10-12AM come in to start your in situ. B&G 3015 • On Monday and Tuesday the lab B&G 3015 will be open at 10AM to start your washes. Biology 4540g Midterm Examination Tuesday February 28, 2012 Room UCC 63 7-10 PM Mosaic Analysis What is a genetic mosaic? Mitotic recombination ww+ - + + Mitotic recombination ww+ - + + Mitotic recombination - - + + Mitotic recombination ww- - - + + w+ w+ Mitotic recombination w- Eye w- - - + + w+ w+ ww+ Mitotic recombination Clones of cells X-rays Indiscriminant crossing over Flip mediated site specific recombination Yeast two micron plasmid A B FRT C FRT D Flip A B FRT FRT D C Flip mediated mitotic recombination FRT FRT Flip mediated mitotic recombination + FRT Sb FRT Flip mediated mitotic recombination + + FRT FRT Sb Sb FRT FRT Flip mediated mitotic recombination hspFLP + + FRT FRT Sb Sb FRT FRT Flip mediated mitotic recombination hspFLP + + FRT FRT Sb Sb FLP FRT FRT Flip mediated mitotic recombination hspFLP + + FRT FRT Sb Sb FLP FRT FRT Flip mediated mitotic recombination + + FRT FRT Sb Sb FRT FRT Flip mediated mitotic recombination + FRT + FRT Sb FRT Sb FRT Flip mediated mitotic recombination + FRT + FRT Sb FRT Sb FRT Mosaic analysis • Assay adult phenotype of an embryonic lethal. • Determine the site of action of a gene product. • Analyze Zygotic lethal/Maternal effect mutants. Pure maternal and zygotic screens Bal Bal + + Zygote Oogenesis Pure zygotic screens Bal Bal + + Zygote Oogenesis Assume pure expression of a gene from the zygotic genome only. Pure maternal screens Bal Bal + + Zygote Oogenesis Assume pure maternal expression of a gene Exceptions to the strict assumptions 1. Maternal rescue 2. Zygotic lethal/Maternal effect Maternal Rescue Zygote Bal + Oogenesis The maternally supplied transcript rescues the zygotic loss of function. Maternal Rescue Hide an essential role for a gene in embryonic pattern formation. Pure zygotic screens may miss important functions. Pure maternal screens Bal Bal + + Zygote Oogenesis Assume pure maternal expression of a gene Zygotic lethal/Maternal effect Bal Bal + + Zygote Oogenesis Dies at late larval stage or pupal stage We can not assay if the gene has a maternal effect phenotype. Zygotic lethal/Maternal effect Since a homozygous adult mother can not be established, we can not assay whether the gene contributes information to the egg. Screen for maternal rescue and zygotic lethal/maternal effect 1/2 to 2/3 of lethal mutations are not embyonic lethal. Norbert Perrimon collected a large # of late larval and pupal lethals. Properties of OvoD OvoD dominant female sterile germ line specific cell autonomous OvoD No eggs Mosaic analysis with OvoD OvoD + No eggs + l Irradiate with X-rays Mosaic analysis OvoD + + l Mosaic analysis OvoD + + l Mosaic analysis OvoD + + l Mosaic analysis OvoD + OvoD + OvoD + + + l + l l Mosaic analysis OvoD + OvoD + + l + l Egg maker Maternal rescue vs. Maternal effect Maternal rescue can be rescued by the paternal allele. Maternal effect is independent of the genotype of the sperm. Maternal effect Egg chamber l l l + Maternal effect l l Embryonic phenotype l + Embryonic phenotype Maternal rescue Egg chamber l l l + Maternal rescue l l Embryonic phenotype l + No phenotype rescued by the paternal allele Non-complementation screen Isolation of more independent mutant alleles in a gene. Non-complementation screen wild-type en1/en1 Non-complementation screen en1/en1 engrailed phenotype en1/+ wild-type phenotype en1/deletion engrailed phenotype en1/enx Non-complementation screen EMS F0 CyO/L virgin females X b pr cn males F1 en1/en1 virgin females X b pr cn*/CyO 4,000 crosses with individual males F2 Screen vials for flies with the en phenotype Vials with no engrailed flies 50% en1 CyO 50% b pr en+cn* en1 Vials with engrailed flies 50% en1 CyO 50% b pr enxcn en1 Cytogenomics Salivary gland chromosome: high resolution cytogenetics. Cytogenomics Obsolete Drosophila genome • 2000 first draft of genome published • More than 200,000 expressed sequence tags available • Large scale transposon mutagenesis • Flybase: computer database and resource Human PP2A Holoenzyme Scaffold Flybase and Modern Drosophila Research 1. No longer have to clone and sequence a gene- reagents readily available. 2. Store of characterized mutants. RNAi 3. Large scale genome screens like 2 hybrid analysis and insitu hybridization are available. 4. Allows researchers to propose and formulate questions with data available. Cytogenomics Salivary gland chromosome: high resolution cytogenetics. Designed deletions FRT FRT Designed deletions FRT FRT Designed deletions FRT FRT Deletion with known breakpoints Gene disruption via homologous recombination Marker Ends out Marker Marker Ends in Marker In vivo recombinant molecules Ends in FRT Marker Gene fragment Rare restriction enzyme site FRT In vivo recombinant molecules Ends in Rare restriction enzyme site Gene fragment FRT Marker FRT In vivo recombinant molecules Ends in Marker FRT Gene fragment Rare restriction enzyme site In vivo recombinant molecules Ends in Marker FRT Gene fragment In vivo recombinant molecules Ends in FRT Marker RNAi in Drosophila 1.) Cell culture RNAi. Any Drosophila gene’s expression can be reduced in cell culture. 2.) RNAi transgenes in flies. 88% of the annotated genes of Drosophila represented in this collection of fly stocks. RNAi in Drosophila RNAi in Drosophila is cell autonomous ie where the RNAi is produced is where the effect will be. C. elegans RNAi is systemic--non cell autonnomous. Can enter the worm via diet. Binary or two component system Fly stock A: UAS line Fly stock B: Driver line Promoter RNAi GAL4 or Enhancer GAL4 binding sites UASGAL4 GAL4 GAL4 RNAi Progeny Dietzl et al., 2007 Nature 448, 151 Dietzl et al., 2007 Nature 448, 151