Lecture 4 Mosaic analysis Maternalzygotic screens Noncomplementation screen

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 -12 AM come in to start your in situ. B&G 3015 • On Monday and Tuesday the lab B&G 3015 will be open at 10 AM to start your washes.

Biology 4540 g 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 D C

Flip mediated mitotic recombination FRT

Flip mediated mitotic recombination + FRT Sb FRT

Flip mediated mitotic recombination + + FRT Sb Sb FRT

Flip mediated mitotic recombination hsp. FLP + + FRT Sb Sb FRT

Flip mediated mitotic recombination hsp. FLP + + FRT Sb Sb FLP FRT

Flip mediated mitotic recombination hsp. FLP + + FRT Sb Sb FLP FRT

Flip mediated mitotic recombination + + FRT Sb Sb FRT

Flip mediated mitotic recombination + FRT Sb FRT

Flip mediated mitotic recombination + 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 + + Zygote Oogenesis

Pure zygotic screens Bal + + Zygote Oogenesis Assume pure expression of a gene from the zygotic genome only.

Pure maternal screens 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 + + Zygote Oogenesis Assume pure maternal expression of a gene

Zygotic lethal/Maternal effect 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 Ovo. D dominant female sterile germ line specific cell autonomous Ovo. D No eggs

Mosaic analysis with Ovo. D + No eggs + l Irradiate with X-rays

Mosaic analysis Ovo. D + + l

Mosaic analysis Ovo. D + + l

Mosaic analysis Ovo. D + + l

Mosaic analysis Ovo. D + + + l l

Mosaic analysis Ovo. D + Egg maker + l

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 en 1/en 1

Non-complementation screen en 1/en 1 engrailed phenotype en 1/+ wild-type phenotype en 1/deletion engrailed phenotype en 1/enx

Non-complementation screen EMS F 0 Cy. O/L virgin females X b pr cn males F 1 en 1/en 1 virgin females X b pr cn*/Cy. O 4, 000 crosses with individual males F 2 Screen vials for flies with the en phenotype

Vials with no engrailed flies 50% en 1 Cy. O 50% b pr en+cn* en 1 Vials with engrailed flies 50% en 1 Cy. O 50% b pr enxcn en 1

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 PP 2 A 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

Designed deletions FRT

Designed deletions FRT Deletion with known breakpoints FRT

Gene disruption via homologous recombination Marker Ends out 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 Gene fragment Rare restriction enzyme site FRT

In vivo recombinant molecules Ends in Marker Gene fragment FRT

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 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 GAL 4 or Enhancer GAL 4 binding sites UASGAL 4 RNAi Progeny

Dietzl et al. , 2007 Nature 448, 151

Dietzl et al. , 2007 Nature 448, 151