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Lecture 4 Mosaic analysis Maternal/zygotic screens Non-complementation screen Cytogenomics to genomics Designer deletions Gene disruption in Drosophila.

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Presentation on theme: "Lecture 4 Mosaic analysis Maternal/zygotic screens Non-complementation screen Cytogenomics to genomics Designer deletions Gene disruption in Drosophila."— Presentation transcript:

1 Lecture 4 Mosaic analysis Maternal/zygotic screens Non-complementation screen Cytogenomics to genomics Designer deletions Gene disruption in Drosophila Systematic RNAi screens

2 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.

3 Biology 4540g Midterm Examination Tuesday February 28, 2012 Room UCC PM

4 Mosaic Analysis What is a genetic mosaic?

5 Mitotic recombination w-w- w+w

6 w-w- w+w

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8 w-w- w+w+ w-w- w+w+

9 w-w- w+w+ w-w- w+w+ Eye w-w- w+w+

10 Mitotic recombination Clones of cells X-rays Indiscriminant crossing over

11 Flip mediated site specific recombination Yeast two micron plasmid A B C D A B C D Flip FRT

12 Flip mediated mitotic recombination FRT

13 Flip mediated mitotic recombination FRT + Sb

14 Flip mediated mitotic recombination FRT + Sb + FRT Sb FRT

15 Flip mediated mitotic recombination FRT + Sb + FRT Sb hspFLP FRT

16 Flip mediated mitotic recombination FRT + Sb + FRT Sb hspFLP FRT FLP

17 Flip mediated mitotic recombination FRT + Sb + FRT Sb hspFLP FRT FLP

18 Flip mediated mitotic recombination FRT + Sb + FRT Sb FRT

19 Flip mediated mitotic recombination FRT + Sb FRT + Sb

20 Flip mediated mitotic recombination FRT + Sb FRT + Sb

21 Mosaic analysis Assay adult phenotype of an embryonic lethal. Determine the site of action of a gene product. Analyze Zygotic lethal/Maternal effect mutants.

22 + Bal + Zygote Oogenesis Pure maternal and zygotic screens

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

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

25 Exceptions to the strict assumptions 1. Maternal rescue 2. Zygotic lethal/Maternal effect

26 + Bal Zygote Oogenesis Maternal Rescue The maternally supplied transcript rescues the zygotic loss of function.

27 Maternal Rescue Hide an essential role for a gene in embryonic pattern formation. Pure zygotic screens may miss important functions.

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

29 + Bal + Zygote Oogenesis Zygotic lethal/Maternal effect Dies at late larval stage or pupal stage We can not assay if the gene has a maternal effect phenotype.

30 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.

31 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.

32 Properties of Ovo D Ovo D dominant female sterile germ line specific cell autonomous Ovo D No eggs

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

34 Ovo D l + + Mosaic analysis

35 Ovo D l + + Mosaic analysis

36 Ovo D l + + Mosaic analysis

37 Ovo D l + + l + + l + + Mosaic analysis

38 Ovo D l + + l + + Egg maker Mosaic analysis

39 Maternal rescue vs. Maternal effect Maternal rescue can be rescued by the paternal allele. Maternal effect is independent of the genotype of the sperm.

40 l l Egg chamber Maternal effect l +

41 l l l + Embryonic phenotype

42 l l Egg chamber Maternal rescue l +

43 l l l + Embryonic phenotype No phenotype rescued by the paternal allele

44 Non-complementation screen Isolation of more independent mutant alleles in a gene.

45 Non-complementation screen wild-type en 1 /en 1

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

47 Non-complementation screen CyO/L virgin females X b pr cn males en 1 /en 1 virgin females X b pr cn*/CyO Screen vials for flies with the en phenotype F0 F1 F2 EMS 4,000 crosses with individual males

48 Vials with no engrailed flies Vials with engrailed flies en 1 CyO en 1 CyO b pr en + cn* en 1 b pr en x cn en 1 50%

49 Cytogenomics Salivary gland chromosome: high resolution cytogenetics.

50 Cytogenomics Obsolete

51 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

52 Scaffold Human PP2A Holoenzyme

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67 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.

68 Cytogenomics Salivary gland chromosome: high resolution cytogenetics.

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70 Designed deletions FRT

71 Designed deletions FRT

72 Designed deletions FRT Deletion with known breakpoints

73 Gene disruption via homologous recombination Marker Ends out Ends in

74 In vivo recombinant molecules Ends in FRT Marker Rare restriction enzyme site Gene fragment

75 In vivo recombinant molecules Ends in FRT Marker Rare restriction enzyme site Gene fragment

76 In vivo recombinant molecules Ends in FRT Marker Rare restriction enzyme site Gene fragment

77 In vivo recombinant molecules Ends in FRT Marker Gene fragment

78 In vivo recombinant molecules Ends in FRT Marker

79 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.

80 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.

81 Binary or two component system Fly stock A: UAS lineFly stock B: Driver line RNAi GAL4 binding sites UASGAL4 GAL4 Promoter or Enhancer RNAi GAL4 Progeny GAL4

82 Dietzl et al., 2007 Nature 448, 151

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