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Drosophila melanogaster

Polytene chromosomes and P-elements

Polytene chromosomes

Deletion mapping

In situ hybridisation and mapping

P-elements

Transgenesis

Applications

In flies larval cell growth andDNA replication occurs withoutcell division

Chromosome banding and polyteney Chromosome banding and polyteney

Polytene chromosomesbands allow a resolution of ~100kb

Chromosome banding and polyteneyPrecise mapping of chromosomal abnormalities

Deletion

Duplication

Inversion

Deletion mapping

If a deletion fails to complement themutation it maps within the deletion

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Deletion mapping

If two overlapping deletions both fail to complement themutation it maps within the region of overlap

Mapping cloned sequences and transposons

Known sequences can be mapped by in situ hybridisationto polytene chromosomes

This has been used to to clone mutants that map to the sameinterval and map the distribution of different classes of transposable element

P-elements are transposable elements

2.9 kb sequence flanked by 31bp inverted repeats

Contains three open reading frames

One codes for P-specific transposase

Transposase binds to 31bp repeats and induces transposition

P-element transposition

Usually P-element transposition is local and an intact copyis left at the original site

Rarer events such as interchromosomal jumps and lossof the original element can be selected for

P-element based vectors lacking the transposase coding sequence are used for transgenesis in Drosophila

P-element transgenesis

Eggs injected with plasmid containingtransgene plus marker gene flanked by P-element 31 bp repeats

Helper plasmid containing transposasecoding sequnce driven by leaky heat shock promoter co-injected

Transposase catalyses the integration of the transgeneand the marker sequence flanked by 31bp repeats

P-element transgenesis

DNA injected into pole plasm of rosy/rosy eggs

Adult germ line may contain some transgenic cells

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P-element transgenesis

Cross adults to rosy/rosy M strain

Offspring with wild type eye colour are stably transgenic

Applications of transgenesis

1) Identification of regulatory elementsElements can be tested in different locations tocontrol for position effects by crossing totransposase expressing strains

2) Misexpression and over expression

3) Insertional mutagenesis and cloning

Identification of regulatory elementsEyeless codes for Drosophila Pax6 homologue

Expressed in the embryonic nervous system

Expressed in eye portion of the eye-antennal imaginal disc

Identification of regulatory elements

Insertions in intron 2 cause recessivereduced eye phenotype

Intron 2-reporter transgenes confer eye-specific expression in flies and mice

Misexpression and overexpression

Transgenic flies which express eyeless in other imaginal discsdevelop ectopic eyes

Cloning by transposon tagging

so1 - no eyes

Null alleles are lethal (solethal)

Deletion mapping of solocated gene in 43C

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Cloning by transposon tagging

NinaE transgenic line has recessive mutant phenotypeassociated with the insertion

Homozygous transgenic flies have slightly rough eyes and optic lobe (brain) defects

In situ hybridisation with a P-element probe locates the insertion in 43C

so1 complements the insertional mutation butsolethal fails to complement it

Cloning by transposon taggingNinaE transgene inserted in 5’UTR of a novel homeobox gene

Gene expressed in embryonic/larval brain and adult/larval eye

Insertion causes reduction in brain expressionso1 causes loss of eye expression

Mammalian Six genes isolated by homology with so proteinare involved in eye development and disease

Summary

Polytene banding patterns allow precise mapping of chromosomal rearrangements

P-element based vectors increase the efficiency of transgenesis

Transposon tagging facilitates gene cloning

ReferencesIntroduction to Genetic Analysis 9th Edition

Chromosomal rearrangements pp 573-583Transposons pp 499-504Transgenesis pp 746-747

P-element home page