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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