drosophila as a model

Drosophila melanogaster�as a model organism�

The essentials of Drosophila: �History, Development, Jargon, �

Reagents, Genetic tricks, and Tools�

Drosophila melanogaster�as a model organism: historical perspective�


Chromosome theory and Mendelian genetics: establishing a�physical basis for heredity �

Thomas Hunt Morgan �

Nobel prize 1933 �


Cytological/genetic map: � The physical position of genes on the chromosomes were mapped relative to �the banding pattern of polytene chromosomes. Cytological maps generated by�Calvin Bridges are still in use today.�X: "1-20 �2L: "21-40 �2R: "41-60 �3L: "61-80 �3R: "81-100 �4: "101 �


Segmental patterning �

cubitus interruptus, wingless, gooseberry, hedgehog, fused, patch, paired, even-skipped, odd-skipped, barrel, runt, engrailed, Kruppel, knirps, and hunchback in 1980 Nature paper



Homeotic genes�


Ed Lewis�


Behavioral genetics�

Seymour Benzer�

Genetic basis of: time (circadian rhythms - ex: period), �love (sexual behavior - ex: fruitless), memory (associative �learning defect ex: dunce).�

Drosophila melanogaster�

-Flies are easy and cheap to rear.�

-10 day life cycle.�

-Can get several hundred progeny per female.�

-No meiotic recombination in males.�

-Only four chromosomes and about 15,000 genes.�

-Many embryonic and adult features provide visible markers for genetic and developmental studies.�

-Genome of melanogaster and 12 related species complete.�

A very practical choice�

The Drosophila melanogaster life cycle�

A very concise description of Drosophila development �


14 mitoses without cytokinesis �


Cellularization �(conversion of multi-nuclear syncitium into �

cellular blastoderm)�


Gastrulation �(formation of germ layers)�


Organogenesis�(example: tracheal development)�


Metamorphosis�(Imaginal disks and histoblasts)�

Drosophila nomenclature�Male�Female�Virgin �

Genes are properly named based on mutant phenotype. Lowercase is used to denote�recessive mutations and capitol letters denote dominant mutations.� examples: "white (w) - eyes are white instead of wild type red (on X or 1st)�

" "Sternopleural (Sp) - extra bristle defect (on 2nd)�" "stripe (sr) - have a black stripe on thorax (on 3rd)�" "ebony (e) - black body color (on 3rd) �

Genotypes are written in order according to chromosomal position. Genes on �different chromosomes are separated by a semicolon, genes on the same chromosome�are separated by a coma. Genes on homologous chromosomes are separated by a �bar or division symbol. � example: "w; Sp/CyO; sr, e�

Generally useful Chromosomal aberrations: � Deficiencies -- " "deletions of genomic DNA, ex: Df(3R)EXEL6276 � Balancers -- ""multiple inversions, used for maintaining recessive�

"" "mutant stocks, ex: FM7; SM6a; TM3 �


Genes are properly named based on mutant phenotype. Lowercase is used to denote�recessive mutations and capital letters denote dominant mutations.� examples: "white (w) - eyes are white instead of wild type red (on X or 1st)�

" "Sternopleural (Sp) - extra bristle defect (on 2nd)�" "stripe (sr) - have a black stripe on thorax (on 3rd)�" "ebony (e) - black body color (on 3rd) �






" "Sternopleural (Sp) - extra bristle defect (on 2nd)�""Curly (Cy) - have curly wings�" "stripe (sr) - have a black stripe on thorax (on 3rd)�" "ebony (e) - black body color (on 3rd)�






" "Sternopleural (Sp) - extra bristle defect (on 2nd)�""Curly (Cy) - have curly wings�" "stripe (sr) - have a black stripe on thorax (on 3rd)�" "ebony (e) - black body color (on 3rd)�

Genotypes are written in order according to chromosomal position. Genes on �different chromosomes are separated by a semicolon, genes on the same chromosome�are separated by a coma. Genes on homologous chromosomes are separated by a �bar or division symbol. � example: "w; Sp, + ; sr, e�

"" + , Cy �



Drosophila nomenclature�

gene names are italicized, Proteins are capitalized�

Certain gene names indicate something specific about gene function: �

fs = female sterile, ex: fs(1)k10 �

l = lethal, ex: l(3)ry82 �

e = enhancer, ex: en(spl)�

su = suppressor, ex: su(H)�

All molecularly defined gene models (predicted genes) have an �“annotation ID”: CG + a number for protein coding genes�CR + a number for non-protein coding genes.�

Drosophila nomenclature�

gene names are italicized, Proteins are capitalized�

Certain gene names indicate something specific about gene function: �

fs = female sterile, ex: fs(1)k10�

l = lethal, ex: l(3)ry82 �

e = enhancer, ex: e(S)�

su = suppressor, ex: su(H)�

All molecularly defined gene models (predicted genes) have an �“annotation ID”: CG + a number for protein coding genes�CR + a number for non-protein coding genes.�

Making transgenic Drosophila melanogaster�P-element transposable element �

Making transgenic Drosophila melanogaster�P-element transposable element �

Alan Spradling and Gerry Rubin �

Gene expression tools: �

Gal4/UAS �

Enhancer trap �

Drosophila: understanding crosses and screens�

Maintaining a recessive lethal mutation as a self-perpetuating stock �

dead- homozygous�Balancer�

or female-sterile�on X�

alive- heterozygous�Stock�

dead - homozygous�mutant �


Complementation tests: are two mutations alleles of the same gene?�

a� b �

a�b �

dead- homozygous�balancer�

alive- heterozygous�a or b/balancer�

dead - mutations in same gene�alive - mutations in different genes�


Mutagenesis strategies: the first decision to make in planning a screen �

Chemical - EMS�"Pros: unbiased, point mutations, high efficiency of mutagenesis�"Cons: must map to identify gene affected�

Transposon insertion - P element (piggyBac, Minos)�"Pros: easy to clone the gene�"Cons: site of insertion bias, often don’t disrupt gene function �

Irradiation - X-rays, gamma-rays�"Pros: often generate null alleles (deletions, etc.)�"Cons: sledgehammer approach often compromises gene function for�""multiple genes or causes chromosomal aberrations.�


EMS = chemical mutagen �DTS: dominant temperature sensitive�

Heat shock �

P�

F1 �

F2 �

F3 �

? - homozygous�mutant �

F3 Screen �

alive - heterozygous�Use to make a stock �

dead - homozygous�balancer�

Permits screening and recovery of recessive lethal mutations�


EMS = chemical mutagen �DTS: dominant temperature sensitive�

Heat shock �

P�

F1 �

F2 �

F3 �

? - homozygous�mutant �

F3 Screen �

alive - heterozygous�Use to make a stock �

dead - homozygous�balancer�


Mis-expression screens�

Identify genes based on an over-expression phenotype.�Can be combined with a sensitized genetic background to �

identify enhancers and suppressors.�


Mis-expression screens�


Several other screening strategies are possible (and comparatively easy) including �mosaic and maternal/zygotic screens, but these will be covered in future seminars.�

Drosophila melanogaster: Reverse genetic approaches�

What if you are starting with a human gene of unknown function and want to use�Drosophila to figure out what it does?�


Imprecise P element excision �

RNAi - injection, UAS-hairpin �

Homologous recombination �




Homologous recombination �

UAS-InR RNAi�α-InR �

α-InR �




Homologous recombination - knock out �




Homologous recombination - knock out �

Homologous recombination and Phi/att - knock in �

Drosophila melanogaster: Mapping and cloning mutants�

Transposable elements: inverse PCR �Chemical mutants: positional cloning �

Drosophila melanogaster: Mapping and cloning mutants�

Transposable elements: inverse PCR �Chemical mutants: positional cloning �

meiotic recombination mapping - visible markers�"" "" SNPs �"" �

complementation tests " - mutants in candidate genes�"" "" Deficiency strains (chromosomal deletions)�

male recombination " - mapping relative to P elements of known �"" "" position �

"sequence analysis of candidate genes�

"rescue of mutant phenotype with transgene�

http://flybase.org/ �

Flybase: a community resource�

drosophila as a model

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