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

Drosophila melanogaster as a model organism: historical perspective

Thomas Hunt Morgan

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

Nobel prize 1933

Drosophila melanogaster as a model organism: historical perspective

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

Drosophila melanogaster as a model organism: historical perspective

Segmental patterning

Nobel prize 1995

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

Drosophila melanogaster as a model organism: historical perspective

Homeotic genes

Ed Lewis Nobel prize 1995

Drosophila melanogaster as a model organism: historical perspective

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

A very practical choice

-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.

The Drosophila melanogaster life cycle

A very concise description of Drosophila development

A very concise description of Drosophila development

14 mitoses without cytokinesis

A very concise description of Drosophila development

Cellularization (conversion of multi-nuclear syncitium into cellular blastoderm)

A very concise description of Drosophila development

Gastrulation (formation of germ layers)

A very concise description of Drosophila development

Organogenesis (example: tracheal development)

A very concise description of Drosophila 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: Deciencies -- " "deletions of genomic DNA, ex: Df(3R)EXEL6276 Balancers -- "multiple inversions, used for maintaining recessive " "mutant stocks, ex: FM7; SM6a; TM3

Drosophila nomenclature
Male Female Virgin 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) 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: Deciencies -- " "deletions of genomic DNA, ex: Df(3R)EXEL6276 Balancers -- "multiple inversions, used for maintaining recessive " "mutant stocks, ex: FM7; SM6a; TM3

Drosophila nomenclature
Male Female Virgin 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) "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/CyO; sr, e Generally useful Chromosomal aberrations: Deciencies -- " "deletions of genomic DNA, ex: Df(3R)EXEL6276 Balancers -- "multiple inversions, used for maintaining recessive " "mutant stocks, ex: FM7; SM6a; TM3

Drosophila nomenclature
Male Female Virgin 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) "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/CyO; sr, e Generally useful Chromosomal aberrations: Deciencies -- " "deletions of genomic DNA, ex: Df(3R)EXEL6276 Balancers -- "multiple inversions, used for maintaining recessive " "mutant stocks, ex: FM7; SM6a; TM3

Drosophila nomenclature
Male Female Virgin 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) "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 Generally useful Chromosomal aberrations: Deciencies -- " "deletions of genomic DNA, ex: Df(3R)EXEL6276 Balancers -- "multiple inversions, used for maintaining recessive " "mutant stocks, ex: FM7; SM6a; TM3

Drosophila nomenclature
Male Female Virgin 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) "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 Generally useful Chromosomal aberrations: Deciencies -- " "deletions of genomic DNA, ex: Df(3R)EXEL6276 Balancers -- "multiple inversions, used for maintaining recessive " "mutant stocks, ex: FM7; SM6a; TM3

Drosophila nomenclature
gene names are italicized, Proteins are capitalized
Certain gene names indicate something specic 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 dened 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 specic 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 dened 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 specic 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 dened 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:

Enhancer trap

Gal4/UAS

Gene expression tools:

Enhancer trap

Gal4/UAS

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

Drosophila: understanding crosses and screens

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

a b dead- homozygous balancer alive- heterozygous a or b/balancer dead - mutations in same gene alive - mutations in different genes

Drosophila: understanding crosses and screens

Mutagenesis strategies: the rst decision to make in planning a screen Chemical - EMS "Pros: unbiased, point mutations, high efciency 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 dont 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.

Drosophila: understanding crosses and screens

F3 Screen

P F1 Permits screening and recovery of recessive lethal mutations

Heat shock

F2 F3
dead - homozygous alive - heterozygous balancer Use to make a stock ? - homozygous mutant

EMS = chemical mutagen DTS: dominant temperature sensitive

Drosophila: understanding crosses and screens

F3 Screen

P F1
Heat shock

F2 F3
dead - homozygous alive - heterozygous balancer Use to make a stock ? - homozygous mutant

EMS = chemical mutagen DTS: dominant temperature sensitive

Drosophila: understanding crosses and screens

F3 Screen

P F1
Heat shock

F2 F3
dead - homozygous alive - heterozygous balancer Use to make a stock ? - homozygous mutant

EMS = chemical mutagen DTS: dominant temperature sensitive

Drosophila: understanding crosses and screens

F3 Screen

P F1
Heat shock

F2 F3
dead - homozygous alive - heterozygous balancer Use to make a stock ? - homozygous mutant

EMS = chemical mutagen DTS: dominant temperature sensitive

Drosophila: understanding crosses and screens

Mis-expression screens

Identify genes based on an over-expression phenotype. Can be combined with a sensitized genetic background to identify enhancers and suppressors.

Drosophila: understanding crosses and screens

Mis-expression screens

Drosophila: understanding crosses and 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 gure out what it does?

Drosophila melanogaster: Reverse genetic approaches

Imprecise P element excision RNAi - injection, UAS-hairpin Homologous recombination

Drosophila melanogaster: Reverse genetic approaches

Imprecise P element excision RNAi - injection, UAS-hairpin Homologous recombination

-InR UAS-InR RNAi

-InR

Drosophila melanogaster: Reverse genetic approaches

Imprecise P element excision RNAi - injection, UAS-hairpin Homologous recombination - knock out

Drosophila melanogaster: Reverse genetic approaches

Imprecise P element excision RNAi - injection, UAS-hairpin 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

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 " " Deciency strains (chromosomal deletions) male recombination " " position " - mapping relative to P elements of known

"sequence analysis of candidate genes "rescue of mutant phenotype with transgene

Flybase: a community resource

http:/ /ybase.org/