Safe Operating Procedure (SOP) Prepared by: Simon Wells

School of Molecular & Biomedical Science Date: 15/07/08

Approved by:

Signature:

As per 7is* Assessment all persons e3amining Drosophila must wear (a "inimum 889# There is a limitation of 01 car on dio3ide outlets to e on at any time for a ma3imum of 0 hours# Demonstrators to ensure ample &entilation : open all doors and windows# 9nsure outlets are turned off when not in use# +hen using morgues ma*e sure all near y ignition sources are off# Familiarisation it! Drosophila _____________________________________________________________________ The Drosophila life cycle Anaesthetisation and microscope techniques Distinguishing males and females Distinguishing mutant phenotypes from wildtype o white eyes from wildtype (red) eyes o Glazed eyes from wildtype eyes o Curly wings from wildtype (flat) wings

"able 1# Symbols and mutant p!enotypes $or genes used in t!is pra%ti%al#
Gene sym ol w Cy wgGla Tb !ull allele name white118 Curly Glazed (actually a dominant allele of wg, wingless)# Tubby "utant phenotype white (pigmentless) eyes# ($ote% allele is w118) wings cur&ed'curled upwards (oss of photoreceptor cells in the eye# This changes the surface appearance of the eye so that it appears glassy# )t also alters the normally round shape of the eye# (ar&ae% pupae and adults are short and thic*set#

+hich of these mutations ha&e dominant and which ha&e recessi&e phenotypes, (see $omenclature notes later in this document)# (Guidance- (ist the different mutations and state whether they are dominant or recessi&e and how you *now this to e so#)

"!e Drosophila li$e %y%le The life cycle of Drosophila% from fertilised egg to reproducti&ely mature adult% ta*es ./ days when cultured at 012C (!igure .)# De&elopment ta*es less time at slightly higher temperatures (Drosophila can sur&i&e only rief e3posure to temperatures higher than 042C)% and longer at lower temperatures (e#g# a full life cycle ta*es o&er 5 wee*s at .62C)# The standard temperature for Drosophila crosses is 012C% and thus a cross you set up in prac class one wee* will not yield results until two wee*s later#

Figure 1# "!e li$e %y%le o$ Drosophila# Arrows are la eled with the num er of days during which the animal remains in the stage at the eginning of the arrow# The transition from an em ryo to a first instar lar&a is called hatching. The transitions etween lar&al instars are moults. The process that con&erts a third instar lar&a to a pupa is pupariation# 9mergence of the adult from the pupal case is called eclosion# Ta*en from ;artwell et al. (0///)#

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Anaest!etising Drosophila !lies are anaesthetised on a pad through which car on dio3ide (C<0) flows% and then o ser&ed under a dissection microscope% using a des* lamp or other light source to pro&ide sufficient illumination# +atch your demonstrator to see how to anaesthetise the flies#

"!ere is a limitation o$ &5 %arbon dio'ide outlets to be on at any time $or a ma'imum o$ & !ours# Demonstrators to ensure ample ventilation ( open all doors and indo s# )nsure outlets are turned o$$ !en not in use#
C<0 is hea&ier than air% and thus the flies on the pad will stay anaesthetised while the C<0 remains turned on# The flies will remain ali&e li*e this for o&er an hour# (They are in much more danger of eing a*ed y the des* lamps than dying from C<0#) <nce flies are remo&ed from the C<0% they will wa*e up in 1=0/ min% depending on how long they were e3posed to the C<0# (oo* at the flies under a dissection microscope% lea&ing them on the C<0 pad# >se a paint rush to gently manipulate the flies while you are e3amining them# !lies that you no longer need are disposed of into a ?fly morgue@% where they meet death y A/B ethanolC

W!en using morgues ensure all nearby ignition sour%es are o$$#
A folded piece of card can act li*e a funnel to help in sweeping the flies into the morgue# )f you are selecting flies to set up a cross% gently rush the flies onto the side of a new &ial# )t is important not to place anaesthetised flies onto stic*y fly food% ecause they will pro a ly get stuc*# Do% when setting up a cross% always place the flies on the side of the &ial and *eep the &ial horizontal until the flies ha&e wo*en up# Chec* that the sides of the &ial are not wet (or the flies will still get stuc*) : you can wipe the inside of the &ial with paper towel to ma*e sure that it is dry#

Distinguis!ing se'es There are three main features that distinguish male and female flies- differences in oth a dominal pigmentation and e3ternal genitalia% and presence or a sence of se3 com s ( (Guidance- "a*e your drawings as carefully as you can# Draw only what you can seeC (AE9( your drawings to indicate the different parts of the anatomy#)0)# "a*e sure that you can distinguish males and females y each of these characteristics# Fou@ll pro a ly find that the first two differences are the easiest to see# ;owe&er% e aware that the a dominal pigmentation is not present in young flies% and can e hard to see in some mutant phenotypes# < ser&e wild type flies and draw in your prac write=up oo* the main features of the structure of the Drosophila ody% including the distinguishing features of males and females# (Guidance"a*e your drawings as carefully as you can# Draw only what you can seeC (AE9( your drawings to indicate the different parts of the anatomy#) Figure &# )'ternal visible distinguis!ing $eatures o$ male and $emale Drosophila# A# Dorsal a dominal pigmentation# !emales ha&e a series of stripes on the dorsal ( ac*) side of the a domen% while males ha&e only a couple of stripes and then a continuous dar* patch of pigmentation at the posterior (tail) end of the a domen# E# The e3ternal genitalia of males and females are &isi ly different# The posterior end of the &entral a domen of a male has dar* rown structures including ?se3 hoo*s@% while e3ternal genitalia in females is not easily &isi le (!igure 6E)# C# 9ach front leg of males only has a ?se3 com @% a small region of dar*% dense ristles a out two thirds of the way down the leg# Adapted from "# Demerec and E#8# Gaufman (.45A) Drosophila Guide# Hth 9d# Carnegie )nstitute of +ashington#

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Distinguis!ing mutant p!enotypes Fou will o ser&e &arious phenotypes in Drosophila : wildtype% an eye colour mutation (white)% an eye surface'shape mutation (Glazed) and mutations affecting wing shape (Curly) and ody shape (Tubby)# )nclude in your prac write=up oo* drawings and'or a written description of the phenotypes you o ser&e# (Guidance- Descri e in words and% if possi le% images the different phenotypes that you o ser&e#)

Setting up a simple %ross Anaesthetise a &ial of w;wgGla'Cy Tb flies and separate out 1 males# Det up a cross with 1 of these males and 1 &irgin white females# "a*e sure you la el the &ial with the date% cross and your name# $e3t wee* you will need to remo&e the parents from the cross (sa&e them in a separate &ial for comparison with their progeny) and then you will score the phenotypes of the progeny the wee* after that# +hen scoring the phenotypes of the progeny% note the eye colour'surface te3ture'shape and the se3 of each fly as well as the ody and wing shape# Compare these to the parents# 7eport these results in your practical notes# +hat can you say a out the inheritance of the Curly% Tu y and white phenotypes, (Guidance- Analyse at least 1/ flies from each cross# "a*e up a ta le where you note the characteristics of each fly you analyse# Then loo* at the pattern of data in the ta le and what this tells you a out the inheritance of each phenotype#)

*omen%lature - Drosophila pra% >se of a standard nomenclature is in&alua le when communicating information a out genes and genotypes# )t is standard in any genetic discussion to use italics when referring to a gene name or genotype# 8lain te3t will denote protein or phenotype depending on the conte3t# 9&ery Drosophila gene has a unique name and a re&iation% or sym ol (see Ta le .)# Genes initially characterised y a recessi&e mutant phenotype are gi&en gene names eginning in lower case% while genes initially characterised y a dominant mutant phenotype or named after an enzyme are gi&en gene names eginning in upper case# Drosophila geneticists use ?I@ to denote a wildtype allele# !or e3ample% a white mutant allele and white wildtype allele can e referred to as w and + respecti&ely# Alternati&ely% if it might e unclear which gene the ?I@ is referring to% you should use w- and w+ respecti&ely# Drosophila has J chromosomes% which are num ered .% 0% 6 and J (or .st% 0nd% 6rd and Jth)# Chromosome . is the K chromosome (one of the two se3 determining chromosomes)% and can e referred to as either . or K# The F chromosome (the other se3 chromosome) has no other name# +hen writing the genotype of a strain% the different chromosomes are separated y semicolons% and it is usual to represent the chromosomes in order from left to right (though only chromosomes rele&ant to the cross are represented)# ;eterozygous genotypes are represented as a fraction% while it is usual to only show one line for homozygotes (!igure 6A)# !inally% when writing genotypes it is usual to only include genes that ha&e mutant alleles in that fly# ;owe&er% in cases where one parent of a cross is wildtype for a particular gene% while the other parent is mutant% it is generally clearer to specify the genotype at this gene for oth flies (!igure 6E)#
;+ A. +; dp pr + ; e B. +; x + Y Figure +# )'amples o$ genotypes to demonstrate %onventions o$ Drosophila nomen%lature# A# The fly represented y this genotype is heterozygous for v% and homozygous wildtype for all other genes located on the same chromosome as v (the K chromosome)# <n the chromosome 0% it is homozygous mutant for dp and pr ut heterozygous for b# Chromosome 6 is homozygous mutant for e# E# !emales (L) heterozygous for Sb are crossed to males (M) that ha&e sn% an K=lin*ed mutation% ut are wildtype for the Sb gene#
v dp pr b

Sb

sn

)t is often useful to identify specific generations% in addition to se3 and genotype# The !. generation% or first filial generation% is the hy rid progeny of any single cross# The parents of an !. generation are denoted as 8.# The progeny of !. indi&iduals allowed to mate amongst themsel&es ecomes the !0% or second filial generation# NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN