DrosophilaGenetics
by
Michael Socolich
I
II III IV V VI		 General Information and Fly Husbandry Nomenclature
Genetic Tools Available to the Fly Geneticists Example Crosses
P-element Transformation
References and Figures
This review is intended to be a primer of Drosophilagenetics for the beginning graduate or post-doctoral student, and any interested researcher. Those interested in a more in depth discussion on the topic are referred to the sources in the reference section.

I. General Information and Fly Husbandry

The fruit fly Drosophila melanogasterhas 3 pairs of autosomal chromosomes and an X and Y chromosome. Each autosome has two arms that are simply referred to as left (L) and right (R). Each chromosome arm is numbered as follows:X (1-20), 2L (21-40), 2R (41-60), 3L (61-80), 3R (81-100), and chromosome 4 (101-102). Each chromosome arm is also numbered by recombination units, thus allowing one to know the expected recombination frequency between two genes located on the same chromosome arm. The chromosomal locations of individual genes are identified either by numerical location or by recombination units. Sex determination in Drosophilais based on the ratio of X chromosome to autosomal sets. Therefore, females, which have two X chromosomes, have a ratio of 1 whereas; males, which have only 1 X chromosome, have a ratio of 0.5. Fortunately, for the fly geneticists recombination does not occur in male flies. This quality can be exploited in certain genetic crosses as will be explained later.
The life cycle of Drosophilais quite simple: eggs are layed which develop into larvae, which develop into pupae, which develop into adult flies. Flies do not "hatch" from pupal cases they "eclose". The above life cycle is dependent on the temperature at which the flies are grown. The generation time for flies grown at 25o C is 10 days, at room temperature (21-22oC) it is 12-13 days, and at 18 o C the generation time is 19 days.
It is a fact of life that female fruit flies can mate with more than one male and store sperm from multiple matings. This forces the geneticists to use virgins when conducting a genetic cross. Using non-virgins results in progeny that do not have the expected genotypes or phenotypes predicted by simple Mendellian genetics. When collecting virgins it is useful to remember that newly eclosed virgins will remain virgins for 6 hours at 25o C, 12 hours at 21-22oC, and 18 hours at 18 oC.
An important source of information for the fly researcher is FlyBase (1). FlyBase is a website that allows one to browse for information regarding individual genes, chromosomal rearrangements, fly anatomy, fly stocks, cytologic maps, and a host of other information useful to the fly researcher.
Proper care and attention are necessary to ensure that one's fly stocks remain healthy. The following points need to be remembered and attended to at all time to maintain healthy stocks:
1.

2.

3.

 Ensure that fly food remains moist at all times. Dry food will inhibit larval growth and result in few flies eclosing.
Do not expose fly stocks to extremes in temperature for extended periods of time, that is less than 18o C or greater than 25o C.
Always transfer stocks every 3 weeks to prevent and minimizethe chance of mite infestation.
For the researcher working with fruit flies the pest one must always be vigilant against is mites! Mites come in many different varieties:mites that eat eggs, mites that eat fly food, and mites that feed off of adult flies (see the attached figures of various mites). Mites can decimate fly stocks!The threat of mites can be minimized by transferring stocks frequently, inspecting vials and bottles regularly, and quarantining any stock(s) that are received from another lab. Proper quarantining of a new stock involves the following procedure:
1. 2.

3. 4.

 Transfer flies to a fresh vial upon receipt of new stock. Keep original vial. Continue to transfer flies everyday for 5 days. Throw out vials from days 1 to 4. On the sixth day dump flies and keep the day 5 vial.
When flies eclose from day 5 vial transfer to new vial and repeat steps 1 and 2 again. Inspect the original vile for the presence of any mites. Also, inspect the day 5 vial for mites.
If after two generations no mites are found in any of the above vials it is safe to introduce this stock into flyroom. However, if mites are found then the above procedure must be continued until two successive generations of mite free vials are generated!

II. Nomenclature

The nomenclature used in Drosophilagenetics is fairly straightforward yet to the initiated can be daunting. It is important that one follows the standard rules of nomenclature to properly and clearly describe the complete genotype of a fly stock.

*Chromosomes are written in order, as follows, with a semi-colonseparating each chromosome.
X/Y; 2; 3; 4
*Genotypes are listed only when a mutation is present and are italicized.
*Recessive mutations are written in lower case (e.g. wfor white gene),
*Dominant mutations are capitalized (e.g. Roifor rough eye).
*If a particular allele is present that allele is superscripted (e.g. norpAP41 ).
*Anything not listed is assumed to be wild type.
*If more than one mutation is present on a chromosome, the mutations are listed from left to right
corresponding to the left and right arms of each chromosome.
arr1 cn bw
*If a mutation is homozygous then the mutation is written only once as follows:
cn bw
*If heterozygous then written as follows:
cn bworcn bw /+ +orcn bw /+
++

*Deficiencies:Df(2L)VA= Deficiency of the left (L) arm of chromosome 2 that includes the gene Venea
abnormeis (VA).
*Transpositions:Tp(1;3)HF308= Transposition involving the X and 3 rdchromosome.
*Inversions:In(2LR)SMC8= Inversion of the left (L) and right (R) armof the 2nd chromosome.
*Translocations:T(1;3)Th1= Translocation between the 1st (X) chromosome and 3rd chromosome.
*Commas follow rearrangements and indicate mutations present:
e.g. In(2LR)SM1, al 2Cy cn2sp2= Inversion involving the
left (L) and right (R) arms of chromosome 2 with the
following mutations present: aristaless ( al ), Curly
(Cy), cinnabar (cn ), and speck (sp).

The above information can be obtained either through FlyBase ( 1) or The Genome of Drosophila melanogaster(2 ).

III. Genetic Tools Available to the Fly Geneticists

There are three tools that one can utilize when performing genetic crosses:(1) balancers, (2) phenotypic markers, and (3) non-recombination in males.
Balancer chromosomes have the following properties that make them powerful:multiple inversions within the same chromosome, one or more dominant markers, usually 2-4 recessive markers, and lethality as homozygotes. Each chromosome in Drosophilahas its own set of balancers. These balancers are numbered numerically and each has its own dominant and recessive markers, though some balancers are "upgrades" of earlier balancers and often share the same markers. The table below is a partial list of the major balancers for each chromosome and the dominant marker associated with each balancer. Due to its small size chromosome 4 has no balancers since homologous recombination does not occur or is infrequent.
Chromosome Balancer Dominant marker
IMAGE imgs/fly01.gif
An example of the multiple inversions present in a balancer is exemplified by TM6B (In(3LR)TM6B, Hu Tb e) that has the following chromosomal numbering 61A[!]87B-86C[!]84F-86C[!]84B-84F[!]84B-75C[!]94A-100F[!]92D-87B[!]61A- 63B[!]72E-63B[!]72E-75C[!]94A-92E[!]100F-100F. The presence of these inversions within the same chromosome prevents homologous recombination. Since most recessive mutations have no phenotype, balancers allow the geneticists to indirectly follow the recessive mutation (by scoring dominant markers) without losing the mutation (due to inhibited recom-bination). Balancers are also used to maintain chromosomal deficiencies that would otherwise be lethal due to the deficiency.
The number of phenotypic markers available to the geneticists is one factor that makes genetics in Drosophilaso unique and easy compared to other organisms. Drosophilagenetics is replete with both recessive and dominant markers that allow one to select flies by eye phenotype, body phenotype, bristle phenotype, larval phenotype, wing phenotype, etc. Phenotypic markers are especially valuable when using balancers. By using Mendel's laws and following the dominant phenotypic markers associated with a balancer one is able to confidently and successfully produce a homozygous recessive (non-phenotypic) stock throughout a multiple generation cross.
The last genetic tool is the previously mentioned lack of recombination in male fruit flies. This phenomenon can be used in certain genetic crosses without worrying about losing the gene of interest while in the unbalanced state. More importantly, since most large scale mutagenesis screens start with potentially mutagenized male flies one need not worry about losing the mutation during meiosis.

IV. Example Crosses

Before presenting any examples of mating crosses, the issue of recombination between two genetic loci needs to be addressed. When trying to either separate or recombine two genetic loci on the same chromosome arm it is necessary to determine beforehand the expected recombination frequency between the two loci and the number of progeny one must screen to find the desired genotype. The expected recombination frequency between two loci can be ascertained by simply determining the recombination frequency difference between two loci.
Recombination frequencies can be obtained from either The Genome of Drosophila melanogaster(2) or FlyBase (1). Deciding how many progeny to screen can be calculated using Mather's (3) formula:
IMAGE imgs/fly02.gif
where N = number of progeny one needs to screen
p = probability of obtaining a recombinant
f= expected recombination rate between two loci

The more closely two loci are located the greater the number of progeny that must be screened before a recombinant is found. Conversely, if two loci are distant from each other, then one need screen only a small number of progeny. Two genes located on different arms of the same chromosome will sort independently from each other as follows from Mendel's law of independent assortment.
Below are several examples of crosses or mating schemes. Brief explanations will be given at each step explaining what is being done and why. Also, the phenotype of the expected flies will be given. Only the genotypes of the desired classes are listed for each mating scheme. The reader is encouraged to determine all theoretical classes that can be produced in each example below by making Punnett squares of each mating.

Cross 1:During the course of conducting some electrophysiological studies it is necessary to test a y w norpAP41fly
that does not exist. This fly can be created from the following two stocks: y w(yellow body color and
white eyes) and norpA P41(red eyes and mutation in the norpA gene). To generate the desired fly, y w
norpAP41, one must find a recombinant between the white ( w) and norpA loci.

All three genes are located on the X chromosome in the following order (starting from the tip of the X chromosome):y w norpA . The recombination value associated with each gene is as follows:y(0.0%), w (1.5%), and norpA(6.5%). Therefore the expected recombination rate, between white and norpA,is 5% (6.5% - 1.5%). The number of progeny that one must screen to have 95% confidence of getting the desired fly is:

N = IMAGE imgs/fly03.gif = 60 progeny!
The mating scheme is:
F1 Red eyed norpA male. norpAP41
Y		 yw
yw		 White eye, yellow body color
virgin female.
X
IMAGE imgs/fly06.gif
F2 X chromosome Balancer male. FM7A
Y		 norpAP41
y w		 Red eyed female.
Recombination occurs here
during meiosis.
X
IMAGE imgs/fly06.gif
F3 White eyed, yellow body male. Potential recombinant ywnorpAP41?
Y		 FM7A
FM7A		 X chromosome virgin female
balancer. FM7A is not. Set up 60
single pair matings.homozygous
lethal.
X
IMAGE imgs/fly06.gif
F4 X chromsome male balancer sibling. FM7A
Y		 y w norpAP41 ?
FM7A		 Yellow body, white eye
potential norpAfemale
recombinant. Crossto
FM7A male sibling.
X
IMAGE imgs/fly06.gif
F5 Potential male recombinant ywnorpAP41?
Y		 ywnorpAP41?
FM7A		 Potential recombinant sibling
Virgin female.
X
IMAGE imgs/fly06.gif
ywnorpAP41?
Y		 y w norpAP41 ?
y w norpAP41 ?		 Potential homozygous
recombinant stock.
Test for norpAby Western
or electrophysiology.
X
In the above cross one can select against recombination between the yellow and white loci by selecting only those males having white eyes and a yellow body at the F3 stage. FM7a unlike most balancers is not homozygous lethal.
Cross 2: Given the following two stocks (w; arr1 cnbw; arr2and w;; ninaE e) produce the following fly: w; arr1 cn bw; arr2 ninaE e.
Again one is trying to find a recombinant between arr2and ninaE, however, since they are on different arms of the same chromosome they will sort independently from each other and there will simply be a 50/50 chance that a particular progeny will contain both mutations.

The mating scheme is:
F1 w; SM6B; TM6B e
Y;ScoMKRS