Download Drosophila Genetics

Drosophila Genetics
by
Michael Socolich
May, 2003
I. General Information and Fly Husbandry
II. Nomenclature
III. Genetic Tools Available to the Fly Geneticists
IV. Example Crosses
V. P-element Transformation
VI. References and Figures
Preface
This review is intended to be a primer of Drosophila genetics 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 melanogaster has 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 Drosophila is 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 Drosophila is 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 25oC is 10 days, at
room temperature (21o-22oC) it is 12-13 days, and at 18oC the generation time is 19 days.
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 25oC, 12 hours
at 21-22oC, and 18 hours at 18oC.
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,
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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. Ensure that fly food remains moist at all times. Dry food will inhibit
larval growth and result in few flies eclosing.
2. Do not expose fly stocks to extremes in temperature for extended
periods of time, that is less than 18oC or greater than 25oC.
3. Always transfer stocks every 3 weeks to prevent and minimize
the 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. Transfer flies to a fresh vial upon receipt of new stock. Keep original
vial.
2. 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.
3. When flies eclose from day 5 vial transfer to new vial and repeat steps
1 and 2 again.
4. 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 one's 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 Drosophila genetics is fairly straightforward yet to the
uninitiated 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-colon
separating 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. w for white gene),
dominant mutations are capitalized (e.g. Roi for rough eye). If a
particular allele is present that allele is superscripted (e.g. norpAP41).
Anything not listed is assumed to be wild type.
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• 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 bw or cn bw/+ + or cn 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 3rd
chromosome.
• Inversions: In(2LR)SMC8= Inversion of the left (L) and right (R) arm
of 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, al2 Cy cn2 sp2 = 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 Drosophila
has 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
X
Balancer
FM3
FM4
FM6
FM7a, b, c
Dominant marker
B=Bar eye
"
"
"
3
2
3
CyO
SM1
SM2
SM5
SM6a, b
TM1
TM2
TM3
TM6
TM6B
TM6C
TM8
TM9
Cy=Curly wings
"
"
"
a=Curly wings, b=Curly wings and rough eye (Roi)
Me=Moiré
Ubx=Ultrabithorax (bristles on the halteres)
Sb=Stubble (short bristles)
Ubx=Ultrabithorax (bristles on the halteres)
Hu=Humeral, Tb=Tubby
Tb=Tubby
DTS=Dominant temp. sensitive, Sb=Stubble
DTS=Dominant temp. sensitive, Sb=Stubble
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87B86C84F-86C84B-84F84B-75C94A-100F92D-87B61A-63B72E-63B72E75C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
recombination). 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 Drosophila so unique and easy compared to other organisms.
Drosophila genetics 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 following a multi-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 by recombination 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 fly with the desired genotype. The expected
recombination frequency between two loci can be ascertained by simply determining the
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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:
N = -log(1-p)
log(1-f)
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 norpAP41 fly. This fly can be created from the following
two stocks: y w (yellow body color and white eyes) and norpAP41 (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 recombinants that one must screen to
have 95% confidence of finding the desired fly is:
N = -log(1-.95) = -log(.05) = -1.301 = 60 progeny!
log(1-.05)
log(.95) -0.022
The mating scheme is:
F1
Red eyed norpA
male.
norpAP41
Y
X
5
yw
yw
White eye, yellow body color
virgin female.
F2
X chromosome balancer
male.
FM7A
Y
F3
White eyed, yellow body
male. Potential norpA recombinant. Set up 60 single
pair matings.
y w norpAP41?
Y
X
F4
X chromosome balancer
male sibling generated
from F3 cross.
FM7A
Y
y w norpAP41?
FM7A
F5
Potential male recombinant.
y w norpAP41?
Y
X y w norpAP41?
FM7A
Potential recombinant sibling
Virgin female.
y w norpAP41?
Y
X y w norpAP41?
y w norpAP41?
Potential homozygous
recombinant stock.
Test for norpA by Western or
electrophysiology.
X
X
norpAP41
yw
Red eyed female.
Recombination occurs here
during meiosis.
FM7A
FM7A
X chromosome virgin female
balancer. FM7A is not
homozygous lethal.
Yellow body, white eye
potential norpA female
recombinant. Cross to
FM7A male sibling.
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 other balancers is not
homozygous lethal.
Cross 2: Given the following two stocks (w; arr1 cn bw; arr2 and w;; ninaE e)
produce the following fly: w; arr1 cn bw; arr2 ninaE e.
Again one is trying to find a recombinant between arr2 and 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 Sco
MKRS
F2
arr1, arr2 over balancers.
White eyes, curly wings,
humeral, tubby, rough
eyes.
X
w; arr1 cn bw; arr2
w arr1 cn bw; arr2
w; arr1 cn bw; arr2
Y SM6B
TM6B e
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w; SM1; TM2
Y Sco MKRS
X
X
w; +; ninaE e
w; +; ninaE e
w; SM1; TM2
ninaE virgin over
w +
ninaE e double balancer.
White eyes, curly
wings, Ubx.
F3
Independent assortment
occurs here during
meiosis. White eyes,
curly wings.
w; arr1 cn bw; ninaE e
w
SM1
arr2
F4
Potential arr2, ninaE
recombinant. Ebony
body color, white eyes,
curly wings, humeral,
tubby, rough eyes. Set up
individual crosses using
these males.
w; arr1 cn bw; arr2? ninaE e
Y
SM6B
TM6B e
X
w; SM6B; TM6B e Backcross males
w; Sco MKRS
to double balancer
virgin females.
F5
Sibling mating. White
eyes, curly wings, rough
eyes, tubby, humeral,
ebony body color.
w; arr1 cn bw; arr2? ninaE e
Y
SM6B
TM6B e
X
w; arr1 cn bw; arr2? ninaE e
w
SM6B
TM6B e
Homozygous stock.
X
w; SM6B; TM6B e Double balancer
Y Sco MKRS
male.
w; arr1 cn bw; arr2? ninaE e
In the above cross the following trick was used: ninaE and e are closely linked genetically and by selecting
an ebony F4 potential recombinant male ones knows that ninaE is present. Therefore, one need only screen
for the absence of arr2 in the final stock instead of having to screen for the absence of both arr2 and ninaE.
Since arr2 and ninaE have sorted independently of each other one need only screen a few lines to find a
recombinant (remember each line has a 50/50 chance of being a double mutant).
Cross 3: One wishes to make a w; inaD; trp fly. The available stocks are w; inaD; +
and w; +; trp. Because both mutations are recessive and therefore do not have
a phenotype to follow it is necessary to outcross each stock to different double
balancer lines. This allows one to indirectly follow the mutations via the
dominant markers for each balancer.
The mating scheme is:
F1
w; inaD; +
Y inaD; +
X
w; SM6B; TM6B
w Sco MKRS
w; +; trp
Y + trp
X
w; SM1; TM2
w Sco MKRS
White eyes, curly
wings, Ubx.
F2
White eyes, curly wings,
rough eyes, tubby,
humeral.
w; SM6B; TM6B
Y inaD
+
X
w; SM1; TM2
w +; trp
F3
White eyes, curly wings,
tubby, humeral. Sibling
cross.
w; SM1; TM6B
Y inaD trp
X
w; SM1; TM6B
w; inaD trp
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w; inaD; trp
inaD; trp
Double mutant stock.
As stated previously, each line was outcrossed to different double balancer stocks so that the individual
mutants could be followed by selecting for the dominant markers from each balancer. To produce the double
mutant stock one switches the balancers for both the second and third chromosome as was done when
selecting progeny from the F2 cross.
V. P-element Transformation
A powerful technique available to the researcher is the use of P-elements to
transform flies. Using P-elements one can reintroduce a wild type copy of a mutated
gene into a null or mutated background. One can also introduce genes that have been
altered (e.g. destroying a phosphorylation site) or tagged (e.g. GFP fusions).
P-elements are transposable pieces of DNA that randomly insert themselves into
genomic DNA. P-elements that are used to generate transgenic animals are engineered
without the transposase gene that catalyzes DNA insertion. A separate piece of DNA
(∆2-3) that contains the transposase gene must be co-injected with the P-element in order
for the P-element to insert itself within the genomic DNA. Transformants are selected
using a variety of phenotypic markers such as eye color, body color, resistance to
antibiotics, etc. Genes expression, from the incorporated P-element, is driven by a
variety of different promoters depending on the experimental design.
P-element DNA and ∆2-3 DNA are injected into the posterior pole of the
developing embryo where the germline cells are located. The intention is to transform
these germline cells so that a stable, transmissable transformant is created.
Embryos that survive the injection procedure are allowed to develop into flies that
represent unique P-element insertion events. Individual flies are backcrossed to the
injection stock to select for germline transformants. Outcrossing to the injection stock is
necessary because not every insertion is into the germline cells but rather into somatic
cells. Backcrossing to the injection stock is performed twice to ensure the selection of
stable germline transformants.
Isolation of germline transformants allows one to proceed with generating a
homozygous stock. A generic homozygous producing mating scheme is shown below for
a P-element that is marked with the white gene (this will produce a fly with red eyes) and
was injected into w1118 (a white eyed stock) flies.
F1
Red eyed male
transformant. Location
of P-element unknown.
w1118
Y
F2
Red eyed male over
double balancers
w; SM6B; TM6B
Y
+
+
F3
Red eyed homozygous stock.
Location of P-element unknown.
X
w; SM6B; TM6B
w; Sco MKRS
X
w; +; +
+ +
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w; SM6B;TM6B
w
+
+
Double balancer
virgins
Red eyed sibling
female virgin over
double balancers.
Generation of transgenic flies is not complete without determining which chromosome
the P-element inserted into. This information is necessary if one anticipates crossing
transgenic animals into various mutant backgrounds. By using male transformant flies
one can immediately determine if the P-element is on the X chromosome (see the
following mating scheme). If the P-element is on the X chromosome than all F2 females
will have red eyes and all F2 males will have white eyes. Both F2 males and F2 females
are crossed against each other and the balancers selected against to generate a
homozygous stock.
F1
Red eyed male
transformant. P-element
is on X chromosome.
w1118 P[w+]; +; +
Y
+ +
F2
All males white eyed
w; SM6B; TM6B
Y
+
+
X
and
w; SM6B; TM6B
w; Sco MKRS
w1118 P[w+]; SM6B; TM6B
w
+
+
Double balancer
virgin female.
All females red
eyed.
If the P-element is not on the X-chromosome then both F2 males and F2 females will
have red eyes. Determining whether the P-element is on the 2nd or 3rd chromosome
requires outcrossing the F2 double balancer males, from the above cross, to the injection
stock and following how the phenotypic marker segregates in the resulting progeny. The
male transformant fly used below is generated from the outcross to the double balancer
stock.
Red eyed male
transformant. In
reality only one
P-element is present
w1118; SM6B; MKRS
Y
P[w+] P[w+]
X
w1118; +; +
w1118 + +
Virgin injection
stock females.
Three classes of flies are produced from the above cross. Depending on how the red eye
phenotype segregates one can determine the chromosome that the P-element is on. The
three classes are:
w1118/Y; SM6B/+; +/P[w+]
All males of this class have red eyes which
means P-element on 3rd chromosome.
w1118/Y; SM6B/+; MKRS/+
All males of this class have white eyes.
No insertion information is revealed.
w1118/Y; +/P[w+]; MKRS/+
All males of this class have red eyes which
means P-element on 2nd chromosome.
One uses MKRS instead of TM6B because the dominant marker on MKRS (stubble bristles) is easier to score
than the dominant marker on TM6B (humeral). MKRS is generally not used as a balancer because it does
not suppress recombination very well even though it does contain inversions.
For an exact location of where the P-element has inserted itself one can map the Pelement by chromosomal in situs. Homozygous P-element transformants which are lethal
or are sterile require the presence of a balancer for continuous propagation. Though Pelements do insert themselves randomly there are "hotspots" within the Drosophila
genome that disproportionately attract P-element insertions.
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Insertion location greatly effects the expression level of the transformed gene and
hence any resulting phenotype. Accordingly, a minimum of two independent P-element
lines must be evaluated to control for any variance in expression. Two independent lines
must also be tested to ensure that any observed phenotype is due to the introduced gene
and not to a neomorphic phenotype resulting from where the P-element inserted into the
genome.
VI. References and Figures
1. FlyBase: http://flybase.bio.indiana.edu:82/
2. The Genome of Drosophila melanogaster. Dan L. Lindsley and Georgianna G.
Zimm. Academic Press, 1992.
3. Mathers, K. The measurement of linkage in heredity, 2nd edition. Methuen, 1951.
The following are excellent sources of fly genetics and fly biology.
Fly Pushing: The theory and practice of Drosophila genetics. Ralph Greenspan. Cold
Spring Harbor Laboratory Press, 1997.
Drosophila, A laboratory handbook. Michael Ashburner. Cold Spring Harbor Press,
1989.
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