Download A BIT ON DROSOPHILA GENETICS AND NOMENCLATURE

COMPLEMENTATION ANALYSIS
Complementation analysis is one of the most simple and yet extremely informative tools
in a geneticist’s arsenal. The analysis is used when you wish to find out if two separate mutations
occur in the same gene or different ones. Complementation analysis is based on the mendelian
concept of one gene - one characteristic so that two mutations which affect the same
characteristic are assumed to affect the same gene, i.e., they are allelic. Today we know that
many characteristics are determined by multiple genes so this concept does not always hold true.
In some cases, mutations in two separate genes fail to complement one another suggesting that
the products of those genes act coordinately. Despite this caveat, complementation analysis is
still a useful first approximation to establish if mutations are allelic or on separate genes.
In the example below, the two mutations in question are referred to as m1 and m2. If m1
and m2 are mutant alleles of the same gene, then the cell will lack either the product of m1/m2
gene, or express a mutated version that may be non-functional or poorly functional. This will be
manifested as a mutant phenotype, a change in a property or characteristic of the organism,
perhaps even death. In this case m1 and m2 do not complement one another and the results are
taken as evidence that m1 and m2 are the same gene. However, it is also possible that m1 and m2
are different genes and yet fail to complement one another.
NO COMPLEMENTATION
m1
B
+
m2
B+
X
A+
B+
A+
m1
m2
B
+
B+
both A alleles are mutant
normal A product is missing
mutant phenotype or death
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B+
In the example above, a fly with a mutation m1 is mated to a fly with a mutation m2.
Note that both m1 and m2 are recessive so neither parent exhibits a mutant phenotype. However,
25% of the progeny will have the genotype m1m2 and will exhibit a mutant phenotype because
they lack wild-type A gene product. What is the genotype and phenotype of the remaining 75%
of the progeny? Utilize a Punnett square to illustrate your answer.
On the other hand, if the two mutations affect different genes, then each cell will have at
least one functional copy of each gene along with the mutant version. For this reason the cell can
still produce a normal gene product and it will not manifest a mutant phenotype. In this situation
we say the mutant m1 complements mutant m2 so m1 and m2 are likely to be mutations in
different genes.
COMPLEMENTATION
B+
m1
A+
m2
X
A+
m1
A +
A+
B+
B
B +
+
m2
both A and B loci have at least one
normal allele. M1 and M2 complement
For this research project, you will be performing monohybrid crosses, that is, you will be
looking at the inheritance of a single gene. The crosses you will do are also known as F1 crosses
because you will be examining the first generation from the parental flies that where mated. The
parents are usually referred to as P or F0 generation.
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A BIT ON DROSOPHILA GENETICS AND NOMENCLATURE
Drosophila has four pairs of chromosomes: one pair of sex chromosomes, designated X
(or chromosome 1) and Y, and three pairs of autosomes, designated chromosome 2, chromosome
3 and chromosome 4. The mutations you will be analyzing are found in chromosome 3 so we
will simplify the analysis by only considering this chromosome. One chromosome from each pair
is inherited from the mother and the other chromosome from the father. The presence of two
homologous chromosomes means that each gene is represented by two alleles, one on each
chromosome. So for every gene, each fly has a maternally inherited allele and a paternally
inherited allele. The exceptions to this rule are the sex-linked genes, those found on the X and Y
chromosomes. In Drosophila, sex is determined by the ratio of X chromosomes to autosomal
sets. A ratio of 0.5 (one X to two autosomes) produces males while a ratio of 1.0 (two X to two
autosomes) produces females. The Y chromosome contains few genes and is not required for
most aspects of male development, only for proper sperm motility. Nevertheless, under most
circumstances flies will inherit the Y chromosome from their fathers only while the X
chromosome is inherited from both father and mother.
Many genes in Drosophila have been assigned names based either on the function of the
gene or, as it’s most often the case, based on the phenotype of a mutation in that gene. For
example, the first ever Drosophila mutation identified was one which affects eye color,
producing white eyes instead of the normal red. For this reason, the gene was named white
although the gene itself does not make the eye white. Instead, the product of the gene makes the
eye red; when the gene is mutated, the eye turns white because the gene product is absent or nonfunctional. To avoid confusion, the natural version of the gene (i.e., the non-mutant form or
‘wild-type’ allele) is often identified with the symbol ‘+’. We will use the symbol w+ or simply
‘+’ to refer to the wild-type allele and the symbol w to refer to the mutant allele. Below is a list of
the most common markers you will encounter throughout this investigation.
GENE
PHENOTYPE
white
Tubby
ebony
red
Serrate
Stubble
eye color white
body shape round
body color dark
eye color dark red
wing margins serrated
thorax bristles short & thick
NORMAL
ALLELE
w+ or +
Tb+ or +
e+ or +
red+ or +
Se+ or +
Sb+ or +
MUTANT ALLELE
w
Tb
e
red
Se
Sb
There are two additional things you should note from the list above. The first is that the
names of genes and their symbols are italicized. The second is that the name and symbol begins
with a lowercase letter if the mutant phenotype is recessive and with an uppercase letter if it is
dominant. For example, a fly with the genotype w / + has red eyes because the w mutation is
recessive. Flies that have white eyes are w / w. On the other hand, the Sb mutation is dominant
so Sb / + flies show the “stubble” phenotype, despite the fact that one of the alleles is normal.
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EXERCISE II
This exercise will introduce you to the fruit fly, Drosophila melanogaster, one of
geneticists’ best friend and closest ally. Here you will learn basic skills in handling Drosophila
and you will learn to identify important anatomical features and genetic markers.
Goals
(i) become familiar with the microscope
(ii) become familiar with “fly pushing” techniques
(iii) learn to distinguish the different life stages of Drosophila
• embryos
• larval instars
• pupal stages
• virgins and newly eclosed adults
(iv) learn to distinguish males from females
• differences in body size and pigmentation
• differences in genital organs
• presence of sex combs in males
• differences in shape of the abdomen
(v) learn to identify the following genetic markers
• Stubble (Sb)
• Serrate (Ser)
• ebony (e)
• red (red)
• white (w)
(vi) become familiar with the following anatomical features
• wing morphology and position
• haltere morphology
• antenna (arista) morphology
• male and female genitalia
• first, second and third leg bristle pattern
• eye pigmentation
• abdominal pigmentation
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(1) Select a fly vial from the center tray. To empty flies into the flynap container, gently tap the
fly vial on a cushioned surface (your book, notebook or a stack of paper towels) and in
one swift movement place the flynap on top of the vial as you remove the cotton plug.
Once the container is securely placed on top of the vial, invert the combo so the fly vial is
now on top and gently tap a second time so the flies fall into the nap container. Keep
tapping the vial on the table if you see flies crawling out of the container into the fly vial.
Hold the vial firmly in place for 20-30 seconds or until all flies have been anesthetized,
remove the vial and immediately cover it with the cotton plug.
(2) Transfer the sleepy flies from the container to an index card under the dissecting scope. The
flies will remain asleep for approx. 1 hr so you can work at your leisure. Adjust the
microscope height, magnification and focus to your liking and comfort. A lower
magnification is better when sorting through many flies. You will require higher
magnification when scoring for the Sb marker or checking for homeotic phenotypes.
(3) Examine the adult flies for all the characteristics listed above using a fine brush to move the
flies about. When finished, return the sleepy flies to an empty vial from the same tray
from which you took them. Make sure the empty vial is labeled with the same name as
the vial where the flies came from.
(4) Examine embryo plates, larval vials and pupal vials. Learn to distinguish all the
developmental stages. Compare to the drawings in the lab manual. Can you recognize all
life stages?
(5) Once you become familiar with the anatomical features and genetic markers, pick two vials
from the “Unknown” rack. For each vial, fill out the form below and turn in at the end of
class. For phenotype, score eye and body color, wing shape, bristle pattern, antenna and
haltere morphology.
--------------------------------------------------------------------------------------------------------------------Your name
Vial name
Sex
Vial name
sex
Phenotype
Phenotype
Abnormal anatomy?
Abnormal anatomy?
NOTES
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