Identifying the recessive mutations on Drosophila melanogaster by malj

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									         Identifying the recessive mutations on Drosophila melanogaster



               rosophila melanogaster and other Drosophila species have been an important
               model organisms ever since they were first introduced into laboratory settings in
               1900 by William Castle at Harvard University who needed a small, fast-
               producing organism for embryological studies. However, it was not until 1909
that the fruit fly made its first debut in genetics. A spontaneous change in eye color from brick
red wild type to white caught the eye of Columbia University professor Thomas Hunt Morgan
who decided to test whether this small-scale mutation would follow the hereditary patterns
predicted by Gregor Mendel. After mating the white-eyed male (w) with a wild type female
(w+), he discovered that the offspring ratios followed the predicted patterns, and as a result, the
life of Drosophila was changed forever.

Throughout the 20th century, Drosophila has been indispensable from discovering the basic
principles of genetics to behavioral and developmental studies. Through the countless
spontaneous and later deliberately introduced mutations, it has helped us to understand how
genes are linked and use this to determine the order and relative distances between the genes,
understand how homeotic genes control the development of the embryo, discover the genes
that control the long-term memory acquisition, alcoholism and circadian rhythms, understand
factors that contribute to aging, help to define the concept of genetic species and speciation,
and make countless other discoveries.

However, for 21st century genetics student, Drosophila melanogaster (literally, "the black-
bellied dew-lover") remains a constant source of trouble by requiring tedious schedule and a
sharp eye to identify the necessary phenotypic traits. Although the easily identifiable
phenotypic traits has been one of the reasons Drosophila has been such a popular study object
for over 100 years, for someone not familiar with Drosophila these traits might not seem as
obvious. The following little guideline is meant to help the novice geneticist to get better
accustomed with few of the common traits of the organism and to be part of the enormous
legacy that has shaped the principles of biology.

1. Telling the males from females:

There are several characteristics that can be used to tell the male flies from females. First of all,
females are generally larger than the males, although this characteristic is not necessarily
always reliable. Observing the flies from the dorsal view (from the top), the males will have
rounded abdomen with dark pigmented tip, while the abdomen of the females has clearly
banded segments all the way to the tip, which is also more pointed (figure 1).

For more reliable sexing, look at the genitals on the ventral side (underside) of the abdomen.
The males have a well-pronounced dark genital arch, surrounded by heavy dark bristles, while
females have more flattened genitalia without much pigment (figures 2 and 3).

             ♂♀

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Figure 1:                                                   Sexing
Drosophila: dorsal view.


            ♂                                               ♀




Figure 2: Sexing Drosophila: ventral view.


           ♂                                                ♀




Figure 3: Sexing Drosophila: genitalia details.




2. Eye pigments: brick red, bright red, brown, and white:


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Drosophila eye color is a multigenic trait - there are approximately 100 genes that control the
pigment synthesis, transport and expression. The wild type brick red color is a result of mixing
two pigments, red and brown, each of which a produced in multiple biosynthetic steps, and
then transported to the pigment cells of ommatidia (the unit eyes that make up the compound
eyes). Mutation in any of the enzymes that catalyze the pigment synthesis will lead to a
defective pathway where one or both of the pigments are not produced. This results in either
bright red, brown, or white eyes. Mutation in the transport pathway will block the transport of
synthesized pigments into the pigment cells, resulting also in white eyes (figure 4).


                               brown pathway (ommochromes)

             tryptophane

                                                                         brick red pigment


                  guanine        red pathway (pteridines)

                       transport pathway

Figure 4: Fly eye color is a result of multiple biosynthetic steps to produce the pigments as
well as enzymes that transport these pigments to the ommatidia.

Looking at the flies individually, it is often hard to say whether the flies have brick red, bright
red or brown eyes, especially since the eye pigment darkens with the age and in some cases,
the fly will have darker areas in the middle of the eye. The best way to distinguish between the
colors is to have a 'comparison fly'. Simply anesthetize a wild type (OR) fly, and compare that
color to your mutants. When placed side to side, the difference between brick red, bright red
and brown are quite clear (figure 5).




Figure 5: Eye colors clockwise from top left: brick red, bright red, white, and brown.

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3. Body color: yellow vs. gray/brown:

Wild type Drosophila body color is influenced by three different pigments - yellow, brown and
black. The yellow gene (y), located on the X-chromosome, blocks the synthesis of brown
pigment, resulting in flies with yellow bodies. Newly hatched yellow-bodied flies have hardly
any pigment at all which may cause confusion when trying to sex the flies. In this case, the best
strategy is to let the young fly mature a few hours in a separate vial after which the black
pigment will be expressed in the segments and genitalia. If in doubt whether the flies are
yellow or gray/brown bodied, look at the wings. If the wings are yellow, the fly is also yellow -
and if the wings are gray, the fly has wild-type body color (figure 6 a and b).


     A
B




          Figure 6: A: yellow-bodied fly (y); B: wild-type fly (+)



4. Wings: normal, crossveinless, cut:

Normally, the wings of a fruit fly have five longitudinal veins and two crossveins as well as
well established wing margins. Several developmental mutations can alter these characteristics;
for example crossveinless wings (cv), alas, lack the crossveins, and cut wings (ct) lack wing
tissue from the rear wing margin. These traits are generally easy to score, however, the cut
wings can also result from rough treatment with the brush under the microscope, and the
crossveinless mutants can sometimes still possess the upper, smaller crossvein. Therefore,
unless the fly has two well-established crossveins, you should score it as cv, and treat your flies
gently to avoid ‘false positive’ cut wings (see figures 7 and 8).

               A                              B                              C




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Figure 7: A: Crossveinless wing (cv); B: Normal wing (+); C: Cut wing (ct
Figure 8: From the left: crossveinless wings, normal wings, cut wings.


5. Bristles: normal vs. forked:

Bristles on Drosophila are not analogous to animal hair (and there are too few to really keep
them warm), but serve the purpose of sensory organs, hosting the sensory nerve endings. The
bristles are composed of bundles of actin filaments, and the pattern at which they are arranged
and bound to each other determine whether the bristles look long, slender and slightly curved
(wild type), or small and crooked (several mutants, including forked, f). The forked mutant
blocks the bundle formation in early development, resulting in small, twisted bristles with split
ends. Although it is a relatively clearly identifiable phenotypic trait, the bristles are small and
can be hard to see at lower magnifications, so at least 20x magnification is necessary. Look for
the bristles in the dorsal and lateral surfaces of the thorax, and keep in mind that even one
forked bristle means that the fly has the defective f gene (figure 9).




       A.                                                     B.                     C.


Figure 9: A: forked bristles (f) on the back of the thorax; B: forked bristles (f); C: normal
bristles (+).




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Note for students: This is written to help you differentiate and understand the recessive
mutations on Drosophila melanogaster for lab exercises 1 and 2. Please do not cite this as a
reference for your lab report. If you need the original references, please check out the list
below.

Story: Anni Moore
Photographs: Tyler White.




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