1

Project 1 - Fruit Fly Genetics

Drosophila, the fly that geneticists love Drosophila melanogaster, the fruit fly, is a cosmopolitan, holometabolous insect found in all

warm countries, while in cooler regions, it is established by migrants during the summer. Thomas Hunt

Morgan, who worked at Columbia University in the early 1900’s, was the first researcher to use the fruit

fly, Drosophila melanogaster, in the study of gene inheritance patterns. Through their work Morgan and

his students identified 80 separate mutants and were the first to demonstrate through experimentation,

that one gene can be isolated to one chromosome. In 1920s, Hermann Muller used X-ray to as a

mutagen to create more kinds of fly mutants. Both Morgan and Muller were awarded the Nobel Prize

for their work on fruit flies.

Table 1: Taxonomy of Drosophila melanogaster

Rank Taxon Common name

superkingdom Eukaryota eucaryotes

Kingdom Metazoa metazoans

Phylum Arthropoda

Superclass Hexapoda Insects

Class Insecta true insects

Subclass Neoptera

Infraclass Endopterygota

Order Diptera flies

Suborder Brachycera

Infraorder Muscomorpha

Superfamily Ephydroidea

Family Drosophilidae pomace flies

Genus Drosophila fruit flies

Species Drosophila melanogaster fruit fly

Drosophila life cycle

For over 100 years Drosophila is has been used as a model organism in genetic analyses. In fact

much of our current knowledge on genes, development and genetic interactions originates from work

with this system. One reason to choose Drosophila is its short life cycle.

2

At room temperature (25oC), generation time is about 10 days:

1 day embryogenesis

1 day first instar larvae

1 day second instar larvae

2-3 days third instar larvae

5 days pupal stage

Once a fly has eclosed from the pupal case a few

hours pass before they become fertile.

At 18°C development is about half as fast and

generation time is about 20 days. But keep in mind that only

healthy stocks will tolerate low temperature. At 29°C

development is accelerated and the generation time lasts

about 8 days. However, temperatures above 25°C are

generally not well tolerated by the most stocks. Temperatures

above 37°C are only tolerated for short times (up to a few

hours), a longer exposure initially causes a heat shock reaction

and finally leads to death.

Female vs. male

In order to do a cross you will need to

distinguish males from females. How to recognize a

female? How to recognize a male? The females are

on average bigger than males. However, since the

difference in size is not significant, you will have to

use further criteria. The abdomen of male bears a

round terminus exhibiting a brownish genital

apparatus on the ventral side. Females have a

pointed abdomen that displays a less noticeable

genital apparatus on the ventral side. In the male

situation the two posterior most sternits (dorsal

cuticle plates) visible from the dorsal side are

completely black, while in females only the last

visible segment is black. A very certain feature to

distinguish males from females is the sex combs.

This dark comb-like structure of the basotarsus

(the most proximal tarsal joint) is only found on the

male foreleg. With a little practice, Drosophila

males and females can be easily recognized.

3

Drosophila genetics

The Drosophila genome

Drosophila has four pairs of chromosomes: the X/Y sex chromosomes and the autosomes 2,3,

and 4. The fourth chromosome is quite tiny and rarely heard from. The size of the genome is about 165

million bases and contains and estimated 14,000 genes (by comparison, the human genome has 3,300

million bases and may have about 25,000 genes; yeast has about 5800 genes in 13.5 million base bases).

The genome was (almost) completely sequenced in 2000, and analysis of the data is now mostly

complete. Two other insect genomes - those of the mosquito and the honey bee - have now also been

sequenced, and several related Drosophila species are also sequenced. All the data is stored in FlyBase

(http://flybase.org/), which is a database of Drosophila genes and genomes.

Polytene chromosomes are the magic markers that first put Drosophila in the spotlight. As the

fly larva grows, it keeps the same number of cells, but needs to make much more gene product. The

result is that the cells get much bigger and each chromosome divides hundreds of times, but all the

strands stay attached to each other. The result is a massively thick polytene chromosome, which can

easily be seen under the microscope.

The standard map of the polytene chromosome divides the genome into 102 numbered bands

(1-20 is the X, 21-60 is the second, 61-100 the third and 101-102 the fourth); each of those is divided

into six letter bands (A-F) and those are subdivided into up to 13 numbered divisions (the picture above

shows band 57). The location of many genes is known to the resolution of a letter band, usually with a

guess to the number location (e.g. 42C7-9, 60A1-2). The polytene divisions don't have exactly the same

length of sequence in them, but on average, a letter band contains about 300kb of DNA and 15-25

genes.

Nomenclature

The first fruit fly mutant ever identified is the white (w) eyed mutant by Thomas Morgan’s

group. Normally fruit flies have red colored eyes, and the white mutant fly has white colored eyes.

Morgan named the gene associated with the white eyed phenotype as the white (w) gene. This means

that the wild type flies have w+ gene and have red colored eyes. Since then, almost all the genes in

Drosophila are named according to the mutant phenotype first discovered. For instance, the yellow (y)

mutant shows light brown colored body cuticles, whereas the wild type fly has y+ gene and show black

colored body cuticles.

The nomenclature used in Drosophila genetics 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.

4

Nine rules for Drosophila nomenclature:

a. The gene receives the mutant name/symbol.

b. Once established, gene symbols do not change. Particular alleles are specified by superscripts

(e.g. w1118). The wild type allele is indicated by a + superscript (e.g. w+).

c. Recessive mutations are written in lower case (e.g. w for white gene),

d. Dominant mutations are capitalized (e.g. Roi for rough eye).

(NOTE: The gene/mutant symbol is determined by the first mutant allele discovered)

e. Chromosomes are written in order, as follows, with a semi-colon separating each chromosome:

X/Y; 2; 3; 4

f. Genotypes are listed only when a mutation is present and are italicized.

g. Anything not listed is assumed to be wild type.

h. If a fly is homozygous for an entire chromosome, that chromosome is written only once.

i. If heterozygous then written as follows (see in-class examples):

cn bw

cn

5

Project 1 Exercises:

Exercise #1: Virtual Fly Lab Perform crosses in Virtual Fly Lab to identify the characteristics of the following mutants.

Mutation Symbol Dominant

vs Recessive

Autosomal or Sex-linked

Lethal or non-lethal when homozygous

Vestigial Wings -VG

Lobed Eyes - L

Aristapedia Antennae - AR

Sable Body - S

Bar Eyes - B

Dumpy Wings - DP

Star Eyes - ST

Follow this procedure when analyzing each of the mutants:

Step 1 - Parental Cross on VFL (Mutant Female x WT Male)

Step 2 - Reciprocal Parental Cross on VFL (Mutant Male x WT Female)

Step 3 - Initial Hypothesis

Step 4 - Draw Punnett squares and offspring proportions for Parental Crosses and revise hypothesis as needed

Step 5 - Predict Outcome of a Self Cross

Step 6 - Perform self cross on VFL to verify hypothesis

6

Example: Vestigial VG

------------------------------------------------------- Step 1 - Parental Cross on VFL (Mutant Female x WT Male)

Parents (Female: VG) x (Male: +) Offspring Phenotype Number Proportion Ratio Female: + 496 0.5216 1.090 Male: + 455 0.4784 1.000 Total 951 Step 2 - Reciprocal Parental Cross on VFL (Mutant Male x WT Female)

Parents (Female: +) x (Male: VG) Offspring Phenotype Number Proportion Ratio Female: + 516 0.5039 1.016 Male: + 508 0.4961 1.000 Total 1024 Step 3 - Initial Hypothesis

The vg mutation is recessive autosomal and non-lethal when homozygous.

Step 4 - Draw Punnett squares and offspring proportions for parental crosses and revise hypothesis as needed

Cross 1 vg vg

+ +/vg +/vg

+ +/vg +/vg

Cross 1 Offspring Proportions: Phenotypes - 100% WT Genotypes - 100% +/vg Do these match the VFL results?

7

Cross 2 + +

Vg +/vg +/vg

Vg +/vg +/vg

Step 5 - Predict Outcome of a Self Cross

Cross 3 + vg

+ +/+ +/vg

Vg +/vg vg/vg

Step 6 - Perform self cross on VFL to verify hypothesis

Parents (Female: +) x (Male: +) Offspring Phenotype Number Proportion Ratio Female: + 376 0.3661 2.848 Male: + 385 0.3749 2.917 Female: VG 132 0.1285 1.000 Male: VG 134 0.1305 1.015 Total 1027

Do these results match the predicted results in Step 5? If so...you're DONE!

Cross 2 Offspring Proportions: Phenotypes - 100% WT Genotypes - 100% +/vg Do these match the VFL results?

Cross 2 Offspring Proportions: Phenotypes - 75% WT 25% VG Genotypes - 100% +/+ 100% +/+ 100% +/+

8

Exercise #2: Mutant Observation A wild type organism is one which contains characteristics that prevail among individuals in natural conditions, as distinct from an atypical mutant type. In other words, the wild type Drosophila is the “normal” fly to which all mutant flies are compared.

In addition to the wild type flies, each station contains mutant flies with six mutant characteristics.

Carefully observe the mutant flies and determine the six mutant phenotypes:

3. Use the brush at each station to select one male and one female fly and place them side by side.

1. Carefully draw a wild type Drosophila in this box. Initials

2. Six Mutant Characteristics

#1 _____________________________________

#2 _____________________________________

#3 _____________________________________

#4 _____________________________________

#5 _____________________________________

#6 _____________________________________

Initials

Initials 3. Male on left Female on right

9

Exercise #3: Live Crosses

Cross #1: Set up the following cross and release the adults after 3 days.

Male X+/Y

Female Xw/Xw

Male Xw/Y

Female X+/Xw

Count: Count:

Initials

Draw a Punnett Square to predict the outcome of this cross:

Day ___

Day ___

Day ___ Day ___

Day ___

Day ___

10

Cross #2: You will be assigned two mutant fly stocks. Select 3-5 males from Mutant 1 and cross with 3-5

females from Mutant 2.

Mutant 1 ___________________

Mutant 2 ___________________

Collect virgin females from this cross as soon as they emerge and save for Cross #3.

Fill in the pedigree below:

Draw a Punnett Square to predict the outcome of this cross:

Initials

11

Cross #3: Use virgin females from cross #2 and cross with 3-5 wild type males. Pass flies 4 times and

release adults on day 6.

What is the genotype of the female? Male?

What possible gametes will the female produce? (Circle the recombinant gametes)

What possible gametes will the male produce?

Collect all of the male offspring from your cross and sort

them according to their phenotypes. Combine the numbers

from your entire group and fill in the table to the right. Use

results from the entire class to draw a chromosomal map of

the 5 mutants used.

Non-recombinant

Males

Recombinant

Males

Total

Males

Recombination

Frequency

Draw the pedigree for this cross below:

Draw a Punnett Square to predict the outcome of this cross:

Initials

12

Appendix 1: How to order virtual fly lab online

Students can purchase the FlyLab on-line, but they must have a credit card for the purchase. Directions:

1. Go to: http://www.biologylab.awlonline.com

2. Click on: BUY NOW (at bottom left).

3. Click on: Student Online Subscriptions (at top row of the page).

4. Scroll down to the section of “Individual Student Labs”, click on: FlyLab $7.00.

5. This will take you to the OnLine Purchase form page.

6. Follow the directions for new user.

13

Appendix 2: Cross Terminology