Project 1 - Fruit Fly Genetics - Discover Development · 2015-01-16 · Project 1 - Fruit Fly...
Transcript of Project 1 - Fruit Fly Genetics - Discover Development · 2015-01-16 · Project 1 - Fruit Fly...
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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.
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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.
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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.
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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
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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
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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?
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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% +/+
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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
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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 ___
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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
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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
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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.