Fly Genetics (fall 2012)
Pat O’Farrell ofarrell@cgl.ucsf.edu - 6-4707
Lecture 1 Overview of the fly and its role in genetics
Frame of reference:
Organisms so far in this course
Phage/E. coli/yeast/C. elegans – differ in biology, but use the same fundamental genetic
mechanisms. Nonetheless, the study of genetics in the systems differ because
1. They use different methods to obtain cross progeny where genes are exchanged.
2. Each has particular strengths for certain types of genetic studies and biological features
that allow pursuit of different questions.
3. History has lead to elaboration of organism specific genetic methods, and identified
hallmark mutations that have influenced how the organisms have been used.
Objectives
• Introduce Drosophila – organism and genome
• Polytene chromosomes and genetics - the first physical map
• A little bit of history – the first mutation, chromosomal basis of inheritance etc
• Implications of an obligate sexual lifestyle to genetics
• Balancer chromosomes and their importance
General reading:
A powerful brief essay describing Morgan, his group, and their influence on science and genetics:
http://www.columbia.edu/cu/alumni/Magazine/Morgan/morgan.html .
Drosophila genetics: The course web site has references (pdf) on.
– Drosophila Genetics Primer: basic classical
– Morgan/early discoveries: An excellent didactic presentation of how Morgan solved puzzles
– Modern tech: an excellent rev with a neurobiol slant describing new genetic techiniques
Also the following web sites are recommended:
Eric Wieschaus Nobel Lecture – excellent description of thought behind the extraordinary screen
for all of the genes that acted zygotically to control embryonic patterning.
http://nobelprize.org/nobel_prizes/medicine/laureates/1995/wieschaus-lecture.html
Anatomy and development “Atlas of Drosophila Development”
http://www.sdbonline.org/fly/atlas/00atlas.htm
And Fly movies at
http://flymove.uni-muenster.de/Homepage.html
A brief synopsis of the important early findings.
http://www.genomenewsnetwork.org/resources/timeline/1910_Morgan.php
In addition, while I don’t have the means to make it available to the class, Ralph Greenspan’s book
“Fly Pushing” from CSHL Press is an excellent practical guide to doing Drosophila genetics.
Fly – its life cycle
Attributes for Genetics
Growth - Cheap to grow and can be
crowded. Room temperature is good
and the organism is not finicky.
Fast - 10 to 14 days generation
Fecund - Hundreds of progeny per
female at a rate of up to 100 per day.
Characters – many visible features
can be easily scored for phenotype
Crosses - Easy sexing and mating
Basic life style
Specialized for rapid growth on
transient supplies of rotting fruit.
Speed – eggs hatch after 24
Growth – the larva is the growth
stage. They feed for about 3.5 days
during which they grow nearly 1,000
X. They molt twice during this growth
under influence of the steroid
hormone ecdysone – the molting
hormone. Molting divides the larval
life into three stages which are called
instars. After a short period of
wandering at the end of the feeding
stage, the larva picks a spot (usually
high and dry) and forms a pupa that is
stuck to the walls of the culture
vessel. About 4 days later, after an
extraordinary transformation, a fly
emerges – called eclosion. Males are
immediately fertile, females are fertile
in less than 2 d.
Concepts
1. Most instructive experiment in
genetics (biology) – remove a specific
gene product and determine what
does not work.
2. Onset of a mutant phenotype
depends on context. How is onset of
a mutant phenotype influenced by
development/life cycle?
A little development – genetic consequences
Embryo
Embryonic division is fast
and supported by
maternally expressed
genes whose products
supply the embryo with all
its needs for 2 h and 13 cell
cycles. Maternally RNA
and protein can contribute
to gene function well into
development. The duration
of maternal contribution is
gene specific.
Genetic consequences:
The major contribution of
maternal genes to early
embryonic development has
influenced genetic screens.
1) The onset of phenotypes
for many mutants can be
delayed because maternally
deposited gene product
supplies function until late in
development (purdurance).
For example, mutants in an
essential replication protein
(PCNA) hatch.
2) Maternal effect embryonic
lethal mutations produce
viable adults, and females lay
eggs that are fertilized but
don't hatch (look sterile). Most
genes providing essential
embryonic function are also
needed to make viable
mothers. Many genes that
readily give maternal effect
lethality are special regulators
of embryonic pattern.
3) Most of the household
functions used by the embryo are maternally supplied, and many of the genes whose expression is required
during embryogenesis (zygotic) regulate embryonic patterning. Mutations in these zygotic genes show
particularly clear phenotypes because there is no pre-existing gene product to purdure, and they tend to
have striking alterations in embryonic pattern.
Larva
Natural history - The larva is a specialized feeding/growing machine. The fly is a reproducing machine that
converts food into eggs and does not grow. Cells localized in specialized sacs called discs proliferate in the
larvae. In the pupa, the large larval cells die and their substance supports the growth and development of
the discs, which form the different parts of the adult. The adult used to be called the imago and the tissues
that produce it are still called imaginal tissues.
Many genes required for adult development are required earlier, complicating their identification. Special
techniques allow the identification of mutations affecting later processes despite and early requirement.
Fly Chromosomes – Karyotype
4 pairs of chromosomes
Sex Pair
Chromosome I = X red, carries ~ 20 % of the genes, the centromere is close to
one end (by convention the right)
Female XX Male XY red/black
Y is heterochromatic – few genes, fertility factors XO is a viable sterile male
2 Pairs of large autosomes = chromosome II and III
Big autosomes have a central centromere (below).
The equal left and right arms are called II L and II R, and III L and III R
Each arm carries ~20% of the gene of the fly
And small IV – mostly heterochromatin
In summary, most of the genes are on three chromosomes I, II and III. Furthermore, to
first approx. the genes can be considered to be evenly divided among 5 chromosomal
arms XL (or simply X), II L, II R, III L and III R. Roughly 1% of the genes are on IV.
Three types of maps of the genes
1) Recombinational map – this is classical genetic map/there are about 50
centimorgans per major chromosome arm (I am assuming you know what this is
from lectures from Hiten and Kaveh).
2) Physical map of genes along the chromosomes (polytene chromosomes/below)
3) DNA sequence map
Polytene Chromosomes
The first physical genetic map
The larvae grows predominantly by cell enlargement. During the growth the cells go
through an endocycle (or endoreduplication cycle) in which the DNA is replicated in
regulated S phases without intervening mitosis. The products of replication remain paired.
Some of the cells get very large and go through many endocycles. The salivary gland
cells go through the largest number of cycles to create chromosomes that are amplified as
much as 512 to 1024 fold. The resulting strands
of DNA/chromatin are perfectly aligned and
easily visible in the light microscope.
Whole polytene nucleus squashed to spread
out the large chromosomes. Note banding.
Note size compared to diploid mitotic
chromosomes (upper left).
Features:
1) Heterochromatin (eg. Y chromosome and
centromeric regions) is not amplified
2) The arms are connected by unamplified
centromeric regions (“chromocenter”).
3) Separately amplified homologs pair – see only
one of each chromosome arm.
4) Pairing can be incomplete – eg. When homology is interrupted by a deletion etc.
5) These are INTERPHASE chromosomes – eg transcribed, replicated and nuclear
These chromosomes played an important role in genetics – notably, providing the first
physical map of the layout of genetic traits and irrefutable proof of the chromosomal basis
of inheritance.
The bands – chromomers
Polytene chromosomes are
finely banded (picture) in a
stereotyped pattern. Calvin
Bridges (a Morgan protégé)
discovered these
chromosomes, drew detailed
images (diagram, left) of them
and invented a
naming/numbering system to
allow easy reference to any position
along the chromosomes.
#LETTER# - E.g. the Ubx gene is
at 89E1-2. The first number gives
a rough position in the genome.
There are 102 units. 20 for
each of the major chromosome arms (fig, right) and 2 for chr IV. Each
numbered unit is divided into 6 lettered divisions, and within this
bands are numbered as needed.
Conceptual versus physically real maps
Chromosomal “aberrations” such as deletions, insertions, translocation, and inversions
interrupt or disrupt the normal arrangement of genes. They are often lethal when
homozygous but viable as heterozygous. Many useful rearranged chromosomes have
been “created”. For example, are small deletions that together cover the entire genome
can be ordered from the stock center.
Deletion mapping – note genes were classically defined by mutation without physical identity
Chromosomal aberrations were useful for aligning the recombinational genetic map to the
bands on polytene chromosomes. For example, deletions will cause loss of specific
bands, and in the heterozygote the two homologs will fail to align with the normal
chromosome showing a loop out across from the deletion. The bands deleted can be
identified and the extent of the deletion indicated by giving its end points using Bridges
nomenclature For example, Df(2R)bw5, which is read as deficiency (Df) located on 2R and
removing the gene bw (brown), is located between a position 59D10-E1 (D10-E1 indicates
the accuracy the cytology could be interpreted) and a position 59E4-F1. The deletion
removes more than the brown gene but was probably named this way because the
investigator simply isolated a mutant allele of brown and later discovered it was a
deficiency. By locating the deletion on the polytene chromosomes the investigator can
now say brown lies within the “deficiency interval” as specified by the mapping. Because
deficiencies often remove several genes, the deletion will fail to complement all of those
genes. Genes that fail to complement a deficiency/deletion are said to lie under the
deficiency or to lie within the deficiency interval. At high resolution it is sometimes worth
worrying about the fact that a partial deletion of gene will give a genetic signature leading
to a statement that it lies within an interval, whereas only part of the gene is within the
region deleted. By testing a series of mutations linked to bw the investigator can
determine whether some of the flanking genes also lie within the deficiency interval.
Many associations of genetic and physical defects have allowed a pretty detailed
alignment of the recombinational map and the polytene map. However, there are many
mutations that have only been roughly mapped by recombination and never precisely
localized on a physical map.
Sequence map
Many tools can be used to align the sequence map with the
genetic map and I will not review all of these. One method that is
particular to Drosophila is hybridization to polytene chromosomes
(the example shows hybridization of a sequence of large BAC
clones of sequences near the II L telomere). Cloned genes used
to be commonly aligned with the genetic and physical map this
way. Now it is sequence based.
Importantly, detailed annotated maps can be accessed online and
the maps are linked to a wealth of data about the genes, homologs
etc. Remember such online resources are models of reality and
often the proposed transcripts are based on informatics, although
recent refinements have added data on transcripts including extensive deep seq data that
are displayed for the entire genome.
Some code:
Genotypes are written for each chromosome in the order X/Y; 2; 3; 4, but the chromosome
number is not indicated. Usually genotypes are only given for mutant alleles and assumed
to be + if not indicated, however to indicate heterozygosity at a locus a plus will be used. If
more than one mutation is present on a chromosome they are written from left to right
according to map order without punctuation (but often writers inappropriately separate
genes with commas). The sequence ;cn bw/cn bw; ; ; would indicate a fly wildtype on X, 3
and 4 and with a homozygous cn (cinnabar) and bw (brown) on II. Unfortunately for
beginners, this would usually be written as just cn bw and the reader would have to know
that these mutations are on chromosome II and that they are homozygous in the absence
of other designation. Importantly, dominant mutations begin with an upper case font. A
number of commonly used chromosomes such as balancers are designated by a special
name. (Confusing – sorry, I did not develop this.) See primer on line for more detail.
single male
multiple males
single female
virgin female
multiple virgin females
For my handout:
Just my symbol for mutagenesis. X-rays when one wants rearrangement mutations
(deletions, translocations etc) and EMS when one wants simpler mutations.
* is used to indicate a chromosome that has been mutageneized and is a candidate
chromosome in a screen.
Just a symbol I use to show that progeny class dies.
Diploidy with sexAlthough the basic principles of genetics are the same in different systems, the way you
think about genetics, use genetics and the problems one studies change. The mode of
genetic exchange has a big impact on how to think about the genetics. Phage, yeast and
C. elegans have different modes of genetic exchange that you’ve learned about.
Organisms like us, are diploid with obligate sex (no parthenogenic procreation) and
progeny are necessarily cross progeny with two sources of genetic material.
The organisms that you have dealt with so far escape the full consequence of obligate
male-female matings by having haploid stages or by self fetilization. Genetically, sex and
diploidy have great advantages in terms of moving genes around but require new
approaches to deal with the complications of following mutations in diploids.
Let’s try a couple of exercises to enhance awareness of diploid genetics with obligate
sexual exchange so that we know what the problems are that we need to deal with.
Brown eyes B is dominant over blue eyes b.
If you have Brown eyes and one of parents had blue eyes, what is your genotype? B/b
If you have brown eyes and both your parents had brown eyes, what is your genotype?
Either B/B or B/b? How would you determine your genotype? Find a blue-eyed mate and
have plenty of children. What do you look for among your children? Obviously blue eyes.
Let’s say the second kid has blue eyes – your genotype is…. B/b. If your first 2 kids are
brown eyed what is your genotype – still don’t know but it is looking like B/B. After 10
brown-eyed kids you can be pretty sure it is B/B. This is a test cross. Cross to reveal
genes masked by diploidy. Most commonly cross to recessive.
The point
Diploidy – masks traits.
Advantage: can carry lethal mutants
Disadvantage: genotype often cannot be read out directly from phenotype, often requiring
test crosses
Efficient genetics requires a solution to the problem of following genes in crosses!
A solution to the problem of following genes was discovered in flies and flies
remain unique in having exceptional tools for following genes in crosses despite the
complications of diploidy. We will look at how the solution was uncovered and
learn how to use the resulting tools.
A little history
In the early 1900’s de Vries proposed that phenotypic variants were the result of rare
changes that he called mutation, but no one had ever seen one nor was there any idea
what these were.
In 1907 Thomas Hunt Morgan began to study flies and he looked hard for a mutation. In
1910 he found the first mutant – a white-eyed fly due to a recessive mutation.
What chromosome was it on?
Inferred the chromosomal basis of heredity! Comparison of cytological observation
and segregation – first this was based only on sex linkage and X Y pattern of inheritance.
Calvin Bridges (1913) offered as “proof” of the chromosomal theory of inheritance an
explanation for exceptional progeny produced by nondisjunction. Can you infer what his
observation was?
They isolated many mutants and realized they could not all segregate independently if it
was the 4 chromosomes that segregated independently (Sutton predicted that some
mutations would segregate in a dependent way (linked) but did not predict recombination).
Cytology showed (somewhat inaccurately) that meiotic chromosomes exchanged and
Morgan deduced recombination would reduce “linkage” in proportion to the separation
of the mutations.
By 1915, eighty-five mutations had been put into the first genetic map, which had four
linkage groups corresponding to the four Drosophila chromosomes.
For interesting brief account of Morgan his group and their influence on science and
genetics see http://www.columbia.edu/cu/alumni/Magazine/Morgan/morgan.html .
Also, for an excellent didactic presentation see chapter on web site (Morgan/early
discoveries).
Picture below shows white yellow double mutant fly (two pictures on left) and a WT (right)
http://images.google.com/imgres?imgurl=http://pharyngula.org/images/white_drosophila_field.jpg&imgrefurl=http://pharyngula.or g/index/weblog/comments/white_lady/&h=300&w=400&sz=21&hl=en&start=1&um=1&tbnid=1SZJdZ6GjKKNdM:&tbnh=93&tbnw=124&prev=/ima
ges%3Fq%3Ddrosophila%2Bwhite%26svnum%3D10%26um%3D1%26hl%3Den%26client%3Dfirefox-a%26rls%3Dorg.mozilla:en-US:official%26sa%3DN
Early years  many interesting mutations
FYI-explanation: The Antennapedia gene is
usually expressed in the thorax where it
directs thoracic patterning. The mutation
causes the gene to be expressed in the
head (dominant addition of new function =
“neomorphic” allele) but it also disrupts
normal expression in thorax (recessive
lethality results).
Antp – 2 phenotypes
Dominant: legs develop in place of
antennae
Recessive: lethal
Keeping the mutant
Evolution of a technology
The tedious solution – periodically select out and discard the +/+ flies
After years of tedium +/+ almost stopped appearing in one stock.
Sturtevant (student of Morgan’s) asked why?
A lethal mutation had appeared on the other
chromosome and as result only the
heterozygote was viable.
#1. Balanced lethals
Occasionally recombination restores a wild type chromosome
That would again take over

!
lethal
+

Inversions suppress recombination
!

Invert segment
!
lethal +

!
lethal
No viable recombinant – get one dicentric and
one acentric chromosome if recombination
occurs (diagram below, left)
Balanced lethals are stable when an inversion prevents recombination.
#2. Multiple inversions on a chromosome
block recombination with a normal homolog
Modern Balancer Chromosomes
The commonly used balancer chromosomes have been refined over the years.
They have three things
1. A lethal mutation * (Balanced lethals principle)
2. Multiple inversions to prevent recombination at any point in the chromosome
3. A Dominant mutation with an easily visible phenotype.
* The behavior of the X-chromosome (first) Balancers is different than I described.
The first
chromosome Balancers do not carry a lethal (except for FM3 that has special applications) because X
chromosomes with a lethal cannot be carried in male. These chromosomes carry a recessive mutation
that makes the females (specifically) sterile so that the viable fertile flies in balanced X linked lethal
situation would be females that are for example Xlethal/FM1 and males that are FM1.
Naming Standard Balancers
Available for all of the big chromosomes (IV is very small and has no
recombination).
•
First Multiple + #
eg. FM1
Bar eye marker
Second Multiple + #
eg. SM5
Curly wing marker
Third Multiple + #
eg. TM3
Stubble bristle marker
Now Keeping a Mutant is Simple
Example:
Now the stock is stable: the surviving progeny = parents generation after generation.
Keeping track of chromosomes without visible markers
Mendel’s laws tell us that whenever the progeny do not get one chromosome from a parent they
must get the other one (simple fact that is easy to forget – important for balancer use). Thus,
when you cross a lethal/Balancer (e.g. lethal/SM5), the progeny that do not get the balancer (e.g.
the progeny without Curly wings) must have received the lethal chromosome. NO TEST
CROSSES ARE NEEDED!
Balancer use can get quite fancy, but it really involves no more than knowing Mendelian segregation
and being able to follow the markers.
An important peculiarity of Drosophila that also can help you keep track of genes even without a balancer.
No Recombination in Males: Useful peculiarity of flies: There is no recombination during male
meiosis. Since there is no recombination there are no chiasmata to hold the meiotic chromosomes
together. Consequently, another pairing mechanism is required and does exist (but we will not
discuss this). The important thing here is that alleles of various genes on the same chromosome
will behave as if they are 100% linked in male – that is there is no shuffling of maternal and
paternal markers in males.
If we have time we will work through the next two pages in class. If not you should work through this material.
If you do you should be able to figure out your popsicle stick problem.
The Importance of Lethal Mutations
Any genetic element that is especially important is likely to be essential. Mutation of essential
sequences is likely to be lethal. Lethal mutations are among the most interesting candidates for
genetic analysis because they target important functions. In most systems they are hard to isolate
in a forward mutagenesis screen – i.e. it is hard to isolate something dead.
What’s wrong with ts lethal mutations? The production of ts allele requires a peculiar allele, and
many yeast genes (~75%) are not mutable to temperature sensitivity at a practical frequency ^.
Here is how one isolates lethal mutations?
Remember * indicates a collection of mutagenized chromosomes in a population. When a
particular mutagenized chromosome is isolated I have tried to designate it, usually as *1, but many
independently mutagenized chromosomes (different) are in the original pool *. The first letter in the
names of dominant mutations are UC. Here L designates the Lobe mutation. It is dominant visible
(eye shape defect) that is recessive lethal. And remember the SM5 balancer is marked with Cy.
1. Mutagenize +/+
and cross to
L/SM5
= */L & */SM5
2. Pick one male progeny, here I assume you pick one with Cy (since you pick one fly for step 2,
you are isolating and effectively “cloning an individual * chromosome”, here indicated as *1) and
cross to fly with marked chromosomes.
*1/SM5
X L/SM5
= *1/SM5 & *1/L & L/SM5 & SM5/SM5
dead
1
3. Pick males and females carrying the cloned candidate chromosome (* ) and a Balancer
chromosome (e.g. any Cy fly that is not L) and cross these (this is a self cross).
*1/SM5
X *1/SM5
=
*1/*1
& *1/SM5 & SM5/SM5
not Curly
Curly
dead
NonCurly progeny show that the *1 chromosome is homozygous viable & lacks a lethal, but if all
progeny are Curly, the cloned *1 chromosome is homozygous lethal (presumes enough progeny
that absence is statistically significant).
If you do steps 2 and 3 many times (a separate vial for each) one can clone many * chromosomes
and if you discard all vials that yield straight winged flies, you will be left with a bunch of vials each
containing a particular * chromosome over a balancer and each of these retained
chromosomes will carry an isolated lethal mutation.
*
Note that you mutagenized whole flies, but you specifically collect lethals on the second
chromosome. Other lethals will be present in the mutagenized population, but these will not be
cloned and made homozygous and they will soon vanish because they are not balanced. Here, we
have focused on chromosome II. Drosophila geneticists use this strategy to “mutagenize one
chromosome at a time”, but of course they are not selectively mutagenizing one chromosome at a
time, but isolated the mutants one chromosome at a time.
^ Harris et al., (1992) Molecular analysis of Saccharomyces cerevisiae chromsome I. On the number of genes and the
identification of essential genes using temperature=sensitive-lethal mutations. J. Mol. Biol. 225, 53-65.
Doing a screen
Mutagenesis technique: Males are mutagenized because they so fecund that even high doses of
mutagen leave adequate fertility. While treatment of sperm can induce DNA damage that is ultimately
mutagenic, the damage can persist into the egg and mutations only “fixed” (as in stabilized) in some of the
cells of the embryo. To avoid (reduce) such mosaic progeny time is allowed after mutagen treatment to
allow damage in sperm progenitor cells to be fixed as mutations and to make their way into mature sperm. If
you wait too long, many sperm may develop from a few mutant stem cells and one ends up with a “jackpot”
in which the same mutant is isolated many times (a bad thing).
Allele frequency and mutagen dose: The choice of level of mutation is a matter of balancing practical
issues. Too high a dose and you kill everything. Too low a dose and you have to work very hard to get your
mutants. But a higher mutation frequency is not always better, because you then have multiple mutations in
each progeny and even each chromosome of the progeny. Then you have to work hard to resolve the lesion
that is responsible for the phenotype from the background. The allele frequency is the frequency at which
you get new alleles of a given gene (or in other words the frequency with which you hit a gene). In flies one
often assess the allele frequency by scoring for white eyes in the progeny (F2 males or F1 females after
mating with w/w females). A good EMS mutagenesis will give an allele frequency of about 1/1000.
Why would you ignore the males with white eyes in this assay?
A screen for mutations on chromosome II (the same as cloning many mutagenized
chromosomes – previous page)
Simply scoring for the absence of straight winged flies identifies flies without a Balancer.
These are necessarily homozygous for the “cloned” mutagenized chromosome (the only nonBalancer chromosome in the last cross). Lack of such straight winged flies indicates the presence
of a lethal (as above).
You can score for viable phenotypes in the straight winged flies – for example an eye color
mutant like cinnabar would show up. Indeed any recessive mutation giving a scorable defect could
be isolated.
When there is a specific lethal phenotype is of interest (e.g. larvae that arrest before
pupariation with an enlarged red ring gland – just a strange one that we have a current interest in),
you can screen for it directly and establish a stock from the surviving heterozygotes.
Popsicle stick Problem #1
a. You are interested in studying the organization of epithelia and have a mutation called multiple
wing hair (mwh) that disturbs the ordinarily very organized arrangement of the cell layer that forms
the surface of the wing. Each cell usually makes one tiny hair – the mutant makes more, usually
three. The mutant is healthy and fertile.
a) You want to make more alleles of this gene (new mutations of the same gene). How do
you do it? **
b) You want to do the above in a way that recovers the new mutant so that you can make a
stock of this mutant. What is the complication? How do you deal with it?
c) You have another mutation in a different gene that severely disrupts the organization of the
epithilium, l(epi). It is lethal. How would you isolate alleles of l(epi)? (note that this much
harder)##
** Starting Info — You have a protocol to EMS mutagenize males so that mutations arise in the
sperm. Usual frequency of mutations is such that a given gene will be mutant once per 1,000
progeny (allele frequency). The mwh gene is on the third chromosome. You have available many
other common mutations with different phenotypes – recessive viable mutants with visible
phenotypes, recessive lethals, mutations with Dominant visible phenotypes that are lethal when
homozygous. You know the map positions of all available mutants (you can make up names for
them). You have a stock carrying a third chromosome balancer marked with Sn (Singed).
Expectation – everyone should get an answer to a) even if they didn’t listen to me. I hope that you
all can come up with some sort of answer to b) and maybe it will be an easy and efficient one. c)
will be the test of whether you can work with the new material presented in lecture. If you have
trouble consult the last two pages of the handout.