Fly Genetics (fall 2011)
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 laval
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. For example,
mutants in an essential
replication protein (PCNA)
hatch.
2) Maternal effect embryonic
lethal mutations. When the
mother is homozygous mutant
in a gene that is essential for
the early development of the
embryo but not for the adult,
she will lay eggs that are
fertilized, but don't hatch
(looks sterile). Looking at the
eggs of ‘sterile’ mutants has
been very productive.
3) While most of the
household functions used by
the embryo are provided by maternally supplied gene products, new (zygotic) expression of regulatory genes
at very particular times and places underlies development of embryonic patterning. Thus, identification of
genes required zygotically for embryogenesis has been a treasure trove of developmental regulators.
Larva
Natural history - The larva is a specialized feeding/growing machine that acts as an incubator for
the production of a fly. The fly is a reproducing machine that converts food into eggs and does not
grow.
The larva grows by increase in cell size not number while small cells localized in specialized sacs
called discs proliferate. In the pupa, the large larval cells give up their lives and their substance to
support 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.
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.
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.
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
The polytene chromosomes
are finely banded in a
stereotyped pattern. Calvin
Bridges (a Morgan’s protégé)
discovered these
chromosomes, drew detailed
images 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) 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, there are small deletions that together cover the entire
genome.
Deletion mapping
These chromosomal aberrations are 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 the 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 that can be accessed on line
and the maps are linked to wealth of data about the genes,
homologs etc. However, a warning is in order. All such maps
should be viewed as work in progress. Often the proposed transcripts are based only on
an informatics search for reasonable promoters and start sites, and even when there is
sound evidence that a particular transcript exists, there might be other transcripts
expressed in other tissues etc.
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 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 85 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).
And here is a site with nice pictures and tribute to Morgan and his white fly.
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
In Class Problem #1
a. You are interested in studying the organization of epithelia and have 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.
You want to isolate more alleles of this genes. How do you do it?
Starting info – 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). Isolate new mutations of the same gene.
Much harderb. You have another mutation in different gene that severely disrupts the organization of the
epithilium, l(epi). It is lethal.
How would you isolate alleles of l(epi)?
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
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.
Lobe is a dominant visible on II. Eye is kidney shaped. The chromosome carrying Lobe
does not have multiple inversions – i.e. recombination can occur in female).
e.g.
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
Inversions suppress 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. Inversions to prevent recombination
3. An 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.
In Class Problem #1 (with some notes)
a. You are interested in studying the organization of epithelia and have 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.
You want to isolate more alleles of this genes. How do you do it?
Starting info – 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). Isolate new mutations of the same gene.
Much harderb. You have another mutation in different gene that severely disrupts the organization of the
epithilium, l(epi). It is lethal.
How would you isolate alleles of l(epi)?
a. Mutagenize males (shoot for an allele frequency of 1/1000). Cross to homozygous mwh flies.
Screen for progeny with Mwh. About 1/1000 progeny should carry a new mutation in the gene and
show the phenotype, while all others will be heterozygous and show no mutant phenotype.
— important subtlety: You will not be able to isolate the new mutation if you do the cross as
above, because the fly you identify with the phenotype will have one chromosome with the old
mutation and one with the new and won’t know which is which. How, can you follow the
chromosomes so that you can isolate the new mwh allele?
b. Unless you’ve already learned some of things I am planning to teach you, the answer to this is
not likely to be apparent. What you should realize is that:
– You can have flies that carry the lethal mutation, if the lethal is rescued by a wild-type allele and
usually in diploid genetics this means the mutation is heterozygous. So you have l(epi)/+ flies.
– Also, if you mutagenize males and cross to wild-type females, about 1/1000 progeny will have
new allele of l(epi). But how can you find it?
We will solve the same problem in class as problem 2, but try and solve this one at home and I will
hand out a solution at next class.
In Class Problem 2 + notes: (Taken from real life – see Regulski et al., 2004 and Yakubovich et al., 2010)
Nitric oxide (NO) is a very simple but reactive molecule that has been ascribed numerous biological roles
(the literature on NO is larger than the literature on yeast). An enzyme that makes NO, called nitric oxide
synthase or NOS, is conserved from bacteria to human. You (Regulski) wanted to isolate a mutation in the
fly enzyme, whose coding sequence was readily recognizable in the genome sequence at 69F (toward the
middle of the right arm of chromosome II).
You have at your disposal a deletion (Df (69F)) of a little more than 20 kb of sequence that removes the NOS
promoter region and 3 N-terminal exons, as well as five other upstream genes (deleted in whole or in part).
At least one of the upstream genes is essential (there are lethal alleles), so this deletion is necessarily also
lethal.
If you assume (as Regulski did) that a NOS mutation would be lethal, how might you isolate a mutant.
Step 1
Mutagenize males. (en mass)
Step 2
Cross mutagenized males with second chromosome balancer flies, CyO KrGFP, (en mass) and collect
progeny that have mutagenized II over balancer (individuals). Choose males (more fecund and easier in the
next cross).
Step 3
In vials – cross individual males that are */CyO KrGFP with females that are Df(69F)/CyO and score the vials
2 weeks later to determine whether there are straight winged progeny (these are presumed to have good
copies of all essential genes within Df(69F) and these must have come from the male). Straight winged flies
shows that no lethal has been generated in the region of the deletion and the flies/vial are discarded (there
were 4823 of these), while the absence of straight winged flies suggests that the combination of */Df(69F) is
lethal. 32 vials of this type where found.
Step 4 Set up stocks of candidate mutants. Collect non GFP flies that are Curly– these have * chromosome
opposite CyO. Either cross with virgins of same or cross to Sp/Cyo and then cross non Sp, Cy progeny inter
se.
Step 5 – Place lethals into complementation groups – you should be able to describe how this would be
done.
Table S1. Screen summary.
Total lines screened
Lethal hits within Df(2L)69F
Lethal Complementation Groups within Df(2L)69F:
Group 1
Mitochondrial Porin
Group 2
Group 3
Group 4
Group 5
4855
32
3 alleles
17 alleles
6 alleles
2 alleles
4 alleles
We screened 4855 lines and identified 32 mutations, which were lethal over the Df(2L)69F deficiency. Inter se complementation analysis split them into 5
groups. Group 1 failed to complement the l(2)k08405 P-element insertion and, thus, represents 3 new alleles of the mitochondrial porin gene. Group 2, the
largest with 17 alleles, corresponds to dNOS (see below). Inter se complementation between all groups indicated a genetic interaction between Groups 2
and 3. Some heteroallelic combinations result in sterile or almost sterile flies, suggesting that mutations in Groups 2 and 3 may affect the same gene.
Groups 4 and 5 do not show any genetic interactions.
Step 6 Assay (in their case one mutant) for NOS activity. Find a deficient allele and sequence to show a
base change in conserved residue of NOS. They expressed the mutant protein in bacteria and showed that
it lacked NOS activity while a wild-type sequence produced NOS activity.
Is this enough prove that they have isolated NOS mutants?
Exercise – I do not intend to cover the next three pages of notes in class, and I would like you to work through
these notes on your own before the next class. Hopefully you will understand them fully, but if not please bring
questions to the next class which will begin by addressing any questions on this material. Please feel free to
consult with each other, and the TA's should try their hand at it.
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. As in the notes but I
think omitted from the first lecture, 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.
1. Mutagenize +/+
and cross to
L/SM5
= */L & */SM5
2. Pick one male fly (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
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 many progeny).
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
time, but isolating the mutants one 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 derived from a few mutant stem cells and one ends up with “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 and 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 – two pages previous)
Simply scoring for the absence of straight winged flies isolates any lethal as covered 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 and enlarged red ring gland – just a strange one that we have current interest in),
you can screen for it directly and establish a stock from the surviving heterozygotes.