Download Nov07-BalancersFinal

Nov07, 2011
BALANCER CHROMOSOMES
If you wish to isolate a mutant allele of an essential gene (lethal mutation), you can isolate
ts alleles. On the other hand, only a small subset of mutations can give you temperature
sensitivity.
Such mutations have to be maintained in heterozygous strains with easily visible marker
nearby on chromosome. This is called balancing the mutation. The effectiveness of this
method is affected by recombination and linkage. If the mutation is recombined away from
the marker, it will be lost eventually.
If recombination happens: You will not be able to figure out whether your recessive
mutation and marker are on one chromosome or the on two homologs unless you
genotyping all of the fly progeny.

Marker
Balancer chromosomes are used in Drosophila, C.elegans and mice genetics.
A balancer chromosome allows any mutation, including lethal to be maintained in a
heterozygous form as well as to identify a lethal mutant in diploid background by isolating
a genetic contribution of one homolog that carries the mutation.
Properties of balancer chromosome
A good balancer chromosome characteristics
I.
Crossover suppression -- inversions
Must greatly reduce or eliminate crossing over in the region of the genome being
investigated. With crossover suppression, the balanced mutation will not be lost
even if the visible marker is not close by on the chromosome.
Inversion heterozygote can form loop structure upon meiosis pairing. A
crossover that occurs within the loop will produce gametes with duplication and
deletion chromosomes, including acentric and dicentric fragments.
Both acentric and dicentric
gametes will fail to complete
meiosis and produce non-viable
gametes.
The most widely used balancer
chromosomes have multiple
inversions induced by repeated
irradiation over many years.
These multiply inverted chromosomes eliminate crossing over in large regions
of the chromosome.
II.
Distinguishing marker mutations -- dominant markers
Homozygous for the balancer need to be distinguished from heterozygous,
which in turn need to be distinguishable from the homozygous non-balancer
mutants.
Some of the dominant markers used are themselves lethal when homozygous or
even inversions can be lethal when homozygous. Lethal marker can be a
completely separate locus from the inversion regions or dominant markers.
III.
Lethal mutations – enable selection
Another innovation is the insertion of gfp on the balancer chromosome. The advantage:
 You can follow a mutation by distinguishing between balancer chromosomes
 The fluorescence arising from GFP can be identified in early embryos long before
any adult visible phenotype such as curly wings can be spotted
Embryos can be sorted by fluorescence using flow
Naming Standard Balancers
Available for all of the big chromosomes (IV is very small and has no
recombination).
 First Multiple
FM1
 Second Multiple
SM5
All carry some sort of dominant marker
 Third Multiple
TM3
 Lobe—is dominant mutation
found on chII. Does not have
inversions.
Balancers:
FM1
Possible dominant markers:
Bar eye
Curly wing
SM5
Stubble Bristle Some code:
TM3
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
these mutations are on chromosome II and that they are homozygous in the absence
thatcytometer
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
NOMENCLATURE
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,
indicates the
collection of mutagenized chromosomes in a population
translocations etc) and EMS when one wants simpler mutations.
n
* - a particular mutagenized chromosome
Males segregate their chromosomes in meiosis without chiasmata formation and
* is used to indicate a chromosome that has been mutageneized and is a candidate
recombination.
chromosome
in amate
screen.
Female
fruit flies can
with more than one male and store sperm from multiple matings. This
forces the geneticists to use virgins when conducting a genetic cross. Using non-virgins results in
a symbol
I use to show
that progeny
class predicted
dies.
progeny that doJust
not have
the expected
genotypes
or phenotypes
by simple Mendellian
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.
Here is how one isolates lethal mutations?
Remember * indicates a collection of mutagenized chromosomes in a population. When a
1
particular mutagenized chromosome is isolated I have tried to designate it, usually as * , 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.
genetics.
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
1
individual * chromosome, here indicated as * ) and cross to fly with marked chromosomes.
*1/SM5
= *1/SM5 & *1/L & L/SM5& SM5/SM5
X L/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
=
NonCurly progeny show that the
progeny are Curly, the cloned
*
1
*
1
*1/*1
& *1/SM5 & SM5/SM5
not Curly
Curly
dead
chromosome is homozygous viable & lacks a lethal, but if all
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.