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Thursday, October 16, 2008

Balancer Chromosomes

Monday's Molecule #92 was a depiction of an inversion in a Drosophila chromosome (right).

The chromosomes shown here are the large polytene chromosomes of the salivary glands. They are made up of 1000-2000 aligned stands of DNA that form when successive rounds of DNA replication are not followed by separation of cell division. Flies that are heterozygous for a wild type chromosome and one with a large deletion inversion, will form the loop structure shown in the diagram.

In normal cells, you won't see this structure as the chromosomes align during mitosis and meiosis, but it still exists. What the structure tells us is that the presence of an inversion, or any other type of chromosomal rearrangement for that matter, doesn't have much effect on chromosomal alignment and segregation during cell division.

Today we want to focus on another point. Imagine that a recombination event (crossover) occurs when the chromosomes are aligned like this. If the crossover takes place in the inverted region then each of the recombined chromosomes will be missing some genes and the cells that are produced from such an event will die.

Imagine that the crossover occurs between point C and D. If we trace the new chromosome staring from A on the black chromosome (the AR chromosome) then you get A B G F E D on the black chromosome followed by C B A on the normal white chromosome. The other product of the crossover will begin with A B C from the normal white chromosome and end with D E F G B A from the black homologue.

There won't be any viable crossovers in the region covered by the mutation. We will see what this has to do with balancer chromosomes in a minute.

Imagine that you are working with an important mutation (x) that affects embryonic development in Drosophila. Flies that are homozygous for the mutation (x/x) are blocked at a particular stage of development and the visible phenotype of the mutations tells you a great deal about the genes that control development. These mutations are recessive lethals. The heterozygous flies with one mutant chrmosomes and one normal chromosome (x/+) are viable.

You want to maintain a stock of these flies so you can have mutant flies whenever you need to do an experiment. If you put a heterozygous male and female together in a fly bottle and leave them for a few weeks, you won't be surprised to find that there are no flies that are homozygous for the lethal mutation. However, repeated crossings of heterozygotes will result in 25% wild-type flies (+/+) and these flies will continue to mate with each other and with the heterozygous flies. You won't be able to tell which flies carry your valuable mutation.

One way around this is to mark your mutant chromosomes with a visible marker. Let's say that your mutation is on chromosome 2. (There are three autosomes and one pair of sex chromosomes in Drosophila.) You will need a dominant marker for reasons that will soon become apparent so let's choose detached (Dt), a mutation that effects the vein pattern on the wings. The chromosome carrying your valuable developmental mutation (x) will also carry the Dt allele.

Now all you have to do is look in your stock bottle for flies with the detached phenotype and you know that those flies should be heterozygous for x (Dt x/+ +). Flies that have normal wings can be recognized and killed. (Drosophila genetics is the ultimate blood sport.)

Problem solved, right?

No. It won't be long before a recombination event separates Dt and x, especially if they are far apart on chromosome 2. After a while you still won't know which flies carry your valuable mutation.

This is where balancers come in handy.

Let's look at the experiment done by Christiane Nüsslein-Volhard and Eric Wieschaus in the late 1970's. This is the experiment that won them the Nobel Prize.

In one of their experiments they were looking for mutations on chromosome 2 that affected embryo development.

They started with a line of flies that had eye color markers on chromosome 2; cinnabar (cn) and (bw). This is the red chromosome in the diagram. They treated males from this line with the potent mutagen ethyl methanesulfonate (EMS) and crossed them to a strain carrying a balancer chromosome (black) and a non-balancer homologous chromosome (blue).

The balancer chromosome is called CyO and it has several interesting features. First, it has a large inversion called In(2LR)O that covers most of the chromosome leaving only the ends intact. Second, it carries a dominant mutation called Curly (Cu) that produces flies with curly wings. This is a homozygous lethal mutation. Third, it carries another homozygous lethal mutation called dumpy-lethal (dp1vI). Finally it carries cn. (Also purple (pr).)

The blue chromosome carries an allele called DTS-91. DTS stands for dominant temperature sensitive. Flies that carry even a single copy of this alleles will die at high temperature. DTS also stand for David T. Suzuki, the man who created these alleles but that's just a coincidence.

The first cross is done at high temperature. The flies produced from the first cross are the F1 generation. None of them will carry the blue chromosome because those flies will be killed at high temperature. All of them will carry one mutagenized chromosome 2 and the CyO balancer chromosome. These flies are crossed again with DTS-91/CyO flies at high temperature to get the second generation (F2) of flies that all contain one mutagenized chromosome 2 and CyO. The second cross helps eliminate other mutagenized chromsomes so that the workers will only be looking at mutations that affect chromosome 2.

Now the cn x bw/CyO flies are allowed to mate with each other until it's time to look at the effect of the mutations. The stock will never produce homozygous cn bw flies as long as the mutated chromosome carries a recessive lethal. Stocks that have flies with cinnibar eyes and not curly wings are discarded.

The stock will never produce flies that are homozygous for the balancer chromosome since it carries two recessive lethal mutations. All flies will have curly wings because they carry the balancer chromosome with Cu. There will never be a recombination event that transfers the developmental mutation to the balancer because the balancer contains a large deletion inversion.

This is why balancer chromosomes are so important n Drosophila genetics. They are essential for maintaining fly stocks carrying homozygous lethal mutations. Such mutations have been extremely important in sorting out fly development.

Christiane Nüsslein-Volhard and Eric Wieschaus created thousands of lines carrying recessive lethal mutations on chromosome 2, and thousands on the X chromosome and chromosome 3, each of which have their own balancers. Then they examined each line to look for embryos that were blocked during early development. (25% of the eggs will be homozygous for the mutant chromosome.)

[Lower Figure credit: St Johnston (2002)]

St. Johnston, D. (2002) THE ART AND DESIGN OF


Anonymous said...

awesome, crystal clear explanation! Thanx :)

Anonymous said...

I ve a small clarification. Wont a few of the F1 flies have one normal (without mutation) chromosome 2 and the balancer chromosome? Why aren't they considered?

Unknown said...

Thanks, I understand now.

Unknown said...

About the previous question "Wont a few of the F1 flies have one normal (without mutation) chromosome 2 and the balancer chromosome? Why aren't they considered?":
I think the answer lies in the sentance "Stocks that have flies with cinnibar eyes and not curly wings are discarded." If we chose the F1 with one normal chromosome 2 and a balancer chromosome to produce F2, we will get alive flies with cinnibar eyes and not curly wings", and we could discard this stock.

Anonymous said...

A word "deletion" in the first paragraph should be changed to "inversion" to match the diagram.

patty said...


pls check the sequence after crossing over

The gene sequence on another recombinant chromosome should read H G F E D from the white chromosome followed by CH from the dark chromosome.

Anonymous said...

Dear Professor Larry Moran:
My name is Rami Al-Jiab, I am a honors student at Massey University. I have been doing some Microinjections and some crossings in which fly (Drosophila melanogaster) strain used contain the TM3Sb balancer chromosome. Upon observation it seems that TM3Sb containing flies are less viable than their conterparts. Is this true, and if so, does this effect both sexes equally, and if not, which sex does it most effect, because i am also assessing male viability in these lines, so I need to have some control for this issue. Thank you for your efforts and helpful site.

KidAcid said...

Also in the last but two paragraph the "deletion" has to be changed to inversion. Anyways thanks for the really good explanation.