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Let us discuss the mutants that were isolated from the screen. We found genes that are
lineage defective or lin, and one such is lin-26, it is the 26th gene in the order in which it was
identified. But this does reveal its role in vulval development. So to understand its function,
firstly, we need to identify the chromosomal location of the gene. So that process is called a
genetic mapping, so mapping a given mutant. So, now let us focus on how that is done.
(Refer Slide Time: 03:37)

There are multiple ways of mapping; I will go through an the most common way that is done in
standard genetics.
(Refer Slide Time: 03:53)

So, first, we need to find out on which chromosome that particular mutant allele is located. So,
for that, we can take a known mutant allele of a gene; whose chromosomal location is already
known. So, in our particular example, since we have six chromosomes, we take six different
markers, each one well known for its position on let us say, chromosome 1, 2, 3, 4, 5, and X.
Now using Morgan's experiment, we can try to see the recombination frequency between the
new mutant allele and the known mutant allele. So based on the recombination frequency, if they
are independently assorting, then they are not on the same chromosome. If they show linkage,
Let us say lin-26 shows tight linkage to a gene located on chromosome 1 and independently
assorts with the rest of the five, then you know lin-26 is probably on chromosome 1. So this is
called two-factor analysis, where you find the recombination frequency between the new mutant
and the known marker mutation. It is the same as what we had already discussed when we
learned about Morgan's experiment with the black and vestigial. Now we know that the
chromosomes have millions of base pairs. C. elegans has about 100 million base pairs divided
into six chromosomes; each one of them is having more than 10 million base pairs. An average
gene is under 2 kb, so you need to know out of the 10 million bases which 2 kb is your gene. So,
there you should try to narrow down within the chromosome to a particular genetic interval,
meaning you can select two known positions. The distance between the two positions is called as
the genetic interval. So, for that, the three-factor analysis should be done. This is a very useful
analysis; it gives a very specific position for your gene at the end of the three-factor analysis. It is
the same as the two-factor analysis except that you are determining the recombination frequency
between your new mutant allele and two different known markers located on the same
chromosome at known intervals.
Now let us take Morgan's example, here we determined the recombination frequency of black
and vestigial is 17%. Now, this 17% can be on either side of black. One percentage is
considered as one centimorgan in memory of Morgan. So recombination frequency does not tell
the side of a given reference point. Now let us take cn, the recombination frequency of black is
9%. Now, this 9% could be on the left or right of cn, and the recombination frequency of
vestigial is 9.5%. This tells that cn is between the two, and vestigial is on the right of black.
So, you give a specific position for the known new mutation, and here we are assuming black
and vestigial as known markers, and the cn is the unknown. So this is called three-factor
analysis. So, the three-factor analysis gives a very specific position, but the resolution and the
accuracy of your conclusion depend on the number of progeny you observed. So higher the
number higher the accuracy. So, once that is done, then we need to do a giant leap that is earlier,
they had to continue this.
Like what do you do is first, you take markers that are far apart and therefore, your allele is
somewhere in between; then you select newer known markers that are closer, by repeating it then
you narrow down to a small region. So, that is very tedious, and that is why gene mapping took a
few years to map a gene. But nowadays, we do it in a month or so if you are entirely on it.
(Refer Slide Time: 10:32)

So, before we go into this, I need to introduce what is called a genetic map and a physical map.
So, a genetic map is purely an imaginary map based on recombination frequency. There is no
physical basis for it whereas the actual DNA sequence is called the physical map.
So historically, people developed methodologies to determine the C. elegans genome sequence.
And one of the methods was breaking down the chromosome into smaller pieces and cloning
them in vector. It is the same concept as the usual plasmid except that in the plasmid, you are
having few 100 base pairs to a few 1000's of bases. So, the biggest insert you might clone is
probably about 10 KB to 12 KB size. But there are vectors called bacterial artificial
chromosomes, yeast artificial chromosomes, and then the cosmids. So these are large vectors,
and large size insert can be cloned in these vectors. For example, you can have a 100 KB range
in yeast artificial chromosomes or BAC's; in cosmids, usually about 40 KB to 50 KB in size can
be cloned. So, the broken chromosome is cloned in these vectors, and transformed into the
respective organism like; for example, if you take BAC and cosmid, you can use bacteria, and
then you get the colonies. Now each colony will have the required vector. Let us say if you
isolate a cosmid and since the cosmid sequence is known, you can design primers using the
flanking sequence, and we can use those Primers as a template for sequencing. So, you do not
need to know the insert sequence in the vector portion, using those vector-specific primers you
get some sequence information about the insert. Then you could either go by sequencing or take
smaller fragments from that insert, radiolabel it, and screen the library again to find newer
cosmid clones that light up with that probe. Like for example, here in the slide, if you take the
first orange one and below light blue one; now randomly I pick the orange piece, and in that, I
took one of the ends to say the right end, and I made a radiolabeled probe and screened again that
cosmid library. Then I find a new one that matches with the probe and then I go and take the
other end of that new one and search for another piece and so on. Since they are overlapping I
will be able to find all the DNA pieces and this is called chromosome walking. So, you walk
along the chromosome in terms of identifying the cosmic clones that are sequentially arranged
along the length of the chromosome. And then you make a map where cosmid 1 is followed by
cosmid 2 followed by cosmid 3 and so on and that is a physical map. So, what is shown here in
the slide is a physical map.

So, now in our mapping approach, we know that cn 9 map units right of black, so now you need
to use the physical map to find in which cosmid that map unit 9 is present. So, for that again you
need to know the known markers physical positions. And once you know that you jump from the
genetic map to the physical map. So the first step, correlation of the genetic and physical maps
near the gene of interest. For that essentially you need to know the physical map. If you know
those two cosmids, so let us say black is in one cosmid and vestigial is in another cosmid. Then
you know your gene is within these cosmids. Then you can do DNA mediated transformation
where you are going to take each one of these cosmids and inject, introduce into the organism,
let us say here in C. elegans you can inject those cosmids. And then see which one rescues the
mutant phenotype because these are made from the wild type organism so wild type copy of that
gene is present in those cosmids. Now you introduce the cosmids and then see which one
rescues.
(Refer Slide Time: 17:26)

So now let us take an example that we saw earlier, so we know the let-60, the dominant allele
that was found in the screen. So, the genetic mapping that is the two-factor, three-factor analysis
put let-60 between dpy-20 (dpy-20 is a known marker and its physical map is known, the cosmid
number is C35H3) and the left breakpoint of nDF27. nDF27 is a deficiency; deficiency means
larger deletions of chromosomes instead of point mutations or a few 100 bases of deletions. They
are often used to determine whether an allele that you got is a null allele or not. So you can
consider it as another known gene like dpy-20 its genetic position is also known, this cosmid
number is F58B3.
(Refer Slide Time: 18:33)

Now you take all the cosmid between dpy-20 and nDf27. So in the slide it just the representative
list of cosmids and all the cosmids numbers between the two are not mentioned. So you inject the
cosmids and see which one of the injected one rescues dpy-20 and let-60. So when you inject
cosmid C35H3, you find it rescuing dpy-20 but not let-60. So, somewhere in that cosmid, dpy-20
is covered but not let-60. Then inject the next one that is C10F4 and then you find it rescues
both. This tells that let-60 is present left to dpy-20. And that gets confirmed when you go to the
next one C33B3 which covers that full region and that also rescues. And then when you go
further that is ZK205 you lost the dpy-20 portion but then you have the portion where let-60 is
present and therefore let-60 is rescued. So, then you get to know where the let-60 gene sequence
is present. So, now you take C10F4 cosmid and look at its left side and check what kind of open
reading frames are there, how many are there, and then you inject each open reading frame and
then you will find the actual let-60 portion.
(Refer Slide Time: 19:45)

So in this particular case when they sequenced the open reading frame that rescued, they found it
encoded a protein that was known already as RAS. Earlier RAS was known only as an oncogene
where its expression caused cell proliferation but people did not know what it normally did. And
this finding revealed that RAS is involved in normal growth and development. So the loss-of-
function allele of let-60 was lethal. So it is a recessive lethal and it is essential for embryonic and
larval development. So therefore RAS is not going to cause cancer all the time, without it you are
not even going to live; only when its expression is out of control you get cancer. So, that is how
they discovered the growth and development role of RAS. So this gives you an idea of how you
would map a gene. So, this is not the only way of identifying the function of a gene; there are
multiple ways.
I will give you an example, what if during the cosmid rescue you find that the DNA injection for
whatever reason is not rescuing and the fragment of the chromosome that you have taken as an
open reading frame or as a cosmid insert need not always express when taken out of the
chromosome context. So, how do you proceed further? So, one approach is you can use an
antisense RNA. So, whatever is the copy of normal mRNA now you are suspecting as a
particular open reading frame, for that particular open reading frame you make antisense RNA
and inject it. So during the 90’s period, this was a common practice. So, in plants even they made
commercially grown crops where antisense RNA expressed against a particular polysaccharide
hydrolyzing enzyme allows the tomato to mature but not to ripen because when the
polysaccharide is degraded you get monosaccharides which make it sweet and then it becomes
softer and ripened tomato. So they used antisense RNA to block that particular enzyme’s
expression, the polygalacturonase and it worked, it is proven and it is commercial. So, in C.
elegans if you remember the embryonic division, the first division is asymmetric; one cell is
larger and another cell is small. So, there was a lab that focused on finding out genes involved in
this asymmetric division. They mutagenized the worms and looked for mutants, like the way our
mutagenesis screen was done, only difference is instead of looking for a bag of worms they were
looking for dead embryos. Once they found dead embryos, they did a detailed observation, like
what we did by looking at the Pn.p fate whether they become primary secondary, etc., they
looked at when does the embryo die, at how many cells stage, then they were looking for
whether that dead embryo allele was having problem in the asymmetric division. So they found
one gene that when mutated led to symmetrical division, the first division. So, they did this
standard experiment, using the cosmid to look for rescue and it did not work. So, they suspected
an open reading frame and they decided to make antisense RNA and inject it and it worked. So,
in the wild-type, blocking that one particular ORF by giving antisense led to symmetrical
division. So they were convinced they mapped the actual gene. But then usually people do a
control, control is sense. So, if you inject the sense RNA you should not get that effect. So, if you
get that effect then the effect is nonspecific. Maybe just injuring the gonad caused that effect, it
could be anything or any RNA sequence could do that. So, they did the sense injection and sense
also had the same phenotype. So, they were worried and that is when Andy Fire, who worked in
Phillip Sharp’s lab(the lab that won Nobel Prize for discovering splicing), who knew very well
about RNA chemistry reasoned out that when you are making single-strand RNA you are
probably making trace amounts of double-stranded RNA and that is probably somehow
interfering. That is how RNAi was discovered. So, while simply mapping a gene and while
trying to fix an experimental artifact they discovered a new phenomenon. Then they decided to
make double-strand RNA and inject it intentionally instead of having trace contamination and it
worked. So this happened in 98.
So, this tells there are multiple ways of mapping a gene, you could either block it or you could
rescue it.
(Refer Slide Time: 25:45)

So, now we are moving away from mapping. So, we will revisit a little bit of mapping to the very
end when we have a complex screen. So, the next is once you find the loss-of-function allele of a
gene and its role in a particular function, like for example, in this example, we are considering
multi-vulva(Muv), now you want to find out additional genes involved in it. So, you still do not
have the confidence that by crushing the worm and making it into a lysate you will be able to
purify the protein that is involved in this function. So, you do not still put a lot of weightage on
that technology and you want to still rely on genetics. So now what you do is, you take the
mutant and mutagenize and screen for new mutants in which the old mutant phenotype is lost.
Meaning can I make a new mutation and rescue the old mutant phenotype back to wild-type and
they are called suppressor screens. So, here you do not do just 10000 gametes like you do not
simply screen 5000 mutagenize worms, here you go large number because you are looking for a
specific genetic interaction, a new mutation that rescues or suppresses the original phenotype.
This will show that these two genes must have an interaction. The mechanism is not known at
this point but they somehow interact. So that interaction in terms of the phenotypic outcome is
called genetic interaction. So, you want to find such genetic interactions. So, this method helps
you to find additional genes and that is why this is a powerful method.
So, here you take Muv/Muv, it is homozygous for that loss of function. The new mutation, the
new gene that you are looking for let us say it is wild-type here +/+. Now you mutagenize and
allow it to self by cloning single worms and repeat the same thing and look for in F2 where some
of them have the phenotype being suppressed. Since you are starting with worms that are
homozygous for Muv, and not doing any cross with wild-type, this Muv/Muv must remain
always, which means you should always get multi-vulva and the moment you do not get multi-
vulva then you found the new mutation that is suppressing multi-vulva. So it should be made
sure that the new mutation for this phenotype follows the Mendelian inheritance pattern like 3:1
for the single phenotype segregation and so on to ensure that you have hit a single gene that is
suppressing it. And here the key is to screen a large number. And if you are going to screen a
large number, the mutant phenotype should be readily visible. The difference between the rest
and the mutant should be obvious then only you can identify it. It cannot be like you had mount
of worms on a slide and go and sit in a microscope and look at the staining pattern of something,
then you will not be able to screen millions of them, in a plate if I have 10000 worms and if I
look at it, the mutant should distinct. So that is the key.
(Refer Slide Time: 29:43)

So, several suppressors were found from the screen among them are the two multi-vulva genes,
lin-15 and let-60. So, special alleles of new genes not recovered in the earlier screen because
they were lethal or null phenotype, have been recovered now. So, like people in our lab know
this because this is the only kind of screen we have done and we found a lot of alleles where if
you cause a total loss of function they would be lethal and a specific partial loss-of-function
would have allowed its function during the early development. Then in a special specific
situation, you find a defect particularly in the background of another mutation. So, you find those
kinds of different alleles having different effects. So you can imagine that an amino acid
substitution in one region versus another region might have different consequences on the
protein function. So that is how you get partial loss of functions. And sometimes you get new
alleles of known genes for example several suppressors of lin-15 multi-vulva mutations were
new alleles of let-60 with the vulva less phenotype. So, worms do not make vulva only when
they lose let-60 function during vulval development. If they lose it in embryonic development
then it results in larval lethal and that is why the term let, larval lethal is used. So they ended up
finding that some special alleles of let-60 made it not doing its job during vulval development so
therefore whether lin-15 is there or not it did not matter, let-60 stops the vulva from developing
and that is how it ended up suppressing lin-15. So, these are the kind of alleles that you get when
you do the suppressor screen. So, these kinds of variant screens there are many, so we are not
going to go through all of them because we just want to get some good idea of genetics to
continue on our development. So, this is just one example of a complex screen.
(Refer Slide Time: 32:03)

So after this sort of a screen they ended up discovering this for let-60, a complete loss means it is
lethal and antimorphic means dominant-negative like vulva less it does not allow other cells to
be in the uninduced fate and hypermorphic, if it is continuously on so it makes multi-vulva.
(Refer Slide Time: 32:32)

So that is a suppressor screen. So, here the original mutant gave a weak phenotype and not a
strong phenotype so we want to mutagenize and look for new mutants that gives a stronger
phenotype; or sometimes the original mutant did not have a particular phenotype, so we discover
because of some other phenotype defect. But when you combine with another mutant mutation in
another gene the double mutant gives a new phenotype. So those are called synthetic phenotypes.
And a genetic screen looking for that kind of phenotypes is called a synthetic screen. So, here I
am using an example that did not come from a synthetic screen. This is something that I did and
therefore it fits as a good example, so I am telling this. So here nos-1 and nos-2 are closely
related genes. So, in the wild-type when you have both of them it is fertile and it is totally fine.
And when you have a mutation in nos-2, let say the nos-2 expression is extremely low, the
activity in y-axis and nos-1 is fine it is wild-type and the worm is again fertile. Now you do the
reverse nos-1’s activity is reduced and nos-2’s activity is high and it is still fertile. Now you
bring both of them down, it is sterile. So this is an example of a synthetic phenotype. So in this
situation what you are going to say is, these two seem to function redundantly. So, like
suppressor mutations, synthetic mutation helps us identify genes that function in a single
pathway. So, redundancy is what you are discovering in this kind of an example. So, we stop
here, and in the next class we will go on to find the hierarchy or the sequence in which a given
set of genes function.

Like for example, we found 20 genes involved in vulval development. Two of them were multi-
vulva and out of which one was the dominant allele. So let us say we ignore those two. Then we
have 18 of them giving vulva less, now we do not know which one of them acts first and which
one of them acts as the 18th and which one of them is in a parallel pathway and so on. So we
want to find an order, that is doable using genetics and it is not always done using biochemical
methods.
In biochemistry, the order of genes, for example, we know A gets converted to B gets converted
C, and so on. That order was determined using epistasis analysis. So that is what we are going to
discuss in the next class, that is one and second to be fair there are non-genetic methods like
radioactive labeling of a specific atom. Like you label the particular carbon and then you see
where that carbon goes. Like for example in glucose if we label the 6th carbon, now in pyruvic acid we see what is there at the end of glycolysis. So, this radioactive tracer as well as this epistasis analysis is how all the biochemical pathways worked out but of course, biochemical pathways were not worked out by doing genetics in C. elegans or Drosophila. They were done mostly using E. coli bacterial genetics. But the basic rules apply in all organisms the same way.