Module 2

Lecture 1 → From Genotype to Phenotype

Chromosomes
In Eukaryotes, we have chromosomes, where DNA is packaged with specialized type of
histones. There are 4 kinds of histones → H2A,H2B,H3 and H4. Two molecules of each
of these proteins make a histone octamer which is a pulley like structure. DNA is
wrapped around this octamer in twice, in total 146 base pairs DNA are wrapped around.
Altogether this structure is known as a nucleosome:

Hence, nucleosome is the fundamental unit of chromosomes in Eukaryotes. Multiple

nuclosomes are then attatched like beads in a string to form chromosome.
In Prokaryotes, such packaging isn’t present. The DNA is just present as a one big
circular chromosome.
Plasmids
Circular DNA molecules present along with the linear DNA inside prokaryotes. Plasmids
do not depend on the chromosomal DNA for replication since they have their own origin
of replication.

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 Genome
All the genes present in a cell.
The genome leads each unique organism to have a common as well as a distinct set of
proteins → Proteome

In a DNA, there are 10 base pairs for each turn. That is, whenever a sequence of 10
base pairs is covered, the DNA must have turned.

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 DNAs also have a major and minor groove which are essential for the proteins to
attatch.

The total length of DNA present in a single cell if we were to linearlize all of it would be 2
metres. While the nucleus is just 10 micrometres in diameter which shows the coiling,
supercoiling etc. of DNA is integral for it to fit so compactly.

Variation in Genotypes (specific combinations of genes) leads to variations in

Phenotypes (appearance)

DNA or proteins the hereditary molecule?

Bacteriophages

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 Normally, when we introduce bacteriophage ( a bacteria-eating virus ) with E.coili
(bacteria), bacteriophage

1. Attatches to the surface of the E.coli and injects its DNA

2. Viral genes take over the host’s synthetic machinery

3. After using the bacteria’s synthetic machinery to replicate many times, the
bacterium breaks open, releasing about 200 viruses.

Hershey-Chase experiment

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 Experiment:

They knew phosphate can be used to label the DNA since it contains a phosphate
backbone, while sulphur can be used to label protein since amino acids like methionine
and cysteine contain sulphur in them.
1. Label one set of Bacteriophage T2 with S-35 and other set with 32-P.These
bacteriophages are then used to infect separate sets of bacteria

2. After a short while, detatch the viruses from the bacterias by mixing them together.

3. Centrifuge the resulting mixture to separate Bacteria and Viruses to study their
resulting compositions.

Observations:
The bacterias being heavier, settle down (called the pellet) while the viruses settle

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 up(called as the supernatal fluid)
It was seen that when viruses were

i. S-35 labelled, most of the viruses were found to be labelled

ii. P-32 labelled, most of the bacteria were found to be labelled

Conclusion:
DNA must be the hereditary material since when the T2 viruses were labelled by P-32,
most of the bacteria were found to be labelled. This meant that the Virus had injected its
DNA which would later lead to the synthesis of viruses inside the DNA. Proteins were
not injected by T2, and hence they weren’t found to be present in the bacteria.

How is information stored?

In each cell with 46 chromosomes, there are 6.4 billion base-pairs of DNA!

DNA contains all the information in the form of its specific base sequences which then
code for proteins.
It is due to the two DNA strands running antiparallel which allows for Hydrogen bonding
as well as helical structure of the DNA.

How is information copied and transferred on?

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 However, this central dogma can be violated in cases of viruses. For example, HIV
normally has RNA instead DNA. The RNA has to be converted to DNA first and then
back to RNA which then forms the protein.

Once again, remember:

DNA/RNA syntehsis always occurs in 5’→3’ direction
Protein synthesis always occurs N→C terminal

DNA replication is SEMI-CONSERVATIVE → replication produces molecules with both

old and new DNA, but each molecule would contain one complete old strand and one
new one.

DNA replication

Nucleotides are added to the growing end. The enzyme DNA polymerase III adds the
next nucleotide to the OH group at the 3’ end of the growing trand.

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 However, to first initate the replication process we need primers. Primers are short
segments of RNA which are synthesized in a 5’→3’ fashion by the enzyme Primase.
The DNA pol III then starts replication of DNA right after where the RNA primer ends.

Alright so, when we initate DNA replication of a double strand, we first need to break the
Hydrogen bonds between the two strands which is done by helicase.
Meanwhile, along the two strands the replication process begins. We know that
synthesis can only take place in the 5’→3’ fashion, so our newly forming DNA is easily
formed complementary to one of the strands which runs 3’→5’. However, for the other
strand running 5’→3’ our synthesis of DNA would have to run 3’→5’ if it were to go
along in the direction the DNA is being unzipped, but as we know, this doesn’t happen.

To cater this ordeal, synthesis along the original strand running 5’→3’ occurs in the
opposite direction to that in which the DNA is being unwinded, that too in short pieces
which are called the ‘Okazaki’ fragments. This strand that is formed is called the

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 discontinuous strand since it’s formed in segments which are then joined together.
Here too, a short primer is first formed with the help of primase, after which the DNA is
segment starts forming right after it with the help of DNA pol III until it encounters the
primer on the previous okazaki fragment (or until the end of the strand)

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 Now to remove these RNA segments on DNA, DNA pol I comes in, hydrolyzes the
primer and replaces it DNA.
And lastly, DNA ligase comes in and joins the two okazaki fragments together by
catalyzing the formation of the phosphodiester bond. (this too runs in the 5’→3’
direction).

DNA proofreading

Chance of an incorrect base being added is 1 in a million. (slides say 1 change per 1
billion base pairs of DNA????)

In short, either an incorrect base might’ve been added or a base could’ve been
damaged.
1. It could either be removed excised during the DNA replication process by the proteins
of the replication complex, and the correct base would be added by DNA pol and
replication proceeds.
2. Mispaired DNA is excised by mismatch repair proteins and some adjacent bases,
while DNA pol adds the new bases.
3. Damaged base is excised by excision repair proteins along with some adjacent
bases, while DNA pol adds the correct bases.

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 It’s even more complex in reality

Takeaway points from this:

Linear DNA has multiple origins of replications, circular DNA ha only one.
Uncoiling of the complex DNA forms is done by topoisomerase

Lecture 2
Transcription

Sense strand/coding strand→ running 5’→3’

Anti-sense strand/non-coding strand → running 3’→5’
mRNA that is eventually formed is identical to the sense strand except for the usual
DNA RNA differences.

How do we know which segment of DNA can be decoded into a protein?→ Promoter
Promoter region in a DNA strand is a very special sequence that is recognized by RNA

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 polymerase. This promoter region has a specific TATAA sequence at ‘-10’ position
which is commonly called as a TATAA box.

So, RNA polymerase recognizes this promoter region after which it starts moving
upstream. Just after the promoter sequence ends, there comes a Transcription start site
(TSS) and it is given a position +1, and it is this site which determines the positions of all
other sequences. Anything downstream (i.e. towards the 5’→3’ direction of the
Transctiption start site.) has a positive position while those upstream (towards the
promoter region i.e. 3’→5’ position) ar given a negative position.

The synthesis of mRNA starts from the +1 site i.e. the TSS site in the 5’→3’ direction.

Similar to the TSS, there comes a Transcription termination site (TTS) downstream of
TSS

Moreover, there are specific start and stop codons. Start codons are present just
downstream of TSS, and stop codons are present just upstream of TTS.

(Which strand is going to be anti-sense and which is sense depends on which of the
strands do we need to copy, and so in which direction is the RNAP moving. The RNAP
will always moves 5’→3’ direction, so the anti-sense will always be the one that is
3’→5’)

RNA polymerase recognizes the promoter sequence and initates its job, however RNAP
itself is controlled by several other Transcription factors
Note: no primer is needed in transcription.

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 In short, transcription has 3 phases→
1. Initation → RNAP recognizes the promoter sequence and begins to synthesize DNA
from the +1 TSS position.
2. Elongation → RNAP keeps adding complementary nucleotides in the 5’→3’ direction
3. Terminantion → Transcription stops whenever RNAP hits the TTS.

Therefore, the requirements for transcription were:

1. rGTP,rCTP,rATP,rUTP (rNTPS)
2. RNA polymerase (RNA pol II)
3. DNA
No primer or primase was required as in the case of DNA replication.
Introns and Exons
In Prokaryotes, mRNA is formed as explained and it moves on to be translated into a
protein.
In Eukaryotes however, there are certain segments which are non-coding which we call
as Introns while the coding segments are called Exons.
The introns are removed from mRNA after it is formed through a process called
splicing, and only all the axons are joined together to form a mature mRNA which then
moves onto translation.

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 So in prokaryotes, translation and transcription occur simultaneously since no splicing is
required, and the procedure is called Co-transcription-translation.

Memorize:

Open Reading Frames (ORF)

Depending on which frame you choose, the triplets and their corresponding amino acids
can change as well. For instance, see this

Normally, the frames are decided with the advent of AUG that appears as the
Translation start site, after which each subsequent triplet is considered to code for an
amino acid.

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 Translation

In translation, the Translation initiation site is usually a start codon i.e. AUG (Methionine)
and the Translation initation site is usually a stop codon i.e. UAG,UAA, UGA.

In total there are 43 = 64 options of total triplets to be formed from the 4 bases. That
said, since multiple triplets can code for the same amino acids, naturally only 20 amino
acids exist.
mRNA after copying the required info from the DNA inside the nucleus, moves out into
the cytoplasm through the nuclear pore and arrives at a ribosome where the translation
process starts.

Transfer RNA (tRNA)

tRNA is just another mRNA but is folded in such a way that its base pairs form
Hydrogen bonds with each other to form a ‘clover-leaf’ model. (tRNA has a secondary
structure).
RNA pol III is used to form tRNA
This tRNA has some important sites:
1. Amino acid attatchment site which always has the code CCA
1. anticodon site→ the triplet code specific for an amino acid that is attached to it, and is

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 complementary to the codon on the mRNA whose triplet interacts with this anticodon on
tRNA

Before tRNAs can go and attatch to a ribosome as well along with mRNA, they need to
charged first i.e. the amino acid they code for needs to be attatched to them before they
proceed.
This is done by an enyme tRNA synthase.

1. First of all, the amino acid that needs to be attatched to its specific tRNA is
activated. So the enzyme catalyzes a reaction whereby an AMP-amino acid
molecule is formed with the help of ATP. A pyrophosphate (the 2 P atoms released
from ATP) too is released, which then converts into 2 inorganic phosphate.

2. Next, the tRNA specific to that amino acid and enzyme arrives at the active site and
the enzyme catalyzes a reaction whereby the amino acid is attatched on the CCA
site of the tRNA, while the AMP is released.

3. The charged tRNA with its amino acid is now ready to move towards ribosome.

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 As stated earlier, both mRNA and tRNA now arrive at the Ribosome
Ribosome
RNA pol I is used to form rRNA which then plays its part in the formation of ribosome.
Note the structure and functions of the 4

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 Initiation

Now, the small subunit first recognizes the ribosome binding site on mRNA, which are
all the sequence on mRNA before the AUG start codon. This sequence is called as an
untranslated region.
The small subunit then moves a bit, recognizes Methionine(AUG), and now the large
subunit binds.
The large subunit attatched in such a way that the Methionine carrying tRNA is placed
on the P site of the ribosome.

Note that the interactions b/w tRNA and mRNA occur only at the P and A sites.

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 Elongation
So by now we have a Methionine carrying tRNA at the P site. Now a new tRNA
complementary to the mRNA codon at the ribosome A site arrives at the A site with its
amino acid. The two amino acids carried by the two tRNAs side by side are linked
together by a peptide bond formation with the help of peptidyl transferase. With this
done, the tRNA which at the P site leaves free. The ribosome now moves forward such
that the tRNA initially at the A site is now at the P site. Again, a new charged tRNA
arrives at the A site and the process repeats.

Terminantion
Elongation continues until there’s a stop codon on mRNA at the A site. At this point, a
release factor binds to the complex and frees the last tRNA on the P site to which all the
polypetide chain is connected. The tRNA now leaves, and so do the remaining
components mRNA and ribosomal subunits separate.

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 Note: At the same time, multiple ribosomes could be attatched to the mRNA!
Notice ribosome is also moving 5’→3’

Distribution of Newly Synthesized Proteins

Each protein has a specialized sequence within its amino acids which tells the cellular
machinery about its presence. The cell then tells the protein on where does it have to go
or what functions does it need to perform etc.

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 The proteins can then be transported to organelles, nucleus etc anywhere or even
Rough endoplasmic reticulum or Golgi apparatus for further modifications

Phosphorylation
Remembe from module 1, protein kinase domains are: Serine, Theronine, and Tyrosine
to which the gamma phosphate of ATP can be added with the help of Protein Kinase,
which can activate or deactivate the molecule.
Also, phosphate group can be removed with the help of protein phosphatase

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 (All the enzymes are proteins with a few exceptions)
The rates at which enzymes work differ between enzymes.
Mutations
Changes in the sequence of a gene.

1. Silent mutation → the change in sequence doesn’t impact the final polypeptide
chain in any way. This can happen since we know multiple triplets can code for the
same amino acid, so the mutation might be such that the triplet still codes for the
same DNA.

2. Missense mutation→ The change in sequence is such that the amino acid codes for
some amino (it now becomes any other triplet other than the original or the
start/stop codons). The length of polypeptide chain isn’t effected, but its function
might be.

3. Nonsense mutation → The change is such that a triplet in a reading frame now
codes for a stop codon. This causes the transcription process to halt middle-way. A
shortened polypeptide is now formed.

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 Lecture 3→ Genetics
Genes cause variation and genes dictate inherent properties of species.
Charachter: an observable feature, such as flower color
Trait→a particular form of charachter, such as white flowers
A heritable charachter is one that is passed from parent to offspring

Production and function of protein

Production and function of protein are dictated by an interaction
between genes and environment
• Any one gene may exist in several forms that
differ from one another- in small ways- Alleles
• Alleles of genes are designated by letter A, a etc.
• Allelic variation- Heredity variation- variation in
species
Predominant forms of genes are caled wild-types
Alternate forms of a gene are called alleles
There are mutations which change one allelic form to another- i.e. mutations are the
source of variation
In each cell (except reproductive) there are 46 sets of chromosomes, where 23 come
from mother and 23 from the father. Together the two pair of chromosomes (one from
mother, and the other from father which contain the same set of genes and so are
similar to each other) are called homologs. Though they have the same set of genes,
both can have different alleles for those genes.
Mendel’s monohybrid crosse

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 He crossed purple plants with white (i.e. cross-pollination), and analyzed the physical
charachteristic over 2 generations to see hereditary information is coming from both
plants.
Variation in Genotypes (specific combinations of genes) leads to variations in
Phenotypes (appearance)
Again, by crossing a plant with round seeds to that of wrinkled seeds, it was found that
in the first generation F1, only plants with round seeds were present. When two such
round seed plants were crossed again, it was found that the wrinkled seeds appeared
again. The rough phenotypic ratio b/w wrinkled and round seeds was found to 3:1. The
Genotypic ratio however was found to be 1:2:1

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 Therefore, he prepared a model that there are 2 factors which control the inheritance,
i.e. these factors exist in pairs.
Mendel’s first law of segregation states that only half the factor of genes is transmitted
(which was later found to be due to meiosis 1)

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 Crossing F1(heterozygous) with homozygous recessive is called a Test Cross
Crossing F1 to one of the homozygous plants is called a Back Cross

Mendel's First Law

the law of segregation; during gamete formation each member of the allelic pair

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 separates from the other member to form the genetic constitution of the gamete
Dihybrid crosses → Analyzing 2 traits at the same time

Crossing a Yellow winrkled with a Green round seed

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 So he found that 9:3:3:1 is the phenotypic ratio for a dihybrid cross. At the same time,
the two 3:1 hidden ratios show that the two different genes don’t interfere with the
inheritance of each other (i.e. the Law of Indepedent Assortment)
When Mendel discovered all this through his experiments, the scientific world was
unaware about genes, alleles, mitosis, meiosis etc. So later in the beginning of 20th
century when Meiosis was discovered, it was found to corroborate Mendel’s theory.
For Monohybrid:

For Dihybrid:
Important thing to note here is that we know in Meiosis I, the homologs separate. But
which combination of two different chromosomes which have a different set of genes is
going to form is random due to independent assortment (considering both the genes we
are considering are on different chromosomes)

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 But what if the two genes we are considering are on the same chromosome?
This time, we just expect two possible gamete compositions supposing our original
chromosome was RrYy i.e. R and y are on the same chromosome, and r and Y are on
the same. So the only two gametes that can be formed are Ry and rY.

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 That said, when we cross two F1 where the two genes are on the chromosome, you
would expect only the parentals to return the results are bit unexpected.
We crossed a wild fly (BbVv) having gray body, normal wings with a recessive(bbvv) fly
having black body and vestigial wings. If chromosomes were to assort independently,
you would expect:

But if we suppose B and V are on the same chromosome and so are b and v, i.e. the
two genes are linked, we would expect the following cross

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 In reality however, when we cross the two flies (BbVv and bbvv), we get the following
results

There are two takeaway points:

1. The 4 ratios are not same as we were expecting with indepdenet assortment, so it is
safe to say the results are closer to the non-independent assortment cross.

2. If you see closely, the parental pheno+genotypes are almost in a 1:1 ratio, as well
as are in majority when compared with the newly resulting genotypes that differ
from their parents. These differing types are known as recombinants

Henceforth, it was found that indeed, the two genes were on the same chromosome
which led to this deviation. However, the presence of recombinant types was explained
with the help of crossing over i.e. when homologous chromosomes pair in meiosis, the

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 chromosomes occasionally break and exchange parts in a process called Crossing over
The point where the two homologous chromosomes overlap is called the chiasmata.

Moreover, we can find the frequency of recombinants by dividing the number of

recombinants with the total number of F2 we have, and multiplying the ratio by 100. In
the case of flies as above, the frequency is given by:

This frequency represents the physical distance between the the two genes on the
same chromosome! In words, the 17% let’s say over here tells that out of 100 meiotic
event,s 17 times non-parental recmombinants will be formed. The frequency percentage
is given the units cM i.e. centi Morgans to represent the physical distance b/w the
genes.
In short, the recombinant frequency is inversely proportional to the distance between
the two genes that are linked(i.e. are on the same chromosome)

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 (Btw fruit-flies were chosen for these experiments since their lifecycle is of only 10 days
and so we can quickly observe the phenotypes)
Also, just remember to get the physical distance between the genes, we need (using
arbritary notations for alleles, where BV and bv are linked)
FO = BBVV x bbvv → BbVv
F1 = BbVv x bbvv → BbVv and bbvv
F2 = BbVv and bbvv (majority) and Bbvv and bbVv (minority, recombinants)
In short, keep noticing if F2 returns you the parental phenotypes in a manner that they
have a very similar ratio→easy identificiton of genes being linked.
Now onto impacts of genes residing on sex chromosomes
In each cell, out of the 23 pairs of chromosomes, only 1 is not identical and that being
the pair of sex chromosomes. Often on the Y chromosome, a gene is missing that is
otherwise present on the X chromosome. This fact leads to deviations from our
expected data. Consider the following cross for instance, where W represents the
dominant red eye colour, and w the mutant white colour. The gene for eye colour in this
case is present only on the X chromosome!

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 Moreover, there can sometime be abnormal inheritance of sex chromosomes. But these
different combinations can lead to different sexes in different organisms.

But why?
In humans, the gene responsible for development of male charachteristics resides on
the Y chromosome and is called the SRY gene. So in XXY, the presence of Y gives you
a male. Similarly, If X alone is present, a female forms.

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 In Drosphila, sex determinantion is dependent on the ratio of:
No. of X chromosomes/ No. of autosomal chromosomes.
If the ratio is 1, a female develops. If it’s 0.5, you get a male
A drosphila has 4 chromosomes altogether, 2 sex and 2 autosomal.
So in the abnormal case of XXY, there are 2 X chromosomes so 2/2 = 1 which gives a
female
In XO, the ratio is 0.5 so you get a male.

Incomplete Dominance
Another deviation. Everything we’ve considered uptil now were cases of complete
dominance, where atleast one of the alleles was dominant over the other and so
expressed itself even in heterozygous forms. However, that’s not always the case.
Sometimes, the two alleles are co-dominant due to which after the cross, a new
phenotype emerges.
Consider the following cross for instance. Here none of the R of red or r of white was
dominant. The resulting Rr ends up coding for a completely new phenotype which is
pink!

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 Differential Gene Expression in Development
If each cell contains the same number of chromosomes, what makes them different?

A diploid udder cell was fused with an egg cell from which nucleus was removed which
was then used for fertilization.
It was found that the diploid nucleus in a fully differntiated cell has not underwent
irreversible changed; it can be reprogrammed back to its inital state which was
totipotent.
However, it was not the original udder cell that was totipotent, but the resulting cell from
nucleus of udder cell in the egg was totipotent; this shows the cytoplast had certain
messages which could reprogram the udder cell into a totipotent cell!
What was noticed in this experiment was that a single cell fertilizied cell can give rise to
many different type of cells such that it can even form a complete individual.

Totipotency→When a cell type has the potential to give rise to the devlopment a
completely normal individual

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 Developmental Processes

1. Determination → determination of cell fates. Which cell is going to form the brain,
nerves, heart etc. etc. Determined cell fates are maintained generation after
generation. For instance let’s say a group of cells decides to become an eye. In
them, the eye specific genes will remain on, while those for other organs etc remain
off.

2. Differentiation

3. Morphogenisis → organization of cells in a specific 3-D order

4. Growth

Embryonic stem cells have the ability to differentiate into any different type of cells.
Differentiation → non-comitted cell giving rise to different type of cells. Differentiation
comes after determination.
Morphogenesis→ conc gradient resulting in different organs
Terminal commitment is the point where cells finally commit.
Metamorphosis → development after an egg has hatched.
Senescence → point after the cell has fully grown and divided, that is the point in old
people where cell division becomes very slow.

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 How is cell fate determined?

1. Cytoplasmic segrergation: asymmetrical cell division

2. Induction: cell to cell communication

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 What’s happening here is essentially that a cell produces an inducer. The diffusion
of the inducer forms such a concentration gradient that one cell receives alot of
such inducers on its receptors while the other doesn’t. Imagine cells on a line side
by side to visualize such a situation. Other explanations can be such that there
could be barriers for the inducer to the receptor of one of the cells which reduces
the amount of inducers getting onto the cell receptors.
Now due to the inducers, the cell receiving alot of them undergoes certain changes
such as the inducer might cause a transcription factor in the cell to activate, or might
the inducer signal might in turn send a signal to the nucleus through translocation.
These effects can then end up activating a segment of a gene which is then
transcribed to produce certain proteins resposible for differentiating the type of cell
that received the most inducers.
On the other hand, the cell that received very few inducers still has that gene
segment inactivated, and so is differentiated in some other way.
So here’s a distinction between two cells!
Consider another example,

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 Pretty self-explanatory. Imagine a source of morphogen towards the cell 1, and so
across the cells from 1 to 6 the morphogen concentration decreases. Different
concentrations of morphogen in each cell leads to different cell fates. Such signals
are called Paracrine signals since they are diffusible.
Several morphogens are transcription factors themselves, their concentration
deiciding

Now for a real-life example, check this

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 Here, the Morphogen’s name is Shh (Sonic hedgehog...) which is prouced by ZPA. The
thumb is formed with the lowest concentration of Shh, while the pinky finger is fomed
with highest concentration of Shh.
Lessons from Fly development

In the fertilizied egg in the very beginning, only the nucleus divides, not the cytoplast.
All these nuclei divide at the same time and start DNA replication, and the cycle
repeats.
While all these nuclei share the same cytoplast, we call this a Syncytial blastoderm.
Then comes a time that all these nuclei move towards the periphery such that you start
seeing grooves forming towards the periphery of the egg. It’s just the time when all
nuclei are going to form separate cells.
Now before we even proceed into gastrulation, from the very beginning, there were
morphogens present in the egg from the mother. Before the fertilization, the mother
deposits maternal genes in the egg, so when fertilization takes place, those
morphogens get activated and released.
By the time we reach gastrulation, we see the embryo becoming segregated into 14
segments. First 3 from the left form the head, the next 3 form the thorax, and the
remaining 8 towards the right form the abdomen.

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 Mother deopsits a layer of genes on the embryo which lead to activation of the zygote.
Only in the fertilized egg do these genes get activated.
Now explaining the morphogensis process in detail,
The egg has a maternal gene in the form of Bicoid mRNA present at the anterior end of
the egg.
There’s also another mRNA which codes for a morphogen named nanos at the
posterior side of the egg. Now whenever fertilization happens, these mRNAs are
translated into their proteins- bicoid at the anterior end and nanos at the posterior.
These two proteins act as morphogen and diffuse across the egg. Now as we said
eariler, the syncitial blastoderm is largely empty with only a number of nuclei inside.
Therefore, these proteins are able to diffuse freely.
Nanos and Bicoid proteins form gradients from their end to the next.

Varying bicoid concentrations lead to varying genes being activated in different parts of
the embryo which eventually leads to different body parts being formed from the same
set of cells.

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 Now as for nanos, after it gets diffused from the posterior to the anterior side, it also
alters the diffusion gradient of the hunchback protein that was formerly uniformly spread
across the egg.

All these varying morphogens arer determining different cell fates, even though the cells
haven’t yet committed to anything.

Bicoid, nanos etc. proteins are called Transcription Factors

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 What next?

1. Maternal effect genes as we saw earlier code for transcription factors like bicoid and
nanos which are diffused across the egg easily (due to absence of fully developed
cells being present inside the egg) and form a diffusion gradient.

2. Now based on the concentration of bicoid, nanos etc. the nuclei will activate certain
genes known as the gap genes which again are transcription factors that are going
to define broad areas in the egg. Absence of this gene’s expression somehwere can
even cause that certain gap to vanish!

3. Gap genes again are transiet, but before they go they end up activating pair rule
genes which are once again transcription factors. Pair rule genes are expressed in
alternating stripes, and it is due to pair rule genes that 14 separate segments of the
embryo are formed as we talked about earlier.
Two major pair rule genes are Eve and Ft2.
Eve is expressed in the odd segments (1,3,5,...13) while Ft2 is expressed in the
even segments (2,4,6,...14)

4. Once again, pair rule genes too disappear but activate segment polarity genes
before they vainsh.
By the time these genes are activated, the embryo has become Cellular

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 Blastoderm i.e. the nuclei present in the embryo as before in the Syncitial
Blastoderm have converted into cells. Now, diffusion gradient can no longer help us
with morphogenesis since cells are covered with their cell surface membrane!
So, many of the segment polarity genes are cell-signalling genes which send
signals to the individual cells inside the embryo.
These segment polarity genes give polarity within the segment. In each segment
let’s say, there’s an aneterior and a posterior compartment. There forms some sort
of an unknown compartmental boundary between the two due to which the cells of
both segments do not mix, even though they interact! So when further development
will take place, they will contribute to tissue and organs of the same fate, but due to
the different pattern they will differ just like we saw earlier in limb formation where
although the limb bud forms the hand, a certain region forms a thumb while the
other a finger.

5. Segment polarity genes now activate Hox genes (Master Regulatory genes/Cell
identity genes/selector genes)
They are the ones which finally Lock the specific cell type into what it’s going to
differentiate.

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 Note the inital region towards the left is called the Antennapedia complex and the
genes including and after Ubx are called the Bithorax complex
Moreover, we surprisngly found that the expression pattern of these hox genes is
identical in mouse and fruitfly!

Homeotic transformations → Cases where a certain body part gene is expressed

somewhere else, such as the genes that supposed to be activated in the leg region are
activated in the head region causing the formation of legs over there.

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 More onto Hox Genes

Homeobox 180 bp DNA sequences in all Hox genes, so whenever a hox gene is
translated, the polypeptide formed has 60 amino acids.

Homeodomain is encoded by homeobox region

Homeodomains bind to specific sequence in promoter regions of key development

genes in DNA, turning them on or off

Conserved in mammals and plants

Pattern formation in flowering plants

How Transcription is regulated in Eukaryotes?

How epigenetics contribute to Differential gene expression?
Basically, how do the genes that have been specified to stay on and off in certain
regions remain that way throughout mitotic divisions? For instance, the set of genes in
different cells is still the same, it is only the expression which is different, and as we
know, the expression of genes is inheritable.

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 Transcription

We have already went through this in detail, but just to revise a bit:

Promoter sequence is the region that is recognized by RNA pol II after which it starts
the formation of mRNA, downstream starting from the TSS
Introns were non-coding sequences in mRNA, and exons coding sequences. Introns
have to be removed by splicing after transcription.
Transcription terminantion sequences come just upstream of the stop codon
(UAG,UAA,UGA)
Enhancers/Regulatory elements : In addition to the promoter sequence, in the DNA
there can be other sequences either upstream or downstream of the gene which
positively or negatively impact the RNA pol II machinery which can either lead to
activation or repression of the gene.
The enhancers attract Activator proteins which can positively influence the RNA pol II to
start transcribing

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 Similarly, there can be repressor proteins which if bound on the promoter sequence can
stop the RNA pol II from transcribing

Note either the enhancer can direct these proteins towards RNA pol II to bring about the
desired effect, or these proteins can be attatched to the enhancer sequence after which
the DNA bends in such a way that the activator/represso proteins comes in direct
contact with the RNA pol II while staying connected to the enhancer

The TATA box on the promotoer region can also be recognized by TBP, TFIID, GTFs
other than by RNA pol II.

DNA methylation as Epigentic factor

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 One way to possibly turn off a gene would be to simply remove it→ but this is risky since
the cell might need it sometime in life.
A much convenient solution is methylation. That is, 5-methyly Cytosine is added in the
promoter region which turns the gene off no matter how strong the promoters are. This
is a chemical, covalent and heritable modification.
When methylation initally occurs with the help of DNA methylase, it happens
symmetrically on both DNA strands but when the DNA replicates, only one DNA has the
methylation.
However, this semi-methylated state is recognized at the replication fork by an enzyme
called as DNA mainetnance methylase which also methylates the newly synthesized
strand. Henceforth, the metyhlation is fully passed onto daughter cells.

Histone Modifications and gene expression

Another interesting discovery was the relation of repression/activation of genes on DNA
with the amino acids of Histone proteins (i.e. the Proteins forming the nucleosome
around which the DNA wraps)
First of all, they saw that individual amino acids can be methylated, acetylated,
phosphorylated etc. as well. Note: previously we were talking about DNA modifications,
not amino acids.

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 They discovered that generally, if in H4(histone of nucleosome) , K5,K8,K12 and K16
are acetylated, the gene associated on that nucleosome is turned on. Similarly,
deactylation correlates to the gene on that nucleosome being off. It was also discovered
that K4 methylation corresponds to acetylation of the mentioned amino acids on H4. So
both these states together are marks for gene activation.
Later they also discovered that wherever DNA will be methylated, H3’s K9 will be
methylated too. Together they correspond to repressed/silent gene.
Methylation of K27 on H3 is involved in silencing of Hox genes. To switch them on K27
is acetylated.
This Methylation of K27 or K9 is detected by proteins called Polycombgroup (PCG)
which are then responsible for silencing the gene, while methylation of K4 is detected by
proteins called Trithorax genes which then activate the gene.
(The numbering of amino acids is being done from the N terminal)

Epigenetic in development
A female has to X chromosomes while a male has 1 which could lead to a higher
expression of genes on X in females. To balance this out, one of the X chromosomes on
the female is inactivated.

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 The inactive sex chromosome is called the barr body.
There is present the Xist gene on the X chromosome, whose trancription forms
interference RNA. This RNA itself attatches o the X chromosome which formed it. Now
with the help of methylation and histone deacetylation, chromosomal proteins containing
heterochromatin are attracted which inactivate this X chromosome

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