Part 1: Central Dogma

Griffiths Experiment:

Involves mice and pneumococcus bacteria

Proved molecules can change properties of bacteria, but did not know exactly what kind of
molecules

Avery Et Al. Experiment:

Proved disabling DNA can prevent bacterial transformation from occurring, hence DNA has
a role in hereditary

Hershey-Chase Experiment:

Bacteriophage: a virus attaches to bacteria and injects genetic material into the cell,
releasing new generation of virus when the bacteria bursts but the protein coat of
bacteriophage remains outside the bacterial cell

T2 bacteriophage properties: reproduce quickly, and only made of DNA and protein coat

To check whether there was protein or DNA responsible for hereditary, they added
separately radioactive phosphate – only found in DNA, and radioactive sulfur 35 – only
found in proteins into the bacteriophage

Then they detected whether radioactive signal was inside or outside the bacteria by using
waring blender to separate bacteriophage virus from bacteria

Then they centrifuged, and bacteria sinks downwards in the form of pellets, while the
smaller size virus remains upwards in the supernatant

Results:

By checking if the pellet was more radioactive or the supernatant (since they knew all the
genetic material should have been transferred to the bacteria by the virus):

Sulfur: The supernatant would be more reactive. Most radioactivity remained with
bacteriophage and did not enter bacteria, less than 1% of the protein radioactivity was
found in the newly replicated viruses proving protein is not being inherited.

Phosphate: The pellet would be more radioactive. Much of the radioactive labelled DNA
was passed to the bacteria and the newly replicated viruses proving DNA is hereditary
material.
Replication of DNA:

Replication means making more DNA by making daughter cells

DNA structure: 2 strands twisted around each other in a double helix

Each strand is made up of 4 chemical bases (ACGT complimentary)

One strand runs from 3’ to 5’, while other runs from 5’ to 3’ and this tells how DNA is
replicated

Enzyme called helicase unzips the 2 strands and separates them by breaking hydrogen
bonds between them and a replication fork in formed

Single stranded binding proteins bind to keep the strand separated

The enzyme topoisomerase keeps the DNA from supercoiling

Each of the separated strand provides a template for replication and the enzyme primase
makes a piece of RNA called primer

Primer marks the starting point for construction of DNA

An enzyme called DNA polymerase 3 binds to the primer and makes new strand of DNA

DNA polymerase 3 can only add DNA bases in one direction (from 5’ to 3’) by moving with
the help of a sliding clamp so:

The 5’ to 3’ strand is called the leading strand; bases are added one after another

The 3’ to 5’ strand runs in opposite direction and is called lagging strand so bases cannot
be added one after another, so this strand is made in small chunks called ‘Okazaki
Fragments’ (these chunks are made in the 5’ to 3’ direction)

The enzyme exonuclease removes all the RNA primers from both DNA strands

DNA polymerase 1 enzyme then fills in the gaps left behind by DNA

The enzyme DNA ligase seals up both strands to form double continuous strand again

It is called semi-conservative because each DNA consists of one old, conserved strand
and one new one

Transcription in prokaryotes

From DNA to mRNA

Gene starts with a promoter region and ends with a terminator region

Promoter region has a recognition site for RNA polymerase to bind – this is where gene
expression is controlled by allowing or blocking the access to the site by the RNA
polymerase

Binding causes the DNA double helix to unwind and open

Then during elongation, the RNA polymerase slides down the template DNA strand in the
5’ to 3’ direction

In the mRNA, there is uracil instead of thymine, and it is complementary to template strand

The RNA polymerase stops working at the terminator region because the mRNA forms a
hairpin shape, stopping the RNA polymerase from working further

Now to process mRNA (for eukaryotes), modified guanine attaches at 5’ end and a poly-A
tail (bunch of adenines) attaches to the 3’ end

These attachments not only help in translation but prevent damage to stored information

The mRNA has sequence that are not coding for protein and make no sense, called introns
so they are spliced out of the mRNA so only exons (useful) are left

This results in a mature mRNA

RNA polymerase 1 and 2 deals with mRNA, and RNA polymerase 3 deals with tRNA

Translation:

Synthesis of protein from mRNA, contains codons to encode for specific amino acids

Poly-A tail exists on the 3’ end of the mRNA

Translation initiation begins when the small subunit of the ribosome attaches to the
modified guanine cap at 5’ and moves towards the initiation site

tRNA contains anticodon complementary to mRNA codons to which it binds

Typically, AUG is the first codon sequence

Attached to the end of the tRNA is the corresponding amino acid for AUG: methionine
The large subunit of the ribosome now binds to the peptidyl (P) site and the aminoacyl (A)
site

The first tRNA occupies P site while second tRNA enters the A site, and is complementary
to the second codon

Methionine is then transferred to the A site amino acid

The ribosome moves along the mRNA and another tRNA enters, and this process of
elongation continues

When a stop codon enters the A site, a release factor enters, translation terminates

Ribosome disassociates and newly formed protein is released

Reading Frames:

A way to divide the sequence of nucleotides

Open reading frame can be translated into protein, they are continuous stretch of codons
beginning at start codon and ending at stop codon

If transcription ceases before stop codon, incomplete protein is made

Example is sickle cell anemia, where one GAG codon has mutated to a GTG codon, hence
the transcribed mRNA will have a GUG codon

Hence, normally there is GLU amino acid, now there will be VAL amino acid

Long ORFs are used to identify protein coding regions in a DNA sequence

Part 2: Genetics
Monohybrid Cross:

Allele is a form of gene inherited from mother and father

If there is a dominant and a recessive allele, the dominant (H) characteristic will be shown
(e.g., HH, Hh)

If there are all recessive alleles, and no dominant allele, only then will the recessive (h)
characteristic be shown (e.g., hh)

Genotype is the genetic makeup of an organism

Phenotype is the physical traits of an organism

HH = homozygous dominant and Hh = heterozygous dominant – show same traits

hh = homozygous recessive

When two heterozygous dominants (Gg and Gg) are crossed, the result obtained is:

x G g

G GG Gg

g Gg gg

This shows that there will be 1 homozygous dominant, 2 heterozygous dominant and 1
homozygous recessive genotype (1:2:1 ratio)

3 dominant and 1 recessive phenotypes (3:1 ratio)

Dihybrid cross:

Two pairs of alleles

Gametes are sex cells – sperm in men and egg in women

Gametes only carry one allele for a gene

Mendel's Law of independent assortment: Two alleles for different traits are not linked

Example: Using Ss and Hh where capital letter is dominant, and small is recessive

Crossing an organism with HhSs and another having hhss

Combinations of parent 1: HS, Hs, hS, hs

Combinations of parent 2: hs only

x HS Hs hS hs
hs HhSs Hhss hhSs hhss
hs HhSs Hhss hhSs hhss
hs HhSs Hhss hhSs hhss
hs HhSs Hhss hhSs hhss
Genotype ratio: 4/16 HhSs, 4/16 Hhss, 4/16 hhSs and 4/16 hhss so 1:1:1:1 ratio

Phenotype ratio: 8/16 Hh, 8/16 hh, 8/16 Ss and 8/16 ss so 1:1:1:1 ratio

Gene Linkage

Crossing over occurs in meiosis to produce gametes, with unique information, different
from parent cells

This means that not all the alleles from a chromosome will be passed when a gamete is
fertilized

More DNA there is between two genes, more likely that crossing over exists between them

Homologous chromosome pair is replicated to form two chromosomes each having sister
chromatids (e.g., HhBb may form HHBB and hhbb)

During prophase 1 of meiosis, synapsis (overlapping) between homologous (non-sister)

chromatids may exchange genetic information to form genetically recombinant
chromatids

In metaphase 1, these genetically recombinant chromatids align on the center of the cell

Recombination:

Without crossing over, gametes aB or Ab are not possible

recombinant frequency = recombined gametes / total gametes x 100

AaBb x aabb

X without crossing over after crossing over

Possible
AB, ab ab AB, aB, Ab, ab ab
gametes
Predicted
AaBb + aabb (1:1) AaBb, Aabb, aaBb, aabb (1:1:1:1)
offspring
Recombinant
0% 50%
frequency

Example:
BbCc – 1000 since BbCc and bbcc exist in greater numbers, they are the parentals

Bbcc – 200 rf = 400 / 2400 x 100 = 16.67%

bbCc – 200

bbcc – 1000

By removing red letters common to all due to bbcc,

we get BBCc and bbCc,

if recombined they will form BBcc and bbCC - which are the parentals for BbCc

Sex Linkage

Carrier of a trait means it does not show, and is recessive

Men have XY chromosomes and women have XX chromosomes

Example: A disease is only carried by X chromosome where X H means hemophilia disease

is present and Xh means no hemophilia disease and Xh is dominant

The mother has XHXh and father has XHY

x XH Xh

XH X HX H X HX h

Y X HY XhY

Combinations present: A girl with disease (XHXH), a girl without disease (XHXh), a boy with
disease (XHY), and boy without disease (XhY)

Genotype vs Phenotype:

Genotype is the exact pairing of alleles, so if we have B and b, three genotypes are present:
BB, Bb, and bb
Phenotype is the expression of a trait, so if we have B and b, two phenotypes are present
because BB and Bb both will have the same dominant traits B showing up, while recessive
bb will have the b trait

Humans inherit 23 chromosomes from mother and 23 from father

Test Cross:

To determine the genotype of organisms with the same phenotype

We test cross an organism showing dominant phenotype (it can be Bb or BB) to an

organism showing recessive phenotype (bb)

x B B x B b

b Bb Bb b Bb bb

b Bb Bb b Bb bb

As seen by the table: crossing BB with bb gives all dominant phenotypes in offspring but
crossing Bb with bb gives 1:1 ratio of both phenotypes

If the cross yields 100% the same phenotype, parent is homozygous dominant (BB)

If the cross yields 50% of each phenotype, parent is heterozygous dominant (Bb)

Back Cross:

We purposefully cross two such organisms that a recessive phenotype we do not want will
not show up

Example: We have bb but do not want that phenotype in the next generation, so we cross it
with BB where B is the dominant phenotype

X b b

B Bb Bb

B Bb Bb
As seen, all the offspring will be heterozygous dominant, and the phenotype of the
dominant will show for all

Hence, the unnecessary recessive phenotype will not show

Mutations:

Mutation is a change in genetic material within the nucleic acid, but show their effect at
protein level

Anything with RNA or DNA can have a mutation (Humans, Plants, Bacteria, Fungi, Archaea,
Viruses)

Mutations are random but factors like chemical exposure or problem in DNA replication
during interphase may cause them

Mutations that have no effect are called silent mutations. For example, if CUU mRNA
codon is written as CUC, it still codes for leucine amino acid and has no effect

Mutations are conservative when the new amino acid formed is the same type as what
should have been formed, not making a great problem (Glu and Asp)

Mutations are non-conservative when the new amino acid formed is different than what it
should have been (Ser (polar, small) and Phe (non-polar, large))

Mis-sense mutations are when a change in base leads to a different amino acid forming

Non-sense mutations are when a change in base leads to the generation of a stop codon
instead of an amino acid – causing premature termination of polypeptide synthesis

Gene mutation can occur due to substitution (exchange of bases), addition (extra base)
or deletion (removal of bases)

Substitution causes point mutation, since different amino acid formed due to exchange

Insertion and deletion are more dangerous as they can affect all bases in the sequence
after them, and are reading frame shift mutations

Chromosome mutation can occur due to duplication (extra genes are copied), deletion
(some genetic material breaks off), inversion (broken chromosome segments gets
inversed) and translocation (fragment from one chromosome breaks off and attaches to
another)
Mutation can also occur when chromosomes do not separate correctly during meiosis.
Resulting in non-disjunction (one cell with extra chromosomes and one with less)

Example, in sickle cell anemia the gene coding for hemoglobin is mutated so:

Val forms instead of Glu amino acid, hence it is a point and non-conservative mutation

If offspring inherits two copies of anemic gene, one from each parent, they will have it

If offspring inherits one copy, they are a carrier since the gene is recessive

Post Translation Modification:

Help to regulate folding of protein, stabilization, and localization

Phosphorylation is when a phosphate is taken from GTP or ATP and added to the protein
by an enzyme called kinase (usually added to Serine or Threonine)

Phosphorylation changes how fast the protein acts – usually make them faster

Used in signal transduction, transcription, mediating metabolism, cell differentiation

Methylation is when a methyl group is added to the amino acid (usually Arginine and
Lysine), and it does not affect the charge on the molecule

Lysine can be methylated up to three times and Arginine two times

Glycosylation is when sugar or carbohydrates in a specific sequence or pattern is added

to the protein

N-linked glycosylation involves attachment of sugar to nitrogen, which is the amide

nitrogen of Asparagine (Asn)

O-linked glycosylation involves attachment of sugar to oxygen in amino acid

Part 3: Development
Dolly Experiment:

Clones are genetically identical and may be formed by asexual reproduction

Dolly, a sheep did not have parents, but 3 mothers: 1 was the unfertilized egg cell donor

The nucleus of the egg cell was removed

From sheep 2, an isolated somatic cell was taken, and nucleus was removed

The nucleus was then transferred to the oocyte from sheep 1, making a hybrid

Electric stimulus causes the cells to divide making a blastocyst

The blastocyst was implanted into the 3 rd sheep – surrogate

Dolly had the DNA of sheep 2 that had donated the DNA

Embryology:

1) Ovulation is when ovary produces egg after every menstrual cycle (The egg has a
surrounding layer called Zona pellucida)

2) in Fertilization, the sperm from the male fertilizes the egg

3) Day 1: A zygote – fertilized egg is formed which is diploid

4) Sperm and Oocyte DNA mix to form one cell that keeps on dividing rapidly through
mitosis, this process is called cleavage (number of cells increase but the overall structure
mass and size remains same)

5) Day 4: Merulla forms (consists of 16 cells)

6) Day 5: Merulla divides again to form blastocyst with 32+ embryonic cells

7) Day 7: Blastocyst goes to implant the uterine wall and sheds the zona pellucida layer

Differentiation in plant cells:

Differentiated cells are totipotent, meaning they can be used to generate any type of cell

Clumps of differentiated plant cells are grown in nutrient media where they lose their
differentiation

Then these dedifferentiated cells divide into mass cells called callus

Callus is planted in specialized medium with hormones and nutrients, allowing embryo to
form and develop

After transplanting it to soil, a fertile plant develops

Human development:

Humans have 23 stages of embryonic development

3 germ layers of embryo

The outermost layer is ectoderm, which forms skin, nervous system, hair, nails, sensory
organs, lining of mouth and anus

The middle layer is mesoderm, which forms muscles, bones, connective tissue, blood
vessels, heart, kidneys and contribute to body’s cavity lining

The innermost layer is endoderm, which forms epithelial linings of respiratory tract,
digestive tract, urinary bladder, reproductive system and contributes to formation of
glands

During organogenesis, these layers give rise to organ formation

Cell differentiation:

The process of generating specialized cells, in which cells ceases to divide and instead,
starts acquiring specialized properties and structural elements

It is irreversible, cell cannot revert once differentiated

It has 2 stages: specification, where alteration is still possible and determination where
the functionality of the cell is now fixed, and transcription begins

After differentiation, cell fate becomes visible as they start working differently

Cytokines and master regulator factors affect what differentiation results in

In mammals totipotency (ability to form any type of cell) exists in early embryonic stage

Summary of basic development stages order:

Fertilization - [sperm (23) + egg (23) = zygote (46)]

Cleavage – formation of multiple cells, establishing gene expression, formation of inner

mass cells

Gastrulation – no division, establishment of germ layers, cell division ends here

Organogenesis – germ layers make organs

Morphogenesis – cells organize and space themselves specially

Specialization of gene expression:

To make cells different from each other functionality wise through difference in protein
formats and gene expressions

Done by asymmetric cell division, which causes cytoplasmic segregation

Segregation affects cell communication, cells with receptors will get signals

Transcription factors will change gene expression

Hence, if cell does not have receptor, it will not get ligand signal, no transcription factor
and hence no gene expression (induction process)

Morphogen Gradient:

Diffusible biochemical molecules to determine the fate of a cell

Cells exposed to higher morphogen have different fates than cells exposed to lower levels

Different morphogen levels lead to different gene/cell expression

Drosophila Embryogenesis:

Anterior is front, posterior is back end of the fly

Upon fertilization, the mRNA gets translated to proteins to establish gradient

Bicoid mRNA and protein:

Maternal cells of the ovary transcribe the maternal gene Bicoid

Bicoid mRNA is transported to anterior end of egg and fixed there

Bicoid mRNA is associated with proper head formation

When translation occurs, the Bicoid protein forms a gradient with most concentration
around mRNA (at anterior end)

The Bicoid protein is a transcriptional activator of hunchback and represses caudal

Nanos mRNA and protein:

It is responsible for the posterior end patterning, and with the help of caudal, helps
determine the identity of each segment

It primarily works to repress hunchback

Hunchback mRNA and protein:

Hunchback mRNA is constant throughout the embryo from anterior to posterior

Hunchback protein makes a gradient throughout the embryo, but becomes less when
nanos levels increase

Caudal mRNA and protein:

Caudal mRNA is constant throughout the embryo from anterior to posterior

Caudal protein makes a gradient throughout the embryo, but becomes less when Bicoid
levels increase

Examples:

A Bicoid mutant fly would be headless since Bicoid is responsible for head formation at the
anterior end

If cytoplasm from the anterior end of an embryo is injected into the center of the mutant
embryo, the head will start growing in the center (because Bicoid exists at anterior end)

Gene cascade:

Maternal effect genes – Bicoid, Nanos, Hunchback, Caudal

Gap genes – transcription factors, define embryo gaps

Pair rule genes – divide these gaps into segments

Segment polarity gene – Associate anterior and posterior ends to these segments and
define their boundaries
Hox (Homeotic) genes – Define the role of each segment, also control animal
development by containing conserved DNA sequence called hox domain/homeobox

Thes hox domains bind to specific sequences or promoter regions of DNA to form proteins

Hox genes is called MAD box gene in plants

Transcription in eukaryotes:

For RNA polymerase to successfully bind to a eukaryotic promoter, transcription factors

must first assemble on the promoter

Basal factors come and bind to TATA box on the promoter, which is the initiation site

Transcription factor TF2D binds to the promoter

Other transcription factor joins in - coactivators

RNA polymerase binds to the anti-sense strand and starts transcription

Enhancer sites are located away from the promoter site on the DNA

Activator proteins bind to enhancer region, which causes the DNA to bend and meet the
transcription complex, increasing transcription

If a repressor protein comes and binds to the silencer region instead, gene expression is
stopped

Epigenetics:

All the cells in our body have same DNA sequences but different activated genes

DNA is wrapped around nucleosomes made of histone proteins

There are 4 types of histone proteins:

H2A, H2B, H3 and H4, of which the last 2 are more prone to be modified epigenetically as
they contain arginine and lysine residues

DNA methylation:

Cytosine is methylated, a promoter region binds to cause silencing effects which proves to
be an obstacle for the TATA binding protein
Hence, DNA transcription is stopped

DNA methyl transferase adds methyl while DNA demethylase removes it

During inheritance, DNA methylation patterns are conserved and transmitted

Histone methylation and demethylation:

Same mechanisms as that of acetylation

On histone 3 (H3):

K4 – Methyl activates DNA by making it accessible by unwrapping and making it

euchromatin (loosely packed)

K9 and K27 – Methyl deactivates DNA by wrapping it and blocking it from being accessed
by making it heterochromatin

Histone Acetylation:

H3K9 and H3K27 - Responsible for unwrapping, and making DNA accessible

Usually, DNA is negatively charged, and histone is positively charged

Adding negatively charged histone neutralizes the histone

This causes the attraction and interaction between them to decrease

Histone acetyl transferase enzyme transfers the acetyl group to the C terminal section of
histone

Deacetylation:

Responsible for wrapping DNA again

Histone deacetylase enzyme used

Chromatin is DNA and histone combined

Heterochromatins:

Tightly packed – silenced/non-functional

Euchromatins:
Loosely packed - active

X-inactivation:

Females have XX and males have XY chromosomes

In females, X-inactivation prevents cells from getting twice as many gene products

It is random and occurs in early embryonic stage

The inactivated X chromosome has been highly methylated to form a condensed version
called ‘Barr body’ and is heterochromatin, and as a result transcription is stopped

Hence, all transcriptions in females are due to 1 activated X chromosome

De novo synthesis:

Synthesis without relying on promoters